MAL2 drives immune evasion in breast cancer by suppressing tumor antigen presentation

Immune evasion is a pivotal event in tumor progression. To eliminate human cancer cells, current immune checkpoint therapy is set to boost CD8⁺ T cell–mediated cytotoxicity. However, this action is eventually dependent on the efficient recognition of tumor-specific antigens via T cell receptors. One primary mechanism by which tumor cells evade immune surveillance is to downregulate their antigen presentation. Little progress has been made toward harnessing potential therapeutic targets for enhancing antigen presentation on the tumor cell. Here, we identified MAL2 as a key player that determines the turnover of the antigen-loaded MHC-I complex and reduces the antigen presentation on tumor cells. MAL2 promotes the endocytosis of tumor antigens via direct interaction with the MHC-I complex and endosome-associated RAB proteins. In preclinical models, depletion of MAL2 in breast tumor cells profoundly enhanced the cytotoxicity of tumor-infiltrating CD8⁺ T cells and suppressed breast tumor growth, suggesting that MAL2 is a potential therapeutic target for breast cancer immunotherapy.     

Identification of heme oxygenase-1–specific regulatory CD8+ T cells in cancer patients

Treg deficiencies are associated with autoimmunity. Conversely, CD4⁺ and CD8⁺ Tregs accumulate in the tumor microenvironment and are associated with prevention of antitumor immunity and anticancer immunotherapy. Recently, CD4⁺ Tregs have been much studied, but little is known about CD8⁺ Tregs and the antigens they recognize. Here, we describe what we believe to be the first natural target for CD8⁺ Tregs. Naturally occurring HLA-A2–restricted CD8⁺ T cells specific for the antiinflammatory molecule heme oxygenase-1 (HO-1) were able to suppress cellular immune responses with outstanding efficacy. HO-1–specific CD8⁺ T cells were detected ex vivo and in situ among T cells from cancer patients. HO-1–specific T cells isolated from the peripheral blood of cancer patients inhibited cytokine release, proliferation, and cytotoxicity of other immune cells. Notably, the inhibitory effect of HO-1–specific T cells was far more pronounced than that of conventional CD4⁺CD25⁺CD127^– Tregs. The inhibitory activity of HO-1–specific T cells seemed at least partly to be mediated by soluble factors. Our data link the cellular stress response to the regulation of adaptive immunity, expand the role of HO-1 in T cell–mediated immunoregulation, and establish a role for peptide-specific CD8⁺ T cells in regulating cellular immune responses. Identification of potent antigen-specific CD8⁺ Tregs may open new avenues for therapeutic interventions in both autoimmune diseases and cancer.     

Protein tyrosine phosphatase nonreceptor type 2 controls colorectal cancer development

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) recently emerged as a promising cancer immunotherapy target. We set out to investigate the functional role of PTPN2 in the pathogenesis of human colorectal carcinoma (CRC), as its role in immune-silent solid tumors is poorly understood. We demonstrate that in human CRC, increased PTPN2 expression and activity correlated with disease progression and decreased immune responses in tumor tissues. In particular, stage II and III tumors displayed enhanced PTPN2 protein expression in tumor-infiltrating T cells, and increased PTPN2 levels negatively correlated with expression of PD-1, CTLA4, STAT1, and granzyme A. In vivo, T cell– and DC-specific PTPN2 deletion reduced tumor burden in several CRC models by promoting CD44⁺ effector/memory T cells, as well as CD8⁺ T cell infiltration and cytotoxicity in the tumor. In direct relevance to CRC treatment, T cell–specific PTPN2 deletion potentiated anti–PD-1 efficacy and induced antitumor memory formation upon tumor rechallenge in vivo. Our data suggest a role for PTPN2 in suppressing antitumor immunity and promoting tumor development in patients with CRC. Our in vivo results identify PTPN2 as a key player in controlling the immunogenicity of CRC, with the strong potential to be exploited for cancer immunotherapy.     

Coinhibitory receptor PD-1H preferentially suppresses CD4+ T cell–mediated immunity

T cell activation is regulated by the interactions of surface receptors with stimulatory and inhibitory ligands. Programmed death-1 homolog (PD-1H, also called VISTA) is a member of the CD28 family of proteins and has been shown to act as a coinhibitory ligand on APCs that suppress T cell responses. Here, we determined that PD-1H functions as a coinhibitory receptor for CD4⁺ T cells. CD4⁺ T cells in mice lacking PD-1H exhibited a dramatically increased response to antigen stimulation. Furthermore, delivery of a PD-1H–specific agonist mAb directly inhibited CD4⁺ T cell activation both in vitro and in vivo, validating a coinhibitory function of PD-1H. In a murine model of acute hepatitis, administration of a PD-1H agonist mAb suppressed CD4⁺ T cell–mediated acute inflammation. PD-1H–deficient animals were highly resistant to tumor induction in a murine brain glioma model, and depletion of CD4⁺ T cells, but not CD8⁺ T cells, promoted tumor formation. Together, our findings suggest that PD-1H has potential as a target of immune modulation in the treatment of human inflammation and malignancies.     

Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease

Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of dopamine-containing neurons. Mounting evidence suggests that dopaminergic cell death is influenced by the innate immune system. However, the pathogenic role of the adaptive immune system in PD remains enigmatic. Here we showed that CD8⁺ and CD4⁺ T cells but not B cells had invaded the brain in both postmortem human PD specimens and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD during the course of neuronal degeneration. We further demonstrated that MPTP-induced dopaminergic cell death was markedly attenuated in the absence of mature T lymphocytes in 2 different immunodeficient mouse strains (Rag1^–/– and Tcrb^–/– mice). Importantly, similar attenuation of MPTP-induced dopaminergic cell death was seen in mice lacking CD4 as well as in Rag1^–/– mice reconstituted with FasL-deficient splenocytes. However, mice lacking CD8 and Rag1^–/– mice reconstituted with IFN-γ–deficient splenocytes were not protected. These data indicate that T cell–mediated dopaminergic toxicity is almost exclusively arbitrated by CD4⁺ T cells and requires the expression of FasL but not IFNγ. Further, our data may provide a rationale for targeting the adaptive arm of the immune system as a therapeutic strategy in PD.     

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination

The function of antigen-specific CD8⁺ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen–specific CD8⁺ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen–specific effector CD8⁺ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen–specific CD8⁺ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM–mediated inhibition. Thus, BTLA activation inhibits the function of human CD8⁺ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.     

Dynamic single-cell RNA sequencing identifies immunotherapy persister cells following PD-1 blockade

Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8⁺ T cell–mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell–like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α–induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti–PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.     

CTLA4 aptamer delivers STAT3 siRNA to tumor-associated and malignant T cells

Intracellular therapeutic targets that define tumor immunosuppression in both tumor cells and T cells remain intractable. Here, we have shown that administration of a covalently linked siRNA to an aptamer (apt) that selectively binds cytotoxic T lymphocyte–associated antigen 4 (CTLA4^apt) allows gene silencing in exhausted CD8⁺ T cells and Tregs in tumors as well as CTLA4-expressing malignant T cells. CTLA4 expression was upregulated in CD8⁺ T cells in the tumor milieu; therefore, CTLA4^apt fused to a STAT3-targeting siRNA (CTLA4^apt–STAT3 siRNA) resulted in internalization into tumor-associated CD8⁺ T cells and silencing of STAT3, which activated tumor antigen–specific T cells in murine models. Both local and systemic administration of CTLA4^apt–STAT3 siRNA dramatically reduced tumor-associated Tregs. Furthermore, CTLA4^apt–STAT3 siRNA potently inhibited tumor growth and metastasis in various mouse tumor models. Importantly, CTLA4 expression is observed in T cells of patients with blood malignancies, and CTLA4^apt–STAT3 siRNA treatment of immunodeficient mice bearing human T cell lymphomas promoted tumor cell apoptosis and tumor growth inhibition. These data demonstrate that a CTLA4^apt-based siRNA delivery strategy allows gene silencing in both tumor-associated T cells and tumor cells and inhibits tumor growth and metastasis.     

Th9 cells promote antitumor immune responses in vivo

Th9 cells are a subset of CD4⁺ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8⁺ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8⁺ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell–mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8⁺ CTL-mediated antitumor immunity and indicate that Th9 cell–based cancer immunotherapy may be a promising therapeutic approach.     

