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Human CD4 + EOMES + T cells are heterogeneous and contain Th1-cells, Tr1-cells, and CD4 + CTL. Tr1- cells and non-classical EOMES + Th1-cells displayed, respectively, anti- and pro-inflammatory cytokine profiles, but both expressed granzyme-K, produced IFN-γ, and suppressed T-cell proliferation. Diffusion map suggested a progressive CD4 + T-cell differentiation from naïve to cytotoxic cells and identified EOMES + Th1-cells as putative Tr1-cell precursors (pre-Tr1)., (© 2024 Wiley‐VCH GmbH.)

Type 1 regulatory (Tr1) T cells are currently defined all T cells with regulatory functions that lack FOXP3 expression and produce IL-10. Tr1 cells are heterogeneous, and the different reported properties of Tr1-cell populations have caused some confusion in the field. Moreover, understanding the role of Tr1 cells in immune-mediated diseases has been hampered by the lack of a lineage-defining transcription factor. Several independent studies indicated recently that the transcription factor Eomesodermin (EOMES) could act as a lineage-defining transcription factor in a population of IL-10 and IFN-γ co-producing Tr1-like cells, since EOMES directly induces IFN-γ and cytotoxicity, enhances IL-10, and antagonizes alternative T-cell fates. Here, we review the known properties of EOMES + Tr1-like cells. They share several key characteristics with other Tr1 cells (i.e., "Tr1-like"), namely high IL-10 production, cytotoxicity, and suppressive capabilities. Notably, they also share some features with FOXP3 + Tregs, like downregulation of IL-7R and CD40L. In addition, they possess several unique, EOMES-dependent features, that is, expression of GzmK and IFN-γ, and downregulation of type-17 cytokines. Published evidence indicates that EOMES + Tr1-like cells play key roles in graft-versus-host disease, colitis, systemic autoimmunity and in tumors. Thus, EOMES + Tr1-like cells are key players of the adaptive immune system that are involved in several different immune-mediated diseases., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Growing evidence suggests that conventional dendritic cells (cDCs) undergo aberrant maturation in COVID-19, which negatively affects T-cell activation. The presence of effector T cells in patients with mild disease and dysfunctional T cells in severely ill patients suggests that adequate T-cell responses limit disease severity. Understanding how cDCs cope with SARS-CoV-2 can help elucidate how protective immune responses are generated. Here, we report that cDC2 subtypes exhibit similar infection-induced gene signatures, with the upregulation of IFN-stimulated genes and IL-6 signaling pathways. Furthermore, comparison of cDCs between patients with severe and mild disease showed severely ill patients to exhibit profound downregulation of genes encoding molecules involved in antigen presentation, such as MHCII, TAP, and costimulatory proteins, whereas we observed the opposite for proinflammatory molecules, such as complement and coagulation factors. Thus, as disease severity increases, cDC2s exhibit enhanced inflammatory properties and lose antigen presentation capacity. Moreover, DC3s showed upregulation of anti-apoptotic genes and accumulated during infection. Direct exposure of cDC2s to the virus in vitro recapitulated the activation profile observed in vivo. Our findings suggest that SARS-CoV-2 interacts directly with cDC2s and implements an efficient immune escape mechanism that correlates with disease severity by downregulating crucial molecules required for T-cell activation., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., (© 2021 Wiley-VCH GmbH.)

