CD70 and IFN‐1 selectively induce eomesodermin or T‐bet and synergize to promote CD8+ T‐cell responses

CD70‐mediated stimulation of CD27 is an important cofactor of CD4⁺ T‐cell licensed dendritic cells (DCs). However, it is unclear how CD70‐mediated stimulation of T cells is integrated with signals that emanate from signal 3 pathways, such as type‐1 interferon (IFN‐1) and IL‐12. We find that while stimulation of CD27 in isolation drives weak Eomesodermin^hiT‐bet^lo CD8⁺ T‐cell responses to OVA immunization, profound synergistic expansion is achieved by cotargeting TLR. This cooperativity can substantially boost antiviral CD8⁺ T‐cell responses during acute infection. Concomitant stimulation of TLR significantly increases per cell IFN‐γ production and the proportion of the population with characteristics of short‐lived effector cells, yet also promotes the ability to form long‐lived memory. Notably, while IFN‐1 contributes to the expression of CD70 on DCs, the synergy between CD27 and TLR stimulation is dependent upon IFN‐1's effect directly on CD8⁺ T cells, and is associated with the increased expression of T‐bet in T cells. Surprisingly, we find that IL‐12 fails to synergize with CD27 stimulation to promote CD8⁺ T‐cell expansion, despite its capacity to drive effector CD8⁺ T‐cell differentiation. Together, these data identify complex interactions between signal 3 and costimulatory pathways, and identify opportunities to influence the differentiation of CD8⁺ T‐cell responses.     

Interrupting CD28 costimulation before antigen rechallenge affects CD8+ T‐cell expansion and effector functions during secondary response in mice

The role of CD28‐mediated costimulation in secondary CD8⁺ T‐cell responses remains controversial. Here, we have used two tools — blocking mouse anti‐mouse CD28‐specific antibodies and inducible CD28‐deleting mice — to obtain definitive answers in mice infected with ovalbumin‐secreting Listeria monocytogenes. We report that both blockade and global deletion of CD28 reveal its requirement for full clonal expansion and effector functions such as degranulation and IFN‐γ production during the secondary immune response. In contrast, cell‐intrinsic deletion of CD28 in transferred TCR‐transgenic CD8⁺ T cells before primary infection leads to impaired clonal expansion but an increase in cells able to express effector functions in both primary and secondary responses. We suggest that the proliferation‐impaired CD8⁺ T cells respond to CD28‐dependent help from their environment by enhanced functional differentiation. Finally, we report that cell‐intrinsic deletion of CD28 after the peak of the primary response does not affect the establishment, maintenance, or recall of long‐term memory. Thus, if given sufficient time, the progeny of primed CD8⁺ T cells adapt to the absence of this costimulator.     

Optimal stimulation for CD70 induction on human monocyte‐derived dendritic cells and the importance of CD70 in naive CD4+ T‐cell differentiation

Studies in mice have shown that CD70 on dendritic cells (DCs) is sufficient to convert T‐cell tolerance into immunity and hence induce anti‐tumour immune responses. Therefore, it is important to investigate (i) optimal stimuli to induce CD70 on human monocyte‐derived DCs (MoDCs), which are widely used for tumour immunotherapy, and (ii) the role of CD70 in functional differentiation of naive CD4⁺ and CD8⁺ T cells stimulated with MoDCs. We show that interferon‐α (IFN‐α) is a key cytokine to differentiate monocytes into DCs with the capacity to express CD70 upon maturation. CD70 expression on IFN‐α‐induced MoDCs was elicited by different categories of maturation‐inducing factors (Toll‐like receptor ligands, CD40 ligand and pro‐inflammatory mediators), among which prostaglandin E₂ was most effective. Naive T cells stimulated with MoDCs also expressed CD70. Stimulation with MoDCs promoted naive CD4⁺ T cells to acquire the ability to produce T helper type 1 and 2 cytokines in a CD70‐dependent manner. In contrast, the CD70–CD27 interaction diminished the production of an immunoregulatory cytokine IL‐10. The CD27 signal did not play a dominant role in the induction of effector molecules in naive CD8⁺ T cells during the stimulation with MoDCs. This study adds a novel function to the versatile cytokines, type I IFNs, that is, the induction of CD70 on MoDCs. CD70 promotes naive CD4⁺ T cells to acquire immunostimulatory activity through the DC–T‐cell and T‐cell–T‐cell interactions during the stimulation with MoDCs. Hence, the CD70–CD27 interaction may play an important role in inducing effective immune responses in DC‐based immunotherapy.     

