Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function.

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated T cells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated T cells in vitro and promote CD8 T cell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs in vivo. Anti-4-1BB mAbs also promoted effector T cell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by either agonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.

A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.

Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

The immunobiology of CD27 and OX40 and their potential as targets for cancer immunotherapy.

In recent years, monoclonal antibodies (mAbs) able to reinvigorate antitumor T-cell immunity have heralded a paradigm shift in cancer treatment. The most high profile of these mAbs block the inhibitory checkpoint receptors PD-1 and CTLA-4 and have improved life expectancy for patients across a range of tumor types. However, it is becoming increasingly clear that failure of some patients to respond to checkpoint inhibition is attributable to inadequate T-cell priming. For full T-cell activation, 2 signals must be received, and ligands providing the second of these signals, termed costimulation, are often lacking in tumors. Members of the TNF receptor superfamily (TNFRSF) are key costimulators of T cells during infection, and there has been an increasing interest in harnessing these receptors to augment tumor immunity. We here review the immunobiology of 2 particularly promising TNFRSF target receptors, CD27 and OX40, and their respective ligands, CD70 and OX40L, focusing on their role within a tumor setting. We describe the influence of CD27 and OX40 on human T cells based on in vitro studies and on the phenotypes of several recently described individuals exhibiting natural deficiencies in CD27/CD70 and OX40. Finally, we review key literature describing progress in elucidating the efficacy and mode of action of OX40- and CD27-targeting mAbs in preclinical models and provide an overview of current clinical trials targeting these promising receptor/ligand pairings in cancer.

Cleavage of TL1A Differentially Regulates Its Effects on Innate and Adaptive Immune Cells.

TNF superfamily cytokines play major roles in the regulation of adaptive and innate immunity. The TNF superfamily cytokine TL1A (TNFSF15), through its cognate receptor DR3 (TNFRSF25), promotes T cell immunity to pathogens and directly costimulates group 2 and 3 innate lymphoid cells. Polymorphisms in the TNFSF15 gene are associated with the risk for various human diseases, including inflammatory bowel disease. Like other cytokines in the TNF superfamily, TL1A is synthesized as a type II transmembrane protein and cleaved from the plasma membrane by metalloproteinases. Membrane cleavage has been shown to alter or abrogate certain activities of other TNF family cytokines; however, the functional capabilities of membrane-bound and soluble forms TL1A are not known. Constitutive expression of TL1A in transgenic mice results in expansion of activated T cells and promotes intestinal hyperplasia and inflammation through stimulation of group 2 innate lymphoid cells. Through the generation of membrane-restricted TL1A-transgenic mice, we demonstrate that membrane TL1A promotes expression of inflammatory cytokines in the lung, dependent upon DR3 expression on T cells. Soluble TL1A alone was unable to produce this phenotype but was still able to induce intestinal type 2 inflammation independently of T cells. These data suggest differential roles for membrane and soluble TL1A on adaptive and innate immune cells and have implications for the consequences of blocking these two forms of TL1A.

Akt signaling is critical for memory CD8+ T-cell development and tumor immune surveillance.

Memory CD8+ T cells confer long-term immunity against tumors, and anticancer vaccines therefore should maximize their generation. Multiple memory CD8+ T-cell subsets with distinct functional and homing characteristics exist, but the signaling pathways that regulate their development are ill defined. Here we examined the role of the serine/threonine kinase Akt in the generation of protective immunity by CD8+ T cells. Akt is known to be activated by the T-cell antigen receptor and the cytokine IL-2, but its role in T-cell immunity in vivo has not been explored. Using CD8+ T cells from pdk1K465E/K465E knockin mice, we found that decreased Akt activity inhibited the survival of T cells during the effector-to-memory cell transition and abolished their differentiation into C-X-C chemokine receptor 3 (CXCR3)loCD43lo effector-like memory cells. Consequently, antitumor immunity by CD8+ T cells that display defective Akt signaling was substantially diminished during the memory phase. Reduced memory T-cell survival and altered memory cell differentiation were associated with up-regulation of the proapoptotic protein Bim and the T-box transcription factor eomesodermin, respectively. These findings suggest an important role for effector-like memory CD8+ T cells in tumor immune surveillance and identify Akt as a key signaling node in the development of protective memory CD8+ T-cell responses.