Whether human IL-10-producing regulatory T cells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4 + T-cell subsets, including conventional cytotoxic CD4 + T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4 + T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes + GzmK + T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4 + Eomes + T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes + Tr1-like cells are effector cells of a unique GzmK-expressing CD4 + T-cell subset., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Th17 cells are a heterogeneous population of pro-inflammatory T cells that have been shown to mediate immune responses against intestinal bacteria. Th17 cells are highly plastic and can transdifferentiate to Th1/17 cells or unconventional Th1 cells, which are highly pathogenic in animal models of immune-mediated diseases such as inflammatory bowel diseases. A recent European Journal of Immunology article by Liu et al. (Eur. J. Immunol. 2015. 45:1010-1018) showed, surprisingly, that Th1 cells have a similar plasticity, and could transdifferentiate to Th17 cells. Thus, IFN-γ-producing Th1 effector cells specific for an intestinal microbial antigen were shown to acquire IL-17-producing capacities in the gut in a mouse model of colitis, and in response to TGF-β and IL-6 in vitro. TGF-β induced Runx1, and together with IL-6 was shown to render the ROR-γt and IL-17 promoters in Th1 cells accessible for Runx1 binding. In this commentary, we discuss how this unexpected plasticity of Th1 cells challenges our view on the generation of Th1/17 cells with the capacity to co-produce IL-17 and IFN-γ, and consider possible implications of this Th1-to-Th17-cell conversion for therapies of inflammatory bowel diseases and protective immune responses against intracellular pathogens., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

IL-10 is an anti-inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8(+) CTL, IL-10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL-10 produced by human APC subsets in T-cell responses. IL-10 production was restricted to CD1c(+) DCs and CD14(+) monocytes. Interestingly, it was differentially regulated, since R848 induced IL-10 in DCs, but inhibited IL-10 in monocytes. Autocrine IL-10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high-affinity peptides. Nevertheless, it completely blocked cross-priming and priming with low-affinity peptides of a self/tumor-antigen. IL-10 also inhibited CD1c(+) DC-induced CD4(+) T-cell priming and enhanced Foxp3 induction, but was insufficient to induce T-cell IL-10 production. CD1c(+) DC-derived IL-10 had also no effect on DC-induced secondary expansions of memory CTL. However, IL-15-driven, TCR-independent proliferation of memory CTL was enhanced by IL-10. We conclude that DC-derived IL-10 selects high-affinity CTL upon priming. Moreover, IL-10 preserves established CTL memory by enhancing IL-15-dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL-10 promotes CTL responses in humans., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Upon activation, lymphocytes release vesicles containing microRNAs (miRNAs). However, little is known as to whether this release results in modulation of circulating miRNAs (the miRNome) in the serum. The present work aims to identify lymphocyte subset-specific signatures of miRNAs within the serum circulating miRNome. We therefore assessed serum miRNA expression profiles in wild-type mice; in mice lacking either CD4(+) T cells, CD8(+) T cells, invariant natural killer T (iNKT) cells, or B cells; and, as a control, in mice in which Dicer has been ablated in T lymphocytes. We found that specific serum miRNAs are differentially modulated when different lymphocyte subsets are lacking. In particular, the serum level of miR-181b-5p, previously demonstrated to be fundamental for the development of iNKT cells, is specifically reduced in mice in which iNKT cells are absent. Interestingly, our results indicate a direct link between the biological role of a single miRNA in lymphocyte development and its serum level, and prove that even a population composed of relatively few cells in vivo, such as iNKT lymphocytes, has a measurable effect on the serum circulating miRNome., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

