CD39 expression by regulatory T cells participates in CD8+ T cell suppression during experimental Trypanosoma cruzi infection.

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case of Trypanosoma cruzi infection, specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein, we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acute T. cruzi infection. Using the DEREG mouse model, we found that Treg cells play a role during the initial stages after T. cruzi infection, restraining the magnitude of CD8+ T cell responses and parasite control. Early Treg cell depletion increased the frequencies of polyfunctional short-lived, effector T cell subsets, without affecting memory precursor cell formation or the expression of activation, exhaustion and functional markers. In addition, Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell immunity. Crucially, the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses, preventing increased parasite replication in T. cruzi infected mice adoptively transferred with Treg cells. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model.

CD39+ conventional CD4+ T cells with exhaustion traits and cytotoxic potential infiltrate tumors and expand upon CTLA-4 blockade.

Conventional CD4+ T (Tconv) lymphocytes play important roles in tumor immunity; however, their contribution to tumor elimination remains poorly understood. Here, we describe a subset of tumor-infiltrating Tconv cells characterized by the expression of CD39. In several mouse cancer models, we observed that CD39+ Tconv cells accumulated in tumors but were absent in lymphoid organs. Compared to tumor CD39- counterparts, CD39+ Tconv cells exhibited a cytotoxic and exhausted signature at the transcriptomic level, confirmed by high protein expression of inhibitory receptors and transcription factors related to the exhaustion. Additionally, CD39+ Tconv cells showed increased production of IFNγ, granzyme B, perforin and CD107a expression, but reduced production of TNF. Around 55% of OVA-specific Tconv from B16-OVA tumor-bearing mice, expressed CD39. In vivo CTLA-4 blockade induced the expansion of tumor CD39+ Tconv cells, which maintained their cytotoxic and exhausted features. In breast cancer patients, CD39+ Tconv cells were found in tumors and in metastatic lymph nodes but were less frequent in adjacent non-tumoral mammary tissue and not detected in non-metastatic lymph nodes and blood. Human tumor CD39+ Tconv cells constituted a heterogeneous cell population with features of exhaustion, high expression of inhibitory receptors and CD107a. We found that high CD4 and ENTPD1 (CD39) gene expression in human tumor tissues correlated with a higher overall survival rate in breast cancer patients. Our results identify CD39 as a biomarker of Tconv cells, with characteristics of both exhaustion and cytotoxic potential, and indicate CD39+ Tconv cells as players within the immune response against tumors.

Interleukin-17 signaling influences CD8+ T cell immunity and tumor progression according to the IL-17 receptor subunit expression pattern in cancer cells.

IL-17 immune responses in cancer are controversial, with both tumor-promoting and tumor-repressing effects observed. To clarify the role of IL-17 signaling in cancer progression, we used syngeneic tumor models from different tissue origins. We found that deficiencies in host IL-17RA or IL-17A/F expression had varying effects on the in vivo growth of different solid tumors including melanoma, sarcoma, lymphoma, and leukemia. In each tumor type, the absence of IL-17 led to changes in the expression of mediators associated with inflammation and metastasis in the tumor microenvironment. Furthermore, IL-17 signaling deficiencies in the hosts resulted in decreased anti-tumor CD8+ T cell immunity and caused tumor-specific changes in several lymphoid cell populations. Our findings were associated with distinct patterns of IL-17A/F cytokine and receptor subunit expression in the injected tumor cell lines. These patterns affected tumor cell responsiveness to IL-17 and downstream intracellular signaling, leading to divergent effects on cancer progression. Additionally, we identified IL-17RC as a critical determinant of the IL-17-mediated response in tumor cells and a potential biomarker for IL-17 signaling effects in tumor progression. Our study offers insight into the molecular mechanisms underlying IL-17 activities in cancer and lays the groundwork for developing personalized immunotherapies.

High-dimensional analysis of 16 SARS-CoV-2 vaccine combinations reveals lymphocyte signatures correlating with immunogenicity.