T cell expression of IL-18R and DR3 is essential for non-cognate stimulation of Th1 cells and optimal clearance of intracellular bacteria.

Th1 cells can be activated by TCR-independent stimuli, but the importance of this pathway in vivo and the precise mechanisms involved require further investigation. Here, we used a simple model of non-cognate Th1 cell stimulation in Salmonella-infected mice to examine these issues. CD4 Th1 cell expression of both IL-18R and DR3 was required for optimal IFN-γ induction in response to non-cognate stimulation, while IL-15R expression was dispensable. Interestingly, effector Th1 cells generated by immunization rather than live infection had lower non-cognate activity despite comparable IL-18R and DR3 expression. Mice lacking T cell intrinsic expression of MyD88, an important adapter molecule in non-cognate T cell stimulation, exhibited higher bacterial burdens upon infection with Salmonella, Chlamydia or Brucella, suggesting that non-cognate Th1 stimulation is a critical element of efficient bacterial clearance. Thus, IL-18R and DR3 are critical players in non-cognate stimulation of Th1 cells and this response plays an important role in protection against intracellular bacteria.

The Use of Anti-CD40 mAb in Cancer.

Immunomodulatory monoclonal antibody (mAb) therapy is at the forefront of developing cancer therapeutics with numerous targeted agents proving highly effective in selective patients at stimulating protective host immunity, capable of eradicating established tumours and leading to long-term disease-free states. The cell surface marker CD40 is expressed on a range of immune cells and transformed cells in malignant states whose signalling plays a critical role in modulating adaptive immune responses. Anti-CD40 mAb therapy acts via multiple mechanisms to stimulate anti-tumour immunity across a broad range of lymphoid and solid malignancies. A wealth of preclinical research in this field has led to the successful development of multiple anti-CD40 mAb agents that have shown promise in early-phase clinical trials. Significant progress has been made to enhance the engagement of antibodies with immune effectors through their interactions with Fcγ receptors (FcγRs) by the process of Fc engineering. As more is understood about how to best optimise these agents, principally through the fine-tuning of mAb structure and choice of synergistic partnerships, our ability to generate robust, clinically beneficial anti-tumour activity will form the foundation for the next generation of cancer therapeutics.

OX40- and CD27-mediated costimulation synergizes with anti-PD-L1 blockade by forcing exhausted CD8+ T cells to exit quiescence.

Exhaustion of chronically stimulated CD8(+) T cells is a significant obstacle to immune control of chronic infections or tumors. Although coinhibitory checkpoint blockade with anti-programmed death ligand 1 (PD-L1) Ab can restore functions to exhausted T cell populations, recovery is often incomplete and dependent upon the pool size of a quiescent T-bet(high) subset that expresses lower levels of PD-1. In a model in which unhelped, HY-specific CD8(+) T cells gradually lose function following transfer to male bone marrow transplantation recipients, we have explored the effect of shifting the balance away from coinhibition and toward costimulation by combining anti-PD-L1 with agonistic Abs to the TNFR superfamily members, OX40 and CD27. Several weeks following T cell transfer, both agonistic Abs, but especially anti-CD27, demonstrated synergy with anti-PD-L1 by enhancing CD8(+) T cell proliferation and effector cytokine generation. Anti-CD27 and anti-PD-L1 synergized by downregulating the expression of multiple quiescence-related genes concomitant with a reduced frequency of T-bet(high) cells within the exhausted population. However, in the presence of persistent Ag, the CD8(+) T cell response was not sustained and the overall size of the effector cytokine-producing pool eventually contracted to levels below that of controls. Thus, CD27-mediated costimulation can synergize with coinhibitory checkpoint blockade to switch off molecular programs for quiescence in exhausted T cell populations, but at the expense of losing precursor cells required to maintain a response.

