TNFR Family Member Research Articles

Regulation of cell fate by lymphotoxin (LT) receptor signalling: Functional differences and similarities of the LT system to other TNF superfamily (TNFSF) members

The role of TNFR family members in regulating cell fate both in the immune system and in non-lymphoid tissues has been under extensive research for decades. Moreover, the ability of several family members (death receptors) to induce death (mainly via apoptosis) represents a promising target for cancer therapy. Many studies have focused mostly on death receptors such as TNFRI, Fas and TRAIL-R due to their strong pro-apoptotic potential. Yet, cell death can be triggered via non-classical death receptors, and the lymphotoxin (LT) system represents a very good example of such a TNFR subfamily. Here we provide a comprehensive review of intracellular signalling pathways and cellular responses to LT-specific signalling, and compare for the first time the LT system to other TNFRs, such as CD40. Our aim is to highlight that non-classical TNFR-TNFL dyads such as the LT system demonstrate more complex, cell-type and context-specific capabilities. Understanding these complexities will permit a better understanding of the biological mechanisms via which non-death domain-containing TNFRs induce cell death, but may also allow the design of better therapeutic strategies.

Anti-GITR agonist therapy intrinsically enhances CD8 T cell responses to chronic lymphocytic choriomeningitis virus (LCMV), thereby circumventing LCMV-induced downregulation of costimulatory GITR ligand on APC.

The costimulatory TNFR family member GITR can provide important survival signals for CD8 T cells. However, little is known about the regulation of this pathway during a chronic infection. In this study, we show that GITR ligand (GITRL) is maximally induced on APCs at day 2 post-lymphocytic choriomeningitis virus (LCMV) clone 13 infection, but is downregulated to below baseline levels by day 8 postinfection (p.i.), and remains so at the chronic stage of infection. At its peak, GITRL expression is highest on macrophages, with lower expression on conventional and plasmacytoid dendritic cells. GITR expression was highest on T regulatory cells but was also detected on Th1 and LCMV-specific CD8 T cells at day 8 p.i. and was maintained at low, but above baseline levels at the chronic stage of LCMV infection. As GITRL was limiting at the chronic stage of infection, we investigated the potential of therapeutic stimulation of GITR at this stage using agonistic anti-GITR Ab. Anti-GITR treatment at day 21 p.i. increased the frequency and number of LCMV-specific CD8 T cells, resulting in increased in vivo CTL activity and a concomitant decrease in viral load, despite the persistence of PD-1 expression. These effects of anti-GITR were CD8 T cell intrinsic, with no detectable effects on Th1 or T regulatory cells. In contrast to other TNFR agonists, such as anti-4-1BB, which can cause immune pathology, a single therapeutic dose of anti-GITR did not induce splenomegaly or increase serum alanine transaminase. These studies identify GITR as a promising therapeutic target for chronic infection.

Abstract LB-263: Enhancing of CD8+ tumor-infiltrating lymphocyte expansion from triple-negative breast cancer patients using of 41BB costimulation

