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.     

Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues

Expression of the lymph node homing and CC‐chemokine receptor 7 (CCR7), with L‐selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T‐cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62L^high CD44^low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7^high CD62L^high CD44^high (central memory) and CCR7^low CD62L^low CD44^high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short‐term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7^high CD4 T cells produced predominantly interleukin (IL)‐2, whereas CCR7^low CD4 T cells produced both IL‐2 and interferon‐γ (IFN‐γ). However, in contrast to previously published reports, the CCR7^high CD8 T‐cell subpopulation produced both IFN‐γ and IL‐2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down‐regulated CCR7 only after multiple cell divisions, and this coincided with the down‐regulation of CD62L and production of IL‐4 and IFN‐γ. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL‐2‐ and IFN‐γ‐producing cells are CCR7^low, while few cytokine‐expressing CCR7^high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T‐cell subpopulations, but indicate additional complexity in that CCR7^high CD8 T cells also may produce IFN‐γ.     

Changes in histone acetylation and methylation that are important for persistent but not transient expression of CCR4 in human CD4+ T cells

Although regulation of CXCR3 and CCR4 is related to Th1 and Th2 differentiation, respectively, many CXCR3⁺ and CCR4⁺ cells do not express IFN‐γ and/or IL‐4, suggesting that the chemokine receptor genes might be inducible by mechanisms that are lineage‐independent. We investigated the regulation of CXCR3 versus IFNG, and CCR4 versus IL4 in human CD4⁺ T cells by analyzing modifications of histone H3. In naïve cord‐blood cells, under nonpolarizing conditions not inducing IL4, CCR4 was induced to high levels without many of the activation‐associated changes in promoter histone H3 found for both IL4 and CCR4 in Th2 cells. Importantly, CCR4 expression was stable in Th2 cells, but fell in nonpolarized cells after the cells were rested; this decline could be reversed by increasing histone acetylation using sodium butyrate. Patterns of histone H3 modifications in CXCR3⁺CCR4^? and CXCR3^?CCR4⁺ CD4⁺ T‐cell subsets from adult blood matched those in cells cultured under polarizing conditions in vitro. Our data show that high‐level lineage‐independent induction of CCR4 can occur following T‐cell activation without accessibility‐associated changes in histone H3, but that without such changes expression is transient rather than persistent.     

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.     

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.     

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.     

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.     

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.     

CXCR4, but not CXCR3, drives CD8+ T‐cell entry into and migration through the murine bone marrow

The BM serves as a blood‐forming organ, but also supports the maintenance and immune surveillance function of many T cells. Yet, in contrast to other organs, little is known about the molecular mechanisms that drive T‐cell migration to and localization inside the BM. As BM accumulates many CXCR3‐expressing memory CD8⁺ T cells, we tested the involvement of this chemokine receptor, but found that CXCR3 is not required for BM entry. In contrast, we could demonstrate that CXCR4, which is highly expressed on both naive and memory CD8⁺ T cells in BM, is critically important for homing of all CD8⁺ T‐cell subsets to the BM in mice. Upon entry into the BM parenchyma, both naïve and memory CD8⁺ T cells locate close to sinusoidal vessels. Intravital imaging experiments revealed that CD8 T cells are surprisingly immobile and we found that they interact with ICAM‐1+VCAM‐1+BP‐1+ perivascular stromal cells. These cells are the major source of CXCL12, but also express key survival factors and maintenance cytokines IL‐7 and IL‐15. We therefore conclude that CXCR4 is not only crucial for entry of CD8⁺ T cells into the BM, but also controls their subsequent localization toward BM niches that support their survival.     

Tissue‐specific Differences in Immune Cell Subsets Located in the Naso‐oropharyngeal‐associated Lymphoid Tissues

Defining the immune cells within the naso‐oropharyngeal‐associated lymphoid tissues would promote the development of efficient orally and nasally delivered immunotherapies. The aim was to compare murine antigen‐presenting cells (APC s) and T cell subsets in the nose‐associated lymphoid tissues (NALT ), cervical lymph nodes (CLN ), mesenteric lymph nodes (MLN ) and peripheral lymph nodes (PLN ) using flow cytometry and in vitro proliferation assays. Overall, the NALT contained a higher proportion of APC s and a lower proportion of T cells compared to the CLN , MLN and PLN . The APC s of the NALT more often belonged to the CD 11c⁺CD 11b⁺ and the CD 11c^negCD 11b⁺ subsets as compared to the other sites. Both of these APC populations showed little sign of activation, that is low expression of the markers CD 40, CD 86 and IA d. Instead, the APC s of the NALT more often co‐expressed CX 3CR 1 and CD 206, markers associated with a tolerogenic function. No increase in the proportion of regulatory T cells was observed in the NALT . Instead, the T cells frequently exhibited a memory/effector phenotype, expressing the homing markers α4β7, CCR 4 and CCR 9, but rarely the naïve phenotype cell surface marker CD 45RB . In contrast, the T cells at the other sites were mostly of the naïve phenotype. In addition, cells from the NALT did not proliferate upon in vitro stimulation with Con A, whereas the cells from the other sites did. Taken together, these results suggest that the NALT is primarily an effector site rather than one for activation and differentiation, despite it being regarded as a site of induction.     

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.     

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.     

Differential control of CD4+ T‐cell subsets by the PD‐1/PD‐L1 axis in a mouse model of allergic asthma

Studies examining the role of PD‐1 family members in allergic asthma have yielded conflicting results. Using a mouse model of allergic asthma, we demonstrate that blockade of PD‐1/PD‐L1 has distinct influences on different CD4⁺ T‐cell subsets. PD‐1/PD‐L1 blockade enhances airway hyperreactivity (AHR), not by altering the magnitude of the underlying Th2‐type immune response, but by allowing the development of a concomitant Th17‐type immune response. Supporting differential CD4⁺ T‐cell responsiveness to PD‐1‐mediated inhibition, naïve PD‐1^?/? mice displayed elevated Th1 and Th17 levels, but diminished Th2 cytokine levels, and ligation of PD‐1 in WT cells limited cytokine production by in vitro polarized Th1 and Th17 cells, but slightly enhanced cytokine production by in vitro polarized Th2 cells. Furthermore, PD‐1 ligation enhanced Th2 cytokine production by naïve T cells cultured under nonpolarizing conditions. These data demonstrate that different CD4⁺ T‐cell subsets respond differentially to PD‐1 ligation and may explain some of the variable results observed in control of allergic asthma by the PD‐1 family members. As the PD‐1/PD‐L1 axis limits asthma severity by constraining Th17 cell activity, this suggests that severe allergic asthma may be associated with a defective PD‐1/PD‐L1 regulatory axis in some individuals.     

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.     

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.     

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.