Paths to expansion: Differential requirements of IRF4 in CD8+ T‐cell expansion driven by antigen and homeostatic cytokines

Interferon regulatory factor 4 (IRF4) regulates the clonal expansion and metabolic activity of activated T cells, but the precise context and mechanisms of its function in these processes are unclear. In this issue of the European Journal of Immunology, Miyakoda et al. [Eur. J. Immunol. 2018. 48 : 1319–1328] show that IRF4 is required for activation and expansion of naïve and memory CD8⁺ T cells driven by T‐cell receptor (TCR) signaling, but dispensable for memory CD8⁺ T‐cell maintenance and homeostatic proliferation driven by homeostatic cytokines. The authors show that the function of IRF4 in CD8⁺ T‐cell expansion is partially dependent upon activation of the PI3K/AKT pathway through direct or indirect attenuation of PTEN expression. These data shed light upon the differential intracellular pathways required for naïve and memory T cells to respond to self‐antigens and/or homeostatic cytokines, and highlight the potential translational relevance of these findings in the context of immune reconstitution such as following allogeneic stem cell transplantation.     

Low-dose antigen-experienced CD4+ T cells display reduced clonal expansion but facilitate an effective memory pool in response to secondary exposure

The strength and duration of an antigenic signal at the time of initial stimulation were assumed to affect the development and response of effectors and memory cells to secondary stimulation with the same antigen. To test this assumption, we used T-cell receptor (TCR)-transgenic CD4⁺ T cells that were stimulated in vitro with various antigen doses. The primary effector CD4⁺ T cells generated in response to low-dose antigen in vitro exhibited reduced clonal expansion upon secondary antigenic exposure after adoptive transfer to hosts. However, the magnitude of their contraction was much smaller than both those generated by high-dose antigen stimulation and by naïve CD4⁺ T cells, resulting in higher numbers of antigen-specific CD4⁺ T cells remaining until the memory stage. Moreover, secondary effectors and memory cells developed by secondary antigen exposure were not functionally impaired. In hosts given the low-dose antigen-experienced CD4⁺ T cells, we also observed accelerated recall responses upon injection of antigen-bearing antigen-presenting cells. These results suggest that primary TCR stimulation is important for developing optimal effectors during initial antigen exposure to confer long-lasting memory CD4⁺ T cells in response to secondary exposure.     

TLR9 stimulation drives naïve B cells to proliferate and to attain enhanced antigen presenting function

Mechanisms that regulate naïve B cell proliferation and function are incompletely defined. In this study, we test the hypothesis that naïve B cell expansion, survival and ability to present antigen to T lymphocytes can be directly modulated by Toll‐like receptor (TLR) agonists. In the absence of B cell receptor stimulation, CpG oligonucleotide, a TLR9 agonist, was particularly efficient in inducing naïve B cell proliferation and survival. Although the expanded naïve B cells did not mature into CD27⁺ or IgG⁺ memory B cells, these cells did differentiate into IgM‐secreting cells with increased surface expression of HLA‐DR, CD40 and CD80. This was associated with an increased potential for these B cells to activate allogeneic T cells. We propose that the activation and expansion of naïve B cells induced by TLR9 agonists could enhance the potential of these cells to interact with cognate antigens and facilitate cell‐mediated immune responses.     

Prolonged exposure of naïve CD8+ T cells to interleukin-7 or interleukin-15 stimulates proliferation without differentiation or loss of telomere length

Interleukin (IL)‐7 and IL‐15 are cytokines implicated in homeostatic control of the peripheral CD8 T‐cell pool. We compared the effects of IL‐7 and IL‐15 on survival and proliferation of purified human CD8⁺ T‐cell subsets. Low concentrations of either cytokine reduced the spontaneous apoptosis of all subsets, and enhancement of survival corresponded to the extent of Bcl‐2 up‐regulation. Surprisingly, although minimal proliferation of naïve CD8⁺ T cells was observed during the first week of culture with cytokines, a marked expansion of these cells occurred at later time points, particularly in response to IL‐15. This occurred largely without phenotypic change or acquisition of effector function, indicating a dissociation of differentiation from proliferation. Notably, progression of naïve CD8⁺ T cells through several cell divisions resulted in up‐regulation of telomerase and the maintenance of telomere length. These data show that IL‐7 and IL‐15 induce cell proliferation and rescue from apoptosis in a concentration, time and subset‐dependent manner, and have implications for the homeostatic expansion of the naïve CD8⁺ T‐cell pool.     