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive form of breast cancer with few treatment options. Recently, it was shown that infiltration of TNBC with CD8+ tumor-infiltrating lymphocytes (TIL) is associated with improved prognosis, suggesting that T-cell responses at the tumor site can be harnessed as an autologous T-cell therapy for TNBC. Although TIL therapy has been developed for solid tumors such as melanoma, cervical, and ovarian cancer, whether TIL, especially CD8+ cells, with effector-memory properties can be expanded with reasonable yields from primary TNBC is not known. Given the high rates of relapse and refractory nature of recurrent TNBC disease, autologous TIL therapy may offer a life-saving option for these patients. Moreover, methods to facilitate CD8+ TIL expansion from TNBC are also desirable given the cytotoxic potential of these cells. One approach to address this need is to manipulate TNF-R family member signaling, such as 41BB/CD137, to provide potent costimulatory signals for CD8+ T-cell activation and division. In this study, we established a method of isolating and generating TIL from patients with primary TNBC surgical specimens and core biopsies using an agonistic 41BB/CD137 antibody. Patients and methods: Four primary human TNBC tumor samples were obtained by surgical resection or core biopsy after neoadjuvant chemotherapy. Small tumor fragments were cultured in 24-well plates in medium containing 3000 IU/ml IL-2, or 3000 IU/ml IL-2 plus 10 μg/ml added agonistic anti-41BB IgG4 (BMS663513). Viable cell numbers and the expression of CD3, CD8, CD4, CD27, CD28, CCR7, CD45RA, CD56, CD16, Granzyme B, and Perforin was determined by flow cytometry on days 7, 14, 21, 28, and 35 after culture set-up. Cytotoxic function of the TIL was evaluated by measuring Caspase 3 cleavage in target cells. Results: Although TIL were successfully expanded from the tumor fragments in 4/4 tumor samples, IL-2 alone yielded fewer cells than IL-2 together with 41BB costimulation provided early during the culture. Anti-41BB markedly increased the percentage and yield of CD8+CD3+ T cells at all times (6.34x106 vs , 202x106, P<0.01). However, the resulting CD8+ cells had decreased CD27 expression coincident with increased CD70 when anti-41BB was added. 41BB costimulation also increased the cytotoxic T-cell activity of the expanded TIL. The expanded TIL could also be cryopreserved and later expanded using anti-CD3 and IL-2. Conclusions: We have established a method to expand CD8+ TIL with cytotoxic activity with high yield from small samples of remaining primary tumor after neoadjuvant chemotherapy using IL-2. Provision of 41BB costimulation immediately upon tumor fragment culture initiation was critical in enhancing the rate and yield of CD8+ T cells with increased effector function; these could be cryopreserved and later thawed with high viability for secondary expansion. Our results support further development of an autologous TIL expansion protocol after neoadjuvant therapy for use in an adoptive cell therapy approach to treat TNBC recurrence or metastasis. Citation Format: Michiko Harao, Elizabeth A. Mittendorf, Gildy V. Babiera, Naoto T. Ueno, Laszlo G. Radvanyi. Enhancing of CD8+ tumor-infiltrating lymphocyte expansion from triple-negative breast cancer patients using of 41BB costimulation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-263. doi:10.1158/1538-7445.AM2014-LB-263

Abstract 4512: Construction and characterization of novel, human serine protease granzyme B-based cancer therapeutics targeting the TNFR family member Fn14

Abstract The TNF superfamily cytokine TWEAK and its receptor Fn14 have emerged as potentially clinically valuable targets for cancer therapy. The expression of Fn14 is comparatively low in normal tissues but elevated in various human solid tumor types, including brain, breast, lung and melanoma, and has been shown to be a negative prognostic indicator. We generated Granzyme B (GrB) containing, Fn14-targeted constructs using either the extracellular domain (ECD) of the human TWEAK ligand (GrB-TWEAK) or an engineered anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both fusion constructs were expressed in mammalian HEK293T cells and purified to homogeneity from conditioned media. Both GrB-TWEAK and GrB-Fc-IT4 constructs were designed to be relatively large in molecular weight (150 kDa and 160 kDa respectively) to allow prolonged circulation in vivo. BiaCore analysis of GrB-TWEAK and GrB-Fc-IT4 binding to the Fn14 ECD indicated that these proteins bound to Fn14 with Kds of ∼8 nM and ∼18 nM, respectively. Confocal immunofluorescence analysis showed that both proteins specifically and rapidly (within 3 hrs) internalized into Fn14-expressing MDA-MB-231 breast cancer and HT-29 colorectal adenocarcinoma cells. GrB-TWEAK and GrB-Fc-IT4 demonstrated impressive and selective cytotoxicity to a panel of Fn14-expressing human tumor cell lines in the low nanomolar range. The endogenous presence of the GrB proteinase inhibitor 9 (PI-9) had no impact on the cytotoxic effect of either construct. Treatment of cells expressing the multidrug resistance protein MDR1 showed no cross-resistance to the fusion constructs in vitro. Mechanistic studies demonstrated that GrB-TWEAK and GrB-Fc-IT4 activated caspase cascades and cytochrome C-related pro-apoptotic mechanisms consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing well-established HT-29 xenografts with GrB-TWEAK (at 20 or 40 mg/kg total doses) showed significant tumor growth inhibition compared to controls (P< 0.05). GrB-TWEAK and GrB-Fc-IT4 were administered to mice bearing MDA-MB-231 orthotopic human breast xenograft tumors and both constructs displayed significant tumor growth inhibition. Treatment with GrB-Fc-IT4 displayed a more pronounced and prolonged tumor growth inhibition and longer survival than GrB-TWEAK treatment. IHC analysis demonstrates excellent tumor uptake of both agents. These data suggest that Fc-containing fusions with GrB may have a design advantage with respect to efficacy and may form the basis of novel, highly effective and nontoxic constructs for targeted therapeutic applications. Research conducted, in part, by the Clayton Foundation for Research (MGR) and National Institutes of Health grant R01 NS055126 (JAW). Citation Format: Hong Zhou, Khalid A. Mohamedali, Yu Cao, Mary Migliorini, Lawrence H. Cheung, Walter N. Hittelman, Jeffrey A. Winkles, Michael G. Rosenblum. Construction and characterization of novel, human serine protease granzyme B-based cancer therapeutics targeting the TNFR family member Fn14. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4512. doi:10.1158/1538-7445.AM2014-4512