MerTK inhibition in tumor leukocytes decreases tumor growth and metastasis

MerTK, a receptor tyrosine kinase (RTK) of the TYRO3/AXL/MerTK family, is expressed in myeloid lineage cells in which it acts to suppress proinflammatory cytokines following ingestion of apoptotic material. Using syngeneic mouse models of breast cancer, melanoma, and colon cancer, we found that tumors grew slowly and were poorly metastatic in MerTK^–/– mice. Transplantation of MerTK^–/– bone marrow, but not wild-type bone marrow, into lethally irradiated MMTV-PyVmT mice (a model of metastatic breast cancer) decreased tumor growth and altered cytokine production by tumor CD11b⁺ cells. Although MerTK expression was not required for tumor infiltration by leukocytes, MerTK^–/– leukocytes exhibited lower tumor cell–induced expression of wound healing cytokines, e.g., IL-10 and growth arrest-specific 6 (GAS6), and enhanced expression of acute inflammatory cytokines, e.g., IL-12 and IL-6. Intratumoral CD8⁺ T lymphocyte numbers were higher and lymphocyte proliferation was increased in tumor-bearing MerTK^–/– mice compared with tumor-bearing wild-type mice. Antibody-mediated CD8⁺ T lymphocyte depletion restored tumor growth in MerTK^–/– mice. These data demonstrate that MerTK signaling in tumor-associated CD11b⁺ leukocytes promotes tumor growth by dampening acute inflammatory cytokines while inducing wound healing cytokines. These results suggest that inhibition of MerTK in the tumor microenvironment may have clinical benefit, stimulating antitumor immune responses or enhancing immunotherapeutic strategies.     

Hypoxia-inducible factor activity promotes antitumor effector function and tissue residency by CD8+ T cells

Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8⁺ T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8⁺ T cells induced HIF-1α/HIF-2α–dependent differentiation of tissue-resident memory–like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8⁺ TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.     

Definition of target antigens for naturally occurring CD4(+) CD25(+) regulatory T cells

The antigenic targets recognized by naturally occurring CD4⁺ CD25⁺ regulatory T cells (T reg cells) have been elusive. We have serologically defined a series of broadly expressed self-antigens derived from chemically induced mouse sarcomas by serological identification of antigens by recombinant expression cloning (SEREX). CD4⁺ CD25⁺ T cells from mice immunized with SEREX-defined self-antigens had strong suppressive activity on peptide-specific proliferation of CD4⁺ CD25⁻ T cells and CD8⁺ T cells. The suppressive effect was observed without in vitro T cell stimulation. Foxp3 expression in these CD4⁺ CD25⁺ T cells from immunized mice was 5–10 times greater than CD4⁺ CD25⁺ T cells derived from naive mice. The suppressive effect required cellular contact and was blocked by anti-glucocorticoid–induced tumor necrosis factor receptor family–related gene antibody. In vitro suppressive activity essentially disappeared 8 wk after the last immunization. However, it was regained by in vitro restimulation with cognate self-antigen protein but not with control protein. We propose that SEREX-defined self-antigens such as those used in this study represent self-antigens that elicit naturally occurring CD4⁺ CD25⁺ T reg cells.     

Cytomegalovirus mediates expansion of IL-15–responsive innate-memory cells with SIV killing function

Interindividual immune variability is driven predominantly by environmental factors, including exposure to chronic infectious agents such as cytomegalovirus (CMV). We investigated the effects of rhesus CMV (RhCMV) on composition and function of the immune system in young macaques. Within months of infection, RhCMV was associated with impressive changes in antigen presenting cells, T cells, and NK cells—and marked expansion of innate-memory CD8⁺ T cells. These cells express high levels of NKG2A/C and the IL-2 and IL-15 receptor beta chain, CD122. IL-15 was sufficient to drive differentiation of the cells in vitro and in vivo. Expanded NKG2A/C⁺CD122⁺CD8⁺ T cells in RhCMV-infected macaques, but not their NKG2-negative counterparts, were endowed with cytotoxicity against class I–deficient K562 targets and prompt IFN-γ production in response to stimulation with IL-12 and IL-18. Because RhCMV clone 68-1 forms the viral backbone of RhCMV-vectored SIV vaccines, we also investigated immune changes following administration of RhCMV 68-1–vectored SIV vaccines. These vaccines led to impressive expansion of NKG2A/C⁺CD8⁺ T cells with capacity to inhibit SIV replication ex vivo. Thus, CMV infection and CMV-vectored vaccination drive expansion of functional innate-like CD8 cells via host IL-15 production, suggesting that innate-memory expansion could be achieved by other vaccine platforms expressing IL-15.     

The SIRPα–CD47 immune checkpoint in NK cells

Here we report on the existence and functionality of the immune checkpoint signal regulatory protein α (SIRPα) in NK cells and describe how it can be modulated for cell therapy. NK cell SIRPα is up-regulated upon IL-2 stimulation, interacts with target cell CD47 in a threshold-dependent manner, and counters other stimulatory signals, including IL-2, CD16, or NKG2D. Elevated expression of CD47 protected K562 tumor cells and mouse and human MHC class I–deficient target cells against SIRPα⁺ primary NK cells, but not against SIRPα⁻ NKL or NK92 cells. SIRPα deficiency or antibody blockade increased the killing capacity of NK cells. Overexpression of rhesus monkey CD47 in human MHC-deficient cells prevented cytotoxicity by rhesus NK cells in a xenogeneic setting. The SIRPα–CD47 axis was found to be highly species specific. Together, the results demonstrate that disruption of the SIRPα–CD47 immune checkpoint may augment NK cell antitumor responses and that elevated expression of CD47 may prevent NK cell–mediated killing of allogeneic and xenogeneic tissues.     

