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.

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.

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.

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.

PD-1 Blockade and CD27 Stimulation Activate Distinct Transcriptional Programs That Synergize for CD8+ T-Cell-Driven Antitumor Immunity.

Purpose: PD-1 checkpoint blockade has revolutionized the field of cancer immunotherapy, yet the frequency of responding patients is limited by inadequate T-cell priming secondary to a paucity of activatory dendritic cells (DC). DC signals can be bypassed by CD27 agonists, and we therefore investigated if the effectiveness of anti-PD-1/L1 could be improved by combining with agonist anti-CD27 monoclonal antibodies (mAb).Experimental Design: The efficacy of PD-1/L1 blockade or agonist anti-CD27 mAb was compared with a dual-therapy approach in multiple tumor models. Global transcriptional profiling and flow cytometry analysis were used to delineate mechanisms underpinning the observed synergy.Results: PD-1/PD-L1 blockade and agonist anti-CD27 mAb synergize for increased CD8+ T-cell expansion and effector function, exemplified by enhanced IFNγ, TNFα, granzyme B, and T-bet. Transcriptome analysis of CD8+ T cells revealed that combination therapy triggered a convergent program largely driven by IL2 and Myc. However, division of labor was also apparent such that anti-PD-1/L1 activates a cytotoxicity-gene expression program whereas anti-CD27 preferentially augments proliferation. In tumor models, either dependent on endogenous CD8+ T cells or adoptive transfer of transgenic T cells, anti-CD27 mAb synergized with PD-1/L1 blockade for antitumor immunity. Finally, we show that a clinically relevant anti-human CD27 mAb, varlilumab, similarly synergizes with PD-L1 blockade for protection against lymphoma in human-CD27 transgenic mice.Conclusions: Our findings suggest that suboptimal T-cell invigoration in cancer patients undergoing treatment with PD-1 checkpoint blockers will be improved by dual PD-1 blockade and CD27 agonism and provide mechanistic insight into how these approaches cooperate for CD8+ T-cell activation. Clin Cancer Res; 24(10); 2383-94. ©2018 AACR.

Transplantation treatment of spinal cord injury patients.

The minimally manipulated cells from fetal nervous and hemopoietic tissues (gestational age 16-22 weeks) were subarachnoidally implanted into 15 patients (18-52 years old) with severe consequences of traumatic spinal cord injury (SCI) at cervical or thoracic spine level. The times after SCI were from 1 month to 6 years. Each patient underwent from one to four cell transplantations (CT) with various time intervals. In 11 of 15 cases, CT was combined with an operative partial disruption of a connective tissue cyst and with implantation into a spinal cord lesion of a spinal cord fragment together with olfactory ensheathing cells. Before CT the patients showed complete motor and sensory function disorder consistent with a grade A of SCI according to Frankel classification. With CT treatment, six patients improved their neurological status from A to C grade of SCI, exhibiting incomplete restoration of both motor and sensory function. The status of other five CT-treated patients became consistent with SCI grade B and was characterized by appearance of contracting activity in some muscles and incomplete restoration of sensitivity. The remaining four patients did not exhibit any clinical improvements. No serious complications of CT were noted. The results suggest a clinical relevance of the CT-based approach to treating severe consequences of SCI.

CD70-CD27 interaction augments CD8+ T-cell activation by human epidermal Langerhans cells.

Human cutaneous dendritic cells (DCs) from epidermal and dermal compartments exhibit functional differences in their induction of CD4+ T-cell and humoral immune responses; however, differences in the regulation of memory CD8+ T-cell responses by human skin DCs remain poorly characterized. We tested the capacity of human Langerhans cells (LCs) and dermal dendritic cells (DDCs) to induce antigen-specific cytokine production and proliferation of memory CD8+ cells. Although tumor necrosis factor-α-matured human DCs from both epidermal and dermal compartments showed efficient potential to activate CD8+ cells, LCs were constitutively more efficient than DDCs in cross-presenting CD8+ epitopes, as well as direct presentation of viral antigen to Epstein-Barr virus-specific CD8+ T cells. LCs showed greater expression of CD70, and blockade of CD70-CD27 signaling demonstrated that superiority of CD8+ activation by epidermal LC is CD70 dependent. This CD70-related activation of CD8+ cells by LCs denotes a central role of LCs in CD8+ immunity in skin, and suggests that regulation of LC CD70 expression is important in enhancing immunity against cutaneous epithelial pathogens and cancer.

