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Understanding and treating the inflammatory adverse events of cancer immunotherapy

  • Author Footnotes
    5 These authors contributed equally
    Michael Dougan
    Correspondence
    Corresponding author
    Footnotes
    5 These authors contributed equally
    Affiliations
    Division of Gastroenterology and Department of Medicine, Massachusetts General Hospital, and Harvard Medical School, Boston, MA 02114, USA
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  • Author Footnotes
    5 These authors contributed equally
    Adrienne M. Luoma
    Footnotes
    5 These authors contributed equally
    Affiliations
    Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA

    Department of Immunology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA
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  • Stephanie K. Dougan
    Affiliations
    Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA

    Department of Immunology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA
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  • Kai W. Wucherpfennig
    Correspondence
    Corresponding author
    Affiliations
    Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215, USA

    Department of Immunology, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA

    Department of Neurology, Brigham & Women’s Hospital and Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA
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  • Author Footnotes
    5 These authors contributed equally
Open ArchivePublished:March 05, 2021DOI:https://doi.org/10.1016/j.cell.2021.02.011

      Summary

      During the past decade, immunotherapies have made a major impact on the treatment of diverse types of cancer. Inflammatory toxicities are not only a major concern for Food and Drug Administration (FDA)-approved checkpoint blockade and chimeric antigen receptor (CAR) T cell therapies, but also limit the development and use of combination therapies. Fundamentally, these adverse events highlight the intricate balance of pro- and anti-inflammatory pathways that regulate protective immune responses. Here, we discuss the cellular and molecular mechanisms of inflammatory adverse events, current approaches to treatment, as well as opportunities for the design of immunotherapies that limit such inflammatory toxicities while preserving anti-tumor efficacy.

      Introduction

      Over the past decade, immunotherapy has had an enormous impact on the treatment of diverse types of cancer, leading to durable remissions in a subset of patients and significantly extending survival for others. The most broadly effective of these therapies are monoclonal antibodies that block the immune checkpoints cytotoxic T lymphocyte antigen (CTLA)-4, programmed death (PD)-1 or its ligand PD-L1. Chimeric antigen receptor (CAR) T cells can provide profound efficacy against hematological malignancies. Alongside the tremendous clinical benefit of immunotherapy has come a diverse array of inflammatory toxicities that can affect any organ system in the body. These toxicities are an important cause of morbidity, frequently lead to treatment discontinuation, and can have debilitating long-term consequences. For checkpoint blockade in particular, risk factors for predicting these events have not yet emerged; the inability to predict who will develop toxicities that are severe (e.g., myocarditis) or permanent (e.g., autoimmune diabetes) is a challenge where immunotherapy is being developed as an alternative to established treatments (Figure 1). Inflammatory toxicities have also been a substantial barrier to the development of novel immunotherapies such as activating antibodies targeting co-stimulatory receptors and systemically delivered cytokine therapies. Concerns about severe on-target toxicity also substantially limit the choice of antigens targeted by CAR T cells in solid tumors. Here, we will discuss the inflammatory toxicities of all current major types of immunotherapies, specifically checkpoint blockade, adoptive T cell therapies, and cytokine therapies.
      Figure thumbnail gr1
      Figure 1Organs frequently affected by inflammatory toxicities of checkpoint blockade
      (A) Organs representing the most clinically important sites of inflammatory toxicities induced by PD-1/PD-L1 (left) or CTLA-4/combination (right) blockade.
      (B) Relationship between incidence and severity for organs affected by checkpoint inhibitor toxicities.
      (C) Potential factors contributing to susceptibility of checkpoint inhibitor toxicities.

      Adverse events triggered by checkpoint blockade

      Function of CTLA-4 and PD-1 inhibitory receptors

      Checkpoint blockade immunotherapy has transformed the treatment of multiple malignancies, extending survival, and in some cases, producing durable remissions (
      • Ribas A.
      • Wolchok J.D.
      Cancer immunotherapy using checkpoint blockade.
      ). T cells have a central role in the efficacy of checkpoint blockade based on their ability to recognize MHC-bound tumor cell peptides through the T cell receptor (TCR). Co-stimulatory signals through the CD28 and other receptors contribute to full T cell activation, whereas the co-inhibitory receptors CTLA-4 and PD-1 attenuate activation (
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ; Figure 2). The ligands of the CD28 and CTLA-4 receptors, CD80 and CD86, are expressed by activated antigen presenting cells, and CTLA-4 therefore provides an inhibitory feedback signal during the initiation of T cell responses in tissue-draining lymph nodes (Figure 2A). CTLA-4 thus appears to function primarily during early phases of a T cell response but may also inhibit T cell activation by dendritic cells within tumors (
      • Baumeister S.H.
      • Freeman G.J.
      • Dranoff G.
      • Sharpe A.H.
      Coinhibitory Pathways in Immunotherapy for Cancer.
      ).
      Figure thumbnail gr2
      Figure 2Modulation of T cell function by antibodies targeting the inhibitory CTLA-4 and PD-1 receptors
      (A) Biology of CTLA-4 receptor. The CTLA-4 inhibitory and CD28 costimulatory receptors bind to the same ligands (CD80/CD86) on antigen presenting cells in lymph nodes. CTLA-4 binds these ligands with higher affinity, thereby reducing ligand availability for CD28. CTLA-4 is stored in an intracellular compartment and transported to the cell surface following initial T cell activation, thereby serving as a negative feedback mechanism. Antibody-mediated inhibition of CTLA-4 function enhances T cell priming by making more CD80/CD86 ligands available for the CD28 costimulatory receptor. Also, the antibody prevents removal of CD80/86 from antigen presenting cells by Tregs.
      (B) Biology of the PD-1 receptor. PD-1 expression is induced by T cell activation, thereby providing an inhibitory signal that constrains T cell function. IFNγ secreted by activated T cells induces expression of PD-L1 on target cells (such as pancreatic β cells). This pathway inhibits autoimmunity and immunopathology. Antibody-mediated blockade of this pathway enhances T cell activation, resulting in greater cytotoxicity and release of pro-inflammatory cytokines.
      CTLA-4 is constitutively expressed by regulatory T cells (Tregs) (
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ). Although the full function of CTLA-4 in Tregs is incompletely understood, surface CTLA-4 on Tregs can remove CD80/CD86 molecules from the surface of activated dendritic cells, thus reducing the density of these ligands for the CD28 costimulatory receptor (
      • Qureshi O.S.
      • Zheng Y.
      • Nakamura K.
      • Attridge K.
      • Manzotti C.
      • Schmidt E.M.
      • Baker J.
      • Jeffery L.E.
      • Kaur S.
      • Briggs Z.
      • et al.
      Trans-endocytosis of CD80 and CD86: a molecular basis for the cell-extrinsic function of CTLA-4.
      ; Figure 2A).
      PD-1 functions in T cells through pathways that are largely non-redundant with CTLA-4 (Figure 2B). However, similar to CTLA-4, PD-1 expression on T cells is upregulated by activation, and the highest levels occur in T cells that have been repeatedly stimulated, including tumor-infiltrating T cells (
      • Baumeister S.H.
      • Freeman G.J.
      • Dranoff G.
      • Sharpe A.H.
      Coinhibitory Pathways in Immunotherapy for Cancer.
      ;
      • Zhao Y.
      • Lee C.K.
      • Lin C.H.
      • Gassen R.B.
      • Xu X.
      • Huang Z.
      • Xiao C.
      • Bonorino C.
      • Lu L.F.
      • Bui J.D.
      • Hui E.
      PD-L1:CD80 Cis-Heterodimer Triggers the Co-stimulatory Receptor CD28 While Repressing the Inhibitory PD-1 and CTLA-4 Pathways.
      ). The PD-1 ligands, PD-L1 and PD-L2, show distinct expression patterns. PD-L2 is primarily expressed on immune cells, whereas PD-L1 has a much broader distribution. PD-L1 is upregulated by a variety of inflammatory stimuli, including interferon (IFN)γ secreted by activated T cells, thus serving to limit T cell-mediated inflammation and autoimmunity (Figure 2B). PD-1 recruits SHP-2 phosphatase and thus inhibits early steps in T cell activation (
      • Baumeister S.H.
      • Freeman G.J.
      • Dranoff G.
      • Sharpe A.H.
      Coinhibitory Pathways in Immunotherapy for Cancer.
      ;
      • Hui E.
      • Cheung J.
      • Zhu J.
      • Su X.
      • Taylor M.J.
      • Wallweber H.A.
      • Sasmal D.K.
      • Huang J.
      • Kim J.M.
      • Mellman I.
      • Vale R.D.
      T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition.
      ).