Qualitative and quantitative effects of 4-1BBL in boosting pre-existing influenza immunity (VAC2P.923)

Abstract Influenza virus induces an acute respiratory infection responsible for up to 500,000 annual deaths, primarily in older persons. CD8+ T cells against conserved influenza proteins confer heterotypic protection, potentially providing a “universal vaccine.” However, live infection only transiently boosts influenza-specific T cell memory, and immunity further declines with age. To improve the longevity of T cell memory, we investigated the effects of stimulating the TNFR-family member 4-1BB. To mimic vaccination of previously flu-immune adults, mice were infected with influenza A/HK-X31 and boosted intranasally one-month later with control replication-deficient adenovirus or adenovirus encoding influenza nucleoprotein (NP) alone (Ad-NP) or NP with 4-1BBL (Ad-NP-4-1BBL). 4-1BBL dramatically enhanced NP-specific CD8+ T cell responses at a dose where Ad-NP had minimal effects. 4-1BBL induced a remarkably long-lived effector-memory population that protects mice against lethal challenge into old age (>1 year old). Increasing the Ad-NP dose did not replicate these effects, and instead caused partial functional exhaustion. Preliminary analysis indicates that the 4-1BBL-induced effector memory T cells have increased expression of IL-7R as well as the transcriptional factor TCF-1 and an increased ratio of T-bet to Eomesodermin. Thus, 4-1BBL in a vaccine vector administered in flu-immune mice induces a potentially unique protective subset of memory CD8+ T cells.

Secretion of IL-16 through TNFR1 and calpain-caspase signaling contributes to MRSA pneumonia.

Staphylococcus aureus is a major cause of severe pneumonia. Multiple mechanisms of proinflammatory signaling are activated to recruit immune cells into the airway in response to S. aureus. We found that IL-16, a T cell cytokine that binds CD4, is potently activated by S. aureus, specifically by protein A (SpA), and to a much greater extent than by Gram negative pathogens or LPS. IL-16 production involved multiple signals including ligation of TNFR family members or EGFR, both receptors for SpA and generation of Ca2+ fluxes to activate calpains and caspase-3. Although human airway epithelial cells, vascular endothelial cells, THP-1 and Jurkat T cells released IL-16 in response to S. aureus in vitro, in a murine model of pneumonia, CD4+ cells were the major source of IL-16 suggesting the involvement of an autocrine signaling pathway. The production of IL-16 contributed to lung damage as neutralization of IL-16 enhanced S. aureus clearance and resulted in diminished lung pathology in S. aureus pneumonia. Our results suggest that the ability of S.aureus to activate TNFR1 and Ca2+/calpain signaling contribute to T cell activation and excessive inflammation in the setting of acute pneumonia.