The TNF-family ligand TL1A and its receptor DR3 promote T cell-mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9-producing T cells.

The TNF family cytokine TL1A (Tnfsf15) costimulates T cells and type 2 innate lymphocytes (ILC2) through its receptor DR3 (Tnfrsf25). DR3-deficient mice have reduced T cell accumulation at the site of inflammation and reduced ILC2-dependent immune responses in a number of models of autoimmune and allergic diseases. In allergic lung disease models, immunopathology and local Th2 and ILC2 accumulation is reduced in DR3-deficient mice despite normal systemic priming of Th2 responses and generation of T cells secreting IL-13 and IL-4, prompting the question of whether TL1A promotes the development of other T cell subsets that secrete cytokines to drive allergic disease. In this study, we find that TL1A potently promotes generation of murine T cells producing IL-9 (Th9) by signaling through DR3 in a cell-intrinsic manner. TL1A enhances Th9 differentiation through an IL-2 and STAT5-dependent mechanism, unlike the TNF-family member OX40, which promotes Th9 through IL-4 and STAT6. Th9 differentiated in the presence of TL1A are more pathogenic, and endogenous TL1A signaling through DR3 on T cells is required for maximal pathology and IL-9 production in allergic lung inflammation. Taken together, these data identify TL1A-DR3 interactions as a novel pathway that promotes Th9 differentiation and pathogenicity. TL1A may be a potential therapeutic target in diseases dependent on IL-9.

Dysregulation of Antiviral Function of CD8(+) T Cells in the Chronic Obstructive Pulmonary Disease Lung. Role of the PD-1-PD-L1 Axis.

RATIONALE: Patients with chronic obstructive pulmonary disease (COPD) are susceptible to respiratory viral infections that cause exacerbations. The mechanisms underlying this susceptibility are not understood. Effectors of the adaptive immune response-CD8(+) T cells that clear viral infections-are present in increased numbers in the lungs of patients with COPD, but they fail to protect against infection and may contribute to the immunopathology of the disease. OBJECTIVES: CD8(+) function and signaling through the programmed cell death protein (PD)-1 exhaustion pathway were investigated as a potential key mechanism of viral exacerbation of the COPD lung. METHODS: Tissue from control subjects and patients with COPD undergoing lung resection was infected with live influenza virus ex vivo. Viral infection and expression of lung cell markers were analyzed using flow cytometry. MEASUREMENTS AND MAIN RESULTS: The proportion of lung CD8(+) T cells expressing PD-1 was greater in COPD (mean, 16.2%) than in controls (4.4%, P = 0.029). Only epithelial cells and macrophages were infected with influenza, and there was no difference in the proportion of infected cells between controls and COPD. Infection up-regulated T-cell PD-1 expression in control and COPD samples. Concurrently, influenza significantly up-regulated the marker of cytotoxic degranulation (CD107a) on CD8(+) T cells (P = 0.03) from control subjects but not on those from patients with COPD. Virus-induced expression of the ligand PD-L1 was decreased on COPD macrophages (P = 0.04) with a corresponding increase in IFN-γ release from infected COPD explants compared with controls (P = 0.04). CONCLUSIONS: This study has established a signal of cytotoxic immune dysfunction and aberrant immune regulation in the COPD lung that may explain both the susceptibility to viral infection and the excessive inflammation associated with exacerbations.

Characteristics of immunosuppressive regulatory T cells in cutaneous squamous cell carcinomas and role in metastasis.

