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.     

High-frequency and adaptive-like dynamics of human CD1 self-reactive T cells

CD1 molecules present lipid antigens to T cells. An intriguing subset of human T cells recognize CD1‐expressing cells without deliberately added lipids. Frequency, subset distribution, clonal composition, naïve‐to‐memory dynamic transition of these CD1 self‐reactive T cells remain largely unknown. By screening libraries of T‐cell clones, generated from CD4⁺ or CD4^?CD8^? double negative (DN) T cells sorted from the same donors, and by limiting dilution analysis, we find that the frequency of CD1 self‐reactive T cells is unexpectedly high in both T‐cell subsets, in the range of 1/10–1/300 circulating T cells. These T cells predominantly recognize CD1a and CD1c and express diverse TCRs. Frequency comparisons of T‐cell clones from sorted naïve and memory compartments of umbilical cord and adult blood show that CD1 self‐reactive T cells are naïve at birth and undergo an age‐dependent increase in the memory compartment, suggesting a naïve/memory adaptive‐like population dynamics. CD1 self‐reactive clones exhibit mostly Th1 and Th0 functional activities, depending on the subset and on the CD1 isotype restriction. These findings unveil the unanticipated relevance of self‐lipid T‐cell response in humans and clarify the basic parameters of the lipid‐specific T‐cell physiology.     

Atopic asthma: differential activation phenotypes among memory T helper cells

Background T cells have been implicated in the pathogenesis of atopic asthma. We have previously shown that memory T helper cells (CD4⁺CD45RO⁺) are preferentially activated relative to naïve T helper cells (CD4⁺CD45RA⁺) after bronchial allergen challenge. However, specific T helper subpopulations that are activated in atopy and/or asthma remain undefined. Objective To determine the T helper subpopulations and activation phenotypes relevant to acute and stable asthma that may be common with or distinct from atopy. Methods Two groups of atopic asthmatics (ten acute and nine stable asthmatics) and two non‐asthmatic groups (14 non‐asthmatic atopics and eight normal non‐atopic controls) were analysed. Ten acute asthmatics were assessed in the emergency room during an acute episode (FEV₁ 43.6% ± 18.4). Nine stable asthmatics were assessed during a symptom‐free period (FEV₁ 85% ± 6). Using multiple colour flow cytometry we analysed T cell subpopulations and the expression of IL‐2‐receptor (IL‐2R) and MHC‐class II antigens (MHC II) on naïve and memory T helper cells in the peripheral blood of asthmatic and non‐asthmatic groups. Results Atopic asthmatics (acute and stable) had an increased percentage of memory T helper cells expressing IL‐2R compared with normal non‐atopics (mean SD 16.1 ± 6%, 12.4 ± 2% and 7.7 ± 1.8%, P < 0.05) but not compared with non‐asthmatic atopics (10 ± 3.5%). Naïve T helper cells had low expression of IL‐2R and MHC II in all four groups. MHC II antigen expression was increased in memory T helper cells of asthmatics (acute and stable) compared with normal non‐atopics (13.9 ± 7.5, 10.6 ± 5 and 4.9 ± 2.5, P < 0.05) but not compared with non‐asthmatic atopics (7.92 4). A novel finding was that IL‐2R and the MHC II molecules were mainly expressed in non‐overlapping populations and coexpression was found predominantly on memory T helper cells. Asthmatics (acute and stable) had higher proportion of double positive memory T helper cells (IL‐2R⁺MHC II⁺) compared with both non‐asthmatic groups (P < 0.05). Conclusions We demonstrate a differential expression of IL‐2R⁺ and MCH II⁺ on CD45RO⁺ T helper cells that would suggest that there are three subsets of activated memory T helper cells in asthmatics. Two non‐overlapping IL‐2R⁺ or MHC II⁺ CD45RO⁺ T helper cells and a third subpopulation of activated cells that coexpress IL‐2R and MHC II (double positives). This latter subpopulation is significantly higher in asthmatics (acute or stable) compared with both non‐asthmatic groups, suggesting a specific T helper activation phenotype distinct to atopic asthmatics as compared with atopic non‐asthmatics.     

