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T follicular helper (Tfh) and T follicular regulatory (Tfr) cells are the two T cell subsets able to interact with B cells driving germinal center (GC) reactions. These T-B interactions are important for protective immune responses within secondary lymphoid tissue. However, the pathological emergence of ectopic lymphoid structures (ELS) that characterize several autoimmune diseases also involves Tfh and Tfr cells. ELS, often with ectopic GCs, can be identified through biopsies. Sjogren's syndrome (SS) is an example of an autoimmune disease where minor salivary gland (MSG) biopsies are often performed for diagnosis and where ELS can be found. Here, we describe a protocol to identify and isolate T follicular cells from MSGs by flow cytometry and immunohistochemistry.

T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells are crucially involved in the regulation of germinal center reactions. Thus, as key players, the assessment of these cell subsets is necessary for a better understanding of the humoral immune response. Flow cytometry (FC) is one of the most used techniques to perform immunophenotypic analysis, allowing the simultaneous study of different proteins by using multicolor fluorescent panels. Here, we describe an approach to identify Tfr cells from human blood and tissues, namely tonsil and lymph node, by flow cytometry.

In recent years molecules involved on the immune synapse became successful targets for therapeutic immune modulation. CD6 has been extensively studied, yet, results regarding CD6 biology have been controversial, in spite of the ubiquitous presence of this molecule on virtually all CD4 T cells. We investigated the outcome of murine and human antibodies targeting CD6 domain 1. We found that CD6-targeting had a major impact on the functional specialization of CD4 cells, both human and murine. Differentiation of CD4 T cells towards a Foxp3+ Treg fate was prevented with increasing doses of anti-CD6, while Th1 polarization was favoured. No impact was observed on Th2 or Th17 specialization. These in vitro results provided an explanation for the dose-dependent outcome of in vivo anti-CD6 administration where the anti-inflammatory action is lost at the highest doses. Our data show that therapeutic targeting of the immune synapse may lead to paradoxical dose-dependent effects due to modification of T cell fate. Funded by UID/BIM/50005/2019, project funded by Fundação para a Ciência e a Tecnologia (FCT) / Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) throught Fundos do Orçamento do Estado, pela Fundação para a Ciência e a Tecnologia (FCT) ( PTDC/DTP-FTO/3080/2014 ); and by the project SRecognite Infect - ERA/0003/2015 using national funds through FCT . Funders did not have a role in study design, data collection, analysis, and interpretation, or in the writing of the manuscript.

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., Gastric cancer (GC) patients display increased regulatory T cell (Tregs) numbers in peripheral blood and among tumor-infiltrating lymphocytes. Nevertheless, the role of Tregs in GC progression remains controversial. Here, we sought to explore the impact of Tregs in GCs with distinct histology, and whether Tregs can directly influence tumor cell behavior and GC development. We performed a comprehensive immunophenotyping of 82 human GC cases, through an integrated analysis of multispectral immunofluorescence detection of T cells markers and patient clinicopathological data. Moreover, we developed 3D in vitro co-cultures with Tregs and tumor cells that were followed by high-throughput and light-sheet imaging, and their biological features studied with conventional/imaging flow cytometry and Western blotting. We showed that Tregs located at the tumor nest were frequent in intestinal-type GCs but did not associate with increased levels of effector T cells. Our in vitro results suggested that Tregs preferentially infiltrated intestinal-type GC spheroids, induced the expression of IL2Rα and activation of MAPK signaling pathway in tumor cells, and promoted spheroid growth. Accumulation of Tregs in intestinal-type GCs was increased at early stages of the stomach wall invasion and in the absence of vascular and perineural invasion. In this study, we proposed a non-immunosuppressive mechanism through which Tregs might directly modulate GC cells and thereby promote tumor growth. Our findings hold insightful implications for therapeutic strategies targeting intestinal-type GCs and other tumors with similar immune context., This work was supported by grants: (1) “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274), by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020— Operacional Program for Competitiveness and Internationalisation (POCI), PORTUGAL 2020, Portuguese funds through Portuguese Science & Technology Foundation (FCT)/Ministério da Ciência, Tecnologia e Inovação; (2) Project CANCER Ref. NORTE-01-0145-FEDER-000029 and DOCnet Ref. NORTE-01-0145-FEDER-000003), by Norte Portugal Regional Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); (3) Project Ref. PTDC/BBB-ECT/2518/2014, by FCT;(4) FCT PhD Programs and by Programa Operacional Potencial Humano (POCH), specifically by the Biotech Health Programe (Doctoral Program on Cellular and Molecular Biotechnology Applied to Health Sciences); (5) “The role of gastric cancer cell-derived extracellular vesicles”, by IPATIMUP Board of Directors; (6) GenomePT project (POCI-01-0145-FEDER-022184), by COMPETE 2020—POCI, Lisboa Portugal Regional Operational Program (Lisboa2020), Algarve Portugal Regional Operational Program (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through ERDF, and by FCT; (7) Marie Skłodowska-Curie grant agreement No. 722148 to Train-EV project by Euro-pean Union’s Horizon 2020 research and innovation program; 8) Project CANCERSTEM funded by ERDF, POCI and FCT; 9) FCT fellowship PD/BD/128406/2017 to SR, and Junior Research contractto JC (regulated by the decree-law 57/2016 emended by law 57/2017). IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT. NFCCdM has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. 852832). The authors acknowledgethe support of the Advanced Light Microscopy, Bioimaging, BioSciences Screening and Histologyand Electron Microscopy i3S Scientific Platforms members of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122), and the support providedby the Translational Cytometry i3S Scientific Platforms.

