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Immune-related adverse events, particularly severe toxicities such as myocarditis, are major challenges to the utility of immune checkpoint inhibitors (ICIs) in anticancer therapy1. The pathogenesis of ICI-associated myocarditis (ICI-MC) is poorly understood. Pdcd1-/-Ctla4+/- mice recapitulate clinicopathological features of ICI-MC, including myocardial T cell infiltration2. Here, using single-cell RNA and T cell receptor (TCR) sequencing of cardiac immune infiltrates from Pdcd1-/-Ctla4+/- mice, we identify clonal effector CD8+ T cells as the dominant cell population. Treatment with anti-CD8-depleting, but not anti-CD4-depleting, antibodies improved the survival of Pdcd1-/-Ctla4+/- mice. Adoptive transfer of immune cells from mice with myocarditis induced fatal myocarditis in recipients, which required CD8+ T cells. The cardiac-specific protein α-myosin, which is absent from the thymus3,4, was identified as the cognate antigen source for three major histocompatibility complex class I-restricted TCRs derived from mice with fulminant myocarditis. Peripheral blood T cells from three patients with ICI-MC were expanded by α-myosin peptides. Moreover, these α-myosin-expanded T cells shared TCR clonotypes with diseased heart and skeletal muscle, which indicates that α-myosin may be a clinically important autoantigen in ICI-MC. These studies underscore the crucial role for cytotoxic CD8+ T cells, identify a candidate autoantigen in ICI-MC and yield new insights into the pathogenesis of ICI toxicity.

Treatment with immune checkpoint blockade (ICB) has revolutionized cancer therapy. Until now, predictive biomarkers1-10 and strategies to augment clinical response have largely focused on the T cell compartment. However, other immune subsets may also contribute to anti-tumour immunity11-15, although these have been less well-studied in ICB treatment16. A previously conducted neoadjuvant ICB trial in patients with melanoma showed via targeted expression profiling17 that B cell signatures were enriched in the tumours of patients who respond to treatment versus non-responding patients. To build on this, here we performed bulk RNA sequencing and found that B cell markers were the most differentially expressed genes in the tumours of responders versus non-responders. Our findings were corroborated using a computational method (MCP-counter18) to estimate the immune and stromal composition in this and two other ICB-treated cohorts (patients with melanoma and renal cell carcinoma). Histological evaluation highlighted the localization of B cells within tertiary lymphoid structures. We assessed the potential functional contributions of B cells via bulk and single-cell RNA sequencing, which demonstrate clonal expansion and unique functional states of B cells in responders. Mass cytometry showed that switched memory B cells were enriched in the tumours of responders. Together, these data provide insights into the potential role of B cells and tertiary lymphoid structures in the response to ICB treatment, with implications for the development of biomarkers and therapeutic targets.

B7-H4 is a negative regulatory B7 family member. We investigated the role of host and donor B7-H4 in regulating acute graft-versus-host disease (GVHD). Allogeneic donor T cells infused into B7-H4-/- versus WT recipients markedly accelerated GVHD-induced lethality. Chimera studies pointed toward B7-H4 expression on host hematopoietic cells as more critical than parenchymal cells in controlling GVHD. Rapid mortality in B7-H4-/- recipients was associated with increased donor T cell expansion, gut T cell homing and loss of intestinal epithelial integrity, increased T effector function (proliferation, proinflammatory cytokines, cytolytic molecules), and reduced apoptosis. Higher metabolic demands of rapidly proliferating donor T cells in B7-H4-/- versus WT recipients required multiple metabolic pathways, increased extracellular acidification rates (ECARs) and oxygen consumption rates (OCRs), and increased expression of fuel substrate transporters. During GVHD, B7-H4 expression was upregulated on allogeneic WT donor T cells. B7-H4-/- donor T cells given to WT recipients increased GVHD mortality and had function and biological properties similar to WT T cells from allogeneic B7-H4-/- recipients. Graft-versus-leukemia responses were intact regardless as to whether B7-H4-/- mice were used as hosts or donors. Taken together, these data provide new insights into the negative regulatory processes that control GVHD and provide support for developing therapeutic strategies directed toward the B7-H4 pathway.