Invariant NKT cells promote CD8+ cytotoxic T cell responses by inducing CD70 expression on dendritic cells.

Activation of invariant NK T (iNKT) cells with the glycolipid alpha-galactosylceramide promotes CD8(+) cytotoxic T cell responses, a property that has been used to enhance the efficacy of antitumor vaccines. Using chimeric mice, we now show that the adjuvant properties of iNKT cells require that CD40 triggering and Ag presentation to CD8(+) T cells occur on the same APCs. We demonstrate that injection of alpha-galactosylceramide triggers CD70 expression on splenic T cell zone dendritic cells and that this is dependent on CD40 signaling. Importantly, we show that blocking the interaction between CD70 and CD27, its costimulatory receptor on T cells, abrogates the ability of iNKT cells to promote a CD8(+) T cell response and abolishes the efficacy of alpha-GalCer as an adjuvant for antitumor vaccines. These results define a key role for CD70 in linking the innate response of iNKT cells to the activation of CD8(+) T cells.

The Death Receptor 3-TNF-like protein 1A pathway drives adverse bone pathology in inflammatory arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of synovial joints that is associated with cartilage and bone destruction. Death Receptor 3 (DR3), a tumor necrosis factor (TNF) receptor superfamily member, has recently been associated with the pathogenesis of RA. We demonstrate that absence of DR3 confers resistance to the development of adverse bone pathology in experimental antigen-induced arthritis (AIA). DR3(ko) mice exhibited a reduction in all histopathological hallmarks of AIA but, in particular, failed to develop subchondral bone erosions and were completely protected from this characteristic of AIA. In contrast, TNF-like protein 1A (TL1A), the ligand for DR3, exacerbated disease in a dose- and DR3-dependent fashion. Analysis of osteoclast number within AIA joint revealed a reduction in areas susceptible to bone erosion in DR3(ko) mice, whereas in vitro osteoclastogenesis assays showed that TL1A could directly promote osteoclastogenesis in mouse and man. Treatment with antagonistic anti-TL1A mAb protected animals in a systemic model of RA disease collagen-induced arthritis. We therefore conclude that the DR3-TL1A pathway regulates joint destruction in two murine models of arthritis and represents a potential novel target for therapeutic intervention in inflammatory joint disease.

Eradication of lymphoma by CD8 T cells following anti-CD40 monoclonal antibody therapy is critically dependent on CD27 costimulation.

Growing evidence points to the potential of agonistic anti-CD40 mAbs as adjuvants for vaccination against cancer. These appear to act by maturing dendritic cells (DCs) and allowing them to prime CD8 cytotoxic T lymphocytes (CTLs). Although it is well established that optimal T-cell priming requires costimulation via B7:CD28, recent studies emphasize the contribution of TNF receptors to this process. To understand how anti-CD40 mAbs trigger effective antitumor immunity, we investigated the role of TNFR superfamily members CD27 and 4-1BB in the generation of this immunity and showed that, although partially dependent on 4-1BB:4-1BBL engagement, it is completely reliant on CD27:CD70 interactions. Importantly, blocking CD70, and to some extent 4-1BBL, during anti-CD40 treatment prevented accumulation of tumor-reactive T cells and subsequent tumor protection. However, it did not influence changes in DC number, phenotype, nor the activity of CTLs once immunity was established. We conclude that CD27:CD70 and 4-1BB:4-1BBL interactions are needed for DC-driven accumulation of antitumor CTLs following anti-CD40 mAb treatment. Finally, in support of the critical role for CD70:CD27, we show for the first time that agonistic anti-CD27 mAbs given without a DC maturation signal completely protect tumor-bearing mice and provide a highly potent reagent for boosting antitumor T-cell immunity.

Requirement for CD70 in CD4+ Th cell-dependent and innate receptor-mediated CD8+ T cell priming.