T-cell help to B lymphocytes is one of the most important events in adaptive immune responses in health and disease. It is generally delivered by cognate CD4(+) T follicular helper (T(FH)) cells via both cell-to-cell contacts and soluble mediators, and it is essential for both the clonal expansion of antibody (Ab)-secreting B cells and memory B-cell formation. CD1d-restricted invariant natural killer T (iNKT) cells are a subset of innate-like T lymphocytes that rapidly respond to stimulation with specific lipid antigens (Ags) that are derived from infectious pathogens or stressed host cells. Activated iNKT cells produce a wide range of cytokines and upregulate costimulatory molecules that can promote activation of dendritic cells (DCs), natural killer (NK) cells, and T cells. A decade ago, we discovered that iNKT cells can help B cells to proliferate and to produce IgG Abs in vitro and in vivo. This adjuvant-like function of Ag-activated iNKT cells provides a flexible set of helper mechanisms that expand the current paradigm of T-cell-B-cell interaction and highlights the potential of iNKT-cell targeting vaccine formulations., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The hepatitis C virus (HCV) binds to human cells through the interaction of its envelope glycoprotein E2 with the tetraspanin CD81. We have previously reported that engagement of CD81 has opposite effects on T and NK cell function, as it enhances T cell receptor-mediated T cell activation and inhibits CD16- or IL-12-mediated NK cell activation. We further investigated this dichotomy and found that another tetraspanin, CD82, induces the same opposing effects on human primary T and NK cells. Activation by other unrelated stimuli such as NKG2D- and beta-1 integrin is also reduced by CD81 ligation on NK cells. CD81 engagement by monoclonal antibody or HCV-E2 enhances zeta and Erk phosphorylation in T cells and reduces them in NK cells, reflecting the opposite functional outcomes. CD81 engagement induces dramatic morphological changes and local F-actin accumulation in both NK and T cells, indicating rearrangement of the actin cytoskeleton. Pharmacological inhibition of actin polymerization reduces T cell activation, whereas it greatly enhances NK cell activation. Importantly, treatment with actin blockers abolishes the inhibitory effect of CD81 ligation on NK cells. We propose that tetraspanin engagement leads to comparable cytoskeleton reorganization in T and NK cells, which in turn results in opposite functional outcomes.

As the human tetraspanin CD81 binds hepatitis C virus (HCV) envelope glycoprotein E2, we addressed the role CD81 may play in cellular trafficking of HCV envelope proteins. Studies on HCV life cycle are complicated by the lack of a robust cell culture system; we therefore transfected mammalian cells with HCV E1-E2 cDNA, with or without human CD81 (huCD81) cDNA. In the absence of huCD81, HCV envelope proteins are almost completely retained in the endoplasmic reticulum. Instead, when huCD81 is present, a fraction of HCV envelope proteins passes through the Golgi apparatus, matures acquiring complex sugars and is found extracellularly associated with exosomes. These are 60-100-nm membrane vesicles enriched in tetraspanins, released into the extracellular milieu by many cell types and having fusogenic activity. We also report that human plasma contains exosomes and that in HCV patients, viral RNA is associated with these circulating vesicles. We propose that the HCV-CD81 complex leaves cells in the form of exosomes, circulates in this form and exploits the fusogenic capabilities of these vesicles to infect cells even in the presence of neutralizing antibodies.

Binding of the hepatitis C virus (HCV) envelope protein E2 to CD81 provides a costimulatory signal for human T cells. This phenomenon may play a role in liver damage and autoimmune manifestations associated with HCV infection. Here we show that cross-linking of CD81 by HCV E2 induced a calcium flux in T cells that depends on Lck since it was blocked by PP1 and absent in Lck-deficient Jurkat T cells. In wild-type Jurkat cells, Lck was activated by CD81 cross-linking, and CD81, like Lck, was found in lipid rafts. Indeed, the integrity of the raft compartment was required for the induction of a calcium flux by E2, since methyl-beta-cyclodextrin abolished this response. A requirement for TCR/CD3 expression was indicated by the absence of a calcium flux following E2 stimulation of TCR/CD3-deficient Jurkat cells. CD81 cross-linking increased and prolonged the anti-CD3-induced tyrosine phosphorylation of TCR1 and of other proteins, indicating that the CD81-mediated signal converges with the TCR/CD3 signaling cascade at its most upstream step. In conclusion, we propose that the costimulatory effects of HCV E2 on T cells depend on CD81 cross-linking that activates Lck through raft aggregation and thus leads to enhanced TCR signaling.

