Mineralocorticoid receptor signaling reduces numbers of circulating human naïve T cells and increases their CD62L,CCR7, and CXCR4 expression

The role of mineralocorticoid receptors (MRs) in human T‐cell migration is not yet understood. We have recently shown that the MR antagonist spironolactone selectively increases the numbers of circulating naïve and central memory T cells during early sleep, which is the time period in the 24 h cycle hallmarked by predominant MR activation. To investigate whether this effect is specific to spironolactone's blockade of MRs and to study the underlying molecular mechanisms, healthy humans were given the selective MR‐agonist fludrocortisone or placebo and numbers of eight T‐cell subsets and their CD62L and CXCR4 expression were analyzed. Fludrocortisone selectively reduced counts of naïve CD4⁺, central memory CD4⁺, and naïve CD8⁺ T cells and increased CXCR4 expression on the naïve subsets. In complementing in vitro studies, fludrocortisone enhanced CXCR4 and CD62L expression, which was counteracted by spironolactone. Incubation of naïve T cells with spironolactone alone reduced CD62L and CCR7 expression. Our results indicate a regulatory influence of MR signaling on human T‐cell migration and suggest a role for endogenous aldosterone in the redistribution of T‐cell subsets to lymph nodes, involving CD62L, CCR7, and CXCR4. Facilitation of T‐cell homing following sleep‐dependent aldosterone release might thus essentially contribute to sleep's well‐known role in supporting adaptive immunity.     

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.     

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.     

Type 1 diabetes linked PTPN22 gene polymorphism is associated with the frequency of circulating regulatory T cells

Dysfunction of FOXP3-positive regulatory T cells (Tregs) likely plays a major role in the pathogenesis of multiple autoimmune diseases including type 1 diabetes (T1D). Whether genetic polymorphisms associated with the risk of autoimmune diseases affect Treg frequency or function is currently unclear. Here, we analysed the effect of T1D-associated major HLA class II haplotypes and seven single nucleotide polymorphisms in six non-HLA genes [INS (rs689), PTPN22 (rs2476601), IL2RA (rs12722495 and rs2104286), PTPN2 (rs45450798), CTLA4 (rs3087243), and ERBB3 (rs2292239)] on peripheral blood Treg frequencies. These were determined by flow cytometry in 65 subjects who had progressed to T1D, 86 islet autoantibody-positive at-risk subjects, and 215 islet autoantibody-negative healthy controls. The PTPN22 rs2476601 risk allele A was associated with an increase in total (p = 6 × 10⁻⁶) and naïve (p = 4 × 10⁻⁵) CD4+CD25+CD127lowFOXP3+ Treg frequencies. These findings were validated in a separate cohort comprising ten trios of healthy islet autoantibody-negative children carrying each of the three PTPN22 rs2476601 genotypes AA, AG, and GG (p = 0.005 for total and p = 0.03 for naïve Tregs, respectively). In conclusion, our analysis implicates the autoimmune PTPN22 rs2476601 risk allele A in controlling the frequency of Tregs in human peripheral blood.     

HIV‐1 impairs in vitro priming of naïve T cells and gives rise to contact‐dependent suppressor T cells

Priming of T cells in lymphoid tissues of HIV‐infected individuals occurs in the presence of HIV‐1. DC in this milieu activate T cells and disseminate HIV‐1 to newly activated T cells, the outcome of which may have serious implications in the development of optimal antiviral responses. We investigated the effects of HIV‐1 on DC–naïve T‐cell interactions using an allogeneic in vitro system. Our data demonstrate a dramatic decrease in the primary expansion of naïve T cells when cultured with HIV‐1‐exposed DC. CD4⁺ and CD8⁺ T cells showed enhanced expression of PD‐1 and TRAIL, whereas CTLA‐4 expression was observed on CD4⁺ T cells. It is worth noting that T cells primed in the presence of HIV‐1 suppressed priming of other naïve T cells in a contact‐dependent manner. We identified PD‐1, CTLA‐4, and TRAIL pathways as responsible for this suppresion, as blocking these negative molecules restored T‐cell proliferation to a higher degree. In conclusion, the presence of HIV‐1 during DC priming produced cells with inhibitory effects on T‐cell activation and proliferation, i.e. suppressor T cells, a mechanism that could contribute to the enhancement of HIV‐1 pathogenesis.     

