Resistance Mechanisms to Anti-PD Cancer Immunotherapy.

The transformative success of antibodies targeting the PD-1 (programmed death 1)/B7-H1 (B7 homolog 1) pathway (anti-PD therapy) has revolutionized cancer treatment. However, only a fraction of patients with solid tumors and some hematopoietic malignancies respond to anti-PD therapy, and the reason for failure in other patients is less known. By dissecting the mechanisms underlying this resistance, current studies reveal that the tumor microenvironment is a major location for resistance to occur. Furthermore, the resistance mechanisms appear to be highly heterogeneous. Here, we discuss recent human cancer data identifying mechanisms of resistance to anti-PD therapy. We review evidence for immune-based resistance mechanisms such as loss of neoantigens, defects in antigen presentation and interferon signaling, immune inhibitory molecules, and exclusion of T cells. We also review the clinical evidence for emerging mechanisms of resistance to anti-PD therapy, such as alterations in metabolism, microbiota, and epigenetics. Finally, we discuss strategies to overcome anti-PD therapy resistance and emphasize the need to develop additional immunotherapies based on the concept of normalization cancer immunotherapy.

Progestogen-driven B7-H4 contributes to onco-fetal immune tolerance.

Immune tolerance mechanisms are shared in cancer and pregnancy. Through cross-analyzing single-cell RNA-sequencing data from multiple human cancer types and the maternal-fetal interface, we found B7-H4 (VTCN1) is an onco-fetal immune tolerance checkpoint. We showed that genetic deficiency of B7-H4 resulted in immune activation and fetal resorption in allogeneic pregnancy models. Analogously, B7-H4 contributed to MPA/DMBA-induced breast cancer progression, accompanied by CD8+ T cell exhaustion. Female hormone screening revealed that progesterone stimulated B7-H4 expression in placental and breast cancer cells. Mechanistically, progesterone receptor (PR) bound to a newly identified -58 kb enhancer, thereby mediating B7-H4 transcription via the PR-P300-BRD4 axis. PR antagonist or BRD4 degrader potentiated immunotherapy in a murine B7-H4+ breast cancer model. Thus, our work unravels a mechanistic and biological connection of a female sex hormone (progesterone) to onco-fetal immune tolerance via B7-H4 and suggests that the PR-P300-BRD4 axis is targetable for treating B7-H4+ cancer.

Fibrinogen-like Protein 1 Is a Major Immune Inhibitory Ligand of LAG-3.

Lymphocyte-activation gene 3 (LAG-3) is an immune inhibitory receptor, with major histocompatibility complex class II (MHC-II) as a canonical ligand. However, it remains controversial whether MHC-II is solely responsible for the inhibitory function of LAG-3. Here, we demonstrate that fibrinogen-like protein 1 (FGL1), a liver-secreted protein, is a major LAG-3 functional ligand independent from MHC-II. FGL1 inhibits antigen-specific T cell activation, and ablation of FGL1 in mice promotes T cell immunity. Blockade of the FGL1-LAG-3 interaction by monoclonal antibodies stimulates tumor immunity and is therapeutic against established mouse tumors in a receptor-ligand inter-dependent manner. FGL1 is highly produced by human cancer cells, and elevated FGL1 in the plasma of cancer patients is associated with a poor prognosis and resistance to anti-PD-1/B7-H1 therapy. Our findings reveal an immune evasion mechanism and have implications for the design of cancer immunotherapy.

A Paradigm Shift in Cancer Immunotherapy: From Enhancement to Normalization.

Harnessing an antitumor immune response has been a fundamental strategy in cancer immunotherapy. For over a century, efforts have primarily focused on amplifying immune activation mechanisms that are employed by humans to eliminate invaders such as viruses and bacteria. This "immune enhancement" strategy often results in rare objective responses and frequent immune-related adverse events (irAEs). However, in the last decade, cancer immunotherapies targeting the B7-H1/PD-1 pathway (anti-PD therapy), have achieved higher objective response rates in patients with much fewer irAEs. This more beneficial tumor response-to-toxicity profile stems from distinct mechanisms of action that restore tumor-induced immune deficiency selectively in the tumor microenvironment, here termed "immune normalization," which has led to its FDA approval in more than 10 cancer indications and facilitated its combination with different therapies. In this article, we wish to highlight the principles of immune normalization and learn from it, with the ultimate goal to guide better designs for future cancer immunotherapies.

Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression.

How tumor cells genetically lose antigenicity and evade immune checkpoints remains largely elusive. We report that tissue-specific expression of the human long noncoding RNA LINK-A in mouse mammary glands initiates metastatic mammary gland tumors, which phenotypically resemble human triple-negative breast cancer (TNBC). LINK-A expression facilitated crosstalk between phosphatidylinositol-(3,4,5)-trisphosphate and inhibitory G-protein-coupled receptor (GPCR) pathways, attenuating protein kinase A-mediated phosphorylation of the E3 ubiquitin ligase TRIM71. Consequently, LINK-A expression enhanced K48-polyubiquitination-mediated degradation of the antigen peptide-loading complex (PLC) and intrinsic tumor suppressors Rb and p53. Treatment with LINK-A locked nucleic acids or GPCR antagonists stabilized the PLC components, Rb and p53, and sensitized mammary gland tumors to immune checkpoint blockers. Patients with programmed ccll death protein-1(PD-1) blockade-resistant TNBC exhibited elevated LINK-A levels and downregulated PLC components. Hence we demonstrate lncRNA-dependent downregulation of antigenicity and intrinsic tumor suppression, which provides the basis for developing combinational immunotherapy treatment regimens and early TNBC prevention.

VISTA (PD-1H) Is a Crucial Immune Regulator to Limit Pulmonary Fibrosis.

VISTA (V domain immunoglobulin suppressor of T cell activation, also called PD-1H [programmed death-1 homolog]), a novel immune regulator expressed on myeloid and T lymphocyte lineages, is upregulated in mouse and human idiopathic pulmonary fibrosis (IPF). However, the significance of VISTA and its therapeutic potential in regulating IPF has yet to be defined. To determine the role of VISTA and its therapeutic potential in IPF, the expression profile of VISTA was evaluated from human single-cell RNA sequencing data (IPF Cell Atlas). Inflammatory response and lung fibrosis were assessed in bleomycin-induced experimental pulmonary fibrosis models in VISTA-deficient mice compared with wild-type littermates. In addition, these outcomes were evaluated after VISTA agonistic antibody treatment in the wild-type pulmonary fibrosis mice. VISTA expression was increased in lung tissue-infiltrating monocytes of patients with IPF. VISTA was induced in the myeloid population, mainly circulating monocyte-derived macrophages, during bleomycin-induced pulmonary fibrosis. Genetic ablation of VISTA drastically promoted pulmonary fibrosis, and bleomycin-induced fibroblast activation was dependent on the interaction between VISTA-expressing myeloid cells and fibroblasts. Treatment with VISTA agonistic antibody reduced fibrotic phenotypes accompanied by the suppression of lung innate immune and fibrotic mediators. In conclusion, these results suggest that VISTA upregulation in pulmonary fibrosis may be a compensatory mechanism to limit inflammation and fibrosis, and stimulation of VISTA signaling using VISTA agonists effectively limits the fibrotic innate immune landscape and consequent tissue fibrosis. Further studies are warranted to test VISTA as a novel therapeutic target for the IPF treatment.

Interaction of glioma-associated microglia/macrophages and anti-PD1 immunotherapy.

Anti-PD-1-based therapy has resulted in a minimal clinical response in malignant gliomas. Gliomas contain numerous glioma-associated microglia/macrophages (GAMs), reported to contribute to an immunosuppressive microenvironment and promote glioma progression. However, whether and how GAMs affect anti-PD-1 immunotherapy in glioma remains unclear. Here, we demonstrated that M1-like GAMs contribute to the anti-PD-1 therapeutic response, while the accumulation of M2-like GAMs is associated with therapeutic resistance. Furthermore, we found that PD-L1 ablation reverses GAMs M2-like phenotype and is beneficial to anti-PD-1 therapy. We also demonstrated that tumor-induced impairment of the antigen-presenting function of GAMs could limit the antitumor immunity of CD4+ T cells in anti-PD-1 therapy. Our study highlights the impact of GAMs activation on anti-PD-1 treatment and provides new insights into the role of GAMs in regulating anti-PD-1 therapy in gliomas.