Polyfunctional response by ImmTAC (IMCgp100) redirected CD8+ and CD4+ T cells

The success of immune system‐based cancer therapies depends on a broad immune response engaging a range of effector cells and mechanisms. Immune mobilizing monoclonal T cell receptors (TCRs) against cancer (ImmTAC™ molecules: fusion proteins consisting of a soluble, affinity enhanced TCR and an anti‐CD3 scFv antibody) were previously shown to redirect CD8⁺ and CD4⁺ T cells against tumours. Here we present evidence that IMCgp100 (ImmTAC recognizing a peptide derived from the melanoma‐specific protein, gp100, presented by HLA‐A*0201) efficiently redirects and activates effector and memory cells from both CD8⁺ and CD4⁺ repertoires. Using isolated subpopulations of T cells, we find that both terminally differentiated and effector memory CD8⁺ T cells redirected by IMCgp100 are potent killers of melanoma cells. Furthermore, CD4⁺ effector memory T cells elicit potent cytotoxic activity leading to melanoma cell killing upon redirection by IMCgp100. The majority of T cell subsets belonging to both the CD8⁺ and CD4⁺ repertoires secrete key pro‐inflammatory cytokines (tumour necrosis factor‐α, interferon‐γ, interleukin‐6) and chemokines (macrophage inflammatory protein‐1α‐β, interferon‐γ‐inducible protein‐10, monocyte chemoattractant protein‐1). At an individual cell level, IMCgp100‐redirected T cells display a polyfunctional phenotype, which is a hallmark of a potent anti‐cancer response. This study demonstrates that IMCgp100 induces broad immune responses that extend beyond the induction of CD8⁺ T cell‐mediated cytotoxicity. These findings are of particular importance because IMCgp100 is currently undergoing clinical trials as a single agent or in combination with check point inhibitors for patients with malignant melanoma.     

CD122+CD8+ Treg suppress vaccine‐induced antitumor immune responses in lymphodepleted mice

Lymphodeleption prior to adoptive transfer of tumor‐specific T cells greatly improves the clinical efficacy of adoptive T‐cell therapy for patients with advanced melanoma, and increases the therapeutic efficacy of cancer vaccines in animal models. Lymphodepletion reduces competition between lymphocytes, and thus creates “space” for enhanced expansion and survival of tumor‐specific T cells. Within the lymphodepleted host, Ag‐specific T cells still need to compete with other lymphocytes that undergo lymphopenia‐driven proliferation. Herein, we describe the relative capacity of naïve T cells, Treg, and NK cells to undergo lymphopenia‐driven proliferation. We found that the major population that underwent lymphopenia‐driven proliferation was the CD122⁺ memory‐like T‐cell population (CD122⁺CD8⁺ Treg), and these cells competed with Ag‐driven proliferation of melanoma‐specific T cells. Removal of CD122⁺CD8⁺ Treg resulted in a greater expansion of tumor‐specific T cells and tumor infiltration of functional effector/memory T cells. Our results demonstrate the lymphopenia‐driven proliferation of CD122⁺CD8⁺ Treg in reconstituted lymphodepleted mice limited the antitumor efficacy of DC vaccination in conjunction with adoptive transfer of tumor‐specific T cells.     

Pathways of memory CD8+ T‐cell development

CD8⁺ T‐cell responses must have at least two components, a replicative cell type that proliferates in the secondary lymphoid tissue and that is responsible for clonal expansion, and cytotoxic cells with effector functions that mediate the resolution of the infection in the peripheral tissues. To confer memory, the response must also generate replication‐competent T cells that persist in the absence of antigen after the primary infection is cleared. The current models of memory differentiation differ in regards to whether or not memory CD8⁺ T cells acquire effector functions during their development. In this review we discuss the existing models for memory development and the consequences that the recent finding that memory CD8⁺ T cells may express granzyme B during their development has for them. We propose that memory CD8⁺ T cells represent a self‐renewing population of T cells that may acquire effector functions but that do not lose the naïve‐like attributes of lymphoid homing, antigen‐independent persistence or the capacity for self‐renewal.     