Pancreatic ductal adenocarcinoma (PDAC) is associated with marked fibrosis and stromal myofibroblasts, but their functional contribution remains unknown. Transgenic mice with the ability to delete αSMA(+) myofibroblasts in pancreatic cancer were generated. Depletion starting at either noninvasive precursor (pancreatic intraepithelial neoplasia) or the PDAC stage led to invasive, undifferentiated tumors with enhanced hypoxia, epithelial-to-mesenchymal transition, and cancer stem cells, with diminished animal survival. In PDAC patients, fewer myofibroblasts in their tumors also correlated with reduced survival. Suppressed immune surveillance with increased CD4(+)Foxp3(+) Tregs was observed in myofibroblast-depleted mouse tumors. Although myofibroblast-depleted tumors did not respond to gemcitabine, anti-CTLA4 immunotherapy reversed disease acceleration and prolonged animal survival. This study underscores the need for caution in targeting carcinoma-associated fibroblasts in PDAC.

CD28 is a cell surface molecule that mediates a costimulatory signal crucial for T cell proliferation and lymphokine production. The signal transduction mechanisms of CD28 are not well understood. Itk, a nonreceptor protein tyrosine kinase specifically expressed in T cells and mast cells, has been implicated in the CD28 signaling pathway because of reports that it becomes phosphorylated on tyrosines and associates with CD28 upon cross-linking of the cell surface molecule. To determine whether Itk plays a functional role in CD28 signaling, we compared T cells from Itk-deficient mice and control mice for their responses to CD28 costimulation. T cells defective in Itk were found to be fully competent to respond to costimulation. Whereas the CD3-mediated proliferative response was severely compromised in the absence of Itk, the calcineurin-independent CD28-mediated response was significantly elevated when compared with cells from control animals. The augmented proliferation was not due to increased production of interleukin-2. The results suggest that Itk has distinct roles in the CD3 versus the CD28 signaling pathways. By negatively regulating the amplitude of signaling upon CD28 costimulation, Itk may provide a means for modulating the outcome of T cell activation during development and during antigen-driven immune responses.

The generation of productive adaptive immune responses depends on the antigen-specific activation of T and B cells. The outcome of T-cell receptor engagement is influenced by signals from both positive and negative regulatory molecules that can either activate or inhibit T-cell function. CD28 and cytotoxic T-lymphocyte antigen-4 are the prototypical members of an immunoglobulin domain-containing protein family that play important roles in the control of T-cell responses against infection, cancer, and in autoimmune disease. Although the precise molecular details of their functions are still under active investigation, tumors and chronic pathogens seem to have exploited these pathways to achieve immune evasion. Furthermore, malfunction of the inhibitory arm of the immune response appears responsible for the development of multiple autoimmune pathologies. As a result, the negative regulators of T-cell activation have become attractive targets for therapeutic intervention in cancer, chronic infection, and autoimmune disease. The application of findings from basic research has provided insight into the manipulation of these pathways in the clinic and offers promising strategies for the treatment of disease. [ABSTRACT FROM AUTHOR]