Germinal centers (GCs) are anatomic structures where B cells undergo affinity maturation, leading to production of high-affinity antibodies. The balance between T follicular helper (TFH) and regulatory (TFR) cells is critical for adequate control of GC responses. The study of human TFH and TFR cell development has been hampered because of the lack of in vitro assays reproducing in vivo biology, along with difficult access to healthy human lymphoid tissues. We used a single-cell transcriptomics approach to study the maturation of TFH and TFR cells isolated from human blood, iliac lymph nodes (LNs), and tonsils. As independent tissues have distinct proportions of follicular T cells in different maturation states, we leveraged the heterogeneity to reconstruct the maturation trajectory for human TFH and TFR cells. We found that the dominant maturation of TFR cells follows a bifurcated trajectory from precursor Treg cells, with one arm of the bifurcation leading to blood TFR cells and the other leading to the most mature GC TFR cells. Overall, our data provide a comprehensive resource for the transcriptomics of different follicular T cell populations and their dynamic relationship across different tissues.

The absence of alloantibodies is a feature of transplantation tolerance. Although the lack of T cell help has been evoked to explain this absence, herein we provide evidence for B cell–intrinsic tolerance mechanisms. Using a murine model of heart tolerance, we showed that alloreactive B cells were not deleted but rapidly lost their ability to differentiate into germinal center B cells and secrete donor-specific antibodies. We inferred that tolerant alloreactive B cells retained their ability to sense alloantigen because they continued to drive T cell maturation into CXCR5(+)PD-1(+) T follicular helper cells. Unexpectedly, dysfunctional alloreactive B cells acquired the ability to inhibit antibody production by new naive B cells in an antigen-specific manner. Thus, tolerant alloreactive B cells contribute to transplantation tolerance by foregoing germinal center responses while retaining their ability to function as antigen-presenting cells and by actively suppressing de novo alloreactive B cell responses.

This article belongs to the Special Issue Trends in Molecular Research for Transplantation Immunology., Regulatory T cells (Tregs) are essential for the maintenance of tolerance to self and non-self through cell-intrinsic and cell-extrinsic mechanisms. Peripheral Tregs survival and clonal expansion largely depend on IL-2 and access to co-stimulatory signals such as CD28. Engagement of tumor necrosis factor receptor (TNFR) superfamily members, in particular TNFR2 and DR3, contribute to promote peripheral Tregs expansion and sustain their survival. This property can be leveraged to enhance tolerance to allogeneic transplants by tipping the balance of Tregs over conventional T cells during the course of immune reconstitution. This is of particular interest in peri-transplant tolerance induction protocols in which T cell depletion is applied to reduce the frequency of alloreactive T cells or in conditioning regimens that allow allogeneic bone marrow transplantation. These conditioning regimens are being implemented to limit long-term side effects of continuous immunosuppression and facilitate the establishment of a state of donor-specific tolerance. Lymphopenia-induced homeostatic proliferation in response to cytoreductive conditioning is a window of opportunity to enhance preferential expansion of Tregs during homeostatic proliferation that can be potentiated by agonist stimulation of TNFR., This work has been supported by grant FIS PI13/00029 (Fondo de Investigaciones Sanitarias, Ministry of Health, Spanish Government and co-funded by European Union ERDF/ESF, “Investing in your future”), LE093U13 and Unit of Excellence Research UIC #012 (Department of Education of the Regional Government, Junta de Castilla y Leon), and Gerencia Regional de Salud (BIO/01/15) to JIRB. It was also funded by FIS PI16/00002 (Instituto de Salud Carlos III and co-funded by European Union ERDF/ESF, “Investing in your future”) and Gerencia Regional de Salud GRS963/A/2014, GRS1142/A/2015 and GRS 1505/A/2017 to M.L.R.G. This work has been supported with funding from the Spanish National Network CIBER-ONC (oncology research) CB16/12/00480 to JAPS. PS is supported by the Swiss National Science Foundation (grant 31003A-176526).

Copyright © 2021 Matos, Hirakawa, Alho, Neleman, Graca and Ritz. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., CD4+ Regulatory T cells (Treg) play a critical role in maintaining immune homeostasis. Various Treg subsets have been identified, however the heterogeneity of Treg subpopulations during development remains uncharacterized. Using mass cytometry we obtained single cell data on expression of 35 functional markers to examine the heterogeneity of Treg cells at birth and in adults. Unsupervised clustering algorithms FlowSOM and ACCENSE were used to quantify Treg heterogeneity. As expected, Treg in umbilical cord blood were predominately naïve while Treg in adult blood were predominately central memory and effector memory cells. Although umbilical cord blood Treg are mostly naïve cells, we observed multiple phenotypic Treg subsets in cord blood. Nevertheless, peripheral blood in adults contained higher percentages of Treg and the heterogeneity of Treg was significantly increased in adults. We also studied Treg heterogeneity throughout a 2-year period after allogeneic hematopoietic stem cell transplantation (alloHSCT) and in patients with chronic graft-versus-host disease (cGVHD). Treg heterogeneity recovered rapidly after alloHSCT and gradually increased in the first two years post-transplant. However, patients with cGVHD had significantly fewer distinct Treg subpopulations, proposing a correlation between a disrupted Treg heterogeneity and cGVHD. Our study is the first to compare human Treg heterogeneity at birth, in healthy adults and in patients after alloHSCT with and without cGVHD. This approach to characterize Treg heterogeneity based on expression of a large panel of functional markers may enable future studies to identify specific Treg defects that contribute to immune dysfunction., This work was supported by NIH grant P01CA229092, EADV Research Fellowship, Rene-Touraine Fellowship and a generous contribution from the Fundação para a Ciência e a Tecnologia (FCT), FCT SFRH/BD/98980/2013

IL-2 is critical for peripheral tolerance mediated by regulatory T (Treg) cells, which represent an obstacle for effective cancer immunotherapy. Although IL-2 is important for effector (E) T cell function, it has been hypothesized that therapies blocking IL-2 signals weaken Treg cell activity, promoting immune responses. This hypothesis has been partially tested using anti–IL-2 or anti–IL-2R Abs with antitumor effects that cannot be exclusively attributed to lack of IL-2 signaling in vivo. In this work, we pursued an alternative strategy to block IL-2 signaling in vivo, taking advantage of the trimeric structure of the IL-2R. We designed an IL-2 mutant that conserves the capacity to bind to the αβ-chains of the IL-2R but not to the γc-chain, thus having a reduced signaling capacity. We show our IL-2 mutein inhibits IL-2 Treg cell–dependent differentiation and expansion. Moreover, treatment with IL-2 mutein reduces Treg cell numbers and impairs tumor growth in mice. A mathematical model was used to better understand the effect of the mutein on Treg and E T cells, suggesting suitable strategies to improve its design. Our results show that it is enough to transiently inhibit IL-2 signaling to bias E and Treg cell balance in vivo toward immunity.