BACKGROUND: Non-melanoma skin cancer is the most common cancer worldwide, and cutaneous squamous cell carcinomas (SCCs) account for substantial morbidity and mortality because of their potential for metastasis. SCCs are surrounded by an immune cell infiltrate containing regulatory T cells (Tregs). The aim of this study was to characterise Tregs in SCCs and investigate whether increased Treg numbers in primary skin SCCs are associated with subsequent metastasis. METHODS: Lymphocytes were extracted from freshly excised skin SCC tumours and corresponding peripheral blood and normal skin. Flow cytometry was used for T-cell analysis and cell sorting. Tritiated thymidine based lymphocyte proliferation assays and interferon γ (IFNγ) ELISPOT assays were used to assess peritumoral lymphocyte function in vitro. Immunohistochemistry was performed on primary cutaneous SCC sections from tumours that subsequently metastasised and from those that did not after 5-year follow-up. FINDINGS: Increased frequencies of CD3+CD4+CD25hiCD127loFOXP3+ Tregs were found in SCCs (21·5% of CD4+ immune infiltrate, n=60 tumours) compared with corresponding peripheral blood (5·4%) and normal skin (7·6%). SCC Tregs expressed significantly higher levels of the co-stimulatory molecules OX40 (37·2% of FOXP3+ cell population, n=10 tumours) and 4-1BB (12·6%, n=9) than peritumoral non-regulatory T cells and Tregs from peripheral blood and normal skin (p=0·0005). The inhibitory receptor CTLA4 and the transcription factor Helios were expressed at high levels in peritumoral Tregs. SCC Tregs significantly suppressed phytohaemagglutinin-stimulated peritumoral CD4+ T-cell proliferation (p=0·005, n=10), peritumoral CD8+ T-cell proliferation (p=0·015, n=9), and IFNγ secretion by CD4+ effector T cells (p=0·026, n=10). Increased in-vitro proliferation of phytohaemagglutinin-stimulated peritumoral CD4+ T cells was shown after the addition of anti-OX40 antibodies (p=0·0078, n=9 tumours) and anti-4-1BB antibodies (p=0·0039, n=9). Immunohistochemistry showed fewer CD8+ T cells in SCCs that metastasised (n=29) than in non-metastatic SCCs (n=26) (28·5% of immune infiltrate vs 44·6%%, p<0·0001) and more FOX3+ Tregs (28·5% of immune infiltrate vs 49·3%, p<0·0001). INTERPRETATION: Our study shows that immunosuppressive Tregs are present in the immune infiltrate of cutaneous SCCs, and contribute to ineffective anti-tumour immune responses, thereby permitting SCC development and promoting metastasis. FUNDING: Wellcome Trust, National Institute for Health Research.

Differential impact of CD27 and 4-1BB costimulation on effector and memory CD8 T cell generation following peptide immunization.

The factors that determine differentiation of naive CD8 T cells into memory cells are not well understood. A greater understanding of how memory cells are generated will inform of ways to improve vaccination strategies. In this study, we analyzed the CD8 T cell response elicited by two experimental vaccines comprising a peptide/protein Ag and an agonist that delivers a costimulatory signal via CD27 or 4-1BB. Both agonists increased expansion of Ag-specific CD8 T cells compared with Ag alone. However, their capacity to stimulate differentiation into effector and memory cells differed. CD27 agonists promoted increased expression of perforin and the generation of short-lived memory cells, whereas stimulation with 4-1BB agonists favored generation of stable memory. The memory-promoting effects of 4-1BB were independent of CD4 T cells and were the result of programing within the first 2 d of priming. Consistent with this conclusion, CD27 and 4-1BB-stimulated CD8 T cells expressed disparate amounts of IL-2, IFN-γ, CD25, CD71, and Gp49b as early as 3 d after in vivo activation. In addition, memory CD8 T cells, generated through priming with CD27 agonists, proliferated more extensively than did 4-1BB-generated memory cells, but these cells failed to persist. These data demonstrate a previously unanticipated link between the rates of homeostatic proliferation and memory cell attrition. Our study highlights a role for these receptors in skewing CD8 T cell differentiation into effector and memory cells and provides an approach to optimize vaccines that elicit CD8 T cell responses.

Fcγ receptor dependency of agonistic CD40 antibody in lymphoma therapy can be overcome through antibody multimerization.