CD5 levels define functionally heterogeneous populations of naïve human CD4+ T cells

Studies in murine models show that subthreshold TCR interactions with self-peptide are required for thymic development and peripheral survival of naïve T cells. Recently, differences in the strength of tonic TCR interactions with self-peptide, as read-out by cell surface levels of CD5, were associated with distinct effector potentials among sorted populations of T cells in mice. However, whether CD5 can also be used to parse functional heterogeneity among human T cells is less clear. Our study demonstrates that CD5 levels correlate with TCR signal strength in human naïve CD4⁺ T cells. Further, we describe a relationship between CD5 levels on naïve human CD4⁺ T cells and binding affinity to foreign peptide, in addition to a predominance of CD5^hi T cells in the memory compartment. Differences in gene expression and biases in cytokine production potential between CD5^lo and CD5^hi naïve human CD4⁺ T cells are consistent with observations in mice. Together, these data validate the use of CD5 surface levels as a marker of heterogeneity among human naïve CD4⁺ T cells with important implications for the identification of functionally biased T- cell populations that can be exploited to improve the efficacy of adoptive cell therapies.     

IL‐15 is critical for the maintenance and innate functions of self‐specific CD8+ T cells

IL‐15 is a pleiotropic cytokine involved in host defense as well as autoimmunity. IL‐15‐deficient mice show a decrease of memory phenotype (MP) CD8⁺ T cells, which develop naturally in naïve mice and whose origin is unclear. It has been shown that self‐specific CD8⁺ T cells developed in male H‐Y antigen‐specific TCR transgenic mice share many similarities with naturally occurring MP CD8⁺ T cells in normal mice. In this study, we found that H‐Y antigen‐specific CD8⁺ T cells in male but not female mice decreased when they were crossed with IL‐15‐deficient mice, mainly due to impaired peripheral maintenance. The self‐specific TCR transgenic CD8⁺ T cells developed in IL‐15‐deficient mice showed altered surface phenotypes and reduced effector functions ex vivo. Bystander activation of the self‐specific CD8⁺ T cells was induced in vivo during infection with Listeria monocytogenes, in which proliferation but not IFN‐γ production was IL‐15‐dependent. These results indicated important roles for IL‐15 in the maintenance and functions of self‐specific CD8⁺ T cells, which may be included in the naturally occurring MP CD8⁺ T‐cell population in naïve normal mice and participate in innate host defense responses.     

Cardiolipin activates antigen‐presenting cells via TLR2‐PI3K‐PKN1‐AKT/p38‐NF‐kB signaling to prime antigen‐specific naïve T cells in mice

Mitochondrial defects and antimitochondrial cardiolipin (CL) antibodies are frequently detected in autoimmune disease patients. CL from dysregulated mitochondria activates various pattern recognition receptors, such as NLRP3. However, the mechanism by which mitochondrial CL activates APCs as a damage‐associated molecular pattern to prime antigen‐specific naïve T cells, which is crucial for T‐cell‐dependent anticardiolipin IgG antibody production in autoimmune diseases is unelucidated. Here, we show that CL increases the expression of costimulatory molecules in CD11c⁺ APCs both in vitro and in vivo. CL activates CD11c⁺ APCs via TLR2‐PI3K‐PKN1‐AKT/p38MAPK‐NF‐κB signaling. CD11c⁺ APCs that have been activated by CL are sufficient to prime H‐Y peptide‐specific naïve CD4⁺ T cells and OVA‐specific naïve CD8⁺ T cells. TLR2 is necessary for anti‐CL IgG antibody responses in vivo. Intraperitoneal injection of CL does not activate CD11c⁺ APCs in CD14 KO mice to the same extent as in wild‐type mice. CL binds to CD14 (Kd = 7 × 10^?7 M). CD14, but not MD2, plays a role in NF‐kB activation by CL, suggesting that CD14⁺ macrophages contribute to recognizing CL. In summary, CL activates signaling pathways in CD11c⁺ APCs through a mechanism similar to gram (+) bacteria and plays a crucial role in priming antigen‐specific naïve T cells.     