Objective To investigate whether the balance of blood follicular helper T (Tfh) cells and T follicular regulatory (Tfr) cells can provide information about ectopic lymphoid neogenesis and disease activity in primary Sjogren's syndrome (SS). Methods We prospectively recruited 56 patients clinically suspected of having SS. Sixteen of these patients subsequently fulfilled the American-European Consensus Group criteria for SS and were compared to 16 patients with non-SS sicca syndrome. Paired blood and minor salivary gland (MSG) biopsy samples were analyzed to study Tfr cells and subsets of Tfh cells in both compartments. Results Patients with primary SS had normal Tfh cell counts in peripheral blood; however, activated programmed death 1-positive (PD-1+) inducible costimulator-positive (ICOS+) Tfh cells in peripheral blood were strongly associated with disease activity assessed by the European League Against Rheumatism Sjogren's Syndrome Disease Activity Index (r = 0.8547, P = 0.0008). Conversely, the blood Tfr cell:Tfh cell ratio indicated ectopic lymphoid structure formation in MSGs, being strongly associated with B cell, CD4+ T cell, and PD-1+ICOS+ T cell infiltration in MSGs, and was especially increased in patients with focal sialadenitis. Further analysis showed that the blood Tfr cell:Tfh cell ratio allowed discrimination between SS patients and healthy donors with excellent accuracy and was a strong predictor of SS diagnosis (odds ratio [OR] 12.96, P = 0.028) and the presence of focal sialadenitis (OR 10, P = 0.022) in patients investigated for sicca symptoms, thus highlighting the potential clinical value of this marker. Conclusion The blood Tfr cell:Tfh cell ratio and PD-1+ICOS+ Tfh cells constitute potential novel biomarkers for different features of primary SS. While the blood Tfr cell:Tfh cell ratio is associated with ectopic lymphoid neogenesis, activated Tfh cells indicate disease activity.

Elevated levels of immunoglobulin E (IgE) are associated with allergies and other immunological disorders. Sensitization with alum adjuvant favours IgE production while CpG-ODN adjuvant, a synthetic toll-like receptor 9 (TLR9) agonist, inhibits it. The cellular mechanisms underlying in vivo TLR regulation of immunoglobulin production, specially IgE, are still controversial. Specifically, TLR-mediated IgE regulation in vivo is not yet known. In this study we showed that augmented levels of IgE induced by sensitizations to OVA with or without alum adjuvant or with OVA-pulsed dendritic cells (DCs) were inhibited by co-administration of CpG. Notably, CpG-mediated suppression of IgE production required MyD88-expression on DCs but not on B-cells. This finding contrasts with previous in vitro studies reporting regulation of IgE by a direct action of CpG on B cells via MyD88 pathway. In addition, we showed that CpG also inhibited IgE production in a MyD88-dependent manner when sensitization was performed with OVA-pulsed DCs. Finally, CpG signalling through MyD88 pathway was also necessary and sufficient to prevent anaphylactic antibody production involved in active cutaneous anaphylaxis.

As a hallmark of autoimmune rheumatic diseases, autoantibodies have been used in diagnosis for decades. However, the immunological mechanism underlying their generation has only become clear following the identification of T follicular helper (TFH) cells and T follicular regulatory (TFR) cells. TFH cells are instrumental in supporting antibody affinity maturation in germinal centre reactions and humoral memory formation, whereas TFR cells suppress TFH cell-mediated antibody responses. Evidence indicates that patients with autoimmune rheumatic diseases have increased numbers of TFH cells that can be hyperactive, and also potentially have altered numbers of TFR cells with reduced function, suggesting a conceivable dysregulation in the balance between TFH cells and TFR cells in these diseases. Therefore, by identifying the molecular mechanisms underlying the development and function of these cell populations, new opportunities have emerged to develop novel therapeutic targets. An increased knowledge of TFH cells and TFR cells has inspired, and hopefully will inspire more, approaches to reinstate the balance of these cells in the prevention and treatment of rheumatic diseases. T follicular helper cells and T follicular regulatory cells tightly control the production of (auto)antibodies by B cells. Understanding their phenotypes and how the function of these cells is dysregulated in rheumatic diseases will aid future therapeutic development.

Summary The identification that T follicular helper (Tfh) cells is critical for the emergence of germinal centre responses prompted the study of CXCR5-expressing CD4+ T cell subsets in autoimmunity. However, circulating CXCR5-expressing T cells are heterogeneous by containing Forkhead box protein 3 (FoxP3)+ T follicular regulatory (Tfr) cells in addition to bona fide Tfh cells. Such heterogeneity may hamper the analysis of the contribution of specific follicular T cell subsets for autoimmune pathogenesis. Therefore, separate assessment of Tfh and Tfr populations offer greater opportunities for stratification of autoimmune patients, such as Sjögren’s syndrome patients.

© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, Germinal centers (GC) have been known as key anatomic structures in humoral immunity, where isotype switching and affinity maturation occur. As a consequence, elucidation of GC regulation has potential implications for the understanding of autoantibody-mediated diseases. It is now accepted that different regulatory mechanisms coexist, including the action of a specialized population of Foxp3+ regulatory T cells with unique access to the B-cell follicle: the T follicular regulatory (Tfr) cells. Tfr cells develop through a multistep process requiring migration through different compartments of lymphoid tissues. This review discusses the ontogeny and physiology of Tfr cells, their distribution within distinct anatomic compartments, and their function. A greater understanding of Tfr biology and GC regulation is likely to lead to better stratification of patients with autoantibody-mediated diseases, and to the identification of novel therapeutic targets., Research funded by PTDC/IMI-IMU/7038/2014, and LISBOA-01-0145-FEDER-007391, projeto cofinanciado pelo FEDER através POR Lisboa 2020–Programa Operacional Regional de Lisboa, do PORTUGAL 2020, e pela Fundação para a Ciência e a Tecnologia.

Background:Several studies have demonstrated that an immune dysregulation affecting both B and T cells occurs in rheumatoid arthritis (RA). Follicular helper T (Tfh) cells are crucial for B cell maturation, activation and class-switching as well as for germinal center (GC) formation, whereas follicular regulatory T (Tfr) cells can modulate the GC reaction by suppressing Tfh and B cells.Objectives:The main goal of this study was to analyze the phenotype and frequency of circulating follicular T cell subsets in established RA patients.Methods:Blood samples were collected from established RA patients with active disease, treated with methotrexate (n=32) and from a group of age and sex-matched healthy donors (n=11). Peripheral blood mononuclear cells (PBMC) were isolated and Tfh (CD4+CXCR5+CD45RO+) and Tfr (CD4+ CXCR5+CD25+FoxP3+) cells, as well as their three major subsets [CXCR3+CCR6- (Th1-like), CXCR3-CCR6- (Th2-like) and CXCR3-CCR6+ (Th17-like)] were evaluated by flow cytometry.Results:The frequency of circulating Tfh cells was similar between established RA patients and controls. Nonetheless, RA patients had a decreased frequency of Th1-like Tfh cells, and an increased frequency of Th2-like Tfh cells when compared to controls. No significant differences were observed in the frequencies of Th17-like Tfh cells between both groups. The frequency of circulating Tfr cells was significantly increased in RA patients in comparison to controls. Furthermore, Tfr cells from RA patients had significantly increased CD69 median fluorescence intensity (MFI) values when compared to controls. No significant differences were found in the percentages and MFI values of PD-1, ICOS, CD28, CTLA-4, CD40-L and HLA-DR expressed by Tfh and Tfr cells in RA patients when compared to controls.Conclusion:Established RA patients have increased circulating frequencies of Tfr cells, with higher CD69 expression levels, when compared to healthy controls. These results suggest a pre-activation state of Tfr cells in RA and a potential role in the disease physiopathology.*RA Moura, JE Fonseca and L Graca are joint senior authors.Disclosure of Interests:None declared

Background T follicular helper (Tfh) cells are essential for the generation of high affinity neutralising antibodies elicited following vaccination and are involved in the pathogenesis of rheumatoid arthritis (RA). Interleukin (IL)−6 has been shown to be critical for Tfh differentiation in mice, while its importance in humans has been less clear, given the lack of adequate in vivo assessment. Objectives To investigate the importance of IL-6 for the in vivo differentiation of human Tfh cells, taking advantage of influenza vaccination in patients under anti-IL-6R therapy. Methods Blood was collected before, 7 and 28 days after vaccination from established RA patients treated with tocilizumab (TCZ, IL-6R blocker), methotrexate (MTX) ±other DMARDs and age- and sex-matched healthy donors (HD). We analysed the frequency of Tfh (CD3+CD4+CD25-Foxp3-CXCR5+CD45RO+), T follicular regulatory (Tfr, CD3+CD4+CD25+Foxp3+CXCR5+) and B cell populations at each time point. We used non-parametric tests, deemed significant at p Results We included 137 participants (42 TCZ, 42 MTX, 53 HD) with similar age and gender distribution. Patients from the TCZ group had more active and severe disease. At baseline, patients treated with TCZ had higher frequency of Tfh and Tfh-Th2-like cells (CXCR3-CCR6-) and lower frequency of Tfr-Th1-like (CXCR3+CCR6-) and B cells. Following influenza vaccination, the overall blood Tfh and Tfr populations remained unchanged in all groups. However, as previously reported, there were marked changes in specific subsets at day 7 of HD following vaccination. We found a marked expansion of activated CXCR5+ICOS+ Tfh cells at day 7, in HD and MTX-treated patients, but this was impaired in the TCZ group (figure 1). The increase in activated CXCR5+ICOS+ Tfh cells was mainly due to a Tfh-Th1-like subpopulation, greatly increased in HD and MTX-treated patients (figure 1). Of note, CXCR5+ICOS+ Tfh-Th17-like cells also accumulated in HD but not in RA patients. The proliferative capacity of CXCR5+ICOS+ Tfh cells seemed to be partially impaired in patients under IL-6R blockade, that displayed marked reduction of Ki67+CD38+ proliferative cells within that compartment (figure 1). Anti-IL-6R treatment also impaired expansion of CD19+IgD- CD27+CD38hi plasmablasts following vaccination, when compared with both MTX and HD groups (figure 1). Changes in CXCR5+ICOS+ Tfh and plasmablasts were significantly correlated in all groups. Conclusions Anti-IL-6R treatment limits proliferative ability of activated CXCR5+ICOS+ Tfh cells, blocking their emergence as well as plasmablast accumulation following influenza vaccination. Our data suggest that IL-6 is crucial for optimal in vivo generation of activated Tfh cells in humans. Disclosure of Interest None declared

The germinal center (GC) is a specialized microstructure that forms in secondary lymphoid tissues, producing long-lived antibody secreting plasma cells and memory B cells, which can provide protection against reinfection. Within the GC, B cells undergo somatic mutation of the genes encoding their B cell receptors which, following successful selection, can lead to the emergence of B cell clones that bind antigen with high affinity. However, this mutation process can also be dangerous, as it can create autoreactive clones that can cause autoimmunity. Because of this, regulation of GC reactions is critical to ensure high affinity antibody production and to enforce self-tolerance by avoiding emergence of autoreactive B cell clones. A productive GC response requires the collaboration of multiple cell types. The stromal cell network orchestrates GC cell dynamics by controlling antigen delivery and cell trafficking. T follicular helper (Tfh) cells provide specialized help to GC B cells through cognate T-B cell interactions while Foxp3+ T follicular regulatory (Tfr) cells are key mediators of GC regulation. However, regulation of GC responses is not a simple outcome of Tfh/Tfr balance, but also involves the contribution of other cell types to modulate the GC microenvironment and to avoid autoimmunity. Thus, the regulation of the GC is complex, and occurs at multiple levels. In this review we outline recent developments in the biology of cell subsets involved in the regulation of GC reactions, in both secondary lymphoid tissues, and Peyer's patches (PPs). We discuss the mechanisms which enable the generation of potent protective humoral immunity whilst GC-derived autoimmunity is avoided.