Structural insight into T cell coinhibition by PD-1H (VISTA).

Programmed death-1 homolog (PD-1H), a CD28/B7 family molecule, coinhibits T cell activation and is an attractive immunotherapeutic target for cancer and inflammatory diseases. The molecular basis of its function, however, is unknown. Bioinformatic analyses indicated that PD-1H has a very long Ig variable region (IgV)-like domain and extraordinarily high histidine content, suggesting that unique structural features may contribute to coinhibitory mechanisms. Here we present the 1.9-Å crystal structure of the human PD-1H extracellular domain. It reveals an elongated CC' loop and a striking concentration of histidine residues, located in the complementarity-determining region-like proximal half of the molecule. We show that surface-exposed histidine clusters are essential for robust inhibition of T cell activation. PD-1H exhibits a noncanonical IgV-like topology including an extra "H" ß-strand and "clamping" disulfide, absent in known IgV-like structures, that likely restricts its orientation on the cell surface differently from other IgV-like domains. These results provide important insight into a molecular basis of T cell coinhibition by PD-1H.

PD-1 regulates germinal center B cell survival and the formation and affinity of long-lived plasma cells.

Memory B and plasma cells (PCs) are generated in the germinal center (GC). Because follicular helper T cells (T(FH) cells) have high expression of the immunoinhibitory receptor PD-1, we investigated the role of PD-1 signaling in the humoral response. We found that the PD-1 ligands PD-L1 and PD-L2 were upregulated on GC B cells. Mice deficient in PD-L2 (Pdcd1lg2(-/-)), PD-L1 and PD-L2 (Cd274(-/-)Pdcd1lg2(-/-)) or PD-1 (Pdcd1(-/-)) had fewer long-lived PCs. The mechanism involved more GC cell death and less T(FH) cell cytokine production in the absence of PD-1; the effect was selective, as remaining PCs had greater affinity for antigen. PD-1 expression on T cells and PD-L2 expression on B cells controlled T(FH) cell and PC numbers. Thus, PD-1 regulates selection and survival in the GC, affecting the quantity and quality of long-lived PCs.

Defining and Understanding Adaptive Resistance in Cancer Immunotherapy.

Despite the unprecedented tumor regression and long-term survival benefit observed with anti-programmed death (PD) [anti-PD-1 or anti-B7-homolog 1 (B7-H1)] therapy in patients with advanced cancers, a large portion of patients do not benefit from such treatment and a fraction of responders relapse. Current efforts to overcome resistance and improve efficacy of anti-PD therapy require a clear understanding of resistance and should precede current avenues using random combinations with available treatment regimens. Here, we categorized three types of resistance, namely target-missing, primary, and acquired resistance. This categorization requires reliable, accurate tissue sampling and appropriate interpretation of results based on the four classifications of tumor immunity in the microenvironment (TIME). We believe that fundamental understanding of these complex tumor-immune interactions and of the cellular and molecular mechanisms underlying these types of true resistance is the key for targeting the right targets in combination with or beyond anti-PD therapy in the future.

B7-H1 maintains the polyclonal T cell response by protecting dendritic cells from cytotoxic T lymphocyte destruction.

Induced B7-H1 expression in the tumor microenvironment initiates adaptive resistance, which impairs immune functions and leads to tumor escape from immune destruction. Antibody blockade of the B7-H1/PD-1 interaction overcomes adaptive resistance, leading to regression of advanced human cancers and survival benefits in a significant fraction of patients. In addition to cancer cells, B7-H1 is expressed on dendritic cells (DCs), but its role in DC functions is less understood. DCs can present multiple antigens (Ags) to stimulate dominant or subdominant T cell responses. Here, we show that immunization with multiple tumor Ag-loaded DCs, in the absence of B7-H1, vastly enhances cytotoxic T lymphocyte (CTL) responses to dominant Ag. In sharp contrast, CTL responses to subdominant Ag were paradoxically suppressed, facilitating outgrowth of tumor variants carrying only subdominant Ag. Suppressed CTL responses to subdominant Ag are largely due to the loss of B7-H1-mediated protection of DCs from the lysis of CTL against dominant Ag. Therefore, B7-H1 expression on DCs may help maintain the diversity of CTL responses to multiple tumor Ags. Interestingly, a split immunization approach, which presents dominant and subdominant Ags with different DCs, promoted CTL responses to all Ags and prevented tumor escape in murine tumor models. These findings have implications for the design of future combination cancer immunotherapies.

