Gloria H. Y. Lin

Immune regulation by 4-1BB and 4-1BBL: complexities and challenges

Summary:  The tumor necrosis factor receptor family member 4‐1BB plays a key role in the survival of activated and memory CD8+ T cells. Depending on the disease model, 4‐1BB can participate at different stages and influence different aspects of the immune response, likely due to the differential expression of receptor and ligand relative to other costimulatory molecules. Studies comparing mild versus severe influenza infection of mice suggest that the immune system uses inducible receptors such as 4‐1BB to prolong the immune response when pathogens take longer to clear. The expression of 4‐1BB on diverse cell types, evidence for bidirectional as well as receptor‐independent signaling by 4‐1BBL, the unexpected hyperproliferation of 4‐1BB‐deficient T cells, and complex effects of agonistic anti‐4‐1BB therapy have revealed additional roles for the 4‐1BB/4‐1BBL receptor/ligand pair in the immune system. In this review, we discuss these diverse roles of 4‐1BB and its ligand in the immune response, exploring possible mechanisms for the observed complexities and implications for therapeutic applications of 4‐1BB/4‐1BBL.

Endogenous 4-1BB Ligand Plays a Critical Role in Protection from Influenza-Induced Disease

A critical issue during severe respiratory infection is whether it is the virus or the host response that does the most damage. In this study, we show that endogenous 4-1BBL plays a critical role in protecting mice from severe effects of influenza disease. During mild respiratory influenza infection in which virus is rapidly cleared, the inducible costimulatory receptor 4-1BB is only transiently induced on lung T cells and 4-1BB ligand (4-1BBL) is completely dispensable for the initial CD8 T cell response and mouse survival. In contrast, during more severe respiratory influenza infection with prolonged viral load, 4-1BB expression on lung CD8 T cells is sustained, and 4-1BBL-deficient mice show decreased CD8 T cell accumulation in the lungs, decreased viral clearance, impaired lung function, and increased mortality. Transfer of an optimal number of naive Ag-specific T cells before infection protects wild-type but not 4-1BBL-deficient mice from an otherwise lethal dose of influenza virus. Transfer of T cells lacking the proapoptotic molecule Bim extends the lifespan of 4-1BBL-deficient mice by one to three days, suggesting that at least part of the role of 4-1BB/4-1BBL is to prolong effector cell survival long enough to clear virus. Intranasal delivery of 4-1BBL by recombinant adenovirus marginally improves survival of 4-1BBL-deficient mice at low dose, but exacerbates disease at high dose. These findings suggest a rationale for the evolutionary accumulation of inducible costimulatory molecules, thereby allowing the immune system to sustain the expression of molecules such as 4-1BB to a level commensurate with severity of infection.

T‐cell intrinsic effects of GITR and 4‐1BB during viral infection and cancer immunotherapy

Summary:  GITR [glucocorticoid inducible tumor necrosis factor receptor (TNFR)‐related protein] and 4‐1BB are costimulatory TNFR family members that are expressed on regulatory and effector T cells as well as on other cells of the immune system. Here we discuss the role of GITR and 4‐1BB on T cells during viral infections and in cancer immunotherapy. Systemic treatment with agonistic anti‐4‐1BB antibody leads to a number of immune system abnormalities, and clinical trials of anti‐4‐1BB have been terminated. However, other modes of 4‐1BB ligation may be less toxic. To date, similar toxicities have not been reported for anti‐GITR treatment of mice, although anti‐GITR antibodies can exacerbate mouse autoimmune models. Intrinsic effects of GITR and 4‐1BB on effector T cells appear to predominate over their effects on other cell types in some models. Despite their similarities in enhancing T‐cell survival, 4‐1BB and GITR are clearly not redundant, and both pathways are required for maximal CD8+ T‐cell responses and mouse survival following severe respiratory influenza infection. GITR uses TNFR‐associated factor (TRAF) 2 and TRAF5, whereas 4‐1BB recruits TRAF1 and TRAF2 to mediate survival signaling in T cells. The differential use of signaling adapters combined with their differential expression may explain the non‐redundant roles of GITR and 4‐1BB in the immune system.