This deposit is composed by a publication in which the IGC' authors have had the role of collaboration (it's a collaboration publication). This type of deposit in ARCA is in restrictedAccess (it can't be in open access to the public), and could only be accessed by two ways: either by requesting a legal copy to the author (the email contact present in this deposit) or by visiting the following link: http://www.jimmunol.org/content/200/1/101.long#ack-1 This publication hasn't any creative commons license associated. It has been shown that dominant tolerance, namely in transplantation, requires Foxp3+regulatory T cells. Although most tolerance-inducing regimens rely on regulatory T cells, we found that induction of tolerance to proteins in aluminum hydroxide can be achieved in Foxp3-deficient mice using nondepleting anti-CD4 Abs. This type of tolerance is Ag specific, and tolerant mice retain immune competence to respond to unrelated Ags. We demonstrated with chicken OVA-specific TCR-transgenic mice that the same tolerizing protocol (CD4 blockade) and the same target Ag (OVA) achieves Foxp3-dependent transplantation tolerance to OVA-expressing skin grafts, but Foxp3-independent tolerance when the Ag is provided as OVA-aluminum hydroxide. In the latter case, we found that tolerance induction triggered recessive mechanisms leading to elimination of effector cells and, simultaneously, a dominant mechanism associated with the emergence of an anergic and regulatory CTLA-4+IL-2lowFoxp3-T cell population, where the tolerance state is IL-10 dependent. Such Foxp3-independent mechanisms can improve the efficacy of tolerance-inducing protocols. Fundacao para a Ciencia e Tecnologia grant: (HMSP-ICT/0034/2013); FEDER through POR Lisboa 2020–Programa Operacional Regional de Lisboa grant: (LISBOA-01-0145-FEDER-007391). info:eu-repo/semantics/publishedVersion

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works, Germinal center (GC) responses are controlled by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells and are crucial for the generation of high-affinity antibodies. Although the biology of human circulating and tissue Tfh cells has been established, the relationship between blood and tissue Tfr cells defined as CXCR5+Foxp3+T cells remains elusive. We found that blood Tfr cells are increased in Sjögren syndrome, an autoimmune disease with ongoing GC reactions, especially in patients with high autoantibody titers, as well as in healthy individuals upon influenza vaccination. Although blood Tfr cells correlated with humoral responses, they lack full B cell–suppressive capacity, despite being able to suppress T cell proliferation. Blood Tfr cells have a naïve-like phenotype, although they are absent from human thymus or cord blood. We found that these cells were generated in peripheral lymphoid tissues before T-B interaction, as they are maintained in B cell–deficient patients. Therefore, blood CXCR5+Foxp3+ T cells in human pathology indicate ongoing humoral activity but are not fully competent circulating Tfr cells., This study was funded by HMSP-ICT/0034/2013, FAPESP/19906/2014, PTDC/IMI-IMU/7038/2014 research grants, and LISBOA-01-0145-FEDER-007391, projeto cofinanciado pelo FEDER através POR Lisboa 2020–Programa Operacional Regional de Lisboa, do PORTUGAL 2020, e pela Fundação para a Ciência e a Tecnologia. The vaccination study was funded by the European Research Council Starting Grant TWILIGHT (to M.A.L.). W.P. was funded by a Newton International Fellowship from the Royal Society. M.A.L. was funded by the Bioscience and Biotechnology Research Council.

IL-9 is a pro-inflammatory cytokine implicated in certain immune-mediated diseases where chronic or acute inflammation of the mucosa plays an important role. Although initially described as being produced by what was then thought to be Th2 cells, it was later described that specialized lymphocyte populations are involved in IL-9 production. In addition to the classical Th9 effector (subset of CD4+ T cells), IL-9 is also produced by nonconventional lymphocytes, namely invariant natural killer T (iNKT) cells and innate lymphoid cells (ILCs). The identification of IL-9-producing cells by flow cytometry and cytokine measurements are pivotal for assigning and defining functional cellular phenotypes. In this chapter we provide methods for the in vitro polarization of IL-9-producing nonconventional lymphocytes and the best conditions for the detection of IL-9 production by intracellular staining.

The Journal of Rheumatology Copyright © 2013. All rights reserved., Objective: B cells play important roles in rheumatoid arthritis (RA). Given the beneficial effect of B cell depletion therapy in RA as well as the observed alterations in B cell subpopulations in this disease, we evaluated whether changes in the expression of genes related to B cell survival and activation were already present in patients with untreated very early RA (VERA; < 6 weeks of disease duration). Methods: The expression of a group of B cell-related activation and survival genes was quantified in peripheral blood mononuclear cells from patients with VERA by real-time PCR and compared with untreated early RA (< 1 year), established treated RA, and other untreated early arthritis conditions. Serum B cell-activating factor belonging to the tumor necrosis factor family (BAFF) was quantified by ELISA. Results: BAFF gene expression and serum levels were highest in patients with VERA. The expression of BAFF receptor (BAFF-R) increased with disease progression, while transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) was elevated since the first weeks of RA onset. Paired box 5 gene expression was also increased at all RA stages. Chemokine (C-X-C motif) receptor 5 was elevated only in established RA. No differences were observed in B cell maturation antigen, activation-induced cytidine deaminase, B lymphocyte-induced maturation protein, and B cell lymphoma 2 expression. Conclusion: Disturbances in the expression of B cell-related activation and survival genes, particularly BAFF and TACI, occur from the onset of RA and precede changes in BAFF-R. These alterations can lead to the development of autoreactive B cells from the first weeks of RA onset., Supported by a grant from Sociedade Portuguesa de Reumatologia/Schering-Plough 2005. RAM was funded by a fellowship from Fundação para a Ciência e a Tecnologia (SFRH/BD/30247/2006).