The range of vaccines developed against severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) provides a unique opportunity to study immunization across different platforms. In a single-center cohort, we analyzed the humoral and cellular immune compartments following five coronavirus disease 2019 (COVID-19) vaccines spanning three technologies (adenoviral, mRNA and inactivated virus) administered in 16 combinations. For adenoviral and inactivated-virus vaccines, heterologous combinations were generally more immunogenic compared to homologous regimens. The mRNA vaccine as the second dose resulted in the strongest antibody response and induced the highest frequency of spike-binding memory B cells irrespective of the priming vaccine. Priming with the inactivated-virus vaccine increased the SARS-CoV-2-specific T cell response, whereas boosting did not. Distinct immune signatures were elicited by the different vaccine combinations, demonstrating that the immune response is shaped by the type of vaccines applied and the order in which they are delivered. These data provide a framework for improving future vaccine strategies against pathogens and cancer.

Polyfunctional KLRG-1+CD57+ Senescent CD4+ T Cells Infiltrate Tumors and Are Expanded in Peripheral Blood From Breast Cancer Patients.

Senescent T cells have been described during aging, chronic infections, and cancer; however, a comprehensive study of the phenotype, function, and transcriptional program of this T cell population in breast cancer (BC) patients is missing. Compared to healthy donors (HDs), BC patients exhibit an accumulation of KLRG-1+CD57+ CD4+ and CD8+ T cells in peripheral blood. These T cells infiltrate tumors and tumor-draining lymph nodes. KLRG-1+CD57+ CD4+ and CD8+ T cells from BC patients and HDs exhibit features of senescence, and despite their inhibitory receptor expression, they produce more effector cytokines and exhibit higher expression of Perforin, Granzyme B, and CD107a than non-senescent subsets. When compared to blood counterparts, tumor-infiltrating senescent CD4+ T cells show similar surface phenotype but reduced cytokine production. Transcriptional profiling of senescent CD4+ T cells from the peripheral blood of BC patients reveals enrichment in genes associated with NK or CD8+-mediated cytotoxicity, TCR-mediated stimulation, and cell exhaustion compared to non-senescent T cells. Comparison of the transcriptional profile of senescent CD4+ T cells from peripheral blood of BC patients with those of HDs highlighted marked similarities but also relevant differences. Senescent CD4+ T cells from BC patients show enrichment in T-cell signaling, processes involved in DNA replication, p53 pathways, oncogene-induced senescence, among others compared to their counterparts in HDs. High gene expression of CD4, KLRG-1, and B3GAT1 (CD57), which correlates with increased overall survival for BC patients, underscores the usefulness of the evaluation of the frequency of senescent CD4+ T cells as a biomarker in the follow-up of patients.

CD8+ T Cell Immunity Is Compromised by Anti-CD20 Treatment and Rescued by Interleukin-17A.

Treatment with anti-CD20, used in many diseases in which B cells play a pathogenic role, has been associated with susceptibility to intracellular infections. Here, we studied the effect of anti-CD20 injection on CD8+ T cell immunity using an experimental model of Trypanosoma cruzi infection, in which CD8+ T cells play a pivotal role. C57BL/6 mice were treated with anti-CD20 for B cell depletion prior to T. cruzi infection. Infected anti-CD20-treated mice exhibited a CD8+ T cell response with a conserved expansion phase followed by an early contraction, resulting in a strong reduction in total and parasite-specific CD8+ T cell numbers at 20 days postinfection. Anti-CD20 injection increased the frequency of apoptotic CD8+ T cells, decreased the number of effector and memory CD8+ T cells, and reduced the frequency of proliferating and cytokine-producing CD8+ T cells. Accordingly, infected anti-CD20-treated mice presented lower cytotoxicity of T. cruzi peptide-pulsed target cells in vivo All of these alterations in CD8+ T cell immunity were associated with increased tissue parasitism. Anti-CD20 injection also dampened the CD8+ T cell response, when this had already been generated, indicating that B cells were involved in the maintenance rather than the induction of CD8+ T cell immunity. Anti-CD20 injection also resulted in a marked reduction in the frequency of interleukin-6 (IL-6)- and IL-17A-producing cells, and recombinant IL-17A (rIL-17A) injection partially restored the CD8+ T cell response in infected anti-CD20-treated mice. Thus, anti-CD20 reduced CD8+ T cell immunity, and IL-17A is a candidate for rescuing deficient responses either directly or indirectly.IMPORTANCE Monoclonal antibody targeting the CD20 antigen on B cells is used to treat the majority of non-Hodgkin lymphoma patients and some autoimmune disorders. This therapy generates adverse effects, notably opportunistic infections and activation of viruses from latency. Here, using the infection murine model with the intracellular parasite Trypanosoma cruzi, we report that anti-CD20 treatment affects not only B cell responses but also CD8+ T cell responses, representing the most important immune effectors involved in control of intracellular pathogens. Anti-CD20 treatment, directly or indirectly, affects cytotoxic T cell number and function, and this deficient response was rescued by the cytokine IL-17A. The identification of IL-17A as the cytokine capable of reversing the poor response of CD8+ T cells provides information about a potential therapeutic treatment aimed at enhancing defective immunity induced by B cell depletion.