Short‐term cytokine stimulation reveals regulatory T cells with down‐regulated Foxp3 expression in human peripheral blood

The identification of regulatory T cells (Treg cells) in human peripheral blood is an important tool in diagnosis, research, and therapeutic intervention. As compared to lymphoid tissues, the frequencies of circulating Treg cells identified as CD4⁺CD25⁺Foxp3⁺ are, however, low. We here show that many of these cells remain undetected due to transient down regulation of Foxp3, which rapidly decays in the absence of cytokine‐mediated STAT5 signals. Short‐term incubation of PBMCs or isolated CD4⁺ T cells, but not of lymph node cells, with IL‐2, ‐7, or ‐15 more than doubles the frequency of Foxp3⁺CD25⁺ among CD4⁺ T cells detectable by flow cytometry. This increase is not due to cell division but to upregulation of both proteins. At the same time, the uncovered Treg cells up‐regulate CD25 and down‐regulate CD127, making them accessible to viable cell sorting. “Latent” Treg cells have a demethylated FOXP3 TSDR sequence, are enriched in naïve, non‐cycling cells, and are functional. The confirmation of our findings in RA and SLE patients shows the feasibility of uncovering latent Treg cells for immune monitoring in clinical settings. Finally, our results suggest that unmasking of latent Treg cells contributes to the increase in circulating CD4⁺CD25⁺Foxp3⁺ cells reported in IL‐2 treated patients.     

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.     

Expression of IL‐15RA or an IL‐15/IL‐15RA fusion on CD8+ T cells modifies adoptively transferred T‐cell function in cis

IL‐15 and IL‐15 receptor alpha (IL‐15RA) play a significant role in multiple aspects of T‐cell biology. However, given the evidence that IL‐15RA can present IL‐15 in trans, the functional capacity of IL‐15RA expressed on CD8⁺ T cells to modify IL‐15 functions in cis is currently unclear. In the current study, we explore the functional consequences of IL‐15RA, expression on T cells using a novel method to transfect naive CD8⁺ T cells. We observed that RNA nucleofection led to highly efficient, non‐toxic, and rapid manipulation of protein expression levels in unstimulated CD8⁺ T cells. We found that transfection of unstimulated CD8⁺ T cells with IL‐15RA RNA led to enhanced viability of CD8⁺ T cells in response to IL‐15. Transfection with IL‐15RA enhanced IL‐15‐mediated phosphorylation of STAT5 and also promoted IL‐15‐mediated proliferation in vivo of adoptively transferred naïve CD8⁺ T cells. We demonstrated that IL‐15RA can present IL‐15 via cis‐presentation on CD8⁺ T cells. Finally, we showed that transfection with a chimeric construct linking IL‐15 to IL‐15RA cell autonomously enhances the viability and proliferation of primary CD8⁺ T cells and cytotoxic potential of antigen‐specific CD8⁺ T cells. The clinical implications of the current study are discussed.     

Allergen‐specific naïve and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy

Although allergen‐specific CD4⁺ T cells are detectable in the peripheral blood of both individuals with or without allergy, their frequencies and phenotypes within the memory as well as naïve repertoires are incompletely known. Here, we analyzed the DRB1*0401‐restricted responses of peripheral blood‐derived memory (CD4⁺CD45RO⁺) and naïve (CD4⁺CD45RA⁺) T cells from subjects with or without allergy against the immunodominant epitope of the major cow dander allergen Bos d 2 by HLA class II tetramers in vitro. The frequency of Bos d 2_127–142‐specific memory T cells in the peripheral blood‐derived cultures appeared to be higher in subjects with allergy than those without, whereas naïve Bos d 2_127–142‐specific T cells were detectable in the cultures of both groups at nearly the same frequency. Surprisingly, the TCR avidity of Bos d 2_127–142‐specific T cells of naïve origin, as assessed by the intensity of HLA class II tetramer staining, was found to be higher in individuals with allergy. Upon restimulation, long‐term Bos d 2_127–142‐specific T‐cell lines generated from both memory and naïve T‐cell pools from individuals with allergy proliferated more strongly, produced more IL‐4 and IL‐10, and expressed higher levels of CD25 but lower levels of CXCR3 than the T‐cell lines from individuals without allergy, demonstrating differences also at the functional level. Collectively, our current results suggest that not only the memory but also the naïve allergen‐specific T‐cell repertoires differ between individuals with or without allergy.     