Making sense of inflammation, epigenetics, and memory CD8+ T-cell differentiation in the context of infection

Summary: Recent findings suggest a new paradigm that early inflammatory cytokines promote the effector T‐cell response while inhibiting the development of CD8⁺ T‐cell memory. Although this opposing effect may appear paradoxical at first, it makes biological sense in the context of an infection, by ensuring a maximal effector response that will clear the pathogen. Once infection is controlled, the withdrawal of inflammatory cytokines allows the differentiation of effectors into long‐lived memory cells that provide protective immunity against re‐infection. Memory T cells differ from naïve T cells in their responsiveness to stimulation, which leads to the rapid expression of effector functions. The molecular basis for enhanced functionality of memory T cells remains largely unknown. Recent results indicate that certain epigenetic changes are imprinted in memory T cells that play an important role in keeping them poised to respond immediately upon antigen re‐encounter. These epigenetic modifications occur as naïve T cells become activated and are influenced by factors that regulate memory formation. Thus, epigenetic changes are an integral component of memory T‐cell differentiation, while inflammation plays an unexpected regulatory role in the process. These advances in our understanding of T‐cell memory will undoubtedly help design unconventional vaccine strategies for inducing large populations of long‐lived and functional memory CD8⁺ T cells.     

gammadelta T-cell clones from intestinal intraepithelial lymphocytes inhibit development of CTL responses ex vivo

Oral administration of antigen induces a state of tolerance that is associated with activation of CD8⁺ T cells that can transfer unresponsiveness to naïve syngeneic hosts. These T cells are not lytic, but they inhibit development of antibody, CD4⁺ T helper cell, and CD8⁺ cytotoxic T lymphocyte (CTL) responses upon adoptive transfer into naïve, syngeneic mice. In addition, we have shown that depletion of γδ T cells by injection of the anti‐δ chain antibody (GL3) down modulates the expression of γδ T‐cell receptor (TCR) and inhibits the induction of oral tolerance to ovalbumin. Oral administration of antigen also fails to induce tolerance in TCR δ‐chain knockout mice suggesting that γδ T cells play a critical, active role in tolerance induced by orally administered antigen. To further study the contribution of γδ T cells to tolerance, murine γδ T cells were isolated from intraepithelial lymphocytes (IEL) of the small intestine by stimulation with splenic filler cells, concanavalin A and growth factors. γδ IEL lines demonstrated lytic activity in a redirected lysis assay. γδ T‐cell clones express different γδ TCR genes and secrete large amounts of interleukin (IL)‐10, but little or no IL‐2, IL‐4, or interferon‐γ. γδ IEL clones expressed transforming growth factor‐β1 and macrophage migration inhibitory factor, as well as IL‐10, mRNA. Moreover, γδ T‐cell clones potently inhibited the generation of CTL responses by secreted molecules rather than by direct cell‐to‐cell contact.     

Mucosal CD8alpha+ DC,with a plasmacytoid phenotype,induce differentiation and support function of T cells with regulatory properties

Repetitive stimulation of naïve T cells by immature splenic dendritic cells (DC) can result in the differentiation of T‐cell lines with regulatory properties. In the present study we identified a population of DC in the mucosae that exhibit the plasmacytoid phenotype, secrete interferon‐α (IFN‐α) following stimulation with oligodeoxynucleotides containing certain cytosine‐phosphate‐guanosine (CpG) motifs and can differentiate naïve T cells into cells that exhibit regulatory properties. Although these DC appear to be present in both spleen and mesenteric lymph nodes (MLN), only CpG‐matured DC from the MLN (but not the spleen) were able to differentiate naïve T cells into T regulatory 1‐like cells with regulatory properties. The activity of these DC failed to sustain robust T‐cell proliferation and thereby enhanced the suppressive efficacy of CD4⁺ CD25⁺ T regulatory cells. These DC are the major CD8α⁺ DC population in the Peyer's patches (PP). Given their significant presence in mucosal tissue, we propose that these DC may provide a mechanistic basis for the homeostatic regulation in the gut by eliciting regulatory cell suppressor function and poorly supporting T helper cell proliferation at a site of high antigenic stimulation like the intestine.     

The influence of the src-family kinases, Lck and Fyn, on T cell differentiation, survival and activation

The src‐family kinases p56^lck (Lck) and p59^fyn (Fyn) are expressed in T cells and are among the first signaling molecules to be activated downstream of the T cell receptor (TCR). Evidence is emerging that although closely related, these signaling molecules have discrete functions during development, maintenance and activation of peripheral T cells. For example, during thymopoiesis Lck is uniquely able to provide all the signals required for pre‐TCRβ selection, although Fyn can substitute for a subset of these. Positive selection of CD4 single‐positive (SP) cells is also critically dependent on the expression of Lck but not Fyn, while differentiation of CD8 SP cells proceeds relatively efficiently in the absence of Lck. In naïve peripheral T cells either Lck or Fyn can transmit TCR‐mediated survival signals, and yet only Lck is able to trigger TCR‐mediated expansion signals under conditions of lymphopenia. Stimulation of naïve T cells by antigenic stimuli is also severely compromised in the absence of Lck, but more subtly impaired by the absence of Fyn. We discuss recent experiments addressing how these two src‐kinase family members interface with downstream signaling pathways to regulate these diverse aspects of T cell behavior.     