PD-L1+ Regulatory B Cells Are Significantly Decreased in Rheumatoid Arthritis Patients and Increase After Successful Treatment.

Background: B cells play an important role in the development and maintenance of rheumatoid arthritis (RA). Although IL-10-producing B cells represent a major subset of regulatory B cells (Bregs) able to suppress autoimmune and inflammatory responses, recent reports showed that B cell-mediated immune suppression may also occur independent of IL-10. For instance, B cells can modulate T cell immune responses through the expression of regulatory molecules such as PD-L1. So far, PD-L1-expressing B cells have not been analyzed in RA patients. Objective: To analyze the frequency of PD-L1-expressing B cells in the peripheral blood of RA patients compared to healthy controls (HC) matched for sex and age, their function on T cell response and their changes in response to therapy. Methods: Fresh peripheral blood B cells from RA patients and HC were characterized by flow cytometry and their functionality assessed in a co-culture system with autologous T cells. Results: The frequencies of CD19+PD-L1+ B cells, CD24hiCD38-PD-L1+ and CD24hiCD38hiPD-L1+ B cells were significantly lower in untreated RA patients than in HC. In a follow-up study, the frequencies of PD-L1+ B cells (CD19+PD-L1+ B cells, CD24hiCD38-PD-L1+ and CD24hiCD38hiPD-L1+ B cells) increased significantly after treatment in good responder patients, although the frequency of total CD24hiCD38hi B cells decreased. CD19+ B cells from untreated RA patients and HC upregulated PD-L1 expression similarly upon stimulation with CpG plus IL-2 and were able to suppress, in vitro, CD8+ T cell proliferation and cytokine production in a PD-L1-dependent manner. Conclusions: Our results show that PD-L1+ B cells exhibiting T cell suppressive capacity are significantly decreased in untreated RA patients but increase in response to successful treatment. PD-L1 expression on B cells from RA patients can be modulated in vitro and PD-L1+ B cells could thus provide new perspectives for future treatment strategies.

The regulatory role of B cells in autoimmunity, infections and cancer: Perspectives beyond IL10 production.

The term regulatory B cells (B regs) is ascribed to a heterogeneous population of B cells with the function of suppressing inflammatory responses. They have been described mainly during the last decade in the context of different immune-mediated diseases. Most of the work on B regs has been focused on IL-10-producing B cells. However, B cells can exert regulatory functions independently of IL-10 production. Here we discuss the phenotypes, development and effector mechanisms of B regs and advances in their role in autoimmunity, infections and cancer.

FcγRIIb and BAFF differentially regulate peritoneal B1 cell survival.

