CD40 ligand‐expressing recombinant vaccinia virus promotes the generation of CD8+ central memory T cells

Central memory CD8⁺ T cells (T_CM) play key roles in the protective immunity against infectious agents, cancer immunotherapy, and adoptive treatments of malignant and viral diseases. CD8⁺ T_CM cells are characterized by specific phenotypes, homing, and proliferative capacities. However, CD8⁺ T_CM‐cell generation is challenging, and usually requires CD4⁺ CD40L⁺ T‐cell “help” during the priming of naïve CD8⁺ T cells. We have generated a replication incompetent CD40 ligand‐expressing recombinant vaccinia virus (rVV40L) to promote the differentiation of human naïve CD8⁺ T cells into T_CM specific for viral and tumor‐associated antigens. Soluble CD40 ligand recombinant protein (sCD40L), and vaccinia virus wild‐type (VV WT), alone or in combination, were used as controls. Here, we show that, in the absence of CD4⁺ T cells, a single “in vitro” stimulation of naïve CD8⁺ T cells by rVV40L‐infected nonprofessional CD14⁺ antigen presenting cells promotes the rapid generation of viral or tumor associated antigen‐specific CD8⁺ T cells displaying T_CM phenotypic and functional properties. These observations demonstrate the high ability of rVV40L to fine tune CD8⁺ mediated immune responses, and strongly support the use of similar reagents for clinical immunization and adoptive immunotherapy purposes.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

Pro‐inflammatory self‐reactive T cells are found within murine TCR‐αβ+CD4−CD8−PD‐1+ cells

TCR‐αβ⁺ double negative (DN) T cells (CD3⁺TCR‐αβ⁺CD4^?CD8^?NK1.1^?CD49b^?) represent a minor heterogeneous population in healthy humans and mice. These cells have been ascribed pro‐inflammatory and regulatory capacities and are known to expand during the course of several autoimmune diseases. Importantly, previous studies have shown that self‐reactive CD8⁺ T cells become DN after activation by self‐antigens, suggesting that self‐reactive T cells may exist within the DN T‐cell population. Here, we demonstrate that programmed cell death 1 (PD‐1) expression in unmanipulated mice identifies a subset of DN T cells with expression of activation‐associated markers and a phenotype that strongly suggests they are derived from self‐reactive CD8⁺ cells. We also found that, within DN T cells, the PD‐1⁺ subset generates the majority of pro‐inflammatory cytokines. Finally, using a TCR‐activation reporter mouse (Nur77‐GFP), we confirmed that in the steady‐state PD‐1⁺ DN T cells engage endogenous antigens in healthy mice. In conclusion, we provide evidence that indicates that the PD‐1⁺ fraction of DN T cells represents self‐reactive cells.     

Innate and virtual memory T cells in man

A hallmark of the antigen‐specific B and T lymphocytes of the adaptive immune system is their capacity to “remember” pathogens long after they are first encountered, a property that forms the basis for effective vaccine development. However, studies in mice have provided strong evidence that some naive T cells can develop characteristics of memory T cells in the absence of foreign antigen encounters. Such innate memory T cells may develop in response to lymphopenia or the presence of high levels of the cytokine IL‐4, and have also been identified in unmanipulated animals, a phenomenal referred to as “virtual memory.” While the presence of innate memory T cells in mice is now widely accepted, their presence in humans has not yet been fully validated. In this issue of the European Journal of Immunology, Jacomet et al. [Eur. J. Immunol. 2015. 45:1926‐1933] provide the best evidence to date for innate memory T cells in humans. These findings may contribute significantly to our understanding of human immunity to microbial pathogens and tumors.     