      Overview of checkpoint blockade toxicities

      Checkpoint blockade induces the most diverse array of toxicities of any of the current immunotherapies in widespread clinical use (
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ; Figure 1A). These toxicities can present as single organ inflammatory diseases (e.g., dermatitis) or as systemic diseases affecting multiple systems. Interestingly, the precise distribution of toxicities varies widely from person to person, and although most toxicities occur within the first few months of treatment, they can manifest throughout the immunotherapy course (
      • Martins F.
      • Sofiya L.
      • Sykiotis G.P.
      • Lamine F.
      • Maillard M.
      • Fraga M.
      • Shabafrouz K.
      • Ribi C.
      • Cairoli A.
      • Guex-Crosier Y.
      • et al.
      Adverse effects of immune-checkpoint inhibitors: epidemiology, management and surveillance.
      ).
      Although involvement of nearly every organ system has been reported, checkpoint blockade toxicities most often affect barrier tissues such as the skin, gastrointestinal tract, and liver, and the respiratory epithelium, consistent with a fundamental immune regulatory role for CTLA-4 and PD-1/PD-L1 at these barriers, with CTLA-4 having a particularly critical role in the gut (
      • Dougan M.
      Checkpoint Blockade Toxicity and Immune Homeostasis in the Gastrointestinal Tract.
      ; Figure 1A). Outside of these epithelial barriers, most toxicities of checkpoint blockade occur in endocrine organs (
      • de Filette J.
      • Andreescu C.E.
      • Cools F.
      • Bravenboer B.
      • Velkeniers B.
      A Systematic Review and Meta-Analysis of Endocrine-Related Adverse Events Associated with Immune Checkpoint Inhibitors.
      ;
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ). Joint inflammation is also relatively frequent, affecting ∼10% of patients (
      • Cappelli L.C.
      • Gutierrez A.K.
      • Bingham 3rd, C.O.
      • Shah A.A.
      Rheumatic and Musculoskeletal Immune-Related Adverse Events Due to Immune Checkpoint Inhibitors: A Systematic Review of the Literature.
      ).
      Ipilimumab more frequently induces inflammatory toxicities than PD-1/PD-L1 inhibitors, and combination checkpoint blockade with CTLA-4 plus PD-1 antibodies has the highest frequency of toxicities (
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ; Figure 1B). GI mucosal, hepatic, and pulmonary toxicities are all reasonably common and lead to substantial morbidity, including the need to delay or discontinue immunotherapy, however, fatal adverse events involving these organs are uncommon (
      • Dougan M.
      Checkpoint Blockade Toxicity and Immune Homeostasis in the Gastrointestinal Tract.
      ;
      • Wang D.Y.
      • Salem J.E.
      • Cohen J.V.
      • Chandra S.
      • Menzer C.
      • Ye F.
      • Zhao S.
      • Das S.
      • Beckermann K.E.
      • Ha L.
      • et al.
      Fatal Toxic Effects Associated With Immune Checkpoint Inhibitors: A Systematic Review and Meta-analysis.
      ).
      Cardiac and neurologic toxicities from immunotherapy are rare but evolve rapidly and account for a substantial fraction of fatal toxicities from checkpoint blockade (
      • Wang D.Y.
      • Salem J.E.
      • Cohen J.V.
      • Chandra S.
      • Menzer C.
      • Ye F.
      • Zhao S.
      • Das S.
      • Beckermann K.E.
      • Ha L.
      • et al.
      Fatal Toxic Effects Associated With Immune Checkpoint Inhibitors: A Systematic Review and Meta-analysis.
      ; Figure 1B). Inflammation in endocrine organs is rarely fatal but often results in permanent organ dysfunction, necessitating lifelong hormonal supplementation that can impact quality of life (
      • de Filette J.
      • Andreescu C.E.
      • Cools F.
      • Bravenboer B.
      • Velkeniers B.
      A Systematic Review and Meta-Analysis of Endocrine-Related Adverse Events Associated with Immune Checkpoint Inhibitors.
      ;
      • Wang D.Y.
      • Salem J.E.
      • Cohen J.V.
      • Chandra S.
      • Menzer C.
      • Ye F.
      • Zhao S.
      • Das S.
      • Beckermann K.E.
      • Ha L.
      • et al.
      Fatal Toxic Effects Associated With Immune Checkpoint Inhibitors: A Systematic Review and Meta-analysis.
      ). Similarly, toxicities involving the joints can persist long after immunotherapy has stopped, causing pain, reducing mobility, and requiring extended treatment (
      • Cappelli L.C.
      • Gutierrez A.K.
      • Bingham 3rd, C.O.
      • Shah A.A.
      Rheumatic and Musculoskeletal Immune-Related Adverse Events Due to Immune Checkpoint Inhibitors: A Systematic Review of the Literature.
      ). Dermatologic toxicities are the most common toxicities seen with checkpoint blockade but these are often mild and generally respond to topical treatments (
      • Phillips G.S.
      • Wu J.
      • Hellmann M.D.
      • Postow M.A.
      • Rizvi N.A.
      • Freites-Martinez A.
      • Chan D.
      • Dusza S.
      • Motzer R.J.
      • Rosenberg J.E.
      • et al.
      Treatment Outcomes of Immune-Related Cutaneous Adverse Events.
      ).

      Mechanisms of checkpoint blockade toxicities

      Understanding the immune mechanisms driving checkpoint blockade toxicities will have important clinical implications, potentially leading to more targeted therapeutic strategies and methods for identifying high-risk patients prior to treatment initiation. These toxicities also represent an important window into basic immune biology: they are the phenotype of receptor blockade and thus reflect the homeostatic functions of CTLA-4, PD-1, and PD-L1. The ability to study these diseases as they unfold will provide insights into the cell types most directly regulated by these receptor pathways, as well as the downstream inflammatory pathways.

      Effector T cell response

      Multiple histopathologic analyses across a variety of organs have investigated the cellular infiltrates in checkpoint blockade toxicities, identifying an expanded population of CD8+ T cells and a smaller population of CD4+ cells (
      • Cohen J.V.
      • Dougan M.
      • Zubiri L.
      • Reynolds K.L.
      • Sullivan R.J.
      • Misdraji J.
      Liver biopsy findings in patients on immune checkpoint inhibitors.
      ;
      • Johnson D.B.
      • Balko J.M.
      • Compton M.L.
      • Chalkias S.
      • Gorham J.
      • Xu Y.
      • Hicks M.
      • Puzanov I.
      • Alexander M.R.
      • Bloomer T.L.
      • et al.
      Fulminant Myocarditis with Combination Immune Checkpoint Blockade.
      ;
      • Marthey L.
      • Mateus C.
      • Mussini C.
      • Nachury M.
      • Nancey S.
      • Grange F.
      • Zallot C.
      • Peyrin-Biroulet L.
      • Rahier J.F.
      • Bourdier de Beauregard M.
      • et al.
      Cancer Immunotherapy with Anti-CTLA-4 Monoclonal Antibodies Induces an Inflammatory Bowel Disease.
      ).
      In a detailed immune analysis of colon biopsies from patients who developed colitis following treatment with CTLA-4 or CTLA-4/PD-1 blockade, we identified a large population of proliferating, cytotoxic CD8+ T cells that were present in biopsies from colitis but not control patients (
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • Bowling P.
      • Hodi F.S.
      • Rahma O.
      • Sullivan R.J.
      • et al.
      Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.
      ; Figure 3). These CD8 T cells expressed IFNγ, granzyme B, as well as PD-1 and CTLA-4 receptors. Analysis of the clonal rearrangements of T cell receptor genes, which can serve as genetic barcodes to identify individual T cells and their progeny, indicated that the majority of expanded CD8+ T cells shared the same receptors as cells found within the tissue-resident memory cell population from the same patients (
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • Bowling P.
      • Hodi F.S.
      • Rahma O.
      • Sullivan R.J.
      • et al.
      Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.
      ). This finding strongly suggests that these expanded cells were, at least in part, derived from resident memory cells within the colon that were held in check by the CTLA-4 and/or PD-1 receptors and became reactivated following checkpoint blockade. Both colitis-associated CD8+ and CD4+ cells expressed CXCR3, the receptor for chemokines CXCL9 and CXCL10, both of which were highly expressed by colitis-associated myeloid cells. This finding indicates that additional T cells may indeed be entering the colon from the peripheral blood in response to inflammatory chemokine gradients. In this model, initial expansion of resident memory cells reacting to colonic antigens (such as microbial antigens) could induce an inflammatory response, resulting in release of chemokines and cytokines that recruit additional cell populations from the blood into the tissue (Figure 3). Whether similar resident memory cell expansion explains toxicity in other barrier organs such as the skin or lungs remains to be established.
      Figure thumbnail gr3
      Figure 3Inflammatory pathways contributing to colon inflammation following checkpoint blockade
      Tissue-resident memory T cells that may be specific for microbial antigens become reactivated following blockade of CTLA-4 and/or PD-1 inhibitory receptors. Reactivated Trm show elevated cytotoxicity, proliferation, and inflammatory cytokine (IFNγ) programs. Myeloid cells respond to IFNγ and other cytokines by amplifying the inflammatory response and recruiting T cells from the circulation, thereby overwhelming Treg-mediated suppression. Damage to colon tissue and loss of barrier integrity may result from T cell-mediated cytotoxicity and inflammatory cytokine signaling.

      Inhibition of Tregs

      In animal models, CTLA-4 targeting antibodies appear to induce antitumor responses through depletion of Tregs that express the highest level of CTLA-4 (
      • Simpson T.R.
      • Li F.
      • Montalvo-Ortiz W.
      • Sepulveda M.A.
      • Bergerhoff K.
      • Arce F.
      • Roddie C.
      • Henry J.Y.
      • Yagita H.
      • Wolchok J.D.
      • et al.
      Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma.
      ). Whether Treg depletion occurs in humans treated with anti-CTLA-4 antibodies is less clear and has remained a point of controversy (
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ). In colitis induced by CTLA-4 blockade, Treg populations were preserved or even expanded, even when biopsies were taken shortly after the onset of symptoms, indicating that Treg depletion is not the driving factor (
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • Bowling P.
      • Hodi F.S.
      • Rahma O.
      • Sullivan R.J.
      • et al.
      Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.
      ; Figure 3). Transcriptional changes consistent with exposure to IFNγ were found in Tregs from patients with checkpoint colitis (
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • Bowling P.
      • Hodi F.S.
      • Rahma O.
      • Sullivan R.J.
      • et al.
      Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.
      ). Whether the suppressive function of Tregs is impaired in checkpoint colitis remains unknown.