Immunomodulatory mAbs, led by the anti-CTLA4 mAb ipilimumab, are an exciting new class of drugs capable of promoting anticancer immunity and providing durable control of some tumors. Close analysis of a number of agents has revealed a critical yet variable role for Fcγ receptors in their efficacy. In this article, we reveal that agonistic anti-CD40 mAbs have an absolute requirement for cross-linking by inhibitory FcγRIIB when used systemically to treat established BCL1 syngeneic lymphoma, and therapy is lost when using a mouse IgG2a mAb not cross-linked by FcγRIIB. Furthermore, in FcγRIIB-deficient mice the lymphoma itself can provide FcγRIIB to cross-link anti-CD40 on neighboring cells, and only when this is blocked does therapy fail. The dependence on FcγRIIB for immunostimulatory activity was not absolute, however, because when anti-CD40 mAbs were administered systemically with the TLR3 agonist polyinosinic:polycytidylic acid or were given subcutaneously, activatory FcγR could also provide cross-linking. Using this mechanistic insight, we designed multimeric forms of anti-CD40 mAb with intrinsic FcγR-independent activity that were highly effective in the treatment of lymphoma-bearing mice. In conclusion, FcγR-independent anti-CD40 activation is a viable strategy in vivo. These findings have important translational implications, as humans, unlike mice, do not have IgG that binds strongly to FcγRIIB; therefore FcγR-independent derivatives represent an attractive therapeutic option.

CD27 costimulation contributes substantially to the expansion of functional memory CD8(+) T cells after peptide immunization.

Naive T cells require signals from multiple costimulatory receptors to acquire full effector function and differentiate to long-lived memory cells. The costimulatory receptor, CD27, is essential for optimal T-cell priming and memory differentiation in a variety of settings, although whether CD27 is similarly required during memory CD8(+) T-cell reactivation remains controversial. We have used OVA and anti-CD40 to establish a memory CD8(+) T-cell population and report here that their secondary expansion, driven by peptide and anti-CD40, polyI:C, or LPS, requires CD27. Furthermore, antigenic peptide and a soluble form of the CD27 ligand, CD70 (soluble recombinant CD70 (sCD70)), is sufficient for secondary memory CD8(+) T-cell accumulation at multiple anatomical sites, dependent on CD80/86. Prior to boost, resting effector- and central-memory CD8(+) T cells both expressed CD27 with greater expression on central memory cells. Nonetheless, both populations upregulated CD27 after TCR engagement and accumulated in proportion after boosting with Ag and sCD70. Mechanistically, sCD70 increased the frequency of divided and cytolytic memory T cells, conferred resistance to apoptosis and enabled retardation of tumor growth in vivo. These data demonstrate the central role played by CD27/70 during secondary CD8(+) T-cell activation to a peptide Ag, and identify sCD70 as an immunotherapeutic adjuvant for antitumor immunity.

Agonist Antibodies for Cancer Immunotherapy: History, Hopes, and Challenges.

Immunotherapy is among the most promising new treatment modalities to arise over the last two decades; antibody drugs are delivering immunotherapy to millions of patients with many different types of cancer. Initial success with antibody therapeutics came in the form of direct targeting or cytotoxic antibodies, such as rituximab and trastuzumab, which bind directly to tumor cells to elicit their destruction. These were followed by immunomodulatory antibodies that elicit antitumor responses by either stimulating immune cells or relieving tumor-mediated suppression. By far the most successful approach in the clinic to date has been relieving immune suppression, with immune checkpoint blockade now a standard approach in the treatment of many cancer types. Despite equivalent and sometimes even more impressive effects in preclinical models, agonist antibodies designed to stimulate the immune system have lagged behind in their clinical translation. In this review, we document the main receptors that have been targeted by agonist antibodies, consider the various approaches that have been evaluated to date, detail what we have learned, and consider how their anticancer potential can be unlocked.

Death receptor 3 is essential for generating optimal protective CD4⁺ T-cell immunity against Salmonella.