A new mechanism shapes the naïve CD8+ T cell repertoire: the selection for full diversity

During thymic T cell differentiation, TCR repertoires are shaped by negative, positive and agonist selection. In the thymus and in the periphery, repertoires are also shaped by strong inter-clonal and intra-clonal competition to survive death by neglect. Understanding the impact of these events on the T cell repertoire requires direct evaluation of TCR expression in peripheral naïve T cells. Several studies have evaluated TCR diversity, with contradictory results. Some of these studies had intrinsic technical limitations since they used material obtained from T cell pools, preventing the direct evaluation of clonal sizes. Indeed with these approaches, identical TCRs may correspond to different cells expressing the same receptor, or to several amplicons from the same T cell. We here overcame this limitation by evaluating TCRB expression in individual naïve CD8⁺ T cells. Of the 2269 Tcrb sequences we obtained from 13 mice, 99% were unique. Mathematical analysis of the data showed that the average number of naïve peripheral CD8⁺ T cells expressing the same TCRB is 1.1 cell. Since TCRA co-expression studies could only increase repertoire diversity, these results reveal that the number of naïve T cells with unique TCRs approaches the number of naïve cells. Since thymocytes undergo multiple rounds of divisions after TCRB rearrangement and 3–5% of thymocytes survive thymic selection events the number of cells expressing the same TCRB was expected to be much higher. Thus, these results suggest a new repertoire selection mechanism, which strongly selects for full TCRB diversity.     

T-cell dynamics after high-dose chemotherapy in adults: elucidation of the elusive CD8+ subset reveals multiple homeostatic T-cell compartments with distinct implications for immune competence

Recovery of total T cell numbers after in vivo T-cell depletion in humans is accompanied by complex perturbation within the CD8⁺ subset. We aimed to elucidate the reconstitution of CD8⁺ T cells by separate analysis of putative naïve CD95⁻ CD28⁺, memory CD95⁺ CD28⁺ and CD28⁻ T cell compartments after acute maximal depletion by high-dose chemotherapy (HD-ChT) in women with high-risk breast cancer. We found that recovery of putative naïve CD8⁺ CD95⁻ CD28⁺ and CD4⁺ CD95⁻ CD28⁺ T cells, was compatible with a thymus-dependent regenerative pathway since their recovery was slow and time-dependent, their values were tightly related to each other, and their reconstitution patterns were inversely related to age. By analysing non-naïve T cells, a striking diversion between putative memory T cells and CD28⁻ T cells was found. These latter increased early well beyond normal values, thus playing a pivotal role in total T-cell homeostasis, and contributed to reduce the CD4 : CD8 ratio. In contrast, putative memory T cells returned to values not significantly different from those seen in patients at diagnosis, indicating that this compartment may recover after HD-ChT. At 3–5 years after treatment, naïve T cells persisted at low levels, with expansion of CD28⁻ T cells, suggesting that such alterations may extend further. These findings indicate that CD28⁻ T cells were responsible for ‘blind’ T-cell homeostasis, but support the notion that memory and naïve T cells are regulated separately. Given their distinct dynamics, quantitative evaluation of T-cell pools in patients undergoing chemotherapy should take into account separate analysis of naïve, memory and CD28⁻ T cells.     

Marked anti-tumor effects of CD8+CD62L+ T cells from melanoma-bearing mice

CD8⁺CD62L⁺ T cells have been shown to play pivotal roles in anti-viral immunity, chronic myeloid leukemia and renal cell carcinoma. Recently, CD8⁺CD62L⁺ T cells from naïve mice (nCD8⁺CD62L⁺ T cells) have shown superior anti-tumor properties in melanoma-bearing mice. Considering that antigen-specific memory T cells have shown to possess more potent immunity than non-specific memory T cells, we hypothesized that CD8⁺CD62L⁺ T cells from tumor-bearing individuals (mCD8⁺CD62L⁺ T cells) might have superior anti-tumor effect than nCD8⁺CD62L⁺ T cells. Therefore, we investigated phenotypes, functions and the in vivo distribution of mCD8⁺CD62L⁺ T cells in tumor-bearing mice. We found that, while keeping the features of central memory T cells, the frequency of mCD8⁺CD62L⁺ T cell in the spleen of tumor-bearing mice was significantly higher than that the one of nCD8⁺CD62L⁺ T cell in naive mice. Moreover, we demonstrated that mCD8⁺CD62L⁺ T cells had higher proliferation rate and IFN-γ production than nCD8⁺CD62L⁺ T cells, in vitro. We performed adoptive transfer of mCD8⁺CD62L⁺ T cells into melanoma-bearing mice and tracked them in spleen, lymph nodes and in melanoma tissues. Our results show that mCD8⁺CD62L⁺ T cells had stronger in vivo anti-tumoral activity than nCD8⁺CD62L⁺ T cells. This study highlights the therapeutic potential of mCD8⁺CD62L⁺ T cells in the immunotherapy of melanoma and possibly other tumors.     