      Myeloid cells

      Analysis of single-cell RNA-sequencing (scRNA-seq) data also provides evidence for an important role of myeloid cells in colitis. The transcriptome of myeloid cells was altered in colitis, and myeloid cells showed transcriptional changes induced by IFNγ and tumor necrosis factor alpha (TNF-α) (
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • Bowling P.
      • Hodi F.S.
      • Rahma O.
      • Sullivan R.J.
      • et al.
      Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.
      ; Figure 3). TNF-α is likely to play an important role in driving checkpoint colitis based on clinical data (
      • Mooradian M.J.
      • Wang D.Y.
      • Coromilas A.
      • Lumish M.
      • Chen T.
      • Giobbie-Hurder A.
      • Johnson D.B.
      • Sullivan R.J.
      • Dougan M.
      Mucosal inflammation predicts response to systemic steroids in immune checkpoint inhibitor colitis.
      ). The scRNA-seq data also provide evidence for an involvement of other inflammatory cytokines, including interleukin (IL)-1β (
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • Bowling P.
      • Hodi F.S.
      • Rahma O.
      • Sullivan R.J.
      • et al.
      Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.
      ).

      B cells and antibodies

      The role of antibodies in contributing to checkpoint blockade toxicities is not well understood. Key antibody-dependent diseases such as systemic lupus erythematosus (SLE) do not seem to be mimicked by any of the common checkpoint blockade toxicities, nor are antibody-mediated diseases such as pemphigus particularly common in these patients, although some rare toxicities have been reported to respond to B cell-directed therapies (
      • Leonardi G.C.
      • Gainor J.F.
      • Altan M.
      • Kravets S.
      • Dahlberg S.E.
      • Gedmintas L.
      • Azimi R.
      • Rizvi H.
      • Riess J.W.
      • Hellmann M.D.
      • Awad M.M.
      Safety of Programmed Death-1 Pathway Inhibitors Among Patients With Non-Small-Cell Lung Cancer and Preexisting Autoimmune Disorders.
      ;
      • Phillips G.S.
      • Wu J.
      • Hellmann M.D.
      • Postow M.A.
      • Rizvi N.A.
      • Freites-Martinez A.
      • Chan D.
      • Dusza S.
      • Motzer R.J.
      • Rosenberg J.E.
      • et al.
      Treatment Outcomes of Immune-Related Cutaneous Adverse Events.
      ;
      • Shiuan E.
      • Beckermann K.E.
      • Ozgun A.
      • Kelly C.
      • McKean M.
      • McQuade J.
      • Thompson M.A.
      • Puzanov I.
      • Greer J.P.
      • Rapisuwon S.
      • et al.
      Thrombocytopenia in patients with melanoma receiving immune checkpoint inhibitor therapy.
      ). Although inflammation of the thyroid gland (thyroiditis) is often observed in patients treated with PD-1/PD-L1 inhibitors, the classic auto-antibodies seen in Hashimoto’s thyroiditis are not universally present (
      • de Filette J.
      • Andreescu C.E.
      • Cools F.
      • Bravenboer B.
      • Velkeniers B.
      A Systematic Review and Meta-Analysis of Endocrine-Related Adverse Events Associated with Immune Checkpoint Inhibitors.
      ). Similarly, the classic antibodies in autoimmune hepatitis are frequently absent in patients who develop checkpoint hepatitis, potentially because alternative antigens are targeted (
      • Reynolds K.
      • Thomas M.
      • Dougan M.
      Diagnosis and Management of Hepatitis in Patients on Checkpoint Blockade.
      ;
      • Tahir S.A.
      • Gao J.
      • Miura Y.
      • Blando J.
      • Tidwell R.S.S.
      • Zhao H.
      • Subudhi S.K.
      • Tawbi H.
      • Keung E.
      • Wargo J.
      • et al.
      Autoimmune antibodies correlate with immune checkpoint therapy-induced toxicities.
      ). One of the most dangerous toxicities from current checkpoint blockade is a neuromuscular inflammatory syndrome that resembles myasthenia gravis (
      • Safa H.
      • Johnson D.H.
      • Trinh V.A.
      • Rodgers T.E.
      • Lin H.
      • Suarez-Almazor M.E.
      • Fa’ak F.
      • Saberian C.
      • Yee C.
      • Davies M.A.
      • et al.
      Immune checkpoint inhibitor related myasthenia gravis: single center experience and systematic review of the literature.
      ). Some, but not all, of these patients have detectable anti-acetylcholine receptor antibodies suggesting a potential link to classic myasthenia gravis which is defined by these autoantibodies.

      Proposed functional subgrouping of inflammatory toxicities

      We propose a functional subgrouping of inflammatory toxicities based on the immune repertoire present in organs at the steady state: (1) epithelial organs with a local microbiota and large populations of tissue-resident T cells, and (2) sterile internal organs with limited T cell infiltration, including endocrine organs (Figure 4).
      Figure thumbnail gr4
      Figure 4Proposed functional subgrouping of inflammatory toxicities
      (A) Top, barrier organs colonized by microbiota harbor an abundant tissue-resident T cell (Trm) population that may recognize microbial antigens. Bottom, reactivation of Trm within barrier organs by checkpoint blockade represents an important step in the development of inflammatory toxicities.
      (B) Top, internal sterile organs tend to have smaller numbers of infiltrating T cells. Bottom, enhanced de novo priming of T cells specific for self-antigens and activation of latent autoreactive T cell clones may represent initial steps in the development of checkpoint blockade-induced toxicities in sterile organs, such as endocrine organs or the heart.
      Epithelial tissues are colonized by a diverse array of microorganisms and consequently are infiltrated by large populations of tissue-resident T cells (Trm) (
      • Masopust D.
      • Soerens A.G.
      Tissue-Resident T Cells and Other Resident Leukocytes.
      ; Figure 4A). The single-cell data from patients with checkpoint colitis demonstrate that the majority of colitis-associated CD8 T cells with highly proliferative and cytotoxic states originated from Trm based on use of T cell receptors as molecular barcodes (
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • Bowling P.
      • Hodi F.S.
      • Rahma O.
      • Sullivan R.J.
      • et al.
      Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.
      ). Tissue-resident memory T cells are an abundant T cell population in the gut mucosa where they act as first responders and form an integral part of an immune sensing network. They can exert cytotoxic function against infected cells and release cytokines/chemokines that rapidly activate surrounding immune cells and recruit additional immune cell populations from the blood (
      • Masopust D.
      • Soerens A.G.
      Tissue-Resident T Cells and Other Resident Leukocytes.
      ;
      • Rosato P.C.
      • Wijeyesinghe S.
      • Stolley J.M.
      • Masopust D.
      Integrating resident memory into T cell differentiation models.
      ). We hypothesize that the CTLA-4 and PD-1 receptors keep Trm populations in check, and loss of these inhibitory signal can result in rapid clonal expansion and conversion to a highly cytotoxic state (Figure 4A).
      Sterile internal organs have a limited repertoire of tissue-resident memory T cells, although such populations can be present due to prior infections at these sites (Figure 4B). Two different mechanisms could account for the development of these inflammatory toxicities: (1) activation of previously expanded self-reactive lymphocytes, and (2) priming of a de novo T cell response (Figure 4B). Not all self-reactive T cells escape negative selection in the thymus, but the frequency of such cells tends to be very low (
      • Wucherpfennig K.W.
      • Sethi D.
      T cell receptor recognition of self and foreign antigens in the induction of autoimmunity.
      ). Blockade of the PD-1 and/or CTLA-4 inhibitory receptors on T cells is likely to facilitate the activation and expansion of these rare self-reactive T cells. One of the major cytokines released by activated T cells is IFNγ that induces PD-L1 expression on target cells (
      • Alspach E.
      • Lussier D.M.
      • Schreiber R.D.
      Interferon γ and Its Important Roles in Promoting and Inhibiting Spontaneous and Therapeutic Cancer Immunity.
      ). This negative feedback loop is abrogated when cancer patients receive PD-1/PD-L1 blocking antibodies. Genetic studies in murine models have shown that PD-L1 expression on pancreatic islet cells inhibits tissue destruction by T cells, pointing to an important role of PD-L1 in the protection of parenchymal cells against immune attack (
      • Keir M.E.
      • Liang S.C.
      • Guleria I.
      • Latchman Y.E.
      • Qipo A.
      • Albacker L.A.
      • Koulmanda M.
      • Freeman G.J.
      • Sayegh M.H.
      • Sharpe A.H.
      Tissue expression of PD-L1 mediates peripheral T cell tolerance.
      ).