The cellular microenvironment regulates CX3CR1 expression on CD8+ T cells and the maintenance of CX3CR1+ CD8+ T cells

Expression levels of the chemokine receptor CX3CR1 serve as high-resolution marker delineating functionally distinct antigen-experienced T-cell states. The factors that influence CX3CR1 expression in T cells are, however, incompletely understood. Here, we show that in vitro priming of naïve CD8⁺ T cells failed to robustly induce CX3CR1, which highlights the shortcomings of in vitro priming settings in recapitulating in vivo T-cell differentiation. Nevertheless, in vivo generated memory CD8⁺ T cells maintained CX3CR1 expression during culture. This allowed us to investigate whether T-cell receptor ligation, cell death, and CX3CL1 binding influence CX3CR1 expression. T-cell receptor stimulation led to downregulation of CX3CR1. Without stimulation, CX3CR1⁺ CD8⁺ T cells had a selective survival disadvantage, which was enhanced by factors released from necrotic but not apoptotic cells. Exposure to CX3CL1 did not rescue their survival and resulted in a dose-dependent loss of CX3CR1 surface expression. At physiological concentrations of CX3CL1, CX3CR1 surface expression was only minimally reduced, which did not hamper the interpretability of T-cell differentiation states delineated by CX3CR1. Our data further support the broad utility of CX3CR1 surface levels as T-cell differentiation marker and identify factors that influence CX3CR1 expression and the maintenance of CX3CR1 expressing CD8⁺ T cells.     

Differential requirements for IRF4 in the clonal expansion and homeostatic proliferation of naive and memory murine CD8+ T cells

Interferon regulatory factor 4 (IRF4) has critical roles in immune cell differentiation and function and is indispensable for clonal expansion and effector function in T cells. Here, we demonstrate that the AKT pathway is impaired in murine CD8⁺ T cells lacking IRF4. The expression of phosphatase and tensin homolog (PTEN), a negative regulator of the AKT pathway, was elevated in Irf4^?/? CD8⁺ T cells. Inhibition of PTEN partially rescued downstream events, suggesting that PTEN constitutes a checkpoint in the IRF4‐mediated regulation of cell signaling. Despite the clonal expansion defect, in the absence of IRF4, memory‐like CD8⁺ T cells could be generated and maintained, although unable to expand in recall responses. The homeostatic proliferation of naïve Irf4^?/? CD8⁺ T cells was impaired, whereas their number eventually reached a level similar to that of wild‐type CD8⁺ T cells. Conversely, memory‐like Irf4^?/? CD8⁺ T cells underwent homeostatic proliferation in a manner similar to that of wild‐type memory CD8⁺ T cells. These results suggest that IRF4 regulates the clonal expansion of CD8⁺ T cells at least in part via the AKT signaling pathway. Moreover, IRF4 regulates the homeostatic proliferation of naïve CD8⁺ T cells, whereas the maintenance of memory CD8⁺ T cells is IRF4‐independent.     

OX40 and IL‐7 play synergistic roles in the homeostatic proliferation of effector memory CD4+ T cells

T‐cell homeostasis preserves the numbers, the diversity and functional competence of different T‐cell subsets that are required for adaptive immunity. Naïve CD4⁺ T (T_N) cells are maintained in the periphery via the common γ‐chain family cytokine IL‐7 and weak antigenic signals. However, it is not clear how memory CD4⁺ T‐cell subsets are maintained in the periphery and which factors are responsible for the maintenance. To examine the homeostatic mechanisms, CFSE‐labeled CD4⁺CD44^highCD62L^low effector memory T (T_EM) cells were transferred into sublethally‐irradiated syngeneic C57BL/6 mice, and the systemic cell proliferative responses, which can be divided distinctively into fast and slow proliferations, were assessed by CFSE dye dilution. We found that the fast homeostatic proliferation of T_EM cells was strictly regulated by both antigen and OX40 costimulatory signals and that the slow proliferation was dependent on IL‐7. The simultaneous blockade of both OX40 and IL‐7 signaling completely inhibited the both fast and slow proliferation. The antigen‐ and OX40‐dependent fast proliferation preferentially expanded IL‐17‐producing helper T cells (Th17 cells). Thus, OX40 and IL‐7 play synergistic, but distinct roles in the homeostatic proliferation of CD4⁺ T_EM cells.     