Foxp3 + CD4 T cells are a specialized regulatory T (Treg) lymphocyte population that plays a unique role in maintaining peripheral tolerance, preventing autoimmune diseases, and limiting chronic inflammation. However, deregulated action of Treg cells may also limit beneficial responses by suppressing immunity against microbes and tumors. Tregs are generated during the normal process of T cell development in the thymus, but may also differentiate in the periphery from conventional T cells. In addition to development, Treg cells are also heterogeneous regarding functional activity, migratory potential, and activation status. Because they have a great impact on the balance between tolerance and autoimmunity, there is great interest in understanding Treg generation, diversity, activation requirements, stability, trafficking, homeostasis, and mechanisms of action in vivo for the development of therapeutic strategies to modulate Treg function in the clinic. In this chapter, we will discuss these topics in the light of current knowledge of the Treg field.

© 2010, American College of Rheumatology, Objective. CD8+ T cells are part of the T cell pool infiltrating the synovium in rheumatoid arthritis (RA). However, their role in the pathogenesis of RA has not been fully delineated. Using the K/BxN mouse model of spontaneous chronic arthritis, which shares many similarities with RA, we studied the potential of CD8 T cell depletion with monoclonal antibodies (mAb) to stop and reverse the progression of experimental arthritis. Methods. CD8+ T cells from the blood and articular infiltrate of K/BxN mice were characterized for cell surface phenotypic markers and for cytokine production. Additionally, mice were treated with specific anti-CD8 mAb (YTS105 and YTS169.4), with and without thymectomy. Results. CD8+ T cells from the peripheral blood and joints of K/BxN mice were mainly CD69+ and CD62L-CD27+ T cells expressing proinflammatory cytokines (interferon-γ [IFNγ], tumor necrosis factor α [TNFα], interleukin-17a [IL-17A], and IL-4), and granzyme B. In mice receiving anti-CD8 mAb, the arthritis score improved 5 days after treatment. Recovery of the CD8+ T cells was associated with a new increase in the arthritis score after 20 days. In thymectomized and anti-CD8 Ab–treated mice, the arthritis score improved permanently. Histologic analysis showed an absence of inflammatory infiltrate in the anti-CD8 mAb–treated mice. In anti-CD8 mAb–treated mice, the serologic levels of TNFα, IFNγ, IL-6, and IL-5 normalized. The levels of the disease-related anti–glucose-6-phosphate isomerase antibodies did not change. Conclusion. These results indicate that synovial activated effector CD8 T cells locally synthesize proinflammatory cytokines (IFNγ, TNFα, IL-17, IL-6) and granzyme B in the arthritic joint, thus playing a pivotal role in maintaining chronic synovitis in the K/BxN mouse model of arthritis., Mr. Raposo’s work was supported by the Instituto do Emprego e Formação Profissional (grant 266/EP/2006) and the Fundação para a Ciência e Tecnologia (grant SFRH/BD/40658/2007). Ms Carvalheiro’s work was supported by the Fundação para a Ciência e Tecnologia (grant SFRH/BD/60467/2009). Dr. Graça’s work was supported by the Fundação para a Ciência e Tecnologia (grant POCI/SAU-MMO/55974/2004). Dr. Souto-Carneiro’s work was supported by a Marie Curie Intra-European Fellowship (LIF-025885) and a EULAR Young Investigator Award.

Copyright © 2015 R. N. Bárcia et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1β, IL-8, LIF and TGFβ2., This study was partially supported by PORLisboa-FEDER (QREN 30196-ClinUCX; QREN 024765-OSCAR).

To maintain immunological balance the organism has to be tolerant to self while remaining competent to mount an effective immune response against third-party antigens. An important mechanism of this immune regulation involves the action of regulatory T-cell (Tregs). In this mini-review, we discuss some of the known and proposed mechanisms by which Tregs exert their influence in the context of immune regulation, and the contribution of mathematical modeling for these mechanistic studies. These models explore the mechanisms of action of regulatory T cells, and include hypotheses of multiple signals, delivered through simultaneous antigen presenting cell (APC) conjugation; interaction of feedback loops between APC, Tregs and effector cells; or production of specific cytokines that act on effector cells. As the fields matures, and competing models are winnowed out, it is likely that we will be able to quantify how tolerance-inducing strategies, such as CD4-blockade, affect T-cell dynamics and what mechanisms explain the observed behavior of T-cell based tolerance.

© 2013 Australasian Society for Immunology Inc., Non-depleting anti-CD4 monoclonal antibodies (MAbs) induce long-term dominant tolerance mediated by regulatory T cells in several animal models of transplantation, allergy and autoimmunity. However, despite many studies on tolerance induction following CD4 blockade, the consequences of this intervention on T-cell kinetics are still unknown. Mathematical models have been useful to understand lymphocyte dynamics, estimating rates of proliferation and cell death following an intervention. Using the same strategy, we found that CD4(+) T cells activated in vitro in the presence of non-depleting anti-CD4 MAbs are prevented from undergoing optimal proliferation and show a higher frequency of apoptosis. Although the changes are small, during the course of a proliferative response, they lead to very distinct final levels of cell numbers. The importance of these mechanisms, predicted by the mathematical model, was validated by showing that lck-driven Bcl-x(L) transgenic mice, bearing T cells resistant to apoptosis, fail to become tolerant to skin grafts following CD4-blockade. Our data show that, in addition to induction of regulatory T cells, CD4 blockade has a marked effect in the effector T-cell pool by the combined action of hindering proliferation while favoring apoptosis. It is, therefore, the combination of all those mechanisms that leads to stable tolerance., ; ;, This work was funded by grants number PIC/IC/82895/2007 and PTDC/SAU-TOX/114424/2009 from Fundação para a Ciência e Tecnologia, Portugal (to Luis Graca). Ruy M Ribeiro has received funding from the European Union 7th Framework Program under grant n1 PCOFUND-GA-2009-246542 and from Fundação para a Ciência e Tecnologia, Portugal