T‐cell help dependence of memory CD8+ T‐cell expansion upon vaccinia virus challenge relies on CD40 signaling

Due to their capacity to differentiate into long‐lived memory cells, CD8⁺ T cells are able to resolve subsequent infections faster than during the primary response. Among other factors, CD4⁺ T cells play a crucial role during primary and secondary CD8⁺ T‐cell responses. However, the timing and mechanisms by which they influence CD8⁺ T cells may differ in primary and secondary responses. Here, we demonstrate that during both primary and secondary vaccinia virus infection, CD4⁺ T cells are necessary to promote CD8⁺ T‐cell responses. While CD4⁺ T cells contributed to memory CD8⁺ T‐cell development, they were even more important during memory recall responses during challenge, as absence of CD4⁺ T cells during challenge resulted in markedly decreased proliferation and increased apoptosis. T‐cell help during primary and secondary responses was mediated via CD40 signaling, with DCs being an integral part of that pathway. As opposed to primary CD8⁺ T‐cell responses where only a combination of agonistic CD40 signaling and provision of IL‐2 could substitute for T‐cell help, agonistic CD40 triggering alone was sufficient to rescue memory CD8⁺ T‐cell responses in absence of T‐cell help in the context of vaccinia virus infection.     

Modulating numbers and phenotype of CD8+ T cells in secondary immune responses

Prime‐boost regimens are frequently used to increase the number of memory CD8⁺ T cells and thus the protective capacity of experimental vaccinations; however, it is currently unknown how the frequency and phenotype of primary (1°) memory CD8⁺ T cells impact the quantity and phenotype of secondary (2°) memory CD8⁺ T‐cell populations. Here, we show that 2° infections of mice that received different 1° infections and/or immunizations generated similar numbers of 2° effector and memory CD8⁺ T cells. Remarkably, this result was independent of the numbers and phenotype of 1° memory CD8⁺ T cells present at the time of rechallenge. However, after adoptive transfer of low numbers of 1° memory CD8⁺ T cells, a linear correlation between 1° memory CD8⁺ T‐cell input and 2° memory CD8⁺ T‐cell numbers was observed. These data suggest that, above a very low threshold, boosting of 1° memory CD8⁺ T‐cell populations elicits 2° immune responses of similar magnitude. Therefore, our study has important implications for the design of prime‐boost regimens that aim to generate protective CD8⁺ T‐cell‐mediated immunity.     

ICOS is required for the generation of both central and effector CD4+ memory T‐cell populations following acute bacterial infection

Interactions between ICOS and ICOS ligand (ICOSL) are essential for the development of T follicular helper (Tfh) cells and thus the formation and maintenance of GC reactions. Given the conflicting reports on the requirement of other CD4⁺ T‐cell populations for ICOS signals, we have employed a range of in vivo approaches to dissect requirements for ICOS signals in mice during an endogenous CD4⁺ T‐cell response and contrasted this with CD28 signals. Genetic absence of ICOSL only modestly reduced the total number of antigen‐specific CD4⁺ T cells at the peak of the primary response, but resulted in a severely diminished number of both T central memory and T effector memory cells. Treatment with blocking anti‐ICOS mAb during the primary response recapitulated these effects and caused a more substantial reduction than blocking CD28 signals with CTLA4Ig. During the memory phase of the response further signals through ICOS or CD28 were not required for survival. However, upon secondary challenge only Tfh cell expansion remained heavily ICOS‐dependent, while CD28 signals were required for optimal expansion of all subsets. These data demonstrate the importance of ICOS signals specifically for memory CD4⁺ T‐cell formation, while highlighting the potential of therapeutically targeting this pathway.     

IL‐27 enhances the survival of tumor antigen‐specific CD8+ T cells and programs them into IL‐10‐producing,memory precursor‐like effector cells

IL‐27 is a member of the IL‐12 family of cytokines that is comprised of an IL‐12 p40‐related protein subunit, EBV‐induced gene 3, and a p35‐related subunit, p28. IL‐27 functions through IL‐27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8⁺ T‐cell responses. However, the exact mechanisms of how IL‐27 enhances antitumor CD8⁺ T‐cell responses remain unclear. Here we show that IL‐27 significantly enhances the survival of activated tumor antigen‐specific CD8⁺ T cells in vitro and in vivo, and programs tumor antigen‐specific CD8⁺ T cells into memory precursor‐like effector cells, characterized by upregulation of Bcl‐6, SOCS3, Sca‐1, and IL‐10. While STAT3 activation and the CTL survival‐enhancing effects can be independent of CTL IL‐10 production, we show here that IL‐27‐induced CTL IL‐10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL‐27 enhances antitumor CTL responses via programming tumor antigen‐specific CD8⁺ T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.     