Cell surface signaling molecules in the control of immune responses: a tide model.

A large numbers of cell surface signaling molecules (CSSMs) have been molecularly identified and functionally characterized in recent years and, via these studies, our knowledge in the control of immune response has increased exponentially. Two major lines of evidence emerge. First, the majority of immune cells rely on one or few CSSMs to deliver a primary triggering signal to sense their environment, leading to initiation of an immune response. Second, both costimulatory CSSMs that promote the response, and coinhibitory CSSMs that inhibit the response, are required to control direction and magnitude of a given immune response. With such tight feedback, immune responses are tuned and returned to baseline. These findings extend well beyond our previous observation in the requirement for lymphocyte activation and argue a revisit of the traditional "two-signal model" for activation and tolerance of lymphocytes. Here we propose a "tide" model to accommodate and interpret current experimental findings.

B7-h2 is a costimulatory ligand for CD28 in human.

CD28 and CTLA-4 are cell surface cosignaling molecules essential for the control of T cell activation upon the engagement of their ligands B7-1 and B7-2 from antigen-presenting cells. By employing a receptor array assay, we have demonstrated that B7-H2, best known as the ligand of inducible costimulator, was a ligand for CD28 and CTLA-4 in human, whereas these interactions were not conserved in mouse. B7-H2 and B7-1 or B7-2 interacted with CD28 through distinctive domains. B7-H2-CD28 interaction was essential for the costimulation of human T cells' primary responses to allogeneic antigens and memory recall responses. Similar to B7-1 and B7-2, B7-H2 costimulation via CD28 induced survival factor Bcl-xL, downregulated cell cycle inhibitor p27(kip1), and triggered signaling cascade of ERK and AKT kinase-dependent pathways. Our findings warrant re-evaluation of CD28 and CTLA-4's functions previously attributed exclusively to B7-1 and B7-2 and have important implications in therapeutic interventions against human diseases.

B7-H4 Modulates Regulatory CD4+ T Cell Induction and Function via Ligation of a Semaphorin 3a/Plexin A4/Neuropilin-1 Complex.

The potent immune regulatory function of an agonistic B7-H4-Ig fusion protein (B7-H4Ig) has been demonstrated in multiple experimental autoimmune models; however, the identity of a functional B7-H4 receptor remained unknown. The biological activity of B7-H4 is associated with decreased inflammatory CD4+ T cell responses as supported by a correlation between B7-H4-expressing tumor-associated macrophages and Foxp3+ T cells within the tumor microenvironment. Recent data indicate that members of the semaphorin (Sema)/plexin/neuropilin (Nrp) family of proteins both positively and negatively modulate immune cell function. In this study, we show that B7-H4 binds the soluble Sema family member Sema3a. Additionally, B7-H4Ig-induced inhibition of inflammatory CD4+ T cell responses is lost in both Sema3a functional mutant mice and mice lacking Nrp-1 expression in Foxp3+ T cells. These findings indicate that B7-H4Ig binds to Sema3a, which acts as a functional bridge to stimulate an Nrp-1/Plexin A4 heterodimer to form a functional immunoregulatory receptor complex resulting in increased levels of phosphorylated PTEN and enhanced regulatory CD4+ T cell number and function.

Focusing and sustaining the antitumor CTL effector killer response by agonist anti-CD137 mAb.