The introduction in the organism of tissues expressing foreign genes—ranging from major histocompatibility antigens to the products of gene therapy—have in common their ability to elicit protective immune responses leading to their rejection. Different strategies have been proposed to overcome immune rejection. Monoclonal antibodies, targeting molecules involved in the molecular events required for T cell activation, offer the promise of resetting the immune system toward tolerance without compromising overall immune competence. The mechanisms leading to immune tolerance rely not only on the induction of regulatory T cells, but also on the elimination of aggressive clones, and the triggering of specific gene expression programs that contribute to self-defense of the target tissue.

Monoclonal antibodies (mAb) have been shown effective in inducing immune tolerance in a range of animal models of autoimmunity, allergy, and transplantation. We investigated whether CD4-blockade, effective in inducing transplantation tolerance, could prevent systemic immune responses leading to anaphylaxis. We found that treatment with a non-depleting anti-CD4 mAb could prevent peanut-induced anaphylaxis following subsequent systemic exposure to crude peanut extract (CPE). Furthermore, the effect of CD4-blockade did not interfere with overall immune competence, as anti-CD4 treated mice remained fully competent to respond to unrelated antigens. Protection from anaphylaxis correlated with increased frequency of Foxp3+ regulatory T cells (Treg), and was abrogated following Treg depletion. Taken together our data suggest that activation of T cells by CPE in presence of CD4-blockade leads to Treg expansion that can prevent peanut-induced anaphylaxis.

Allergic airways disease is a consequence of a Th2 response to an allergen leading to a series of manifestations such as production of allergen-specific IgE, inflammatory infiltrates in the airways, and airway hyper-reactivity (AHR). Several strategies have been reported for tolerance induction to allergens leading to protection from allergic airways disease. We now show that CD4 blockade at the time of house dust mite sensitization induces antigen-specific tolerance in mice. Tolerance induction is robust enough to be effective in pre-sensitized animals, even in those where AHR was pre-established. Tolerant mice are protected from airways eosinophilia, Th2 lung infiltration, and AHR. Furthermore, anti-CD4 treated mice remain immune competent to mount immune responses, including Th2, to unrelated antigens. Our findings, therefore, describe a strategy for tolerance induction potentially applicable to other immunogenic proteins besides allergens.

©The Author 2010. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved., Objective. To characterize circulating B-cell subpopulations of arthritis patients with, This work was supported by a grant from Sociedade Portuguesa de Reumatologia/Schering- Plough 2005. R.A.M. was funded by Fundação para a Ciência e Tecnologia (FCT) SFRH/BD/30247/2006, and M.M.S.-C. by Marie Curie Intra-European Fellowship LIF-025885 and a EULAR Young Investigator Award.

In the last decade there has been an increasing interest for the action of regulatory T cells (Treg) in preventing transplant rejection, autoimmunity and other inflammatory diseases and in maintaining dominant tolerance. It is becoming clear that such regulatory function does not rely simply on direct inhibition of "aggressive" T cells. In fact, several studies suggest that Treg cells may induce changes in the target tissue, promoting a state of "immune privilege" where protective genes such as heme oxygenase-1 (HO-1) and indolamine 2,3 dioxygenase (IDO) may have a critical role.

Our ability to harness tolerance mechanisms will have a major impact in organ transplantation. It should enable drug minimization, and eventually, the elimination of all immunosuppressive drugs. An improved understanding of the biology of regulatory T cells will make it possible to replace current induction regimens with those favouring the selective vaccination of T cells that prevent graft rejection. Once regulation is established, the continued supply of graft antigens should empower T cell regulation to become the dominant natural mechanism to prevent graft rejection.

BACKGROUND: Allografts are occasionally accepted in the absence of immunosuppression. Because naturally occurring CD4(+)CD25(+) regulatory T cells (natural CD25(+) Treg cells) have been shown to inhibit allograft rejection, we investigated their influence on the outcome of allografts in nonimmunosuppressed mouse recipients. METHODS: We compared survival times of male CBA/Ca skin grafts in female CBA/Ca recipients expressing a transgenic anti-HY T-cell receptor on a RAG-1(+/+) (A1[M]RAG+) or a RAG-1(-/-) (A1[M]RAG-) background. Depletion of natural CD25(+) Treg cells in A1[M]RAG+ mice was achieved by in vivo administration of the PC61 monoclonal antibody. The influence of natural CD25(+) Treg cells on the fate of major histocompatibility complex class II-mismatched (C57BL/6X bm12)F1 skin or bm12 heart transplants in C57BL/6 recipients was also assessed. Finally, we investigated the impact of natural CD25(+) Treg cells on the production of T-helper (Th)1 and Th2 cytokines in mixed lymphocyte cultures between C57BL/6 CD4(+) CD25(-) T cells as responders and bm12 or (C57BL/6X bm12)F1 antigen-presenting cells as stimulators. RESULTS: Male allografts were spontaneously accepted by female A1(M)RAG+ mice but readily rejected by female A1(M)RAG+ mice depleted of natural CD25(+) Treg cells by pretreatment with the PC61 monoclonal antibody. Depletion of CD25(+) Treg cells also enhanced eosinophil-determined rejection of (C57BL/6X bm12)F1 skin grafts or bm12 cardiac grafts in C57BL/6 recipients. Finally, natural CD25(+) Treg cells inhibited the production of interleukin (IL)-2, interferon-gamma, IL-5, and IL-13 in mixed lymphocyte culture in a dose-dependent manner. CONCLUSION: Natural CD25(+) Treg cells control Th1- and Th2-type allohelper T-cell responses and thereby influence the fate of allografts in nonimmunosuppressed recipients., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Alloreactive CD8+ T cells may persist in animals made tolerant of transplanted tissues; their function is controlled through continuous censorship by regulatory CD4+ T cells. We sought to establish the stage at which such censorship operates. We found that monospecific CD8+ T cells introduced into tolerant animals responded to the tolerated tissue antigen as if they had received CD4+ T cell "help": they proliferated and accumulated normally. However, they did show compromised graft rejection, interferon-gamma production and cell-mediated cytotoxicity. These findings suggest that tolerance mediated by regulatory T cells acts by censoring immune effector functions rather than by limiting the induction of T cell responses.