Establishing a balance between the rapid generation of effective immunity and the production of overly exuberant or excessively prolonged responses is critical to the maintenance of the equilibrium between health and disease. The preservation of homeostasis and prevention of inappropriate activation of immunity is safeguarded by systems integrating the influences of T-cell receptor signaling, pro-inflammatory danger signals, and positive costimulatory signals on the one hand with those of several layers of both cell-intrinsic and cell-extrinsic inhibitory checkpoints on the other. Evolution has thus provided an immunological system capable of clearance of pathogens and infected cells but which generally avoids the severe collateral damage that is associated with failure to control immunity. Central tolerance to self-antigens constitutes the first line of defense against self-destruction. Because central tolerance mechanisms fail to eliminate all self-reactive immune effector cells, other immune-regulating (peripheral tolerance) mechanisms are required to prevent excessive immune responses. Dysfunction of these inhibitory pathways in terms of reduced activity can result in the unmasking of self-directed responses and a variety of autoimmune morbidities. Conversely, enhanced inhibitory activity can restrict the generation of clinically useful immunity to cancers and to chronic infectious pathogens. This may manifest not only as inhibition of immunity directed towards what are largely aberrantly or overexpressed ‘self’ targets on malignant cells but also additional exaggeration of inhibitory pathway activity mediated via upregulation on tumor cells or stromal tissues of the ligands for inhibitory receptors expressed by lymphocytes. The selective pressures exerted by immuno-editing will favor the outgrowth of such immuno-evasive malignant clones. These pathways therefore represent significant hurdles to the generation of therapeutic anti-cancer responses. The most active of the T-cell intrinsic inhibitory pathways belong to the immunoglobulin superfamily, which occupies a central importance in the coordination of immune responses. The CD28/cytotoxic T-lymphocyte antigen-4 (CTLA-4):B7-1/B7-2 receptor/ligand grouping represents the archetypal example of these immune regulators. Therapies aimed at overcoming these mechanisms of peripheral tolerance, in particular by blocking the inhibitory checkpoints, offer the potential to generate anti-tumor activity, either as monotherapies or in synergism with other therapies that directly or indirectly enhance presentation of tumor epitopes to the immune system. [ABSTRACT FROM AUTHOR]

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) functions as a negative regulator of endogenous and vaccine-induced antitumor immunity. The administration of fully human anti-CTLA-4 blocking monoclonal antibodies to advanced-cancer patients increases immune-mediated tumor destruction in some subjects. Nonetheless, patients that respond also frequently manifest serious inflammatory pathologies, raising the possibility that the therapeutic and toxic effects of CTLA-4 blockade might be linked. Here we show that periodic infusions of anti-CTLA-4 antibodies after vaccination with irradiated, autologous tumor cells engineered to secrete GM-CSF (GVAX) generate clinically meaningful antitumor immunity without grade 3 or 4 toxicity in a majority of metastatic melanoma patients. The application of this sequential immunotherapy to advanced ovarian carcinoma patients also revealed that tumor destruction and severe inflammatory pathology could be dissociated, although further refinements are required to increase clinical responses and to minimize toxicity in this population. The extent of therapy-induced tumor necrosis was linearly related to the natural logarithm of the ratio of intratumoral CD8+ effector T cells to FoxP3+ regulatory T cells (Tregs) in posttreatment biopsies. Together, these findings help clarify the immunologic and clinical effects of CTLA-4 antibody blockade in previously vaccinated patients and raise the possibility that selective targeting of antitumor Tregs may constitute a complementary strategy for combination therapy. [ABSTRACT FROM AUTHOR]

Despite the importance of programmed death-1 (PD-1) for T cell inhibition, little is known about its intracellular trafficking or requirements for localization to the immunological synapse. Here, we show that in activated T cells, PD-1 is present at the plasma membrane, near the Golgi and in the trans-Golgi network. Unlike CD28 and CTLA-4, PD-1 accumulation at the synapse is extensive only when T cells interact with dendritic cells (DC5) expressing high B7-DC levels. However, B7-H1 is also critically important, especially when the DC5 have little 87-DC. Despite this preference, B7-H1-/- DCs elicit greater cytokine secretion than B7-DC-/- DCs during T cell restimulation, possibly because they also express less B7-DC. PD-1 and CD28 have similar kinetics of synaptic accumulation, suggesting that the process involves T cell receptor-triggered cytoskeletal reorganization followed by ligand binding. [ABSTRACT FROM AUTHOR]