B1 cells produce most natural Abs in unimmunized mice and play a key role in the response to thymus-independent Ags and microbial infection. Enlargement of B1 cell number in mice is often associated with autoimmunity. However, the factors that control peripheral B1 cell survival remain poorly characterized. Mice lacking the inhibitory receptor FcγRIIb exhibit a massive expansion in peritoneal B1 cells, implicating this receptor in B1 cell homeostasis. In this study, we show that peritoneal B1 cells express the highest levels of FcγRIIb among B cell subsets and are highly susceptible to FcγRIIb-mediated apoptosis. B1 cells upregulate FcγRIIb in response to innate signals, including CpG, and the B cell homeostatic cytokine BAFF efficiently protects activated B1 cells from FcγRIIb-mediated apoptosis via receptor downregulation. BAFF-transgenic mice manifest an expansion of peritoneal B1 cells that express lower levels of FcγRIIb and exhibit reduced susceptibility to apoptosis. Whereas both peritoneal B1 cells from wild-type and BAFF-transgenic mice immunized with CpG exhibit an increase in FcγRIIb levels, this change is blunted in BAFF-transgenic animals. Our combined results demonstrate that FcγRIIb controls peritoneal B1 cell survival and this program can be modulated by the BAFF signaling axis.

BAFF mediates splenic B cell response and antibody production in experimental Chagas disease.

BACKGROUND: B cells and antibodies are involved not only in controlling the spread of blood circulating Trypanosoma cruzi, but also in the autoreactive manifestations observed in Chagas disease. Acute infection results in polyclonal B cell activation associated with hypergammaglobulinemia, delayed specific humoral immunity and high levels of non-parasite specific antibodies. Since TNF superfamily B lymphocyte Stimulator (BAFF) mediates polyclonal B cell response in vitro triggered by T. cruzi antigens, and BAFF-Tg mice show similar signs to T. cruzi infected mice, we hypothesized that BAFF can mediate polyclonal B cell response in experimental Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: BAFF is produced early and persists throughout the infection. To analyze BAFF role in experimental Chagas disease, Balb/c infected mice were injected with BR3:Fc, a soluble receptor of BAFF, to block BAFF activity. By BAFF blockade we observed that this cytokine mediates the mature B cell response and the production of non-parasite specific IgM and IgG. BAFF also influences the development of antinuclear IgG and parasite-specific IgM response, not affecting T. cruzi-specific IgG and parasitemia. Interestingly, BAFF inhibition favors the parasitism in heart. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate, for the first time, an active role for BAFF in shaping the mature B cell repertoire in a parasite infection.

Interleukins 1beta and 6 but not transforming growth factor-beta are essential for the differentiation of interleukin 17-producing human T helper cells.

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) have been linked to host defense and autoimmune diseases. In mice, the differentiation of T(H)-17 cells requires transforming growth factor-beta and IL-6 and the transcription factor RORgammat. We report here that for human naive CD4(+) T cells, RORgammat expression and T(H)-17 polarization were induced by IL-1beta and enhanced by IL-6 but were suppressed by transforming growth factor-beta and IL-12. Monocytes and conventional dendritic cells, but not monocyte-derived dendritic cells activated by microbial stimuli, efficiently induced T(H)-17 priming, and this function correlated with antigen-presenting cell production of IL-1beta and IL-6 but not IL-12. Our results identify cytokines, antigen-presenting cells and microbial products that promote the polarization of human T(H)-17 cells and emphasize an important difference in the requirements for the differentiation of T(H)-17 cells in humans and mice.

Cytokines and chemokines shaping the B-cell compartment.

The whole life of a B-cell from a stem cell to a mature plasma cell is governed, among other factors, by cytokines and growth factors in their microenvironment. Remarkable progress in the understanding of the mechanisms of cytokines action on the B-cell compartment was achieved by analysis of gene-targeted mice. The generation of mice deficient for individual cytokines or their receptors has shed light on the in vivo function of cytokines in B-cell responses. This review focuses on the role of cytokines in the development, maturation and differentiation of different B-cell subsets into antibody-secreting cells or memory B-cells.

BAFF and LPS cooperate to induce B cells to become susceptible to CD95/Fas-mediated cell death.