The age-related and T cell-independent immunological properties of most capsular polysaccharides limit their use as vaccines, especially in children under 2 years of age. To overcome these limitations, polysaccharide antigens have been successfully conjugated to a variety of carrier proteins, such as diphtheria toxoid or tetanus toxoid (TT) and the diphtheria mutant (CRM197) to produce very successful glycoconjugate vaccines. The increasing demand for new conjugate vaccines requires the availability of additional carriers providing high and long-lasting T helper cell immunity. Here we describe the design and construction of three recombinant carrier proteins (N6, N10, N19) constituted by strings of 6, 10 or 19 human CD4(+) T cell epitopes from various pathogen-derived antigens, including TT and proteins from Plasmodium falciparum, influenza virus and hepatitis B virus. Each of these epitopes is defined as universal in that it binds to many human MHC class II molecules. When conjugated to Haemophilus influenzae type b (Hib) oligosaccharide, these carriers elicit a potent anti-Hib antibody response in mice. In the case of the N19-Hib conjugate, this response is at least as good as that observed with CRM197-Hib, a conjugate vaccine currently used for mass immunization. We also show that some of the universal epitopes constituting the recombinant carriers are specifically recognized by two human in vitro systems, suggesting that T cell memory is provided by the selected epitopes. The data indicate that rationally designed recombinant polyepitope proteins represent excellent candidates for the development and clinical testing of new conjugate vaccines.

The present study demonstrates that the quality of the virus-specific CD8(+) T cell responses, as detected by both enzyme-linked immunospot assay and specific MHC-peptide tetramers, changed in relation to the different disease activity in chronically hepatitis C virus-infected patients. Indeed, both the serum alanine transaminase and the hepatic flogosis levels were related directly to the frequencies of peripheral memory effector CD8(+) T cells producing IFN-gamma (Tc1), but inversely to the frequencies of those producing both IL-4 and IL-10 (Tc2). Longitudinal studies highlighted that Tc1 or Tc2 responses fluctuate in relation to the different phases of the disease in the same individual. Furthermore, the Tc1 or Tc2 phenotype correlates with tetramer-positive cells expressing either CXCR3 or CCR3, promoting differential tissue localization of these cells and the maintenance of T cell homeostasis. Finally, studies at the level of liver-infiltrating lymphocytes indicated that they produced both IFN-gamma and IL-4 with an evident bias towards the Tc1-like phenotype. Our studies suggest that the progressive fluctuation of Tc1 and Tc2 responses may play a fundamental role in maintaining a long-lasting low-level liver inflammation, and may constitute the basis for new therapeutic strategies of immune regulation.

Chronic hepatitis C virus (HCV) infection frequently develops into liver disease and is accompanied by extra-hepatic autoimmune manifestations. The tetraspanin CD81 is a putative HCV receptor as it binds the E2 envelope glycoprotein of HCV and bona fide HCV particles. Here we show that HCV E2 binding to CD81 on human cells in vitro lowers the threshold for IL-2 receptor alpha expression and IL-2 production, resulting in strongly increased T cell proliferation. HCV E2-induced co-stimulation also enhances the production of IFN-gamma and IL-4 and causes increased TCR down-regulation. This suggests that binding of HCV particles to CD81 on T cells in vivo may lead to activation by otherwise suboptimal stimuli. Therefore, co-stimulation of autoreactive T cells by HCV may contribute to liver damage and autoimmune phenomena observed in HCV infection.

NKT cells are a small subset of T lymphocytes which express an invariant V(alpha24JalphaQ TCR and recognize glycolipids presented by CD1d. In adults, NKT cells have a memory phenotype, frequently associated with oligoclonal expansion, express NK cell markers, and produce TO cytokines upon primary stimulation. Because of these features, NKT cells are regarded as lymphocytes of innate immunity. We investigated NKT cells from cord blood to see how these cells appear in the absence of exogenous stimuli. We found that NKT cells are present at comparable frequencies in cord blood and adult peripheral blood mononuclear cells and in both cases display a memory (CD45RO+CD62L-) phenotype. However, neonatal NKT cells differ from their adult counterparts by the following characteristics: (1) they express markers of activation, such as CD25; (2) they are polyclonal; (3) they do not produce cytokines in response to primary stimulation. Together, our data show that human NKT cells arise in the newborn with an activated memory phenotype, probably due to recognition of an endogenous ligand(s). The absence of oligoclonal expansion and primary effector functions also suggest that neonatal NKT cells, despite their activated memory phenotype, require a further priming/differentiation event to behave as fully functional cells of innate immunity.