      Relationship between checkpoint blockade induced inflammatory toxicities and spontaneous autoimmune diseases

      We will consider the relationship between checkpoint blockade toxicities and spontaneous autoimmunity by comparing ulcerative colitis (UC) to checkpoint colitis (example of epithelial tissue infiltrated by tissue-resident memory cells) and autoimmune type 1 diabetes to checkpoint diabetes (example of a sterile organ with limited immune cell infiltration). UC and checkpoint colitis resemble each other clinically, although checkpoint colitis typically has a more rapid onset. Endoscopically, the diseases are indistinguishable, and although histopathologic characteristics of checkpoint colitis have been identified, they are not consistent enough to readily distinguish it from UC. scRNA-seq datasets were recently reported for both UC and checkpoint colitis (
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • Bowling P.
      • Hodi F.S.
      • Rahma O.
      • Sullivan R.J.
      • et al.
      Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.
      ;
      • Smillie C.S.
      • Biton M.
      • Ordovas-Montanes J.
      • Sullivan K.M.
      • Burgin G.
      • Graham D.B.
      • Herbst R.H.
      • Rogel N.
      • Slyper M.
      • Waldman J.
      • et al.
      Intra- and Inter-cellular Rewiring of the Human Colon during Ulcerative Colitis.
      ). In ulcerative colitis, an increase in IL17+ CD8 T cells was observed, whereas IL17 expression was unchanged in checkpoint colitis. The IL-23 receptor is essential for differentiation of IL-17 producing T cells, and polymorphisms in the IL23 gene are associated with susceptibility to ulcerative colitis (
      • Neurath M.F.
      IL-23 in inflammatory bowel diseases and colon cancer.
      ). Increased expression of IL-17 by immune cells in ulcerative colitis has also been demonstrated by analysis of biopsy specimens and serum samples. In contrast, expression of IFNγ by T cells and IFNγ inducible genes by myeloid cells were key aspects of the immune microenvironment in checkpoint colitis (
      • Luoma A.M.
      • Suo S.
      • Williams H.L.
      • Sharova T.
      • Sullivan K.
      • Manos M.
      • Bowling P.
      • Hodi F.S.
      • Rahma O.
      • Sullivan R.J.
      • et al.
      Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy.
      ).
      In both spontaneous type 1 diabetes (T1D) and checkpoint diabetes, the insulin-producing β cells are destroyed by an immune attack, necessitating insulin replacement therapy (
      • Pugliese A.
      Autoreactive T cells in type 1 diabetes.
      ). T1D occurs most commonly in childhood and progresses slowly, commonly over years, from pre-diabetes to clinically manifest disease, with residual islet function frequently still detectable at disease onset (
      • van Belle T.L.
      • Coppieters K.T.
      • von Herrath M.G.
      Type 1 diabetes: etiology, immunology, and therapeutic strategies.
      ). In contrast, in checkpoint diabetes, insulin-producing β cells are rapidly lost, and patients typically present with hypoglycemia, ketoacidosis, and no detectable β cell function (
      • Stamatouli A.M.
      • Quandt Z.
      • Perdigoto A.L.
      • Clark P.L.
      • Kluger H.
      • Weiss S.A.
      • Gettinger S.
      • Sznol M.
      • Young A.
      • Rushakoff R.
      • et al.
      Collateral Damage: Insulin-Dependent Diabetes Induced With Checkpoint Inhibitors.
      ). All patients who developed checkpoint diabetes had been treated with a PD-1/PD-L1 blocking antibodies or a combination of PD-1 plus CTLA-4 antibodies (
      • Stamatouli A.M.
      • Quandt Z.
      • Perdigoto A.L.
      • Clark P.L.
      • Kluger H.
      • Weiss S.A.
      • Gettinger S.
      • Sznol M.
      • Young A.
      • Rushakoff R.
      • et al.
      Collateral Damage: Insulin-Dependent Diabetes Induced With Checkpoint Inhibitors.
      ;
      • Tsang V.H.M.
      • McGrath R.T.
      • Clifton-Bligh R.J.
      • Scolyer R.A.
      • Jakrot V.
      • Guminski A.D.
      • Long G.V.
      • Menzies A.M.
      Checkpoint Inhibitor-Associated Autoimmune Diabetes Is Distinct From Type 1 Diabetes.
      ). Patients with an early onset of checkpoint diabetes may have already harbored previously expanded populations of islet-reactive T cells with checkpoint blockade enabling rapid reactivation and further expansion of such T cells; this may also be the case in other rapidly progressive toxicities such as myocarditis.
      T1D and checkpoint diabetes also differ in immunological parameters and immunogenetics. Although >95% of spontaneous T1D patients have autoantibodies against at least one islet antigen at diagnosis, autoantibodies were detected in only 40% of checkpoint diabetes patients (
      • Stamatouli A.M.
      • Quandt Z.
      • Perdigoto A.L.
      • Clark P.L.
      • Kluger H.
      • Weiss S.A.
      • Gettinger S.
      • Sznol M.
      • Young A.
      • Rushakoff R.
      • et al.
      Collateral Damage: Insulin-Dependent Diabetes Induced With Checkpoint Inhibitors.
      ). Susceptibility to spontaneous T1D is strongly associated with particular alleles of HLA-DR and HLA-DQ genes (
      • van Belle T.L.
      • Coppieters K.T.
      • von Herrath M.G.
      Type 1 diabetes: etiology, immunology, and therapeutic strategies.
      ). The number of investigated checkpoint diabetes patients is currently too small for a definite assessment, but 3 of 10 patients in one case series had HLA alleles known to provide protection from spontaneous T1D (
      • Tsang V.H.M.
      • McGrath R.T.
      • Clifton-Bligh R.J.
      • Scolyer R.A.
      • Jakrot V.
      • Guminski A.D.
      • Long G.V.
      • Menzies A.M.
      Checkpoint Inhibitor-Associated Autoimmune Diabetes Is Distinct From Type 1 Diabetes.
      ). Taken together, these findings establish important differences in kinetics and immunological parameters between these diseases.

      Approaches to treatment of checkpoint blockade toxicities

      Treatment for checkpoint blockade toxicities must be managed in light of the patient’s cancer diagnosis, and optimal treatment strategies should mitigate the toxicity while preserving anti-tumor activity (Figure 5). Optimal treatment strategies established by randomized, controlled trials are not available for any toxicity at present, and current practice guidelines are all based on retrospective analyses, experience from cancer treatment trials, and anecdotal experience (
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ). Nearly all toxicities from checkpoint blockade respond to high-dose systemic glucocorticoids, with the exception of dermatologic toxicities for which topical agents are generally sufficient and endocrine toxicities that are typically identified after loss of function by the target organ (
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ). In most cases, toxicities that resolved following immune suppression will not recur if immune suppression can be successfully tapered, unless patients are re-challenged with checkpoint blockade targeting the same pathway (
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ). How immunosuppressive glucocorticoids impact antitumor responses requires further investigation. Animal models suggest that glucocorticoids may inhibit antitumor T cell responses, and glucocorticoids are well known to induce apoptosis of activated T cells (
      • Acharya N.
      • Madi A.
      • Zhang H.
      • Klapholz M.
      • Escobar G.
      • Dulberg S.
      • Christian E.
      • Ferreira M.
      • Dixon K.O.
      • Fell G.
      • et al.
      Endogenous Glucocorticoid Signaling Regulates CD8+ T Cell Differentiation and Development of Dysfunction in the Tumor Microenvironment.
      ). Findings from retrospective studies of patients on checkpoint blockade who received systemic glucocorticoids have been mixed, although overall suggestive that they have deleterious effects on optimal antitumor responses, particularly when systemic glucocorticoids are used at initiation of immunotherapy (
      • Arbour K.C.
      • Mezquita L.
      • Long N.
      • Rizvi H.
      • Auclin E.
      • Ni A.
      • Martínez-Bernal G.
      • Ferrara R.
      • Lai W.V.
      • Hendriks L.E.L.
      • et al.
      Impact of Baseline Steroids on Efficacy of Programmed Cell Death-1 and Programmed Death-Ligand 1 Blockade in Patients With Non-Small-Cell Lung Cancer.
      ;
      • Faje A.T.
      • Lawrence D.
      • Flaherty K.
      • Freedman C.
      • Fadden R.
      • Rubin K.
      • Cohen J.
      • Sullivan R.J.
      High-dose glucocorticoids for the treatment of ipilimumab-induced hypophysitis is associated with reduced survival in patients with melanoma.
      ;
      • Horvat T.Z.
      • Adel N.G.
      • Dang T.O.
      • Momtaz P.
      • Postow M.A.
      • Callahan M.K.
      • Carvajal R.D.
      • Dickson M.A.
      • D’Angelo S.P.
      • Woo K.M.
      • et al.
      Immune-Related Adverse Events, Need for Systemic Immunosuppression, and Effects on Survival and Time to Treatment Failure in Patients With Melanoma Treated With Ipilimumab at Memorial Sloan Kettering Cancer Center.
      ). Nevertheless, systemic glucocorticoids are first line therapy for most severe toxicities where a rapid response is necessary to prevent significant morbidity and mortality.
      Figure thumbnail gr5
      Figure 5Potential therapeutic targets for inflammatory toxicities induced by checkpoint blockade
      Potential therapeutic agents for checkpoint inhibitor (CPI) toxicities are shown along with their potential impact on tumor immunity. The upper part of the figure illustrates therapeutic strategies that may not impair tumor immunity. The lower part shows therapeutic approaches that may alleviate inflammatory toxicities but also negatively impact tumor immunity.
      Strategies for management of toxicities that do not respond to glucocorticoids are derived from experience in treating autoimmunity and transplant rejection. These strategies fall into several mechanistic categories that will likely have different effects on antitumor responses.