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.     

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.     

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.     

Molecular mechanisms of apoptosis in the cells of the immune system in human aging

Summary: Aging is associated with progressive decline in immune functions and increased frequency of infections, autoimmunity, and cancer. Among immune functions, a decline in T‐cell functions during aging predominates. In this review, I discuss the molecular signaling of three distinct pathways of apoptosis, namely the death receptor pathway, the mitochondrial pathway, and the most recently described endoplasmic reticulum stress pathway, and the relative sensitivity of naïve, central memory, and effector memory CD8⁺ T‐cell subsets to apoptosis. In addition, I review apoptosis, especially via death receptor pathway, in naïve and various memory subsets of CD4⁺ and CD8⁺ T cells (with primary emphasis on CD8⁺ naïve and memory subsets) in human aging and discuss the role of apoptosis in immune senescence.     

Autophagy plays a protective role as an anti‐oxidant system in human T cells and represents a novel strategy for induction of T‐cell apoptosis

Autophagy is an intracellular degradation system that plays an important role in T‐cell survival. However, the precise mechanism linking autophagy and cell death in primary human T cells is unclear because methods for monitoring autophagy in small numbers of primary human cells remain controversial. We established a novel method for assessing autophagy in activated human T cells using a retroviral GFP–LC3 expression system. We found that autophagy was induced after TCR stimulation and that autophagy‐defective naïve CD4⁺ T cells were susceptible to apoptosis through the intrinsic apoptotic pathway. Enhanced apoptosis of autophagy‐defective T cells resulted from accumulation of ROS due to impaired mitophagy. We also demonstrated that effector memory CD4⁺ T cells had lower autophagic activity than naïve CD4⁺ T cells, which contributed to their enhanced apoptosis due to increased ROS. Moreover, blocking autophagy increased intracellular mitochondrial volume and ROS levels in activated T cells. These results suggest a protective role of autophagy as an anti‐oxidant system in activated human T cells. The combination of an autophagy blocker and a mitochondrial electron transport chain inhibitor has a synergistic effect on T‐cell death, which could be a novel strategy for induction of T‐cell apoptosis.     

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.     

Strong and sustained effector function of memory‐ versus naïve‐derived T cells upon T‐cell receptor RNA transfer: Implications for cellular therapy

Current protocols used to select CMV‐specific T cells for adoptive immunotherapy focus on virus‐specific memory T cells from seropositive donors. However, this strategy is not feasible in patients undergoing allogeneic haematopoietic stem‐cell transplantation (HSCT) from CMV‐seronegative donors. Here, we redirected T cells of CMV‐seronegative donors with a human genetically engineered TCR recognizing an HLA‐A*0201‐binding peptide epitope of CMVpp65. To facilitate clinical translation of this approach, we used a non‐viral expression system based on in vitro transcribed RNA and electroporation. Although memory and naïve‐derived T‐cell subsets were both efficiently transfected by TCR‐RNA, memory‐derived T cells showed much stronger levels of HLA‐A*0201‐restricted cytolytic activity to CMV‐infected fibroblasts and maintained acquired function for 5–10 days. In addition to redirection of CD8⁺ cytotoxic T cells, TCR‐RNA transfection was capable of redirecting CD4⁺ T cells into potent Ag‐specific Th cells that efficiently triggered maturation of DCs. Our data suggest that memory rather than naïve‐derived T cells are the preferred subset for transient TCR expression by RNA electroporation, providing more efficient and sustained virus‐specific CD4⁺ and CD8⁺ T‐cell function. CMV TCR‐RNA may represent a suitable therapeutic ‘off‐the‐shelf’ reagent to be used in severe CMV infections of HSCT patients when endogenous CMV‐specific T‐cell immunity is insufficient.     

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.     

Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2-polarizing conditions

Background Human thymic stromal lymphopoietin (TSLP) is expressed in the human asthmatic lung and activates dendritic cells (DCs) to strongly induce proallergic T‐helper type 2 (Th2) cell responses, suggesting that TSLP plays a critical role in the pathophysiology of human asthma. Th2 cells are predominantly involved in mild asthma, whereas a mixture of Th1 and Th2 cells with neutrophilic inflammation, probably induced by Th17, affects more severe asthmatic disease. Exacerbation of asthmatic inflammation is often triggered by airway‐targeting RNA viral infection; virus‐derived double‐stranded RNA, Toll‐like receptor (TLR)3 ligand, activates bronchial epithelial cells to produce pro‐inflammatory mediators, including TSLP. Objective Because TSLPR‐expressing DCs express TLR3, we examined how the relationship between TSLP and TLR3 ligand stimulation influences DC activation. Methods CD11c⁺DCs purified from adult peripheral blood were cultured in TLR ligands containing media with or without TSLP and then co‐cultured with allogeneic naïve CD4⁺T cells. Results CD11c⁺ DCs responded to a combination of TSLP and TLR3 ligand, poly(I : C), to up‐regulate expression of the functional TSLP receptor and TLR3. Although TSLP alone did not induce IL‐23 production by DCs, poly(I : C) alone primed DCs for the production of IL‐23, and a combination of TSLP and poly(I : C) primed DCs for further production of IL‐23. The addition of poly(I : C) did not inhibit TSLP‐activated DCs to prime naïve CD4⁺ T cells to differentiate into inflammatory Th2 cells. Furthermore, DCs activated by a combination of TSLP and poly(I : C) primed more naïve CD4⁺ T cells to differentiate into Th17‐cytokine–producing cells with a central memory T cell phenotype compared with DCs activated by poly(I : C) alone. Conclusions These results suggest that through DC activation, human TSLP and TLR3 ligands promote differentiation of Th17 cells with the central memory T cell phenotype under Th2‐polarizing conditions.     

Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor

Mesenchymal stem cells (MSCs) inhibit T‐cell activation and proliferation but their effects on individual T‐cell‐effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4⁺ T cells toward the Th17 phenotype was examined. CD4⁺ T cells exposed to Th17‐skewing conditions exhibited reduced CD25 and IL‐17A expression following MSC co‐culture. Inhibition of IL‐17A production persisted upon re‐stimulation in the absence of MSCs. These effects were attenuated when cell–cell contact was prevented. Th17 cultures from highly purified naïve‐ and memory‐phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX‐2 inhibitor. Media from MSC/Th17 co‐cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC‐mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation‐induced IL‐17A secretion by naturally occurring, effector‐memory Th17 cells from a urinary obstruction model was also inhibited by MSC co‐culture in a COX‐dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T‐cell precursors and inhibit naturally‐occurring Th17 cells derived from a site of inflammation. Suppression entails cell‐contact‐dependent COX‐2 induction resulting in direct Th17 inhibition by PGE2 via EP4.     

GPA33 is expressed on multiple human blood cell types and distinguishes CD4+ central memory T cells with and without effector function

The Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively determined GPA33 expression patterns on human blood leukocyte subsets, using mass and flow cytometry. We found that GPA33 was expressed on fractions of B, dendritic, natural killer and innate lymphoid cells. Most prominent expression was found in the CD4⁺ T cell compartment. Naïve and CXCR5⁺ regulatory T cells were GPA33^high, and naïve conventional CD4⁺ T cells expressed intermediate GPA33 levels. The expression pattern of GPA33 identified functional heterogeneity within the CD4⁺ central memory T cell (Tcm) population. GPA33⁺ CD4⁺ Tcm cells were fully undifferentiated, bona fide Tcm cells that lack immediate effector function, whereas GPA33^– Tcm cells exhibited rapid effector functions and may represent an early stage of differentiation into effector/effector memory T cells before loss of CD62L. Expression of GPA33 in conventional CD4⁺ T cells suggests a role in localization and/or preservation of an undifferentiated state. These results form a basis to study the function of GPA33 and show it to be a useful marker to discriminate between different cellular subsets, especially in the CD4⁺ T cell lineage.     

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.