CD28 and CD57 define four populations with distinct phenotypic properties within human CD8+ T cells

After repeated antigen exposure, both memory and terminally differentiated cells can be generated within CD8⁺ T cells. Although, during their differentiation, activated CD8⁺ T cells may first lose CD28, and CD28⁻ cells may eventually express CD57 as a subsequent step, a population of CD28⁺CD57⁺(DP) CD8⁺ T cells can be identified in the peripheral blood. How this population is distinct from CD28⁻CD57⁻(DN) CD8⁺ T cells, and from the better characterized non-activated/early-activated CD28⁺CD57⁻ and senescent-like CD28⁻CD57⁺ CD8⁺ T cell subsets is currently unknown. Here, RNA expression of the four CD8⁺ T cell subsets isolated from human PBMCs was analyzed using microarrays. DN cells were more similar to “early” highly differentiated cells, with decreased TNF and IFN-γ production, impaired DNA damage response and apoptosis. Conversely, increased apoptosis and expression of cytokines, co-inhibitory, and chemokine receptors were found in DP cells. Higher levels of DP CD8⁺ T cells were observed 7 days after Hepatitis B vaccination, and decreased levels of DP cells were found in rheumatoid arthritis patients. More DP and DN CD8⁺ T cells were present in the bone marrow, in comparison with PBMCs. In summary, our results indicate that DP and DN cells are distinct CD8⁺ T cell subsets displaying defined properties.     

TGF‐β induces the differentiation of human CXCL13‐producing CD4+ T cells

In the ectopic lymphoid‐like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4⁺ T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF‐β induces the differentiation of human CXCL13‐producing CD4⁺ T cells from naïve CD4⁺ T cells. The TGF‐β‐induced CXCL13‐producing CD4⁺ T cells do not express CXCR5, B‐cell lymphoma 6 (BCL6), and other Tfh‐cell markers. Furthermore, expression levels of CD25 (IL‐2Rα) in CXCL13‐producing CD4⁺ T cells are significantly lower than those in FoxP3⁺ in vitro induced Treg cells. Consistent with this, neutralization of IL‐2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4⁺ T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4⁺ T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13‐producing CD4⁺ T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13‐producing CD4⁺ T cells. Our findings demonstrate that CXCL13‐producing CD4⁺ T cells lacking Tfh‐cell features differentiate via TGF‐β signaling but not via FoxP3, and exert their function in IL‐2‐limited but TGF‐β‐rich and proinflammatory cytokine‐rich inflammatory conditions.     

Thymus‐resident memory CD8+ T cells mediate local immunity

The thymus is a primary lymphoid organ responsible for production and selection of T cells. Nonetheless, mature T cells and in particular activated T cells can reenter the thymus. Here, we identified memory CD8⁺ T cells specific for lymphocytic choriomeningitis virus or vaccinia virus in the thymus of mice long‐time after the infection. CD8⁺ T cells were mainly located in the thymic medulla, but also in the cortical areas. Interestingly, virus‐specific memory CD8⁺ T cells in the thymus expressed the cell surface markers CD69 and CD103 that are characteristic of tissue‐resident memory T cells in a time‐dependent manner. Kinetic analyses and selective depletion of peripheral CD8⁺ T cells by antibodies further revealed that thymic virus‐specific memory CD8⁺ T cells did not belong to the circulating pool of lymphocytes. Finally, we demonstrate that these thymus‐resident virus‐specific memory CD8⁺ T cells efficiently mounted a secondary proliferative response, exhibited immediate effector functions and were able to protect the thymus from lymphocytic choriomeningitis virus reinfection. In conclusion, the present study not only describes for the first time virus‐specific memory CD8⁺ T cells with characteristics of tissue‐resident memory T (T_RM) cells in a primary lymphoid organ but also extends our knowledge about local T‐cell immunity in the thymus.     