Tumor‐specific CD4+ T cells maintain effector and memory tumor‐specific CD8+ T cells

Immunotherapies that augment antitumor T cells have had recent success for treating patients with cancer. Here we examined whether tumor‐specific CD4⁺ T cells enhance CD8⁺ T‐cell adoptive immunotherapy in a lymphopenic environment. Our model employed physiological doses of tyrosinase‐related protein 1‐specific CD4⁺ transgenic T cells‐CD4⁺ T cells and pmel‐CD8⁺ T cells that when transferred individually were subtherapeutic; however, when transferred together provided significant (p ≤ 0.001) therapeutic efficacy. Therapeutic efficacy correlated with increased numbers of effector and memory CD8⁺ T cells with tumor‐specific cytokine expression. When combined with CD4⁺ T cells, transfer of total (naïve and effector) or effector CD8⁺ T cells were highly effective, suggesting CD4⁺ T cells can help mediate therapeutic effects by maintaining function of activated CD8⁺ T cells. In addition, CD4⁺ T cells had a pronounced effect in the early posttransfer period, as their elimination within the first 3 days significantly (p < 0.001) reduced therapeutic efficacy. The CD8⁺ T cells recovered from mice treated with both CD8⁺ and CD4⁺ T cells had decreased expression of PD‐1 and PD‐1‐blockade enhanced the therapeutic efficacy of pmel‐CD8 alone, suggesting that CD4⁺ T cells help reduce CD8⁺ T‐cell exhaustion. These data support combining immunotherapies that elicit both tumor‐specific CD4⁺ and CD8⁺ T cells for treatment of patients with cancer.     

Generation of highly effective and stable murine alloreactive Treg cells by combined anti‐CD4 mAb,TGF‐β, and RA treatment

The transfer of alloreactive regulatory T (aTreg) cells into transplant recipients represents an attractive treatment option to improve long‐term graft acceptance. We recently described a protocol for the generation of aTreg cells in mice using a nondepleting anti‐CD4 antibody (aCD4). Here, we investigated whether adding TGF‐β and retinoic acid (RA) or rapamycin (Rapa) can further improve aTreg‐cell generation and function. Murine CD4⁺ T cells were cultured with allogeneic B cells in the presence of aCD4 alone, aCD4+TGF‐β+RA or aCD4+Rapa. Addition of TGF‐β+RA or Rapa resulted in an increase of CD25⁺Foxp3⁺‐expressing T cells. Expression of CD40L and production of IFN‐γ and IL‐17 was abolished in aCD4+TGF‐β+RA aTreg cells. Additionally, aCD4+TGF‐β+RA aTreg cells showed the highest level of Helios and Neuropilin‐1 co‐expression. Although CD25⁺Foxp3⁺ cells from all culture conditions displayed complete demethylation of the Treg‐specific demethylated region, aCD4+TGF‐β+RA Treg cells showed the most stable Foxp3 expression upon restimulation. Consequently, aCD4+TGF‐β+RA aTreg cells suppressed effector T‐cell differentiation more effectively in comparison to aTreg cells harvested from all other cultures, and furthermore inhibited acute graft versus host disease and especially skin transplant rejection. Thus, addition of TGF‐β+RA seems to be superior over Rapa in stabilising the phenotype and functional capacity of aTreg cells.     

IL-9 exerts biological function on antigen-experienced murine T cells and exacerbates colitis induced by adoptive transfer

IL-9 is involved in various T cell-dependent inflammatory models including colitis, encepahlitis, and asthma. However, the regulation and specificity of IL-9 responsiveness by T cells during immune responses remains poorly understood. Here, we addressed this question using two different models: experimental colitis induced by transfer of naive CD4⁺CD45RB^high T cells into immunodeficient mice, and OVA-specific T cell activation. In the colitis model, constitutive IL-9 expression exacerbated inflammation upon transfer of CD4⁺CD45RB^high T cells from WT but not from Il9r^−/− mice, indicating that IL-9 acts directly on T cells. Suprisingly, such naïve CD4⁺CD45RB^high T cells failed to express the Il9r or respond to IL-9 in vitro, in contrast with CD4⁺CD45RB^low T cells. By using OVA-specific T cells, we observed that T cells acquired the capacity to respond to IL-9 along with CD44 upregulation, after long-lasting (5 to 12 days) in vivo antigenic stimulation. Il9r expression was associated with Th2 and Th17 phenotypes. Interestingly, in contrast to the IL-2 response, antigen restimulation downregulated IL-9 responsiveness. Taken together, our results demonstrate that IL-9 does not act on naïve T cells but that IL-9 responsiveness is acquired by CD4⁺ T cells after in vivo activation and acquisition of memory markers such as CD44.     