      Cytokine blockade

      The acute inflammatory cytokines TNF-α, IL-6, and IL-1β play important roles in numerous autoimmune diseases, making them attractive targets for the treatment of checkpoint blockade toxicities. Of these, TNF-α inhibitors have been the most directly studied. Based on several retrospective studies as well as limited numbers of patients from cancer immunotherapy trials, blockade of TNF-α appears to be highly effective for treatment of glucocorticoid-refractory enterocolitis induced by either CTLA-4 or PD-1/PD-L1 inhibitors (
      • Dougan M.
      • Wang Y.
      • Rubio-Tapia A.
      • Lim J.K.
      AGA Clinical Practice Update on Diagnosis and Management of Immune Checkpoint Inhibitor (ICI) Colitis and Hepatitis: Expert Review.
      ;
      • Mooradian M.J.
      • Wang D.Y.
      • Coromilas A.
      • Lumish M.
      • Chen T.
      • Giobbie-Hurder A.
      • Johnson D.B.
      • Sullivan R.J.
      • Dougan M.
      Mucosal inflammation predicts response to systemic steroids in immune checkpoint inhibitor colitis.
      ). This is consistent with the established efficacy of TNF-α inhibitors in inflammatory bowel disease and indicates an important role for this cytokine in perpetuating inflammation in the gastrointestinal tract (Figure 5) (
      • Dougan M.
      • Wang Y.
      • Rubio-Tapia A.
      • Lim J.K.
      AGA Clinical Practice Update on Diagnosis and Management of Immune Checkpoint Inhibitor (ICI) Colitis and Hepatitis: Expert Review.
      ).
      The role of TNF-α in antitumor responses is unclear. In animal models, inhibition of TNF-α synergizes with checkpoint blockade, leading to enhanced CD8 T cell immunity (
      • Bertrand F.
      • Montfort A.
      • Marcheteau E.
      • Imbert C.
      • Gilhodes J.
      • Filleron T.
      • Rochaix P.
      • Andrieu-Abadie N.
      • Levade T.
      • Meyer N.
      • et al.
      TNFα blockade overcomes resistance to anti-PD-1 in experimental melanoma.
      ). Whether this is the case in humans is unknown; small retrospective studies of patients with colitis have shown no negative effect on overall survival, but a prospective cohort study from the Netherlands found a correlation between the use of TNF-α blockers and decreased cancer survival after immunotherapy (
      • Mooradian M.J.
      • Wang D.Y.
      • Coromilas A.
      • Lumish M.
      • Chen T.
      • Giobbie-Hurder A.
      • Johnson D.B.
      • Sullivan R.J.
      • Dougan M.
      Mucosal inflammation predicts response to systemic steroids in immune checkpoint inhibitor colitis.
      ;
      • Verheijden R.J.
      • May A.M.
      • Blank C.U.
      • Aarts M.J.B.
      • van den Berkmortel F.W.P.J.
      • van den Eertwegh A.J.M.
      • de Groot J.W.B.
      • Boers-Sonderen M.J.
      • van der Hoeven J.J.M.
      • Hospers G.A.
      • et al.
      Association of Anti-TNF with Decreased Survival in Steroid Refractory Ipilimumab and Anti-PD1-Treated Patients in the Dutch Melanoma Treatment Registry.
      ). This cohort study lacked information on the indication for TNF-α blockade, and precise data were not available on the clinical context (
      • Verheijden R.J.
      • May A.M.
      • Blank C.U.
      • Aarts M.J.B.
      • van den Berkmortel F.W.P.J.
      • van den Eertwegh A.J.M.
      • de Groot J.W.B.
      • Boers-Sonderen M.J.
      • van der Hoeven J.J.M.
      • Hospers G.A.
      • et al.
      Association of Anti-TNF with Decreased Survival in Steroid Refractory Ipilimumab and Anti-PD1-Treated Patients in the Dutch Melanoma Treatment Registry.
      ). Because most patients who receive TNF-α blockade also receive extended courses of high-dose glucocorticoids, this correlation may reflect high-dose glucocorticoid use rather than TNF-α blockade itself.
      Although IL-1β and IL-6 inhbition has not yet been adequately explored as treatment for checkpoint blockade toxicities, their success in treatment of autoimmunity suggests potential therapeutic benefit (
      • Esfahani K.
      • Elkrief A.
      • Calabrese C.
      • Lapointe R.
      • Hudson M.
      • Routy B.
      • Miller Jr., W.H.
      • Calabrese L.
      Moving towards personalized treatments of immune-related adverse events.
      ). This possibility is particularly intriguing for IL-1β, which has already been implicated in the promotion of lung cancer in humans (
      • Ridker P.M.
      • MacFadyen J.G.
      • Thuren T.
      • Everett B.M.
      • Libby P.
      • Glynn R.J.
      CANTOS Trial Group
      Effect of interleukin-1β inhibition with canakinumab on incident lung cancer in patients with atherosclerosis: exploratory results from a randomised, double-blind, placebo-controlled trial.
      ). In a secondary analysis of a phase 3 trial of the anti-IL-1β monoclonal antibody canakinumab designed to test secondary prevention of cardiovascular events in patients who had had a myocardial infarction, canakinumab treatment was associated with a dose-dependent reduction in incident lung cancer and lung cancer mortality (
      • Ridker P.M.
      • MacFadyen J.G.
      • Thuren T.
      • Everett B.M.
      • Libby P.
      • Glynn R.J.
      CANTOS Trial Group
      Effect of interleukin-1β inhibition with canakinumab on incident lung cancer in patients with atherosclerosis: exploratory results from a randomised, double-blind, placebo-controlled trial.
      ). The ability of IL-1β inhibition to synergize with PD-1 blockade is currently undergoing direct testing in clinical trials. IL-6 inhibition has shown efficacy in a limited number of immunotherapy toxicities (
      • Esfahani K.
      • Elkrief A.
      • Calabrese C.
      • Lapointe R.
      • Hudson M.
      • Routy B.
      • Miller Jr., W.H.
      • Calabrese L.
      Moving towards personalized treatments of immune-related adverse events.
      ), and elevated IL-6 correlates with worse tumor outcomes in patients treated with checkpoint blockade, potentially indicating a deleterious role for this cytokine in antitumor immunity (
      • Keegan A.
      • Ricciuti B.
      • Garden P.
      • Cohen L.
      • Nishihara R.
      • Adeni A.
      • Paweletz C.
      • Supplee J.
      • Jänne P.A.
      • Severgnini M.
      • et al.
      Plasma IL-6 changes correlate to PD-1 inhibitor responses in NSCLC.
      ). This is consistent with several mouse models that have shown synergy between checkpoint blockade and IL-6 inhibition (
      • Esfahani K.
      • Elkrief A.
      • Calabrese C.
      • Lapointe R.
      • Hudson M.
      • Routy B.
      • Miller Jr., W.H.
      • Calabrese L.
      Moving towards personalized treatments of immune-related adverse events.
      ). This strategy is now being testing in a multicenter trial in patients with metastatic melanoma.

      Inhibition of T cell migration

      Integrin inhibitors can suppress inflammatory responses with organ selectivity, providing an attractive target for immunotherapy toxicities. Vedolizumab, an inhibitor of α4β7 integrin that prevents immune cell trafficking to the gastrointestinal mucosa and is approved for the treatment of inflammatory bowel disease, has shown efficacy comparable to TNF-α blockade in retrospective analyses of patients with steroid-refractory enterocolitis (
      • Abu-Sbeih H.
      • Ali F.S.
      • Alsaadi D.
      • Jennings J.
      • Luo W.
      • Gong Z.
      • Richards D.M.
      • Charabaty A.
      • Wang Y.
      Outcomes of vedolizumab therapy in patients with immune checkpoint inhibitor-induced colitis: a multi-center study.
      ).

      Inhibition of costimulation

      CTLA-4-Ig is effective in several autoimmune diseases and is the therapy of choice for patients with CTLA-4 haploinsufficiency (
      • Kuehn H.S.
      • Ouyang W.
      • Lo B.
      • Deenick E.K.
      • Niemela J.E.
      • Avery D.T.
      • Schickel J.N.
      • Tran D.Q.
      • Stoddard J.
      • Zhang Y.
      • et al.
      Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4.
      ). CTLA-4-Ig binds to the CD80/CD86 costimulatory proteins and prevents their association with CD28 (
      • Esfahani K.
      • Elkrief A.
      • Calabrese C.
      • Lapointe R.
      • Hudson M.
      • Routy B.
      • Miller Jr., W.H.
      • Calabrese L.
      Moving towards personalized treatments of immune-related adverse events.
      ;
      • Kuehn H.S.
      • Ouyang W.
      • Lo B.
      • Deenick E.K.
      • Niemela J.E.
      • Avery D.T.
      • Schickel J.N.
      • Tran D.Q.
      • Stoddard J.
      • Zhang Y.
      • et al.
      Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4.
      ;
      • Pauken K.E.
      • Dougan M.
      • Rose N.R.
      • Lichtman A.H.
      • Sharpe A.H.
      Adverse Events Following Cancer Immunotherapy: Obstacles and Opportunities.
      ). CTLA-4-Ig has been used in patients with refractory checkpoint blockade toxicities with some apparent success, although the number of treated patients has been very small (
      • Salem J.E.
      • Allenbach Y.
      • Vozy A.
      • Brechot N.
      • Johnson D.B.
      • Moslehi J.J.
      • Kerneis M.
      Abatacept for Severe Immune Checkpoint Inhibitor-Associated Myocarditis.
      ). How this therapy would impact antitumor responses requires further investigation.