CD8 T Cell-Intrinsic GITR Is Required for T Cell Clonal Expansion and Mouse Survival following Severe Influenza Infection

The regulation of T cell expansion by TNFR family members plays an important role in determining the magnitude of the immune response to pathogens. As several members of the TNFR family, including glucocorticoid-induced TNFR-related protein (GITR), are found on both regulatory and effector T cells, there is much interest in understanding how their effects on these opposing arms of the immune system affect disease outcome. Whereas much work has focused on the role of GITR on regulatory T cells, little is known about its intrinsic role on effector T cells in an infectious disease context. In this study, we demonstrate that GITR signaling on CD8 T cells leads to TNFR-associated factor (TRAF) 2/5-dependent, TRAF1-independent NF-κB induction, resulting in increased Bcl-xL. In vivo, GITR on CD8 T cells has a profound effect on CD8 T cell expansion, via effects on T cell survival. Moreover, GITR is required on CD8 T cells for enhancement of influenza-specific CD8 T cell expansion upon administration of agonistic anti-GITR Ab, DTA-1. Remarkably, CD8 T cell-intrinsic GITR is essential for mouse survival during severe, but dispensable during mild respiratory influenza infection. These studies highlight the importance of GITR as a CD8 T cell costimulator during acute viral infection, and argue that despite the similarity among several TNFR family members in inducing T lymphoctye survival, they clearly have nonredundant functions in protection from severe infection.

Loss of the signaling adaptor TRAF1 causes CD8+ T cell dysregulation during human and murine chronic infection

Loss of intracellular TRAF1 expression correlates with CD8+ T cell exhaustion and contributes to increased viral load at the chronic stage of HIV and LCMV infection, a phenotype that can be reversed by IL-7 stimulation together with 4-1BB agonist.

The contextual role of TNFR family members in CD8(+) T-cell control of viral infections.

Immunity to viruses must be tightly controlled to avoid pathology. Receptors and ligands of the tumor necrosis factor (TNF) family play important roles in controlling lymphocyte activation and survival during an immune response. The role of specific TNF receptor (TNFR) family members in antiviral immunity depends on the stage of the immune response and can vary with the virus type and its virulence. Here, we focus on five members of the TNFR family that are prominently expressed on CD8+ T cells during viral infections, namely, 4‐1BB (CD137), CD27, OX40 (CD134), GITR, and TNFR2. 4‐1BB, CD27, OX40, and GITR have primarily prosurvival roles for CD8+ T cells during viral infection, although under some circumstances 4‐1BB, GITR, or CD27 signals can limit immunity. Although TNFR2 can be costimulatory under some circumstances, its main role in CD8+ T‐cell responses during viral infection appears to be in contraction of the response. Several TNF family ligands are being explored as adjuvants for viral vaccines, and agonistic antibodies to TNFR family members are being investigated for immunotherapy of chronic viral infection alone and in combination with checkpoint blockade. Such therapies will require thorough and specific optimization to avoid pathology induced by hyperstimulation of these pathways.

Evaluating the cellular targets of anti-4-1BB agonist antibody during immunotherapy of a pre-established tumor in mice.

Background Manipulation of the immune system represents a promising avenue for cancer therapy. Rational advances in immunotherapy of cancer will require an understanding of the precise correlates of protection. Agonistic antibodies against the tumor necrosis factor receptor family member 4-1BB are emerging as a promising tool in cancer therapy, with evidence that these antibodies expand both T cells as well as innate immune cells. Depletion studies have suggested that several cell types can play a role in these immunotherapeutic regimens, but do not reveal which cells must directly receive the 4-1BB signals for effective therapy. Methodology/Principal Findings We show that re-activated memory T cells are superior to resting memory T cells in control of an 8-day pre-established E.G7 tumor in mice. We find that ex vivo activation of the memory T cells allows the activated effectors to continue to divide and enter the tumor, regardless of antigen-specificity; however, only antigen-specific reactivated memory T cells show any efficacy in tumor control. When agonistic anti-4-1BB antibody is combined with this optimized adoptive T cell therapy, 80% of mice survive and are fully protected from tumor rechallenge. Using 4-1BB-deficient mice and mixed bone marrow chimeras, we find that it is sufficient to have 4-1BB only on the endogenous host αβ T cells or only on the transferred T cells for the effects of anti-4-1BB to be realized. Conversely, although multiple immune cell types express 4-1BB and both T cells and APC expand during anti-4-1BB therapy, 4-1BB on cells other than αβ T cells is neither necessary nor sufficient for the effect of anti-4-1BB in this adoptive immunotherapy model. Conclusions/Significance This study establishes αβ T cells rather than innate immune cells as the critical target in anti-4-1BB therapy of a pre-established tumor. The study also demonstrates that ex vivo activation of memory T cells prior to infusion allows antigen-specific tumor control without the need for reactivation of the memory T cells in the tumor.