Cancer immunotherapy is undergoing significant progress due to recent clinical successes by refined adoptive T-cell transfer and immunostimulatory monoclonal Ab (mAbs). B16F10-derived OVA-expressing mouse melanomas resist curative immunotherapy with either adoptive transfer of activated anti-OVA OT1 CTLs or agonist anti-CD137 (4-1BB) mAb. However, when acting in synergistic combination, these treatments consistently achieve tumor eradication. Tumor-infiltrating lymphocytes that accomplish tumor rejection exhibit enhanced effector functions in both transferred OT-1 and endogenous cytotoxic T lymphocytes (CTLs). This is consistent with higher levels of expression of eomesodermin in transferred and endogenous CTLs and with intravital live-cell two-photon microscopy evidence for more efficacious CTL-mediated tumor cell killing. Anti-CD137 mAb treatment resulted in prolonged intratumor persistence of the OT1 CTL-effector cells and improved function with focused and confined interaction kinetics of OT-1 CTL with target cells and increased apoptosis induction lasting up to six days postadoptive transfer. The synergy of adoptive T-cell therapy and agonist anti-CD137 mAb thus results from in vivo enhancement and sustainment of effector functions.

How Ebola and Marburg viruses battle the immune system.

The filoviruses Ebola and Marburg have emerged in the past decade from relative obscurity to serve now as archetypes for some of the more intriguing and daunting challenges posed by such agents. Public imagination is captured by deadly outbreaks of these viruses and reinforced by the specter of bioterrorism. As research on these agents has accelerated, it has been found increasingly that filoviruses use a combination of familiar and apparently new ways to baffle and battle the immune system. Filoviruses have provided thereby a new lens through which to examine the immune system itself.

Granulocytic myeloid-derived suppressor cells suppress virus-specific CD8+ T cell responses during acute Friend retrovirus infection.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) can suppress T cell responses in several different diseases. Previously these suppressive cells were observed to expand in HIV patients and in a mouse retrovirus model, yet their suppressive effect on virus-specific CD8+ T cells in vitro and in vivo has not been characterized thus far. RESULTS: We used the Friend retrovirus (FV) model to demonstrate that MDSCs expand and become activated during the late phase of acute FV infection. Only the subpopulation of granulocytic MDSCs (gMDSCs) but not monocytic MDSC suppressed virus-specific CD8+ T cell proliferation and function in vitro. gMDSCs expressed arginase 1, high levels of the inhibitory ligand PD-L1 and the ATP dephosphorylating enzyme CD39 on the cell surface upon infection. All three molecules were involved in the suppressive effect of the gMDSCs in vitro. MDSC depletion experiments in FV-infected mice revealed that they restrict virus-specific CD8+ T cell responses and thus affect the immune control of chronic retroviruses in vivo. CONCLUSIONS: Our study demonstrates that MDSCs become activated and expand during the acute phase of retrovirus infection. Their suppressive activity on virus-specific CD8+ T cells may contribute to T cell dysfunction and the development of chronic infection.

PD-L1 Expression on Retrovirus-Infected Cells Mediates Immune Escape from CD8+ T Cell Killing.

Cytotoxic CD8+ T Lymphocytes (CTL) efficiently control acute virus infections but can become exhausted when a chronic infection develops. Signaling of the inhibitory receptor PD-1 is an important mechanism for the development of virus-specific CD8+ T cell dysfunction. However, it has recently been shown that during the initial phase of infection virus-specific CD8+ T cells express high levels of PD-1, but are fully competent in producing cytokines and killing virus-infected target cells. To better understand the role of the PD-1 signaling pathway in CD8+ T cell cytotoxicity during acute viral infections we analyzed the expression of the ligand on retrovirus-infected cells targeted by CTLs. We observed increased levels of PD-L1 expression after infection of cells with the murine Friend retrovirus (FV) or with HIV. In FV infected mice, virus-specific CTLs efficiently eliminated infected target cells that expressed low levels of PD-L1 or that were deficient for PD-L1 but the population of PD-L1high cells escaped elimination and formed a reservoir for chronic FV replication. Infected cells with high PD-L1 expression mediated a negative feedback on CD8+ T cells and inhibited their expansion and cytotoxic functions. These findings provide evidence for a novel immune escape mechanism during acute retroviral infection based on PD-L1 expression levels on virus infected target cells.