      Checkpoint blockade toxicities in high risk patients

      Currently, we have minimal information about the risk factors that predispose patients to develop specific toxicities of checkpoint blockade (Figure 1C). A limited number of reports have investigated the risk of adverse events in patients with underlying autoimmune disease (
      • Abu-Sbeih H.
      • Faleck D.M.
      • Ricciuti B.
      • Mendelsohn R.B.
      • Naqash A.R.
      • Cohen J.V.
      • Sellers M.C.
      • Balaji A.
      • Ben-Betzalel G.
      • Hajir I.
      • et al.
      Immune Checkpoint Inhibitor Therapy in Patients With Preexisting Inflammatory Bowel Disease.
      ;
      • Leonardi G.C.
      • Gainor J.F.
      • Altan M.
      • Kravets S.
      • Dahlberg S.E.
      • Gedmintas L.
      • Azimi R.
      • Rizvi H.
      • Riess J.W.
      • Hellmann M.D.
      • Awad M.M.
      Safety of Programmed Death-1 Pathway Inhibitors Among Patients With Non-Small-Cell Lung Cancer and Preexisting Autoimmune Disorders.
      ;
      • Menzies A.M.
      • Johnson D.B.
      • Ramanujam S.
      • Atkinson V.G.
      • Wong A.N.M.
      • Park J.J.
      • McQuade J.L.
      • Shoushtari A.N.
      • Tsai K.K.
      • Eroglu Z.
      • et al.
      Anti-PD-1 therapy in patients with advanced melanoma and preexisting autoimmune disorders or major toxicity with ipilimumab.
      ). These retrospective studies have had mixed results and indicate that the risk of toxicity is probably dependent on the nature of the underlying autoimmune disease.
      Patients who have developed a treatment-limiting toxicity from immunotherapy represent another important high-risk population. Depending on the specific toxicity, a substantial fraction of patients who develop a toxicity are likely to have recurrence, although this risk drops if the immunotherapy target is changed (e.g., combination checkpoint blockade changed to single agent PD-1 blockade) (
      • Dolladille C.
      • Ederhy S.
      • Sassier M.
      • Cautela J.
      • Thuny F.
      • Cohen A.A.
      • Fedrizzi S.
      • Chrétien B.
      • Da-Silva A.
      • Plane A.F.
      • et al.
      Immune Checkpoint Inhibitor Rechallenge After Immune-Related Adverse Events in Patients With Cancer.
      ;
      • Pollack M.H.
      • Betof A.
      • Dearden H.
      • Rapazzo K.
      • Valentine I.
      • Brohl A.S.
      • Ancell K.K.
      • Long G.V.
      • Menzies A.M.
      • Eroglu Z.
      • et al.
      Safety of resuming anti-PD-1 in patients with immune-related adverse events (irAEs) during combined anti-CTLA-4 and anti-PD1 in metastatic melanoma.
      ). An alternative approach is to treat the toxicity with a concurrent therapy that is not expected to inhibit antitumor immunity when immunotherapy is restarted. This concurrent treatment approach is the standard for endocrine toxicities where hormone replacement is used and is often effective for dermatologic toxicities that can be treated topically (
      • de Filette J.
      • Andreescu C.E.
      • Cools F.
      • Bravenboer B.
      • Velkeniers B.
      A Systematic Review and Meta-Analysis of Endocrine-Related Adverse Events Associated with Immune Checkpoint Inhibitors.
      ;
      • Phillips G.S.
      • Wu J.
      • Hellmann M.D.
      • Postow M.A.
      • Rizvi N.A.
      • Freites-Martinez A.
      • Chan D.
      • Dusza S.
      • Motzer R.J.
      • Rosenberg J.E.
      • et al.
      Treatment Outcomes of Immune-Related Cutaneous Adverse Events.
      ). For checkpoint enterocolitis, TNF-α blockade has been used alongside checkpoint blockade to prevent recurrence, as have local steroids (
      • Badran Y.R.
      • Cohen J.V.
      • Brastianos P.K.
      • Parikh A.R.
      • Hong T.S.
      • Dougan M.
      Concurrent therapy with immune checkpoint inhibitors and TNFα blockade in patients with gastrointestinal immune-related adverse events.
      ;
      • Hughes M.S.
      • Molina G.E.
      • Chen S.T.
      • Zheng H.
      • Deshpande V.
      • Fadden R.
      • Sullivan R.J.
      • Dougan M.
      Budesonide treatment for microscopic colitis from immune checkpoint inhibitors.
      ).

      Adverse events induced by adoptive T cell therapies

      A major advance in the cancer immunotherapy field is the development of cellular therapies in which T cells are engineered to express a tumor-specific antigen receptor. Discovery of the TCR and costimulatory receptors enabled the development of CARs composed of a tumor-directed antibody domain and cytoplasmic signaling domains derived from the TCR (ζ chain) and costimulatory receptors (commonly CD28 or 41BB). Such second-generation CARs engage both TCR and costimulatory signaling pathways required for full T cell activation (
      • Eshhar Z.
      • Waks T.
      • Gross G.
      The emergence of T-bodies/CAR T cells.
      ;
      • Kalos M.
      • June C.H.
      Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology.
      ;
      • Sadelain M.
      • Brentjens R.
      • Rivière I.
      The basic principles of chimeric antigen receptor design.
      ). CAR T cells that recognize the CD19 antigen have shown striking efficacy against relapsed, treatment-refractory disease in multiple B cell malignancies, including acute lymphoblastic leukemia (ALL) and B cell lymphomas. CD19 CAR T cells have now been approved by the Food and Drug Administration (FDA) for the treatment of ALL in children and young adults as well as B cell lymphomas (
      • Maude S.L.
      • Laetsch T.W.
      • Buechner J.
      • Rives S.
      • Boyer M.
      • Bittencourt H.
      • Bader P.
      • Verneris M.R.
      • Stefanski H.E.
      • Myers G.D.
      • et al.
      Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia.
      ;
      • Neelapu S.S.
      • Locke F.L.
      • Bartlett N.L.
      • Lekakis L.J.
      • Miklos D.B.
      • Jacobson C.A.
      • Braunschweig I.
      • Oluwole O.O.
      • Siddiqi T.
      • Lin Y.
      • et al.
      Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma.
      ;
      • Park J.H.
      • Rivière I.
      • Gonen M.
      • Wang X.
      • Sénéchal B.
      • Curran K.J.
      • Sauter C.
      • Wang Y.
      • Santomasso B.
      • Mead E.
      • et al.
      Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia.
      ). Furthermore, CAR T cells specific for B cell maturation antigen (BCMA) have shown promising results for the treatment of multiple myeloma (
      • Raje N.
      • Berdeja J.
      • Lin Y.
      • Siegel D.
      • Jagannath S.
      • Madduri D.
      • Liedtke M.
      • Rosenblatt J.
      • Maus M.V.
      • Turka A.
      • et al.
      Anti-BCMA CAR T-Cell Therapy bb2121 in Relapsed or Refractory Multiple Myeloma.
      ). Serious toxicities remain a major limitation of current CAR T cell therapies, and fall into 3 categories: (1) cytokine release syndrome (CRS), (2) neurotoxicity, and (3) on-target and off-target killing of healthy cells (Figure 6). These toxicities are also relevant for cellular therapies based on tumor-specific TCRs, and to a lesser extent, for bispecific antibodies that target a tumor surface antigen and activate T cells by CD3 binding.
      Figure thumbnail gr6
      Figure 6Inflammatory toxicities induced by CAR T cell therapies
      (A) CAR T cells transduced with a CD19-specific receptor eradicate CD19-expressing leukemia cells while inducing potent myeloid cell activation and release of inflammatory mediators, resulting in cytokine release syndrome (CRS). Indicated CRS therapies are colored according to current clinical use (black) or promising pre-clinical data (gray). NO, nitric oxide.
      (B) CD19-specific T cells can recognize CD19 expressed by perivascular cells at the blood brain barrier. Direct cytotoxicity and indirect cytokine-mediated damage disrupt the blood-brain barrier, resulting in neurological toxicities. Therapies for neurotoxicity are shown colored according to current clinical use (black) or promising pre-clinical data (gray).

      Cytokine release syndrome

      In hematological malignancies, CAR T cell activation and expansion can result in high-level secretion of cytokines/chemokines. CRS can be a severe and potentially life-threatening toxicity of CAR T cell therapy (
      • Teachey D.T.
      • Lacey S.F.
      • Shaw P.A.
      • Melenhorst J.J.
      • Maude S.L.
      • Frey N.
      • Pequignot E.
      • Gonzalez V.E.
      • Chen F.
      • Finklestein J.
      • et al.
      Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia.
      ). For the two FDA-approved CD19 CAR T cell products, CRS occurs in 74%–94% of patients, with grade 3 or higher toxicity in 13%–49% (;
      U.S. Food and Drug Administration
      YESCARTA package insert.
      ). The first presenting symptom of CRS is typically fever, but patients can rapidly progress to develop hypotension and hypoxemia along with impaired function of vital organs. Peak levels of 24 cytokines and soluble cytokine receptors, including IFNγ, IL-6, and GM-CSF are strongly associated with severe CRS (
      • Teachey D.T.
      • Lacey S.F.
      • Shaw P.A.
      • Melenhorst J.J.
      • Maude S.L.
      • Frey N.
      • Pequignot E.
      • Gonzalez V.E.
      • Chen F.
      • Finklestein J.
      • et al.
      Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia.
      ). The IL-6 pathway plays a prominent role in CRS pathogenesis: the IL-6 receptor blocking antibody tocilizumab is widely used to treat severe CRS and can induce rapid clinical improvement (
      • Maude S.L.
      • Barrett D.
      • Teachey D.T.
      • Grupp S.A.
      Managing cytokine release syndrome associated with novel T cell-engaging therapies.
      ). Glucocorticoids are also used to treat severe CRS but could negatively impact CAR T cell function. Contributing factors for development of severe CRS include a high burden of malignant cells, a large dose of transferred CAR T cells, and the choice of costimulatory signaling domain in the CAR construct. Knowledge of these factors has enabled a risk-adapted approach, including transfer of smaller numbers of CAR T cells to patients with higher burden of malignant cells (
      • Brudno J.N.
      • Kochenderfer J.N.
      Recent advances in CAR T-cell toxicity: Mechanisms, manifestations and management.
      ).
      Early clinical, laboratory, and cytokine data pointed to significant similarities between CRS and macrophage activation syndrome, a potentially life-threatening complication of systemic inflammatory disorders (
      • Crayne C.B.
      • Albeituni S.
      • Nichols K.E.
      • Cron R.Q.
      The Immunology of Macrophage Activation Syndrome.
      ;
      • Teachey D.T.
      • Lacey S.F.
      • Shaw P.A.
      • Melenhorst J.J.
      • Maude S.L.
      • Frey N.
      • Pequignot E.
      • Gonzalez V.E.
      • Chen F.
      • Finklestein J.
      • et al.
      Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia.
      ). A humanized mouse model of CRS demonstrated that activated CAR T cells induced massive recruitment of monocytes/macrophages that produced high levels of IL-6, IL-1, and nitric oxide (
      • Giavridis T.
      • van der Stegen S.J.C.
      • Eyquem J.
      • Hamieh M.
      • Piersigilli A.
      • Sadelain M.
      CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade.
      ; Figure 6A). IL-1β may play an important role in the pathogenesis of CRS and could be targeted with FDA-approved IL-1β inhibitors (
      • Cavalli G.
      • Dinarello C.A.
      Treating rheumatological diseases and co-morbidities with interleukin-1 blocking therapies.
      ;
      • Giavridis T.
      • van der Stegen S.J.C.
      • Eyquem J.
      • Hamieh M.
      • Piersigilli A.
      • Sadelain M.
      CAR T cell-induced cytokine release syndrome is mediated by macrophages and abated by IL-1 blockade.
      ;
      • Teachey D.T.
      • Lacey S.F.
      • Shaw P.A.
      • Melenhorst J.J.
      • Maude S.L.
      • Frey N.
      • Pequignot E.
      • Gonzalez V.E.
      • Chen F.
      • Finklestein J.
      • et al.
      Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia.
      ).