Perforin Is a Novel Immune Regulator of Obesity-Related Insulin Resistance

Obesity-related insulin resistance is associated with an influx of pathogenic T cells into visceral adipose tissue (VAT), but the mechanisms regulating lymphocyte balance in such tissues are unknown. Here we describe an important role for the immune cytotoxic effector molecule perforin in regulating this process. Perforin-deficient mice (Prf1null) show early increased body weight and adiposity, glucose intolerance, and insulin resistance when placed on high-fat diet (HFD). Regulatory effects of perforin on glucose tolerance are mechanistically linked to the control of T-cell proliferation and cytokine production in inflamed VAT. HFD-fed Prf1null mice have increased accumulation of proinflammatory IFN-γ–producing CD4+ and CD8+ T cells and M1-polarized macrophages in VAT. CD8+ T cells from the VAT of Prf1null mice have increased proliferation and impaired early apoptosis, suggesting a role for perforin in the regulation of T-cell turnover during HFD feeding. Transfer of CD8+ T cells from Prf1null mice into CD8-deficient mice (CD8null) resulted in worsening of metabolic parameters compared with wild-type donors. Improved metabolic parameters in HFD natural killer (NK) cell–deficient mice (NKnull) ruled out a role for NK cells as a single source of perforin in regulating glucose homeostasis. The findings support the importance of T-cell function in insulin resistance and suggest that modulation of lymphocyte homeostasis in inflamed VAT is one possible avenue for therapeutic intervention.

GITR-Dependent Regulation of 4-1BB Expression: Implications for T Cell Memory and Anti–4-1BB–Induced Pathology

The TNFR family member 4-1BB plays a key role in the survival of activated and memory CD8 T cells. However, the mechanisms that regulate 4-1BB re-expression on memory CD8 T cells after Ag clearance are unknown. In unimmunized mice, ∼10% of CD8 CD44hi memory T cells in the bone marrow (BM) and liver express 4-1BB, with minimal 4-1BB expression in spleen and lymph node. IL-2, IL-15, and IL-7 are collectively dispensable for 4-1BB expression on the memory CD8 T cells. Rather, T cell–intrinsic glucocorticoid-induced TNFR-related protein (GITR) contributes to 4-1BB expression on CD8 T cells upon their entry into the BM or liver. Consistent with its role in regulation of 4-1BB, GITR is required on memory CD8 T cells for their persistence in vivo. These findings reveal site-specific effects of the BM and liver microenvironment on CD8 memory T cells. Previous work has demonstrated that 4-1BB agonists given to unimmunized mice induce splenomegaly, hepatitis, and other immune system anomalies. Moreover, severe liver pathology has been observed in a subset of anti–4-1BB–treated melanoma patients. Remarkably, the absence of GITR in mice almost completely abrogates cellular expansions, splenomegaly, and liver inflammation associated with anti–4-1BB agonist treatment of unimmunized mice. In contrast, lack of CD8 T cells selectively improves liver pathology, but not splenomegaly in the mice. Thus, the regulation of 4-1BB expression by GITR on CD8 T cells, as well as on other cells, contributes to the pathological effects of anti–4-1BB in unimmunized mice.

IL-15–Dependent Upregulation of GITR on CD8 Memory Phenotype T Cells in the Bone Marrow Relative to Spleen and Lymph Node Suggests the Bone Marrow as a Site of Superior Bioavailability of IL-15

CD8 memory T cells are enriched in the bone marrow, a site where these cells are thought to receive homeostatic signals. However, the primary site where CD8 memory T cells receive their cytokine-induced homeostatic signals has recently come under debate. In this study, we demonstrate that the bone marrow contains a fraction of CD8 memory phenotype T cells with elevated expression of glucocorticoid-induced TNFR-related protein (GITR). In contrast, splenic and lymph node memory phenotype T cells have GITR levels similar to those on naive T cells. The bone marrow GITRhi memory T cells have a phenotype indicative of cytokine activation, with higher CD122 and lower CD127 than do the GITRbasal memory T cells. Remarkably, these bone marrow-specific GITRhi cells are almost completely ablated in the absence of IL-15, whereas TNFR2 and 4-1BB expression on the CD8 memory T cells are IL-15 independent. Furthermore, adoptively transferred splenic CD8 memory phenotype T cells show IL-15–dependent GITR upregulation upon entry into the bone marrow. This result implies that the selective appearance of GITRhi memory phenotype T cells in the bone marrow reflects the local microenvironment rather than a different subset of memory T cells. GITR−/− mice have a lower frequency of CD8 memory phenotype cells in the bone marrow, yet the GITR−/− cells hyperproliferate compared with those in wild-type mice. Taken together, these data suggest that GITR plays a role in the survival of CD8 memory phenotype T cells and that GITR upregulation represents a precise marker of cells that have responded to IL-15.