Expression of Epstein-Barr Virus-encoded Latent Membrane Protein (LMP-1), p16 and p53 Proteins in Nonendemic Nasopharyngeal Carcinoma (NPC): A Clinicopathological Study

Although the latent membrane protein type 1 (LMP1) is frequently expressed in Epstein-Barr virus (EBV) malignancies, its contribution to the pathogenesis of nasopharyngeal carcinoma (NPC) is not fully defined. LMP1 functions as a viral mimic of the TNFR family member engaging a number of signaling pathways that induce morphological and phenotypic alterations. This study aimed to investigate the LMP1 expression and EBV infection in relation to clinical outcome and survival in a series of Mexican NPC patients. We also studied expression of p16 and p53 proteins. We analyzed in 25 tumor specimens the expression of LMP1, p16 and p53 by immunohistochemistry (IHC) and EBV presence by IHC/in situ hybridization. Differences in clinical outcome and survival in relation to protein expression were correlated through χ(2) statistics and Kaplan-Meier survival curves. Our results showed a rate of 92% (23/25) of EBV infection. The expressions of LMP-1, p16 and p53 proteins were 40.0, 44.0 and 40.0%, respectively. LMP-1 immunoexpression was more common in older patients (>50 vs. <50 years old, p = 0.02) and with parapharyngeal space invasion (p = 0.02). The presence of metastatic disease at diagnosis (p = 0.03), distant recurrence disease (p = 0.006) and shorter distance recurrence-free survival (p = 0.05) was associated with lack of p16. In our series, EBV infection rates are particularly high for nonendemic NPC, although without a statistically significant difference in overall survival, LMP1 and p16 expression was correlated with poorer clinical prognosis. Probably, LMP1 and p16 detection identify a worse clinical prognosis in NPC patient subgroup.

CD137 Accurately Identifies and Enriches for Naturally Occurring Tumor-Reactive T Cells in Tumor

Upregulation of CD137 (4-1BB) on recently activated CD8(+) T cells has been used to identify rare viral or tumor antigen-specific T cells from peripheral blood. Here, we evaluated the immunobiology of CD137 in human cancer and the utility of a CD137-positive separation methodology for the identification and enrichment of fresh tumor-reactive tumor-infiltrating lymphocytes (TIL) or tumor-associated lymphocytes (TAL) from ascites for use in adoptive immunotherapy. TILs from resected ovarian cancer or melanoma were measured for surface CD137 expression directly or after overnight incubation in the presence of tumor cells and homeostatic cytokines. CD137(pos) TILs were sorted and evaluated for antitumor activity in vitro and in vivo. Fresh ovarian TILs and TALs naturally expressed higher levels of CD137 than circulating T cells. An HLA-dependent increase in CD137 expression was observed following incubation of fresh enzyme-digested tumor or ascites in IL-7 and IL-15 cytokines, but not IL-2. Enriched CD137(pos) TILs, but not PD-1(pos) or PD-1(neg) CD137(neg) cells, possessed autologous tumor reactivity in vitro and in vivo. In melanoma studies, all MART-1-specific CD8(+) TILs upregulated CD137 expression after incubation with HLA-matched, MART-expressing cancer cells and antigen-specific effector function was restricted to the CD137(pos) subset in vitro. CD137(pos) TILs also mediated superior antitumor effects in vivo, compared with CD137(neg) TILs. Our findings reveal a role for the TNFR-family member CD137 in the immunobiology of human cancer where it is preferentially expressed on tumor-reactive subset of TILs, thus rationalizing its agonistic engagement in vivo and its use in TIL selection for adoptive immunotherapy trials.