Dendritic cell (DC) conditioning by CD4+ T cells, or via engagement of innate receptors, is thought to be essential for CD8+ T cell priming. However, the molecular features that distinguish a conditioned DC from an unconditioned DC are poorly defined. In this study, we investigate the role of CD70, a member of the TNF superfamily that is expressed on activated DC, in CD4+ Th-dependent and -independent CD8+ T cell responses. We demonstrate that CD70 is required for CD4+ T cell-dependent priming of CD8+ T cells as well as priming mediated by the viral signature, dsRNA. Accordingly, mice that were subjected to CD70 blockade during the primary response fail to generate a memory CD8+ T cell response. Furthermore, we find that CD70 is dispensable for CD4+ T cell expansion and help for B cells, thus suggesting a direct role for CD70 in CD8+ T cell priming. Our results show that the innate and adaptive (CD4+ T cells) arms of the immune system use a common signaling pathway in driving CD8+ T cell responses and suggest that expression of CD70 on DC represents the hallmark of conditioned DC.

Cutting edge: a critical role for CD70 in CD8 T cell priming by CD40-licensed APCs.

The CD154/CD40 interaction is an important pathway of CD4 T cell help for CD8 T cell responses. In this study, we address the role of CD70, a member of the TNF superfamily and the ligand for the T cell costimulatory receptor CD27, in CD40-mediated priming of CD8 T cells. Using an agonistic anti-CD40 mAb to mimic the CD154/CD40 interaction we demonstrate that the priming of OT-I TCR transgenic or endogenous mouse OVA-specific CD8 T cells is critically dependent on CD70/CD27 interaction. CD70 blockade inhibited CD40-mediated clonal expansion of CD8 T cells and reduced the number of memory CD8 T cells generated. Furthermore, CD70 blockade during the initial priming of CD8 T cells inhibited the ability of memory CD8 T cells to expand in response to a second encounter with Ag. Our data indicate that CD70 expression on APCs plays a key role in CD40-dependent CD8 T cell responses.

Bone Marrow Cells in Suppressing Leukemic Cell Growth.

In this paper we review our experimental findings concerning the capacity of bone marrow cells (BMC) to control leukemic cell growth. It has been shown that the cells isolated from normal bone marrow can provide dose dependent suppression of the proliferative activity of leukemic cells in vitro. BMC cytostatic effect is antigen non-specific and does not associate with cell death. Cytostatic BMC differ from mature macrophages, T and B lymphocytes and have the lower floating density. These cells are detected in both aggregated and non-aggregated fraction of BMC, stimulated by wheat germ agglutinin. During long-term cultivation of bone marrow the cytostatic activity was associated with the radioresistant stromal cells. Both soluble factors and cell-to-cell interactions are involved into the cytostatic process generated by BMC. Based on the obtained results, we suggest that the cytostatic activity of BMC may be increased under the influence of lymphokines, such as IL-2 and IFNgamma.

Expression and costimulatory effects of the TNF receptor superfamily members CD134 (OX40) and CD137 (4-1BB), and their role in the generation of anti-tumor immune responses.

This study addresses the relative importance of CD134 (OX40) and CD137 (4-1BB) in the costimulation of CD4+ and CD8+ T cells under comparable conditions of antigenic stimulation. We demonstrate that CD134 is capable of directly costimulating CD8+ T cells. However, costimulation of CD8+ T cells by CD134 is less potent than that triggered by CD137. The higher costimulatory activity of CD137, when compared with CD134, correlates well with its faster expression kinetics and higher levels on CD8+ T cells. Furthermore, induction of CD137 expression on CD8+ T cells is highly sensitive to low levels of TCR stimulation, which is in contrast with CD134. Conversely, CD134 is more effective than CD137 in costimulating CD4+ T cells. This, however, could not be attributed to differential expression. We also demonstrate that the transient nature of CD134 and CD137 expression on activated CD4+ T cells is the resultof proteolytic shedding. Consistent with the greater ability of CD137 to costimulate CD8+ T cells, stimulation of CD137 in vivo is considerably more effective than CD134 in augmenting anti-tumor immune responses. Therefore, agents that stimulate signaling via CD137 are likely to be more useful in clinical conditions where highly effective CD8+ CTL responses are required.