Microorganisms with pathogen-associated molecular patterns (PAMP) activate B cells directly by binding to TLR and also indirectly by inducing APC to release cytokines such as BAFF that promote B cell survival. We found that murine B cells activated concomitantly with LPS (TLR-4 ligand) and BAFF are protected from spontaneous apoptosis, but are more susceptible to Fas/CD95-mediated cell death. This increased susceptibility to Fas-induced apoptosis is associated with a dramatic coordinated up-regulation of Fas/CD95 and IRF-4 expression through a mechanism mediated, at least in part, by inhibition of the MEK/ERK pathway. Up-regulation of Fas/CD95 by BAFF is restricted to B cells activated through TLR-4, but not through TLR-9, BCR or CD40. TLR ligands differ in the BAFF family receptors (R) they induce on B cells: BAFF-R is increased by the TLR4 ligand, LPS, but not by the TLR9 ligand, CpG-containing oligodeoxynucleotides, which, in contrast, strongly up-regulates transmembrane activator and CAML interactor (TACI). This suggests the up-regulation of Fas by BAFF is mediated by BAFF-R and not by TACI. Consistently, APRIL, which binds to TACI and B cell maturation antigen but not BAFF-R, did not enhance Fas expression on LPS-activated B cells. Increased susceptibility to Fas-mediated killing of B cells activated with LPS and BAFF may be a fail-safe mechanism to avoid overexpansion of nonspecific or autoreactive B cells.

A Trypanosoma cruzi antigen signals CD11b+ cells to secrete cytokines that promote polyclonal B cell proliferation and differentiation into antibody-secreting cells.

Microbial-induced polyclonal activation of B cells is a common event in several forms of infections, and is believed to play a crucial role both for enhancing the production of specific antibodies and for maintenance of B cell memory. Therefore, a major challenge in biomedical research is the identification of pathogen-derived products capable of rapidly mounting B cell expansion and differentiation. Here we report that glutamate dehydrogenase (GDH) stimulates polyclonal proliferation and differentiation of naive B cells. This stimulation was found to be T cell independent, but to absolutely require CD11b(+) cells. Moreover, we demonstrate that stimulation of CD11b(+) cells by GDH leads to the production of IL-6, IL-10 and B cell-activating factor (BAFF), all of which combine to powerfully induce B cell expansion. Importantly, IL-6 and IL-10 further drive B cell terminal differentiation into plasma cells by up-regulating critical transcription factors and immunoglobulin secretion. Our data provide the first evidence that a protozoan antigen can induce BAFF production by accessory cells, which in concert with other cytokines trigger polyclonal B cell activation.

Galectin-3 mediates IL-4-induced survival and differentiation of B cells: functional cross-talk and implications during Trypanosoma cruzi infection.

The role of transcription factors in B cell survival and differentiation has been delineated during the last years. However, little is known about the intermediate signals and the intracellular pathways that control these events. In this study, we provide evidence both in vitro and in vivo, showing that galectin-3 (Gal-3), a beta-galactoside-binding protein, is a critical mediator of B cell differentiation and survival. Although Gal-3 is not expressed in resting B cells from normal mice, its expression is markedly induced after activation with stimuli such as IL-4 and CD40 cross-linking. These signals promote survival and block the final differentiation of these cells, thus allowing the rising of a memory B cell phenotype. In addition, Gal-3 is expressed in B cells from Trypanosoma cruzi-infected mice, which received signals for activation and differentiation in vivo. By using an antisense strategy, we determined that Gal-3 is a critical signal mediating the effects of IL-4 on B cell fate. Blockade of intracellular Gal-3 in vitro abrogated IL-4-induced survival of activated B cells, favoring the differentiation toward a plasma cell pathway. Moreover, B cells with restrained endogenous Gal-3 expression failed to down-regulate the Blimp-1 transcription factor after IL-4 stimulation. Finally, inhibition of Gal-3 in vivo skewed the balance toward plasma cell differentiation, which resulted in increased Ig production and parasite clearance during T. cruzi infection. Thus, the present study provides evidence of a novel role for Gal-3 as an intracellular mediator of B cell survival and a checkpoint in IL-4-induced B cell commitment toward a memory phenotype.