Chronic viral hepatitis is characterized by a dramatic lymphocyte infiltrate in the liver. Although it is one of the most common chronic inflammatory diseases in humans, little information is available on the functional state of these intra-hepatic lymphocytes (IHL). To address this issue, we have optimized cytofluorimetric techniques to assess directly ex vivo the functions, dynamics and repertoires of IHL isolated from biopsies of patients with chronic hepatitis C. We estimate that 1% of the total body lymphocytes infiltrate the inflamed liver and find that, at variance with peripheral blood lymphocytes (PBL) isolated from the same patients, most IHL display an activated phenotype and produce Th1 type lymphokines when stimulated in vitro. Virtually all IHL are found in the G0/G1 state of the cell cycle, while a sizeable percentage of them is undergoing programmed cell death in vivo, as detected by the TUNEL assay performed on freshly isolated cells. In contrast again to PBL from the same patients, IHL show a preferential compartmentalization of NK and TCRgamma/delta+ cells, and a remarkable (up to 20-fold) enrichment for Valpha24+ T cells. Together our data suggest that in a liver injured by chronic hepatitis C, most IHL are pro-inflammatory activated cells which are highly enriched for effectors of innate resistance. These IHL do not undergo clonal expansion in the liver but rather display effector function and die in situ at a high rate, suggesting that maintenance of the IHL pool is dependent on continuous migration from extra-hepatic sites.

DNA immunization is a very promising approach to the formulation of multivalent vaccines. However, little information is currently available on the immunogenicity of multi-plasmid formulations. To address this issue, we immunized mice with a combination of four plasmids encoding malarial antigens and we compared antibody responses with those obtained with single-plasmid injections. We found that when four plasmids encoding Plasmodium falciparum circumsporozoite protein, thrombospondin-related anonymous protein, major merozoite surface protein (MSP)1 and Pfs25 are co-injected into mice, Ab responses against each antigen are elicited at levels at least as high as the level obtained with single-plasmid injection. The quality of antibody production, as determined by isotype analysis, was similar when single- and multi-plasmid administrations were compared, indicating the priming of the same cytokine profile for CD4+ T helper cells. The sera from mice immunized with the four-plasmid formulation specifically recognized sporozoites, blood stage schizonts and gametes, indicating that DNA immunization induced antibody responses relevant to the native conformation. Finally and of particular interest, in the case of MSP1, the antibody response appears to be strongly potentiated by the presence of additional plasmids, indicating an adjuvant effect of DNA.

It has been suggested that loss of uninfected T cells in HIV infection occurs because of lymphocyte activation resulting in cell death by apoptosis. To address the question of whether cross-linking of CD4/HIV gp120 complexes by antibodies were sufficient to induce T cell depletion in vivo, we developed an animal model of continuous interaction between human CD4 (hCD4), gp120 and anti-gp120 antibodies in the absence of other viral factors. Double-transgenic mice have been generated in which T cells express on their membrane hCD4 and secrete HIV gp120. Although these mice have hCD4/gp120 complexes present on the surface of T cells, they do not show gross immunological abnormalities, and they are able to produce anti-gp120 antibodies following immunization with denaturated gp120. However, double-transgenic mice with antibodies to gp120, when immunized with tetanus toxoid, mount an IgG response that is significantly lower than that of double-transgenic mice without antibodies to gp120. Furthermore, the presence of anti-gp120 antibodies leads to CD4+ T cell depletion and immunodeficiency in the absence of HIV infection. Thus, the antibody response to gp120 can lead to CD4+ T cell attrition in vivo.