CD4+ CD25+ Treg regulate the contribution of CD8+ T-cell subsets in repopulation of the lymphopenic environment

Peripheral T‐cell expansion is of major relevance for immune function after lymphopenia. In order to promote regeneration, the process should result in a peripheral T‐cell pool with a similar subpopulation structure as before lymphopenia. We investigated the repopulation of the CD8⁺ central‐memory T cells (T_CM) and effector‐memory T cells (T_EM) pools after adoptive transfer of sorted CD8⁺ T cells from naïve, T_CM and T_EM subsets into T‐cell‐deficient hosts. We show that the initial kinetics of expansion are distinct for each subset and that the contribution to the repopulation of the CD8⁺ T‐cell pool by the progeny of each subset is not a mere function of its initial expansion. We demonstrate that CD4⁺CD25⁺ Treg play a major role in the repopulation of the CD8⁺ T‐cell pool and that CD8⁺ T‐cell subsets impact on each other. In the absence of CD4⁺CD25⁺ Treg, a small fraction of naïve CD8⁺ T cells strongly proliferates, correlating with further expansion and differentiation of co‐expanding CD8⁺ T cells. CD4⁺CD25⁺ Treg suppress these responses and lead to controlled repopulation, contributing decisively to the maintenance of recovered T_CM and T_EM fractions, and leading to repopulation of each pool with progeny of its own kind.     

Effect of standard tuberculosis treatment on naive,memory and regulatory T‐cell homeostasis in tuberculosis–diabetes co‐morbidity

Perturbations in CD4⁺ and CD8⁺ T‐cell phenotype and function are hallmarks of tuberculosis–diabetes co‐morbidity. However, their contribution to the pathogenesis of this co‐morbidity and the effect of anti‐tuberculosis treatment on the phenotype of the T‐cell subsets is poorly understood. In this study, we examined the frequency of different T‐cell subsets in individuals with pulmonary tuberculosis (PTB) with diabetes mellitus (DM) or without coincident diabetes mellitus (NDM) before, during and after completion of anti‐tuberculosis chemotherapy. PTB‐DM is characterized by heightened frequencies of central memory CD4⁺ and CD8⁺ T cells and diminished frequencies of naive, effector memory and/or effector CD4⁺ and CD8⁺ T cells at baseline and after 2 months of treatment but not following treatment completion in comparison with PTB‐NDM. Central memory CD4⁺ and CD8⁺ T‐cell frequencies exhibited a positive correlation with fasting blood glucose and glycated haemoglobin A1c levels, whereas the frequencies of naive and effector memory or effector CD4⁺ and CD8⁺ T cells exhibited a negative correlation. However, the frequencies of CD4⁺ and CD8⁺ T‐cell subsets in individuals with PTB exhibited no significant relationship with bacterial burdens. Finally, although minor alterations in the T‐cell subset compartment were observed at 2 months of treatment, significantly decreased frequencies of central memory and significantly enhanced frequencies of naive CD4⁺ and CD8⁺ T cells were observed at the completion of treatment. Our data reveal a profound effect of coexistent diabetes on the altered frequencies of central memory, effector memory and naive T cells and its normalization following therapy.     

Human antigen‐specific CD4+CD25+CD134+CD39+ T cells are enriched for regulatory T cells and comprise a substantial proportion of recall responses