A Cell-Intrinsic Requirement for NF-κB–Inducing Kinase in CD4 and CD8 T Cell Memory

NF-κB-inducing kinase [(NIK), MAP3K14] is an essential kinase linking a subset of TNFR family members to the noncanonical NF-κB pathway. To assess the cell-intrinsic role of NIK in murine T cell function, we generated mixed bone marrow chimeras using bone marrow from NIK knockout (KO) and wild-type (WT) donor mice and infected the chimeras with lymphocytic choriomeningitis virus (LCMV). The chimeras possess an apparently normal immune system, including a mixture of NIK KO and WT T cells, and the virus was cleared normally. Comparison of the NIK KO and WT CD4 and CD8 T cell responses at 8 d post infection revealed modest but significant differences in the acute response. In both CD4 and CD8 compartments, relatively fewer activated (CD44(hi)) NIK KO T cells were present, but within the CD44(hi) population, a comparable percentage of the activated cells produced IFN-γ in response to ex vivo stimulation with antigenic LCMV peptides, although IL-7R expression was reduced in the NIK KO CD8 T cells. Assessment of the LCMV-specific memory at 65 d post infection revealed many more LCMV-specific WT memory T cells than NIK KO memory T cells in both the CD4 and the CD8 compartments, although the small number of surviving NIK KO memory T cells responded to secondary challenge with virus. These results demonstrate a cell-intrinsic requirement for NIK in the generation and/or maintenance of memory T cells in response to acute viral infection.

Regulation of A1 by OX40 Contributes to CD8+ T Cell Survival and Anti-Tumor Activity

The TNFR family member OX40 (CD134) is critical for optimal clonal expansion and survival of T cells. However, the intracellular targets of OX40 in CD8 T cells are not fully understood. Here we show that A1, a Bcl-2 family protein, is regulated by OX40 in effector CD8 T cells. In contrast to wild-type T cells, OX40-deficient CD8 T cells failed to maintain A1 expression driven by antigen. Conversely, enforced OX40 stimulation promoted A1 expression. In both situations, the expression of A1 directly correlated with CD8 T cell survival. In addition, exogenous expression of A1 in OX40-deficient CD8 T cells reversed their survival defect in vitro and in vivo. Moreover, forced expression of A1 in CD8 T cells from OX40-deficient mice restored the ability of these T cells to suppress tumor growth in a murine model. These results indicate that OX40 signals regulate CD8 T cell survival at least in part through maintaining expression of the anti-apoptotic molecule A1, and provide new insight into the mechanism by which OX40 may impact anti-tumor immunity.

Pathogenic role of Epstein–Barr virus latent membrane protein-1 in the development of nasopharyngeal carcinoma

Undifferentiated nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus-associated malignant tumor. A consistent elevation in EBV antibody titers is a well-established risk factor for the development of NPC. The pathophysiological relationship and molecular mechanisms of EBV-mediated carcinogenesis have not been fully elucidated. While NPC tumors are known to express three EBV-encoded proteins, EBNA1, LMP1, and LMP2, they also express a large number of virus-encoded small RNAs (EBERs) and microRNAs (miRNAs). Among them, LMP1 may be a central player in the development of NPC. LMP1, an EBV-encoded primary oncogene, functions as a viral mimic of the TNFR family member, CD40, and engages in a number of signaling pathways that induce morphological and phenotypic alterations in epithelial cells. LMP1 upregulates EMT, and contributes to the highly metastatic features of NPC. Moreover, LMP1-associated EMT is accompanied by the expression of cancer stem cell (CSC)/cancer progenitor cell (CPC) markers (CD44high/CD24low) and the acquisition of stem cell/progenitor cell-like properties. BART miRNAs, encoded from the BamHI-A region of the viral genome, are the most abundant transcripts. They modulate apoptosis and host innate immune defense mechanisms. Some BART1 miRNAs are considered to negatively regulate LMP1 protein expression. LMP1 is secreted via exosomes, is incorporated into EBV-uninfected cells by endocytosis, and affects the environment surrounding the tumor. Here we reviewed the contribution of EBV gene products to NPC pathogenesis in relation with LMP1.