Early effector maturation of naïve human CD8+ T cells requires mitochondrial biogenesis

The role of mitochondrial biogenesis during naïve to effector differentiation of CD8⁺ T cells remains ill explored. In this study, we describe a critical role for early mitochondrial biogenesis in supporting cytokine production of nascent activated human naïve CD8⁺ T cells. Specifically, we found that prior to the first round of cell division activated naïve CD8⁺ T cells rapidly increase mitochondrial mass, mitochondrial respiration, and mitochondrial reactive oxygen species (mROS) generation, which were all inter‐linked and important for CD8⁺ T cell effector maturation. Inhibition of early mitochondrial biogenesis diminished mROS dependent IL‐2 production – as well as subsequent IL‐2 dependent TNF, IFN‐γ, perforin, and granzyme B production. Together, these findings point to the importance of mitochondrial biogenesis during early effector maturation of CD8⁺ T cells.     

Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8+ memory T cells

Protection against malaria can be achieved by induction of a strong CD8⁺ T‐cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long‐lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8⁺ T‐cell response and sterile protection, which is short‐lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite‐specific antibodies that showed protective capacity. The numbers of CSP‐specific CD8⁺ T cells were not substantially enhanced by sporozoite infections; however, CSP‐specific memory CD8⁺ T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized‐only mice. CD4⁺ T cells contributed to protection as well; but CD8⁺ memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8⁺ effector‐memory T cells with increased cytotoxic activity as well as CD4⁺ memory T cells and neutralizing antibodies.     

Changes in T‐cell subsets identify responders to FcR‐nonbinding anti‐CD3 mAb (teplizumab) in patients with type 1 diabetes

The mechanisms whereby immune therapies affect progression of type 1 diabetes (T1D) are not well understood. Teplizumab, an FcR nonbinding anti‐CD3 mAb, has shown efficacy in multiple randomized clinical trials. We previously reported an increase in the frequency of circulating CD8⁺ central memory (CD8CM) T cells in clinical responders, but the generalizability of this finding and the molecular effects of teplizumab on these T cells have not been evaluated. We analyzed data from two randomized clinical studies of teplizumab in patients with new‐ and recent‐onset T1D. At the conclusion of therapy, clinical responders showed a significant reduction in circulating CD4⁺ effector memory T cells. Afterward, there was an increase in the frequency and absolute number of CD8CM T cells. In vitro, teplizumab expanded CD8CM T cells by proliferation and conversion of non‐CM T cells. Nanostring analysis of gene expression of CD8CM T cells from responders and nonresponders versus placebo‐treated control subjects identified decreases in expression of genes associated with immune activation and increases in expression of genes associated with T‐cell differentiation and regulation. We conclude that CD8CM T cells with decreased activation and regulatory gene expression are associated with clinical responses to teplizumab in patients with T1D.     

Evidence for eomesodermin‐expressing innate‐like CD8+ KIR/NKG2A+ T cells in human adults and cord blood samples

Polyclonal CD8⁺ T cells, with a marked innate/memory phenotype, high eomesodermin (Eomes) expression, and the capacity to generate IFN‐γ rapidly without prior exposure to antigen, have been described in mice. However, even though a pool of human CD8⁺ T cells expressing killer Ig‐like receptors (KIRs) was recently documented, the existence of a human equivalent of murine innate/memory CD8⁺ T cells remains to be established. Here, we provide evidence for a population of KIR/NKG2A⁺CD8⁺ T cells in healthy human adults sharing the same features, namely increased Eomes expression, prompt IFN‐γ production in response to innate‐like stimulation by IL‐12+IL‐18, and a potent antigen‐independent cytotoxic activity along with a preferential terminally differentiated effector memory phenotype. None of the above functional characteristics applied to the KIR/NKG2A^? fraction of the Eomes⁺CD8⁺ T‐cell population, thereby underlining the ability of KIR/NKG2A to distinguish between “innate/memory‐like” and “conventional/memory” pools of CD8⁺ T cells. Remarkably, KIR/NKG2A⁺Eomes⁺CD8⁺ T cells with innate‐like functions and a memory/terminally differentiated effector memory phenotype were also identified in human cord blood, suggesting that their development did not depend on cognate antigens. Taken together, our results support the conclusion that CD8⁺ T cells co‐expressing Eomes and KIR/NKG2A may represent a new, functionally distinct “innate/memory‐like” subset in humans.     