      Neurotoxicity

      Neurotoxicity represents a second major complication of CAR T cell therapy that can be fatal. For the two FDA-approved CD19 CAR T cell products, neurotoxicity is observed in 72%–87% of patients, with grade 3 or higher neurotoxicity in 18%–31% (;
      U.S. Food and Drug Administration
      YESCARTA package insert.
      ). A wide range of clinical symptoms are observed including headache, confusion, language disturbances, seizures, and cerebral edema (
      • Gust J.
      • Taraseviciute A.
      • Turtle C.J.
      Neurotoxicity Associated with CD19-Targeted CAR-T Cell Therapies.
      ). Neurotoxicity is associated with CRS, and cytokines such as IFNγ and IL-6 are elevated not only in the blood but also the cerebrospinal fluid (CSF). Increased blood-brain barrier permeability and local cytokine production by CAR T cells are thought to account for elevated CSF cytokine levels (Figure 6B). Neuropathological investigation demonstrated perivascular infiltration by CAR T cells in the CNS, macrophage infiltration, as well as widespread white matter and neuronal injury. Tocilizumab has limited efficacy for the treatment of neurotoxicity, potentially due to low antibody penetration into the CNS. Therefore, high-dose glucocorticoids are currently used for the treatment of neurotoxicity (
      • Gust J.
      • Taraseviciute A.
      • Turtle C.J.
      Neurotoxicity Associated with CD19-Targeted CAR-T Cell Therapies.
      ). In a humanized model of neurotoxicity, monocytes were identified as the major source of IL-1 and IL-6, and the IL-1 receptor antagonist anakinra, which has a molecular weight of 17 kDa, was shown to inhibit both CRS and neurotoxicity (
      • Norelli M.
      • Camisa B.
      • Barbiera G.
      • Falcone L.
      • Purevdorj A.
      • Genua M.
      • Sanvito F.
      • Ponzoni M.
      • Doglioni C.
      • Cristofori P.
      • et al.
      Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells.
      ).
      Interestingly, rates of neurotoxicity appear to be lower for CAR T cell therapies that do not target the CD19 antigen, although neurotoxicity has also been observed with CAR T cells specific for other antigens (
      • Gust J.
      • Taraseviciute A.
      • Turtle C.J.
      Neurotoxicity Associated with CD19-Targeted CAR-T Cell Therapies.
      ). A recent study indicated that neurotoxicity may not only be mediated by cytokines, but also on-target activity of CD19 CAR T cells against CNS-resident cells. CD19 was thought to have a B cell-restricted expression pattern, but analysis of human brain scRNA-seq data demonstrated CD19 mRNA in perivascular cells critical for blood-brain barrier integrity (
      • Parker K.R.
      • Migliorini D.
      • Perkey E.
      • Yost K.E.
      • Bhaduri A.
      • Bagga P.
      • Haris M.
      • Wilson N.E.
      • Liu F.
      • Gabunia K.
      • et al.
      Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies.
      ; Figure 6B). Targeting of cells by CD19 CAR T cells could result in endothelial activation, increased blood-brain barrier permeability, and myeloid cell recruitment (
      • Parker K.R.
      • Migliorini D.
      • Perkey E.
      • Yost K.E.
      • Bhaduri A.
      • Bagga P.
      • Haris M.
      • Wilson N.E.
      • Liu F.
      • Gabunia K.
      • et al.
      Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies.
      ).

      On-target and off-target toxicity of adoptive T cell therapies

      In adoptive T cell therapies, large numbers of activated cells that express a tumor-reactive CAR or TCR are transferred to patients. The expression patterns of potential target antigens are therefore a critical consideration in the design of adoptive T cell therapies. CAR T cells show two types of on-target activity against healthy cells. (1) Engineered CD19 CAR T cells eliminate not only malignant but also healthy B cells (
      • Deya-Martinez A.
      • Alonso-Saladrigues A.
      • Garcia A.P.
      • Faura A.
      • Torrebadell M.
      • Vlagea A.
      • Catala A.
      • Esteve-Sole A.
      • Juan M.
      • Rives S.
      • et al.
      Kinetics of humoral deficiency in CART19-treated children and young adults with acute lymphoblastic leukaemia.
      ). Similarly, BCMA CAR T cells eliminate malignant and healthy plasma cells that provide long-term protection from infectious agents by high-level antibody production (
      • Raje N.
      • Berdeja J.
      • Lin Y.
      • Siegel D.
      • Jagannath S.
      • Madduri D.
      • Liedtke M.
      • Rosenblatt J.
      • Maus M.V.
      • Turka A.
      • et al.
      Anti-BCMA CAR T-Cell Therapy bb2121 in Relapsed or Refractory Multiple Myeloma.
      ). Loss of B cells or plasma cells can be managed clinically by injection of polyclonal IgG from healthy donors. T cell-mediated elimination of transformed cells and the corresponding healthy cell type represents a major limitation of CAR T cell therapy. This issue has become highly relevant for CAR T cell therapy of solid tumors, many of which originate from essential cell types, such as epithelial cells. (2) Even though a target antigen may appear to have a restricted expression pattern, CAR T cells can also target other healthy cell types expressing the antigen at a lower level, such as brain perivascular cells that express CD19 (
      • Parker K.R.
      • Migliorini D.
      • Perkey E.
      • Yost K.E.
      • Bhaduri A.
      • Bagga P.
      • Haris M.
      • Wilson N.E.
      • Liu F.
      • Gabunia K.
      • et al.
      Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies.
      ). For example, a CAR specific for ERBB2 (Her-2/neu) that was based on the FDA-approved antibody trastuzumab (Herceptin) induced fatal pulmonary edema in a patient with colon cancer following transfer of a large dose of engineered T cells. Further investigation revealed low level expression of ERBB2 by lung epithelial cells (
      • Morgan R.A.
      • Yang J.C.
      • Kitano M.
      • Dudley M.E.
      • Laurencot C.M.
      • Rosenberg S.A.
      Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2.
      ). This example illustrates that a CAR may not be safe even when it is derived from a widely utilized therapeutic antibody, a finding explained by the highly activated state of T cells induced by CAR signaling.
      A number of engineering approaches are being developed to protect healthy cell types from on-target attack by CAR T cells. The general concept is to control CAR T cell activation by combinatorial logic based on differential expression of two antigens by healthy versus transformed cells. For example, T cells can be engineered to express activating and inhibitory CARs directed against different surface antigens. Healthy cells that express the ligand for the inhibitory CAR can thus be protected, even if they also express the antigen for the activating CAR (equivalent to TCR + PD-1 signaling in normal T cells) (
      • Fedorov V.D.
      • Themeli M.
      • Sadelain M.
      PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses.
      ). The SynNotch approach also increases the selectivity of T cell activation by requiring expression of two antigens by tumor cells. SynNotch receptors take advantage of Notch receptor biology: mechanical force exerted by ligand binding induces proteolytic cleavage of the cytoplasmic domain which then acts as a transcription factor. A SynNotch receptor recognizing antigen A induces a transcriptional response, resulting in expression of a CAR specific for antigen B. Tumor cells that express antigens A + B are effectively targeted, but healthy cells that express only antigens A or B are spared (
      • Roybal K.T.
      • Williams J.Z.
      • Morsut L.
      • Rupp L.J.
      • Kolinko I.
      • Choe J.H.
      • Walker W.J.
      • McNally K.A.
      • Lim W.A.
      Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors.
      ;
      • Srivastava S.
      • Salter A.I.
      • Liggitt D.
      • Yechan-Gunja S.
      • Sarvothama M.
      • Cooper K.
      • Smythe K.S.
      • Dudakov J.A.
      • Pierce R.H.
      • Rader C.
      • Riddell S.R.
      Logic-Gated ROR1 Chimeric Antigen Receptor Expression Rescues T Cell-Mediated Toxicity to Normal Tissues and Enables Selective Tumor Targeting.
      ). The transcription factor activated in the SynNotch system can also be used to further engineer the functional program, including release of cytokines or other therapeutic proteins (
      • Morsut L.
      • Roybal K.T.
      • Xiong X.
      • Gordley R.M.
      • Coyle S.M.
      • Thomson M.
      • Lim W.A.
      Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors.
      ).
      The field has recently seen a resurgence of interest in adoptive T cell therapies based on TCRs that perform surveillance of the cellular proteome in the form of short peptides bound to MHC proteins. TCRs thus offer more opportunities for tumor antigen discovery. A substantial effort is underway to identify the antigen specificity of tumor-infiltrating T cells in patients responding to checkpoint blockade. TCRs have a high degree of specificity for the relevant MHC-bound peptide but can cross-react with peptides that meet the sequence requirements for MHC binding and TCR recognition (
      • Wucherpfennig K.W.
      • Sethi D.
      T cell receptor recognition of self and foreign antigens in the induction of autoimmunity.
      ). T cells undergo a rigorous selection process in the thymus that eliminates those cells with high affinity for self-antigens, including highly cross-reactive T cells. This natural selection process is sidestepped when TCRs are selected in vitro from recombinant libraries or subjected to significant mutagenesis. The affinity of TCRs isolated from cancer patients is frequently suboptimal, but mutagenesis to enhance TCR affinity can introduce undesirable cross-reactivity, as illustrated by a TCR specific for a MAGE-A3 peptide presented by HLA-A1. The first two patients who received MAGE-A3 specific T cells died of cardiogenic shock several days following T cell transfer. In both patients, severe myocardial damage with substantial T cell infiltration was observed, but MAGE-A3 expression could not be detected in the heart. Rather, this TCR cross-reacted with titin, a striated muscle specific protein (
      • Linette G.P.
      • Stadtmauer E.A.
      • Maus M.V.
      • Rapoport A.P.
      • Levine B.L.
      • Emery L.
      • Litzky L.
      • Bagg A.
      • Carreno B.M.
      • Cimino P.J.
      • et al.
      Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma.
      ). It is therefore important to systematically examine the cross-reactivity of engineered TCRs prior to initiation of clinical trials.
      Currently, the toxicities of CAR T cell therapies represent a significant challenge, but further engineering of these cells provides an opportunity to significantly improve their safety profile while maintaining anti-tumor activity.