Heat Shock Protein 90 Inhibitors Repress Latent Membrane Protein 1 (LMP1) Expression and Proliferation of Epstein-Barr Virus-Positive Natural Killer Cell Lymphoma

Epstein-Barr virus (EBV) LMP1 is a major oncoprotein expressed in latent infection. It functions as a TNFR family member and constitutively activates cellular signals, such as NFκB, MAPK, JAK/STAT and AKT. We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 Natural Killer (NK) cell lymphoma cells, but also in B and T cells. Tumor formation in immuno-defficient NOD/Shi-scid/IL-2Rγnull (NOG) mice was also retarded. These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

Altered adaptive and TNF-superfamily soluble inflammatory mediators mark impending Systemic Lupus Erythematosus disease flare in European-American lupus patients after influenza vaccination (P6282)

Abstract SLE is a waxing and waning disease characterized by major organ involvement, disease flares and immune dysregulation. Understanding mechanisms and identifying biomarkers of flare would help prevent damage and improve management. Plasma samples were obtained from SLE patients on the day of influenza vaccination (BL) and at 6 and 12 weeks post (FU) in 28 European American SLE patients who exhibited SELENA-SLEDAI defined disease flare at 6 (n=13) or 12 (n=15) weeks post-vaccination. Each SLE patient was matched to a unique SLE patient who did not flare (NF), and to a healthy control (HC). Samples from 13 SLE patients with flare were compared to samples from a non-flare year (self nonflare, SNF). Plasma samples were tested for 52 soluble inflammatory mediators using xMAP technology. SLE patients who exhibited disease flare had significantly (p &amp;lt; 0.01) higher levels of BL pro-inflammatory mediators, including Th1, Th2, and Th17-type cytokines compared to NF/SNF SLE patients and HC. Regulatory cytokines, including IL-10 and TGF-β were increased (p &amp;lt; 0.01) at BL in SLE patients not exhibiting disease flare (NF/SNF) compared to SLE flare and HC. TNFR family members, TNFRI, TNFRII, Fas, FasL, CD40L, were increased at BL in SLE flare patients. Select serum cytokines and chemokines are elevated in patients before SLE disease flare, providing important pathways to dissect for mechanism of flare, therapeutic selection or development.

IL-27Rα is required for shaping the magnitude and repertoire of T cells responses to subunit immunization (P4500)

Abstract A goal of cellular immune vaccines is the generation of large numbers of antigen specific T cells in response to subunit immunization. A broad spectrum of cytokine and cell surface costimulatory molecules are known to shape the magnitude and repertoire of T cells responding to vaccination. We show here the majority of TLR agonist-based vaccine adjuvants unexpectedly depend on IL-27 for eliciting CD4 and CD8 T cells responses. This is in sharp contrast to infectious challenge which generates T cell responses that are IL-27 independent. Mixed bone marrow chimera experiments demonstrate that IL-27 dependency is T cell intrinsic, requiring T cell expression of IL-27Rα. Further, we show that IL-27 dependency is not only necessary for the maximal expression of vaccine elicited T cell responses, but also critical for representation of high affinity T cells in the responding T cell pool. This observation complements the biology from other reports demonstrating dependence of low affinity T cells on the TNFR family member CD27. Collectively our data highlight the unexpected central importance of IL-27 in the generation of robust, high affinity cellular immune response to subunit immunization.

Usurpation of a lymphocyte costimulatory molecule as a novel melanoma cell survival pathway (P2187)

Abstract BTLA (B- and T- lymphocyte attenuator), binds to the TNF-R family member HVEM (herpes virus entry mediator). HVEM through its interaction with BTLA in trans or in cis has been shown to act as a pro-survival signal through activation of NF-κB. In contrast, signaling through BTLA following HVEM engagement has been found to induce a negative signaling pathway by activating SHP1/2 phosphatases. During an initial immunohistochemistry screen for the expression of BTLA in melanoma tumor sections, we unexpectedly found that a many tumor cells also exhibited a uniform and strong positive intracellular expression of BTLA. BTLA was expressed in both primary and metastatic melanoma cells with low to negative expression in benign nevi. PCR analysis also revealed that although most tumor lines expressed isoform 1 of BTLA, some lines expressed both isoform 1 and 2 suggesting BTLA by be secreted. Moreover, we also found that melanoma cells express both surface and intracellular HVEM. Intriguingly, significant pools of BTLA were found to be co-localized with intracellular HVEM in the melanoma cells. This co-localization suggests that tumor cells may use HVEM activation as a mechanism of cell survival. Blockade of BTLA or inhibition of BTLA or HVEM expression on melanoma cells may therefore be a novel approach to inhibit tumor cell survival as well as prevent negative signaling through BTLA in immune cells.