Induction of transplantation tolerance with certain therapeutic nondepleting monoclonal antibodies can lead to a robust state of peripheral “dominant” tolerance. Regulatory CD4+ T cells, which mediate this form of “dominant” tolerance, can be isolated from spleens of tolerant animals. To determine whether there were any extra-lymphoid sites that might harbor regulatory T cells we sought their presence in tolerated skin allografts and in normal skin. When tolerated skin grafts are retransplanted onto T cell–depleted hosts, graft-infiltrating T cells exit the graft and recolonize the new host. These colonizing T cells can be shown to contain members with regulatory function, as they can prevent nontolerant lymphocytes from rejecting fresh skin allografts, without hindrance of rejection of third party skin. Our results suggest that T cell suppression of graft rejection is an active process that operates beyond secondary lymphoid tissue, and involves the persistent presence of regulatory T cells at the site of the tolerated transplant.

Background B cells play several important roles in rheumatoid arthritis (RA). Previous studies by our group have demonstrated that very early rheumatoid arthritis (VERA) patients have disturbances in peripheral blood memory B cells and increased circulating B cell related cytokines. Objectives The main goal of this work was to analyse the expression of genes related with B cell survival and activation in VERA ( Materials and methods BAFF, BAFF-R, TACI, BCMA, AID, CXCR5, Pax5, Blimp-1, β2m and Bcl-2 mRNA levels from peripheral blood mononuclear cell (PBMC) were determined by real-time PCR. Serum BAFF was quantified by ELISA. Results VERA patients had increased BAFF and TACI mRNA levels in comparison with controls. Moreover, BAFF mRNA expression was higher in VERA in comparison with RA and VEA patients. BAFF-R expression was elevated in ERA and RA when compared to controls. Pax5 expression was increased in all RA patients and reduced in VEA in comparison with VERA. β2m levels were elevated in VERA, ERA and VEA patients when compared to controls. No differences were observed in BCMA, AID, CXCR5, Blimp-1 and Bcl-2 expression. Furthermore, VERA patients had the highest serum BAFF levels. Conclusions Disturbances in the expression of B cell related activation genes, particularly BAFF and TACI, occur since the very early phase of RA. The increased BAFF serum and mRNA levels occurring in VERA patients might indicate BAFF as a potential therapeutic target since the first weeks of RA onset.

CD8 T cells are part of the T cell pool infiltrating the rheumatoid synovial membrane. Moreover, CD8 T cells have been linked to the formation of synovial ectopic germinal centres in rheumatoid arthritis (RA), and they produce proinflammatory cytokines. Hence, CD8 T cells appear to be intimately involved in initiating and maintaining the chronic inflammation in the RA synovium. Nevertheless, studies on animal models of …

Background: A major challenge in the application of marrow transplantation as a route to immunological tolerance of a transplanted organ is to achieve hematopoietic stem cell (HSC) engraftment with minimal myelosuppressive treatments. Results: We here describe a combined antibody protocol which can achieve long-term engraftment with clinically relevant doses of MHC-mismatched bone marrow, without the need for myelosuppressive drugs. Although not universally applicable in all strains, we achieved reliable engraftment in permissive strains with a two-stage strategy: involving first, treatment with anti-CD8 and anti-CD4 in advance of transplantation; and second, treatment with antibodies targeting CD4, CD8 and CD40L (CD154) at the time of marrow transplantation. Long-term mixed chimerism through co-receptor and co-stimulation blockade facilitated tolerance to donor-type skin grafts, without any evidence of donor-antigen driven regulatory T cells. Conclusion: We conclude that antibodies targeting co-receptor and co-stimulatory molecules synergise to enable mixed hematopoietic chimerism and central tolerance, showing that neither cytoreductive conditioning nor 'megadoses' of donor bone marrow are required for donor HSC to engraft in permissive strains.

The pathogenesis of multiple sclerosis requires the participation of effector neuroantigen-specific T cells. Thus, T cell targeting has been proposed as a promising therapeutic strategy. However, the mechanism underlying effective disease prevention following T cell targeting remains incompletely known. We found, using several TCR-transgenic strains, that CD4 blockade is effective in preventing experimental autoimmune encephalopathy and in treating mice after the disease onset. The mechanism does not rely on direct T cell depletion, but the anti-CD4 mAb prevents the proliferation of naive neuroantigen-specific T cells, as well as acquisition of effector Th1 and Th17 phenotypes. Simultaneously, the mAb favors peripheral conversion of Foxp3+ regulatory T cells. Pre-existing effector cells, or neuroantigen-specific cells that undergo cell division despite the presence of anti-CD4, are committed to apoptosis. Therefore, protection from experimental autoimmune encephalopathy relies on a combination of dominant mechanisms grounded on regulatory T cell induction and recessive mechanisms based on apoptosis of neuropathogenic cells. We anticipate that the same mechanisms may be implicated in other T cell-mediated autoimmune diseases that can be treated or prevented with Abs targeting T cell molecules, such as CD4 or CD3.