The activation of lymphocytes and development of adaptive immune responses is initiated by the engagement of TCRs by antigenic peptide—MHC complexes and shaped at the clonal level by both positive and negative costimulatory signals. The B7 family members are involved at several stages in this process. In this issue of the JCI, Vogt et al. show that the B7 family—related protein V-set and Ig domain—containing 4 (VSIG4) can act as an inhibitor of T cell activation (see the related article beginning on page 2817). Intriguingly, the same molecule was recently independently identified as a complement receptor of the Ig superfamily (CRIg) and was convincingly demonstrated to be a receptor for complement component 3 fragments. These findings raise interesting questions regarding the physiological roles and mechanisms of action of this molecule. Identification of dual functions of this molecule provides an additional level of complexity in T cell costimulation. [ABSTRACT FROM AUTHOR]

A preface for the volume 90 issue of the journal "Advances in Immunology: Cancer Immunotherapy," is presented.

Despite considerable interest in the modulation of tumor-associated Foxp3+ regulatory T cells (Tregs) for therapeutic benefit, little is known about the developmental origins of these cells and the nature of the antigens that they recognize. We identified an endogenous population of antigen-specific Tregs (termed MJ23 Tregs) found recurrently enriched in the tumors of mice with oncogene-driven prostate cancer. MJ23 Tregs were not reactive to a tumor-specific antigen but instead recognized a prostate-associated antigen that was present in tumor-free mice. MJ23 Tregs underwent autoimmune regulator (Aire)-dependent thymic development in both mate and female mice. Thus, Aire-mediated expression of peripheral tissue antigens drives the thymic development of a subset of organ-specific Tregs, which are likely coopted by tumors developing within the associated organ. [ABSTRACT FROM AUTHOR]

Inflammation of the normally tolerant liver microen-vironment precedes the development of chronic liver disease. Study of the pathogenesis of autoimmune liver diseases, such as autoimmune hepatitis (AIH), has been hampered by a lack of autochthonous chronic animal models. Through our studies of T cell costimulation, we generated transgenic mice expressing a ligand specific for the CD28 receptor, which normally shares ligands with the related inhibitory receptor CTLA-4. The mice spontaneously develop chronic inflammatory liver dis-ease with several pathologies found in AIH, including elevated serum aminotransferases in the context of nor-mal alkaline phosphatase and bilirubin levels, lympho-cytic inflammation, focal necrosis, oval cell hyperplasia, and fibrosis. The prevalence of IFN-7--producing CD8+ T cells in the livers of transgenic mice suggests a role for autoimmune cytotoxicity in the chronic disease state. The CD28 ligand-specific transgenic mice will facilitate evaluation of CD8+ T cell function in liver disease pa-thologies found in AIH. [ABSTRACT FROM AUTHOR]

: The abscopal effect is a phenomenon in which local radiotherapy is associated with the regression of metastatic cancer at a distance from the irradiated site. The abscopal effect may be mediated by activation of the immune system. Ipilimumab is a monoclonal antibody that inhibits an immunologic checkpoint on T cells, cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4). We report a case of the abscopal effect in a patient with melanoma treated with ipilimumab and radiotherapy. Temporal associations were noted: tumor shrinkage with antibody responses to the cancer–testis antigen NY-ESO-1, changes in peripheral-blood immune cells, and increases in antibody responses to other antigens after radiotherapy. (Funded by the National Institutes of Health and others.) [ABSTRACT FROM PUBLISHER]

The reported affinity differences between CD28 and CTLA-4 binding to B7-1 and B7-2 may serve to selectively regulate CD28 and CTLA-4 function by differentially recruiting and/or stabilizing these molecules at the immunological synapse. Here we show that ligand binding is important for the accumulation of both CD28 and CTLA-4 at the synapse. While CD28 is recruited to the synapse in the absence of B7-1 and B7-2 binding, it is not effectively stabilized there, as its localization can be disrupted by CTLA-4. In the case of CTLA-4, ligand binding is critical for its concentration at the synapse. We also demonstrate that the affinity and avidity differences in ligand binding translate into selective recruitment of CD28 or CTLA-4 to the immunological synapse—B7-1 is the major ligand mediating CTLA-4 localization, while B7-2 is the main ligand for CD28 concentration at the synapse. [Copyright &y& Elsevier]