The TNF receptor superfamily member death receptor 3 (DR3) exacerbates Th2- and Th17-cell-mediated inflammatory and autoimmune conditions, yet no role in host defence has been reported. Here, we examined the role of DR3 during infection with Salmonella enterica serovar Typhimurium. Infection resulted in protracted expression of the DR3 ligand TL1A but not the related TNF superfamily proteins OX40L or CD30L. TL1A expression was localized to splenic F4/80(+) macrophages where S. enterica Typhimurium replicates, and temporally coincided with the onset of CD4(+) -cell expansion. To address the relevance of the TL1A-DR3 interaction, we examined immune responses to S. enterica Typhimurium in mice lacking DR3. Infected DR3(-/-) mice harboured reduced numbers of antigen-experienced and proliferating CD4(+) T cells compared with WT mice. Furthermore, the frequency of IFN-γ(+) CD4(+) T cells in DR3(-/-) mice was lower throughout the time of bacterial clearance. Importantly, bacterial clearance, which is dependent on Th1 cells, was also impaired in DR3(-/-) mice. This defect was intrinsic to CD4(+) T cells as evidenced by an increase in bacterial burden in RAG2-deficient mice receiving DR3(-/-) CD4(+) T cells compared with WT CD4(+) -cell recipients. These data establish for the first time a role for DR3 in a host defence response.

The death receptor 3/TL1A pathway is essential for efficient development of antiviral CD4⁺ and CD8⁺ T-cell immunity.

Death receptor 3 (DR3, TNFRSF25), the closest family relative to tumor necrosis factor receptor 1, promotes CD4(+) T-cell-driven inflammatory disease. We investigated the in vivo role of DR3 and its ligand TL1A in viral infection, by challenging DR3-deficient (DR3(KO)) mice and their DR3(WT) littermates with the ß-herpesvirus murine cytomegalovirus or the poxvirus vaccinia virus. The phenotype and function of splenic T-cells were analyzed using flow cytometry and molecular biological techniques. We report surface expression of DR3 by naive CD8(+) T cells, with TCR activation increasing its levels 4-fold and altering the ratio of DR3 splice variants. T-cell responses were reduced up to 90% in DR3(KO) mice during acute infection. Adoptive transfer experiments indicated this was dependent on T-cell-restricted expression of DR3. DR3-dependent CD8(+) T-cell expansion was NK and CD4 independent and due to proliferation, not decreased cell death. Notably, impaired immunity in DR3(KO) hosts on a C57BL/6 background was associated with 4- to 7-fold increases in viral loads during the acute phase of infection, and in mice with suboptimal NK responses was essential for survival (37.5%). This is the first description of DR3 regulating virus-specific T-cell function in vivo and uncovers a critical role for DR3 in mediating antiviral immunity.

Interaction with FcγRIIB is critical for the agonistic activity of anti-CD40 monoclonal antibody.

A high activatory/inhibitory FcγR binding ratio is critical for the activity of mAb such as rituximab and alemtuzumab that attack cancer cells directly and eliminate them by recruiting immune effectors. Optimal FcγR binding profiles of other anti-cancer mAb, such as immunostimulatory mAb that stimulate or block immune receptors, are less clear. In this study, we analyzed the importance of isotype and FcγR interactions in controlling the agonistic activity of the anti-mouse CD40 mAb 3/23. Mouse IgG1 (m1) and IgG2a (m2a) variants of the parental 3/23 (rat IgG2a) were engineered and used to promote humoral and cellular responses against OVA. The mouse IgG1 3/23 was highly agonistic and outperformed the parental Ab when promoting Ab (10-100-fold) and T cell (OTI and OTII) responses (2- to >10-fold). In contrast, m2a was almost completely inactive. Studies in FcγR knockout mice demonstrated a critical role for the inhibitory FcγRIIB in 3/23 activity, whereas activatory FcγR (FcγRI, -III, and -IV) was dispensable. In vitro experiments established that the stimulatory effect of FcγRIIB was mediated through Ab cross-linking delivered in trans between neighboring cells and did not require intracellular signaling. Intriguingly, activatory FcγR provided effective cross-linking of 3/23 m2a in vitro, suggesting the critical role of FcγRIIB in vivo reflects its cellular distribution and bioavailability as much as its affinity for a particular Ab isotype. In conclusion, we demonstrate an essential cross-linking role for the inhibitory FcγRIIB in anti-CD40 immunostimulatory activity and suggest that isotype will be an important issue when optimizing reagents for clinical use.