Patients with lung cancer and paraneoplastic Hu syndrome harbor HuD-specific type 2 CD8+ T cells

Paraneoplastic neurologic disorders (PNDs) offer an uncommon opportunity to study human tumor immunity and autoimmunity. In small cell lung cancer (SCLC), expression of the HuD neuronal antigen is thought to lead to immune recognition, suppression of tumor growth, and, in a subset of patients, triggering of the Hu paraneoplastic neurologic syndrome. Antigen-specific CTLs believed to contribute to disease pathophysiology were described 10 years ago in paraneoplastic cerebellar degeneration. Despite parallel efforts, similar cells have not been defined in Hu patients. Here, we have identified HuD-specific T cells in Hu patients and provided an explanation for why their detection has been elusive. Different Hu patients harbored 1 of 2 kinds of HuD-specific CD8⁺ T cells: classical IFN-γ–producing CTLs or unusual T cells that produced type 2 cytokines, most prominently IL-13 and IL-5, and lacked cytolytic activity. Further, we found evidence that SCLC tumor cells produced type 2 cytokines and that these cytokines trigger naive CD8⁺ T cells to adopt the atypical type 2 phenotype. These observations demonstrate the presence of an unusual noncytotoxic CD8⁺ T cell in patients with the Hu paraneoplastic syndrome and suggest that SCLC may evade tumor immune surveillance by skewing tumor antigen–specific T cells to this unusual noncytolytic phenotype.     

TAP-independent self-peptides enhance T cell recognition of immune-escaped tumors

Tumor cells frequently escape from CD8⁺ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the “self” TRH4 peptide/D^b complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-I^lo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8⁺ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies.     

Characterization of Human CD8+ T Cells Reactive with Mycobacterium tuberculosis–infected Antigen-presenting Cells

Previous studies in murine models, including those using the β2 microglobulin knockout mouse, have suggested an important role for CD8⁺ T cells in host defense to Mycobacterium tuberculosis (Mtb). At present, little is understood about these cells in the human immune response to tuberculosis. This report demonstrates the existence of human Mtb-reactive CD8⁺ T cells. These cells are present preferentially in persons infected with Mtb and produce interferon γ in response to stimulation with Mtb-infected target cells. Recognition of Mtb-infected cells by these CD8⁺ T cells is restricted neither by the major histocompatibility complex (MHC) class I A, B, or C alleles nor by CD1, although it is inhibited by anti–MHC class I antibody. The Mtb-specific CD8⁺ T cells recognize an antigen which is generated in the proteasome, but which does not require transport through the Golgi-ER. The data suggest the possible use of nonpolymorphic MHC class Ib antigen presenting structures other than CD1.     

IL-15 promotes activation and expansion of CD8+ T cells in HIV-1 infection

In HIV-1–infected patients, increased numbers of circulating CD8⁺ T cells are linked to increased risk of morbidity and mortality. Here, we identified a bystander mechanism that promotes CD8 T cell activation and expansion in untreated HIV-1–infected patients. Compared with healthy controls, untreated HIV-1–infected patients have an increased population of proliferating, granzyme B⁺, CD8⁺ T cells in circulation. Vβ expression and deep sequencing of CDR3 revealed that in untreated HIV-1 infection, cycling memory CD8 T cells possess a broad T cell repertoire that reflects the repertoire of the resting population. This suggests that cycling is driven by bystander activation, rather than specific antigen exposure. Treatment of peripheral blood mononuclear cells with IL-15 induced a cycling, granzyme B⁺ phenotype in CD8⁺ T cells. Moreover, elevated IL-15 expression in the lymph nodes of untreated HIV-1–infected patients correlated with circulating CD8⁺ T cell counts and was normalized in these patients following antiretroviral therapy. Together, these results suggest that IL-15 drives bystander activation of CD8⁺ T cells, which predicts disease progression in untreated HIV-1–infected patients and suggests that elevated IL-15 may also drive CD8⁺ T cell expansion that is linked to increased morbidity and mortality in treated patients.