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 CD44(hi) 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.

Co-Stimulation through 4-1BB/CD137 Improves the Expansion and Function of CD8+ Melanoma Tumor-Infiltrating Lymphocytes for Adoptive T-Cell Therapy

Adoptive T-cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) can induce tumor regression in up to 50% or more of patients with unresectable metastatic melanoma. However, current methods to expand melanoma TIL, especially the “rapid expansion protocol” (REP) were not designed to enhance the generation of optimal effector-memory CD8+ T cells for infusion. One approach to this problem is to manipulate specific co-stimulatory signaling pathways to enhance CD8+ effector-memory T-cell expansion. In this study, we determined the effects of activating the TNF-R family member 4-1BB/CD137, specifically induced in activated CD8+ T cells, on the yield, phenotype, and functional activity of expanded CD8+ T cells during the REP. We found that CD8+ TIL up-regulate 4-1BB expression early during the REP after initial TCR stimulation, but neither the PBMC feeder cells in the REP or the activated TIL expressed 4-1BB ligand. However, addition of an exogenous agonistic anti-4-1BB IgG4 (BMS 663513) to the REP significantly enhanced the frequency and total yield of CD8+ T cells as well as their maintenance of CD28 and increased their anti-tumor CTL activity. Gene expression analysis found an increase in bcl-2 and survivin expression induced by 4-1BB that was associated with an enhanced survival capability of CD8+ post-REP TIL when re-cultured in the absence or presence of cytokines. Our findings suggest that adding an agonistic anti-4-1BB antibody during the time of TIL REP initiation produces a CD8+ T cell population capable of improved effector function and survival. This may greatly improve TIL persistence and anti-tumor activity in vivo after adoptive transfer into patients.

FcγRΙΙB controls the potency of agonistic anti-TNFR mAbs.

Isotype plays a crucial role in therapeutic monoclonal antibody (mAb) function, mediated in large part through differences in Fcγ receptor (FcγR) interaction. Monoclonal Abs such as rituximab and alemtuzumab, which bind target cells directly, are designed for efficient recruitment of immune effector cells through their activatory FcγR engagement to mediate maximal target cell killing. In this setting, binding to inhibitory FcγRIIB is thought to inhibit function, making mAbs with high activatory/inhibitory (A/I) FcγR binding ratios, such as mouse IgG2a and human IgG1, the first choice for this role. In contrast, exciting new data show that agonistic mAbs directed against the tumour necrosis factor receptor superfamily member CD40 require interaction with FcγRIIB for in vivo function. Such ligation activates antigen-presenting cells, promotes myeloid and CTL responses and potentially stimulates effective anti-cancer immunity. It appears that the role of FcγRIIB is to mediate mAb hyper-crosslinking to allow CD40 downstream intracellular signalling. Previous work has shown that mAbs directed against other TNFR family members, Fas and death receptor 5 and probably death receptor 4, also require FcγRIIB hyper-crosslinking to promote target cell apoptosis, suggesting a common mechanism of action. In mouse models, IgG1 is optimal for these agents as it binds to FcγRIIB with tenfold higher affinity than IgG2a and hence has a relatively low A:I FcγR binding ratio. In contrast, human IgG isotypes have a universally low affinity for FcγRIIB, but in the case of human IgG1, engineering the Fc to increase its affinity for FcγRIIB can potentially overcome this problem. Thus, modifying the A/I binding ratio of human IgG Fc can be used to optimise different types of therapeutic activity by enhancing cytotoxic or hyper-crosslinking function.