Signals required for programming effector and memory development by CD8+ T cells

Summary: Stimulation of naïve CD8⁺ T cells with antigen and costimulation results in proliferation and weak clonal expansion, but the cells fail to develop effector functions and are tolerant long term. Initiation of the program leading to the strong expansion and development of effector functions and memory requires a third signal that can be provided by interleukin‐12 (IL‐12) or interferon‐α (IFN‐α). CD4⁺ T cells condition dendritic cells (DCs) to effectively present antigen to CD8⁺ T cells, and this conditioning involves, at least in part, CD40‐dependent upregulation of the production of these signal 3 cytokines by the DCs. Upon being fully activated, the cytotoxic T lymphocytes develop activation‐induced non‐responsiveness (AINR), a form of split anergy characterized by an inability to produce IL‐2 to support continued expansion. If antigen remains present, IL‐2 provided by CD4⁺ T cells can reverse AINR to allow further expansion of the effector population and conversion to responsive memory cells following antigen clearance. If IL‐2 or potentially other proliferative signals are not available, persistent antigen holds cells in the AINR state and prevents the development of a responsive memory population. Thus, in addition to antigen and costimulation, CD8⁺ T cells require cytokine signals at distinct stages of the response to be programmed for optimal generation of effector and memory populations.     

Freeze-thaw lysates of Plasmodium falciparum-infected red blood cells induce differentiation of functionally competent regulatory T cells from memory T cells

In addition to naturally occurring regulatory T (nTreg) cells derived from the thymus, functionally competent Treg cells can be induced in vitro from peripheral blood lymphocytes in response to TCR stimulation with cytokine costimulation. Using these artificial stimulation conditions, both naïve as well as memory CD4⁺ T cells can be converted into induced Treg (iTreg) cells, but the cellular origin of such iTreg cells in vivo or in response to more physiologic stimulation with pathogen‐derived antigens is less clear. Here, we demonstrate that a freeze/thaw lysate of Plasmodium falciparum schizont extract (PfSE) can induce functionally competent Treg cells from peripheral lymphocytes in a time‐ and dose‐dependent manner without the addition of exogenous costimulatory factors. The PfSE‐mediated induction of Treg cells required the presence of nTreg cells in the starting culture. Further experiments mixing either memory or naïve T cells with antigen presenting cells and CFSE‐labeled Treg cells identified CD4⁺CD45RO⁺CD25^? memory T cells rather than Treg cells as the primary source of PfSE‐induced Treg cells. Taken together, these data suggest that in the presence of nTreg cells, PfSE induces memory T cells to convert into iTreg cells that subsequently expand alongside PfSE‐induced effector T cells.     

Evidence of the extrathymic development of tyrosinase-related protein-2-recognizing CD8+ T cells with low avidity

The majority of the human tumour‐associated antigens characterized to date are derived from non–mutated self‐proteins. However, nothing is known about the development of autoreactive and tumour‐associated antigen‐recognizing T cells. Tyrosinase‐related protein (TRP)‐2 is a non‐mutated melanocyte differentiation antigen and TRP‐2‐recognizing CD8⁺ T cells are known to show responses to melanoma both in humans and mice. In addition, TRP‐2‐reactive T cells with low avidity have been suggested to be readily induced from the spleen cells of naïve mice. On the other hand, recent reports suggest that self antigen‐reactive CD8⁺ T cells can be positively selected in the periphery. In this study, we tested the possibility that TRP‐2‐reactive CD8⁺ T cells in naïve mice could develop via the extrathymic pathway. As a consequence, TRP‐2‐reactive CD8⁺ T cell precursors in naïve C57BL/6 mice were suggested to express both interleukin‐2 (IL‐2) receptor β chain (IL‐2Rβ) and CD44 molecules, in a manner similar to that of extrathymically developed T cells. Furthermore, IL‐2Rβ⁺ CD44⁺ CD8⁺ T cells were detected in the adult thymectomized and bone marrow‐reconstituted mice, and functional TRP‐2‐reactive T cells were generated from their spleen cells. Overall, these results suggest that low avidity CD8⁺ T cells recognizing TRP‐2 can be developed extrathymically.