      Adverse events induced by cytokine therapies

      Cytokines are natural immune modulatory agents (
      • Dranoff G.
      Cytokines in cancer pathogenesis and cancer therapy.
      ). Often acting in autocrine or paracrine fashion, cytokines typically mediate responses in a spatially restricted context. Nevertheless, some cytokines act systemically on distant organs such as the hypothalamus, liver and bone marrow to augment systemic immunity. Recombinant cytokines known to be important for the effector T cell response to cancer have been administered to cancer patients, often with severe treatment-limiting side effects (
      • Dranoff G.
      Cytokines in cancer pathogenesis and cancer therapy.
      ). Local delivery strategies that more closely mimic the natural production of cytokines have shown promising results in preclinical models and serve to limit the toxicities associated with systemic delivery. Such strategies include delivery by direct intratumoral injection, oncolytic viruses, nanoparticles, and antibody or nanobody conjugates (
      • Dougan M.
      • Dougan S.K.
      Targeting Immunotherapy to the Tumor Microenvironment.
      ). In general, to understand the toxicity associated with a cytokine, one must understand the full array of cell types that express cognate receptors and induced cellular responses. Advances in protein engineering have also allowed the design of cytokines with improved targeting capabilities and reduced signaling by immunosuppressive cells or cell types responsible for systemic toxicities.
      IL-2 provides an excellent example for design strategies that mitigate severe systemic toxicities and simultaneously enhance antitumor activity. IL-2 serves as a critical growth factor for activated T cells, and considerable efforts have been made to harness it for therapeutic use. High-dose IL-2 is curative in a small fraction of patients with renal cell carcinoma and melanoma, although determining maximum dosing is difficult, and fatal capillary leak syndrome has been observed due to expression of CD25 by pulmonary endothelial cells (
      • Dranoff G.
      Cytokines in cancer pathogenesis and cancer therapy.
      ;
      • Létourneau S.
      • van Leeuwen E.M.
      • Krieg C.
      • Martin C.
      • Pantaleo G.
      • Sprent J.
      • Surh C.D.
      • Boyman O.
      IL-2/anti-IL-2 antibody complexes show strong biological activity by avoiding interaction with IL-2 receptor alpha subunit CD25.
      ;
      • Zhu E.F.
      • Gai S.A.
      • Opel C.F.
      • Kwan B.H.
      • Surana R.
      • Mihm M.C.
      • Kauke M.J.
      • Moynihan K.D.
      • Angelini A.
      • Williams R.T.
      • et al.
      Synergistic innate and adaptive immune response to combination immunotherapy with anti-tumor antigen antibodies and extended serum half-life IL-2.
      ). These severe toxicities have greatly limited clinical use of high-dose IL-2.
      The IL-2 receptor is composed of IL2Rβ (CD122) and the gamma common chain (γc), which together form an intermediate-affinity receptor for IL-2 (
      • Leonard W.J.
      • Lin J.X.
      • O’Shea J.J.
      The γc Family of Cytokines: Basic Biology to Therapeutic Ramifications.
      ). The intermediate-affinity receptor is expressed by all T cells and natural killer (NK) cells, and IL-2 is an important growth factor for these lymphocytes. IL-2Rα (CD25) associates with the IL2Rβ and γc to form the trimeric high-affinity IL-2 receptor complex. CD25 is expressed at the highest level by Foxp3+ CD4 Tregs, which preferentially expand when IL-2 is limiting. Given that neither expansion of Tregs nor pulmonary edema are desirable outcomes, several strategies to reduce binding of IL-2 to CD25 while preserving binding to the intermediate-affinity IL-2 receptor have been developed. Using yeast display and targeted evolution, a variant of IL-2 (Super-2) that lacks binding to CD25 has been developed, as has a computationally designed de novo protein that activates the intermediate-affinity IL-2 receptor but lacks CD25 binding (
      • Levin A.M.
      • Bates D.L.
      • Ring A.M.
      • Krieg C.
      • Lin J.T.
      • Su L.
      • Moraga I.
      • Raeber M.E.
      • Bowman G.R.
      • Novick P.
      • et al.
      Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’.
      ;
      • Silva D.A.
      • Yu S.
      • Ulge U.Y.
      • Spangler J.B.
      • Jude K.M.
      • Labão-Almeida C.
      • Ali L.R.
      • Quijano-Rubio A.
      • Ruterbusch M.
      • Leung I.
      • et al.
      De novo design of potent and selective mimics of IL-2 and IL-15.
      ). Alternatively, attachment of polyethylene glycol (PEG) chains to IL-2 increases its serum half-life and inhibits binding to the high-affinity IL-2 receptor. Slow turnover of the PEG chains releases active versions of IL-2, and PEGylated IL-2 (Bempegaldesleukin) is currently in clinical trials in combination with PD-1 blockade (
      • Charych D.H.
      • Hoch U.
      • Langowski J.L.
      • Lee S.R.
      • Addepalli M.K.
      • Kirk P.B.
      • Sheng D.
      • Liu X.
      • Sims P.W.
      • VanderVeen L.A.
      • et al.
      NKTR-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models.
      ).
      Strategies that mimic the localized nature of cytokine signaling are likely to be both less toxic and more effective as cancer therapeutics. Synthetic cytokines and cytokine mimetics also open the possibility of further engineered variants with altered binding properties.

      Conclusions

      Activated T cells serve as the major effector cells of all current cancer immunotherapies. Targeting of negative feedback mechanisms (checkpoint blockade) or engineering of synthetic receptors (CAR T cells) can enable effective T cell-mediated tumor immunity but also unleash severe inflammatory toxicities. Defining the cellular and molecular pathways responsible for major inflammatory toxicities will be important in order to develop therapeutic approaches compatible with sustained antitumor immunity. Available data suggest that recruitment and activation of myeloid cells by T cells can result in local and/or systemic release of proinflammatory cytokines (including IL-6, TNF-α, and IL-1β) that play a central role in these inflammatory toxicities, as illustrated by the significant therapeutic activity of IL-6 receptor blockade in cytokine release syndrome induced by CAR T cells. Targeting of such inflammatory pathways may enable treatment or even prevention of inflammatory toxicities while preserving anti-tumor immunity.

      Acknowledgments

      This work was supported by a Team Award from the American Cancer Society ( MRAT-18-113-01 ), the Melanoma Research Alliance ( 597698 to K.W.W., S.K.D., and M.D.), NIH ( R01 CA238039 , R01CA251599 , and P01 CA163222 to K.W.W. and T32CA207021 to A.M.L. and K.W.W.), a NIH Mentored Clinical Scientist Development Award ( 1K08DK114563-01 to M.D.), and the American Gastroenterological Association Research Scholars Award (to M.D.).

      Declaration of interests

      K.W.W. serves on the scientific advisory board of TCR2 Therapeutics, T-Scan Therapeutics, SQZ Biotech, and Nextechinvest and receives sponsored research funding from Novartis. He is a scientific co-founder of Immunitas Therapeutics. M.D. is a consultant for Tillotts Pharma, Partner Therapeutics, and Genentech-Roche, receives research funding from Novartis, and is on the Scientific Advisory Board for Neoleukin Therapeutics. S.K.D. receives research funding from Novartis, Bristol-Myers Squibb, and Eli Lilly and is a scientific co-founder of Kojin.

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