Human memory T-cell subsets require distinct costimulatory signals to efficiently expand ex vivo

Event Abstract Back to Event Human memory T-cell subsets require distinct costimulatory signals to efficiently expand ex vivo Valentina Volpin1, 2*, Nicoletta Cieri1, 2 and Chiara Bonini1 1 San Raffaele Scientific Institute, Italy 2 Università Vita-Salute San Raffaele, Italy Costimulation plays a critical role for activation of T cells in response to pathogens, cancer and in alloreactive immune responses. However, costimulatory requirements of memory T cell responses have been much less studied than those for primary response generation, especially in humans. In the present work, we evaluated the ability of several costimulatory molecules (the CD28-family member CD28 and ICOS, and the TNFR-family members 4-1BB, OX40, GITR, CD27, CD30, HVEM) in expanding ex-vivo human memory T-cell subsets. We assessed the effect of these costimulatory molecules on central memory (TCM), effector memory (TEM) and on the recently identified memory stem T cells (TSCM). Memory T-cell subsets were FACS-sorted, according to the expression of CD45RA/CD62L and CD95, activated by coated anti-CD3 antibody in the presence of anti-costimulatory molecules antibodies and cultured with low dose homeostatic cytokines. The efficacy of costimulatory molecules was measured in terms of T-cell expansion and preservation of the original surface phenotype. Strikingly, we found that each memory T-cell subset best responded to distinct costimulatory signals. TSCM lymphocytes expanded, while maintaining a higher fraction of cells with the original and less-differentiated phenotype, when stimulated in the presence of OX-40- and GITR-mediated signals, while TCM best performed when CD28- and 4-1BB-costimulated. Finally, TEM cells expanded to higher numbers in response to CD27 costimulation. We are currently evaluating the cytokine secretion profile of differentially costimulated T-cell subsets. Altogether, these findings may help in designing new strategies to boost immunity against immune-evading pathogens and tumors, as well as to increase vaccine efficacy. Keywords: T-cell memory subsets, costimulations, Immunotherapy, T-cell expansion, recall responses Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Adaptive Immunity Citation: Volpin V, Cieri N and Bonini C (2013). Human memory T-cell subsets require distinct costimulatory signals to efficiently expand ex vivo. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00999 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 26 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Valentina Volpin, San Raffaele Scientific Institute, Milan, Italy, volpin.valentina@hsr.it Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Valentina Volpin Nicoletta Cieri Chiara Bonini Google Valentina Volpin Nicoletta Cieri Chiara Bonini Google Scholar Valentina Volpin Nicoletta Cieri Chiara Bonini PubMed Valentina Volpin Nicoletta Cieri Chiara Bonini Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Developmentally regulated availability of RANKL and CD40 ligand reveals distinct mechanisms of fetal and adult cross-talk in the thymus medulla.

T cell tolerance in the thymus is a key step in shaping the developing T cell repertoire. Thymic medullary epithelial cells play multiple roles in this process, including negative selection of autoreactive thymocytes, influencing thymic dendritic cell positioning, and the generation of Foxp3(+) regulatory T cells. Previous studies show that medullary thymic epithelial cell (mTEC) development involves hemopoietic cross-talk, and numerous TNFR superfamily members have been implicated in this process. Whereas CD40 and RANK represent key examples, interplay between these receptors, and the individual cell types providing their ligands at both fetal and adult stages of thymus development, remain unclear. In this study, by analysis of the cellular sources of receptor activator for NF-κB ligand (RANKL) and CD40L during fetal and adult cross-talk in the mouse, we show that the innate immune cell system drives initial fetal mTEC development via expression of RANKL, but not CD40L. In contrast, cross-talk involving the adaptive immune system involves both RANKL and CD40L, with analysis of distinct subsets of intrathymic CD4(+) T cells revealing a differential contribution of CD40L by conventional, but not Foxp3(+) regulatory, T cells. We also provide evidence for a stepwise involvement of TNFRs in mTEC development, with CD40 upregulation induced by initial RANK signaling subsequently controlling proliferation within the mTEC compartment. Collectively, our findings show how multiple hemopoietic cell types regulate mTEC development through differential provision of RANKL/CD40L during ontogeny, revealing molecular differences in fetal and adult hemopoietic cross-talk. They also suggest a stepwise process of mTEC development, in which RANK is a master player in controlling the availability of other TNFR family members.