Progesterone promotes maternal–fetal tolerance by reducing human maternal T‐cell polyfunctionality and inducing a specific cytokine profile

Progesterone is a steroid hormone essential for the maintenance of human pregnancy, and its actions are thought to include promoting maternal immune tolerance of the semiallogenic fetus. We report that exposure of maternal T cells to progesterone at physiological doses induced a unique skewing of the cytokine production profile of CD4⁺ and CD8⁺ T cells, with reductions not only in potentially deleterious IFN‐γ and TNF‐α production but also in IL‐10 and IL‐5. Conversely, production of IL‐4 was increased. Maternal T cells also became less polyfunctional, focussing cytokine production toward profiles including IL‐4. This was accompanied by reduced T‐cell proliferation. Using fetal and viral antigen‐specific CD8⁺ T‐cell clones, we confirmed that this as a direct, nonantigen‐specific effect. Yet human T cells lacked conventional nuclear progesterone receptors, implicating a membrane progesterone receptor. CD4⁺ and CD8⁺ T cells responded to progesterone in a dose‐dependent manner, with subtle effects at concentrations comparable to those in maternal blood, but profound effects at concentrations similar to those at the maternal–fetal interface. This characterization of how progesterone modulates T‐cell function is important in understanding the normal biology of pregnancy and informing the rational use of progesterone therapy in pregnancies at risk of fetal loss.     

T cell receptor alpha variable 12‐2 bias in the immunodominant response to Yellow fever virus

The repertoire of human αβ T‐cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen‐specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA‐A*0201‐restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8⁺ T cell response to the highly effective YF‐17D vaccine. We discover that these A2/LLW‐specific CD8⁺ T cells are highly biased for the TCR α chain TRAV12‐2. This bias is already present in A2/LLW‐specific naïve T cells before vaccination with YF‐17D. Using CD8⁺ T cell clones, we show that TRAV12‐2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline‐encoded complementarity determining region (CDR) 1α loop of TRAV12‐2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T‐cell responses specific for the A2/LLW epitope.     

Differential expression of CTLA-4 among T cell subsets

CTLA‐4 (CD152), the CD28 homologue, is a costimulatory molecule with negative effects on T cell activation. In addition to its role in the termination of activation, CTLA‐4 has been implicated in anergy induction and the function of regulatory cells. As an intracellular molecule, it must first relocate to the cell surface and be ligated, in order to inhibit activation. Although some studies have investigated CTLA‐4 expression on CD4⁺ T cells, evidence is lacking regarding the kinetics of expression, and expression on T cell subpopulations. We have investigated CTLA‐4 kinetics on human purified peripheral CD4⁺, naïve, memory, CD4⁺CD25^–, CD4⁺CD25⁺ regulatory T cells, and T cell clones. Intracellular stores of CTLA‐4 were shown to be very low in naïve T cells, whilst significant amounts were present in memory T cells and T cell clones. Cell surface CTLA‐4 expression was then investigated on CD4⁺CD45RA⁺ (naïve), CD4⁺CD45RO⁺ (memory), CD4⁺CD25^–, and CD4⁺CD25⁺ T cells. CD25 and CD45RO are both expressed by regulatory T cells. On naïve and CD4⁺CD25^– T cells, CTLA‐4 expression declined after four hours. In contrast, on memory and CD4⁺CD25⁺ T cells, high levels of expression were maintained until at least 48 hours. In addition, significant CTLA‐4 expression was observed on T cell clones following anergy induction, indicating the potential involvement of CTLA‐4 also in this form of tolerance.     

The DNA damage response induces antigen presenting cell‐like functions in fibroblasts

The DNA damage response (DDR) alerts the immune system to the danger posed by DNA damage through the induction of damage‐associated molecular pattern molecules, chemokines, and ligands for activating immune receptors such as lymphocyte function‐associated antigen 1 (LFA‐1), NKG2D, and DNAX accessory molecule 1 (DNAM‐1). Here we provide evidence that OVA^257–264‐pulsed fibroblasts gain the ability to activate naïve OT‐I CD8⁺ T cells in response to DNA damage. The ability of fibroblasts to activate OT‐I CD8⁺ T cells depended on the upregulation of ICAM‐1 on fibroblasts and DNAM‐1 expression of CD8⁺ T cells. OVA^257–264‐pulsed fibroblasts were able to induce a protective T‐cell response against B16‐OVA cells in a DDR‐dependent manner. Hence, the DDR may alert the immune system to the presence of potentially dangerous cells by upregulating the expression of ligands that can induce the activation of innate and adaptive immune cells.     

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.     

Memory lapses in graft-versus-host disease

“Faster, better, more” is the conventional benchmark used to define responses of memory T cells when compared with their naïve counterparts. In this issue of the European Journal of Immunology, Mark and Warren Shlomchik and colleagues [Eur. J. Immunol. 2011. 41 : 2782–2792] make the intriguing observation that murine memory CD4⁺ T‐cell populations enriched for alloreactive precursors are fully capable of rejecting allogeneic skin grafts but yet are incapable of inducing significant graft‐versus‐host disease. These observations add to the emerging concept that memory CD4⁺ T‐cell development is more nuanced and complex than predicted by conventional models. In particular, the data suggest that it may be just as important to consider what naïve or effector cells have “lost” in their transition to memory.