Human Ag‐specific CD4⁺ T cells can be detected by their dual expression of CD134 (OX40) and CD25 after a 44 hours stimulation with cognate Ag. We show that surface expression of CD39 on Ag‐specific cells consistently identifies a substantial population of CD4⁺CD25⁺CD134⁺CD39⁺ T cells that have a Treg‐cell‐like phenotype and mostly originate from bulk memory CD4⁺CD45RO⁺CD127^lowCD25^highCD39⁺ Treg cells. Viable, Ag‐specific CD25⁺CD134⁺CD39⁺ T cells could be expanded in vitro as cell lines and clones, and retained high Forkhead Box Protein 3, CTLA‐4 and CD39 expression, suppressive activity and Ag specificity. We also utilised this combination of cell surface markers to measure HIV‐Gag responses in HIV⁺ patients before and after anti‐retroviral therapy (ART). Interestingly, we found that the percentage of CD39^? cells within baseline CD4⁺ T‐cell responses to HIV‐Gag was negatively correlated with HIV viral load pre‐ART and positively correlated with CD4⁺ T‐cell recovery over 96 weeks of ART. Collectively, our data show that Ag‐specific CD4⁺CD25⁺CD134⁺CD39⁺ T cells are highly enriched for Treg cells, form a large component of recall responses and maintain a Treg‐cell‐like phenotype upon in vitro expansion. Identification and isolation of these cells enables the role of Treg cells in memory responses to be further defined and provides a development pathway for novel therapeutics.     

Positive selection of self‐antigen‐specific CD8+ T cells by hematopoietic cells

In contrast to thymic epithelial cells, which induce the positive selection of conventional CD8⁺ T cells, hematopoietic cells (HCs) select innate CD8⁺ T cells whose Ag specificity is not fully understood. Here we show that CD8⁺ T cells expressing an H‐Y Ag‐specific Tg TCR were able to develop in mice in which only HCs expressed MHC class I, when HCs also expressed the H‐Y Ag. These HC‐selected self‐specific CD8⁺ T cells resemble innate CD8⁺ T cells in WT mice in terms of the expression of memory markers and effector functions, but are phenotypically distinct from the thymus‐independent CD8⁺ T‐cell population. The peripheral maintenance of H‐Y‐specific CD8⁺ T cells required presentation of the self‐Ag and IL‐15 on HCs. HC‐selected CD8⁺ T cells in mice lacking the Tg TCR also showed these features. Furthermore, by using MHC class I tetramers with a male Ag peptide, we found that self‐Ag‐specific CD8⁺ T cells in TCR non‐Tg mice could develop via HC‐induced positive selection, supporting results obtained from H‐Y TCR Tg mice. These findings indicate the presence of self‐specific CD8⁺ T cells that are positively selected by HCs in the peripheral T‐cell repertoire.     

CD4+CD25+ regulatory T cells utilize FasL as a mechanism to restrict DC priming functions in cutaneous immune responses

Recent studies have suggested Fas‐mediated elimination of antigen‐presenting cells as an important mechanism down‐regulating the induction of autoimmune responses. It remains unknown whether this mechanism restricts the magnitude of immune responses to non‐self antigens. We used a mouse model of a cutaneous CD8⁺ T‐cell‐mediated immune response (contact hypersensitivity, CHS) to test if CD4⁺CD25⁺ T cells expressing FasL regulate hapten‐specific effector CD8⁺ T cell expansion through the elimination of Fas‐expressing hapten‐presenting DC. In WT mice, attenuation of CD4⁺CD25⁺ T regulatory cell activity by anti‐CD25 mAb increased hapten‐presenting DC numbers in skin‐draining LN, which led to increased effector CD8⁺ T‐cell priming for CHS responses. In contrast, CD4⁺CD25⁺ T cells did not regulate hapten‐specific CD8⁺ T‐cell priming and CHS responses initiated by Fas‐defective (lpr) DC. Thus, restricting DC priming functions through Fas–FasL interactions is a potent mechanism employed by CD4⁺CD25⁺ regulatory cells to restrict CD8⁺ T‐cell‐mediated allergic immune responses in the skin.     

T cells compete by cleaving cell surface CD27 and blocking access to CD70‐bearing APCs

T cells compete against each other for access to molecules on APCs in addition to peptide/MHC complexes. However, the identity of cell surface molecules that influence T‐cell competition, other than peptide/MHC, have yet to be defined. Here, we identify CD70, a TNF ligand expressed on activated APCs, as an important mediator of T‐cell competition for APCs. Upon engagement of CD27 by CD70, CD27 is proteolytically cleaved from the surface of the interacting CD8⁺ T cell and captured by CD70 expressing dendritic cells. The capture of CD27 effectively masks CD70 on APCs, disallowing the interaction with CD27 on other competing T cells. Collectively, our data indicate that T cells compete against each other for access to the TNF‐ligand CD70, an interaction that affects the duration and potency of T cell/DC interactions, thus influencing the repertoire of responding CD8⁺ T cells to self or foreign antigens.