Fcγ receptor binding is required for maximal immunostimulation by CD70-Fc.

Introduction: T cell expressed CD27 provides costimulation upon binding to inducible membrane expressed trimeric CD70 and is required for protective CD8 T cell responses. CD27 agonists could therefore be used to bolster cellular vaccines and anti-tumour immune responses. To date, clinical development of CD27 agonists has focussed on anti-CD27 antibodies with little attention given to alternative approaches. Methods: Here, we describe the generation and activity of soluble variants of CD70 that form either trimeric (t) or dimer-of-trimer proteins and conduct side-by-side comparisons with an agonist anti-CD27 antibody. To generate a dimer-of-trimer protein (dt), we fused three extracellular domains of CD70 to the Fc domain of mouse IgG1 in a 'string of beads' configuration (dtCD70-Fc). Results: Whereas tCD70 failed to costimulate CD8 T cells, both dtCD70-Fc and an agonist anti-CD27 antibody were capable of enhancing T cell proliferation in vitro. Initial studies demonstrated that dtCD70-Fc was less efficacious than anti-CD27 in boosting a CD8 T cell vaccine response in vivo, concomitant with rapid clearance of dtCD70-Fc from the circulation. The accelerated plasma clearance of dtCD70-Fc was not due to the lack of neonatal Fc receptor binding but was dependent on the large population of oligomannose type glycosylation. Enzymatic treatment to reduce the oligomannose-type glycans in dtCD70-Fc improved its half-life and significantly enhanced its T cell stimulatory activity in vivo surpassing that of anti-CD27 antibody. We also show that whereas the ability of the anti-CD27 to boost a vaccine response was abolished in Fc gamma receptor (FcγR)-deficient mice, dtCD70-Fc remained active. By comparing the activity of dtCD70-Fc with a variant (dtCD70-Fc(D265A)) that lacks binding to FcγRs, we unexpectedly found that FcγR binding to dtCD70-Fc was required for maximal boosting of a CD8 T cell response in vivo. Interestingly, both dtCD70-Fc and dtCD70-Fc(D265A) were effective in prolonging the survival of mice harbouring BCL1 B cell lymphoma, demonstrating that a substantial part of the stimulatory activity of dtCD70-Fc in this setting is retained in the absence of FcγR interaction. Discussion: These data reveal that TNFRSF ligands can be generated with a tunable activity profile and suggest that this class of immune agonists could have broad applications in immunotherapy.

Triggering of TNFRSF25 promotes CD8⁺ T-cell responses and anti-tumor immunity.

TNFRSF25 is a member of the TNF receptor superfamily (TNFRSF) that binds to the TNF-like protein TL1A. Although recent studies have demonstrated a role for TNFRSF25 in regulating CD4(+) T-cell responses, it remains to be determined if TNFRSF25 functions as a costimulatory receptor for CD8(+) T cells. Here, we demonstrate that ectopic expression of TL1A on mouse plasmacytomas promotes elimination of tumor cells in a CD8(+) T-cell-dependent manner and renders mice immune to a subsequent challenge with tumor cells. To gain further insight into the role of TNFRSF25 in CD8(+) T-cell responses, we analyzed the effect of TNFRSF25 triggering on OT-I TCR transgenic T cells. We demonstrate that TNFRSF25 triggering in vivo with soluble TL1A promotes the proliferation and accumulation of antigen-specific CD8(+) T cells as well as their differentiation into CTLs. Furthermore, we show that TNFRSF25 also functions as a costimulatory receptor for memory CD8(+) T cells. Thus, TNFRSF25 triggering enhances the secondary expansion of endogenous antigen-specific memory CD8(+) T cells. Our data suggest that TNFRSF25 agonists, such as soluble TL1A, could potentially be used to enhance the immunogenicity of vaccines that aim to elicit human anti-tumor CD8(+) T cells.