Origins and functional basis of regulatory CD11c+CD8+ T cells

Previously, we showed that CD11c defines a novel subset of CD8⁺ T cells whose in vivo activity is therapeutic for arthritis; however, the mechanisms directing their development, identity of their precursors, and basis of their effector function remain unknown. Here, we show that the novel subset develops from CD11c^surface?CD8⁺ T cells and undergoes robust expansion in an antigen‐ and 4‐1BB (CD137)‐dependent manner. CD11c⁺CD8⁺ T cells exist in naïve mice (<3%) with limited suppressive activity. Once activated, they suppress CD4⁺ T cells in vivo and in vitro. Suppression of CD4⁺ by CD11c⁺CD8⁺ T cells is related to an increase in IDO activity induced in competent cells via the general control non‐derepressible‐2 pathway. CD11c⁺CD8⁺ T cells are refractory to the effect of IDO but constrict in a novel 1‐methyl D ,L ‐tryptophan‐dependent mechanism resulting in reversal of their suppressive effects. Thus, our data uncover, for the first time, the origin, development, and basis of the suppressive function of this novel CD11c⁺CD8⁺ T‐cell subpopulation that has many signature features of Treg.     

mTOR signaling promotes a robust and continuous production of IFN‐γ by human memory CD8+ T cells and their proliferation

Human CD8⁺ T cells are functionally heterogeneous and can be divided into phenotypically and functionally distinct subsets according to CCR7 and CD45RA expression levels. Among these, CCR7^lowCD45RA^low effector memory CD8⁺ T cells (Tem) and CCR7^lowCD45RA^high CD8⁺ T cells, which are designated as Temra and considered to be terminally differentiated cells, are Ag‐experienced T cells but show different functionalities. Here, we show that, while Tem proliferate vigorously and produce IFN‐γ persistently and robustly, Temra proliferate poorly and lose the ability to produce IFN‐γ over time after TCR stimulation. Temra showed impaired cell growth upon TCR stimulation, which was associated with defective activation of the mammalian target of rapamycin (mTOR) signaling. Furthermore, rapamycin, an inhibitor of mTOR signaling, interfered with the robust and continuous proliferation of and IFN‐γ production by Tem at later time points after TCR stimulation. Thus, these data collectively indicate that activation of mTOR signaling is required for the robust functions of Tem cells in humans and suggest that defective mTOR signaling in Temra contributes to their functional impairment.     

CD40L expression by CD4+ but not CD8+ T cells regulates antiviral immune responses in acute LCMV infection in mice

CD40‐CD40 ligand (CD40L) signaling plays multiple indispensable roles in cellular and humoral immunity. Impaired memory T‐cell responses in the absence of CD40L have been well documented, but the requirement of this interaction for efficient priming of CD8⁺ T cells especially under inflammatory conditions has been under debate. In contrast to previous publications, we report here that virus‐specific CD8⁺ T‐cell responses as well as viral clearance are affected not only in the memory but also in the effector phase in CD40L^?/? mice infected with lymphocytic choriomeningitis virus (LCMV) Armstrong strain. Interestingly, a considerable part of the LCMV‐specific effector and memory T cells consists of CD40L⁺ CD8⁺ T cells. However, deficiency of CD40L in CD8⁺ T cells did influence neither the quantity nor the quality of primary T‐cell responses in LCMV infection. Virus‐specific CD8⁺ T cells in conditional knockout mice, with a selective deletion of the CD40L in CD8⁺ T cells, were fully functional regarding cytokine production and efficient pathogen clearance. Thus, our results unambiguously demonstrate that while CD40L is critical to generate effective primary CD8⁺ T‐cell responses also under inflammatory conditions, CD40L expression by CD8⁺ T cells themselves is dispensable in acute LCMV infection.