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SARS-CoV-2 omicron subvariants BA.1 and BA.2 became dominant in many countries in early 2022. These subvariants are now being displaced by BA.4 and BA.5. While natural infection with BA.1/BA.2 provides some protection against BA.4/BA.5 infection, the duration of this protection remains unknown.We used the national Portuguese COVID-19 registry to investigate the waning of protective immunity conferred by prior BA.1/BA.2 infection towards BA.5. We divided the individuals infected during the period of BA.1/BA.2 dominance (>90% of sample isolates) in successive 15-day intervals and determined the risk of subsequent infection with BA.5 over a fixed period.Compared with uninfected people, one previous infection conferred substantial protection against BA.5 re-infection at 3 months (RR=0.12; 95% CI: 0.11-0.12). However, although still significant, the protection was reduced by two-fold at 5 months post-infection (RR=0.24; 0.23-0.24).These results should be interpreted in the context of vaccine breakthrough infections, as the vaccination coverage in the individuals included in the analyses is >98% since the end of 2021.This waning of protection following BA.1/BA.2 infection highlights the need to assess the stability and durability of immune protection induced with the adapted vaccines (based on BA.1) over time.

© 2021 Wiley-VCH GmbH, The germinal center (GC) reactions are critical for the production of high-affinity antibodies that comprise the protective humoral response elicited by infection or vaccination. GCs are initiated through the interaction of B cells with T follicular helper (Tfh) cells. While the transcriptional regulation of Tfh differentiation has been studied in great detail, the impact of micro RNAs (miRNAs) on Tfh development and stability has been harder to address. It was previously shown that a complete deletion of miRNAs biogenesis prevents Tfh differentiation. In this issue of the European Journal of Immunology [Eur. J. Immunol. 2021. 51: 408-413], Zeiträg et al. use an inducible gene deletion approach to reveal that miRNAs are also required for the maintenance of Tfh cells induced following viral infection in mice. These results provide new clues to the regulation of GC responses through Tfh and T follicular regulatory cells., This research is funded by grant 081/596653860 from Fundação para a Ciência e Tecnologia (FCT), Portugal (to L. Graca).

© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made., Peptidyl-prolyl cis-trans isomerase C (Ppic) is expressed in several bone marrow (BM) hematopoietic progenitors and in T-cell precursors. Since the expression profile of Ppic in the hematoimmune system was suggestive that it could play a role in hematopoiesis and/or T lymphocyte differentiation, we sought to test that hypothesis in vivo. Specifically, we generated a Ppic-deficient mouse model by targeting the endogenous locus by CRISPR/Cas9 and tested the requirement of Ppic in hematopoiesis. Several immune cell lineages covering BM progenitors, lymphocyte precursors, as well as mature cells at the periphery were analyzed. While most lineages were unaffected, invariant NKT (iNKT) cells were reduced in percentage and absolute cell numbers in the Ppic-deficient thymus. This affected the most mature stages in the thymus, S2 and S3, and the phenotype was maintained at the periphery. Additionally, immature transitional T1 and T2 B lymphocytes were increased in the Ppic-deficient spleen, but the phenotype was lost in mature B lymphocytes. In sum, our data show that Ppic is dispensable for myeloid cells, platelets, erythrocytes, αβ, and γδ T lymphocytes in vivo in the steady state, while being involved in B- and iNKT cell differentiation., This work was supported by the Instituto Gulbenkian de Ciência (IGC) of the Calouste Gulbenkian Foundation, and the Portuguese National Research Council (Fundação para a Ciência e Tecnologia [FCT]) Grant PTDC/BIA-BID/30925/2017 to VCM, that also supports the salary of RSP. VCM is supported by an individual contract awarded by FCT (CEECIND/03106/2018). CVR is a PhD student of the IGC Integrative Biology and Biomedicine (IBB) PhD Program and supported by an individual FCT PhD Fellowship ref. PD/BD/139190/2018. This work had the support of the research infrastructures Congento LISBOA-01-0145-FEDER-022170 and PPBI-POCI-01-0145-FEDER-022122, both cofinanced by FCT and Lisboa2020, under PORTUGAL2020 agreement (European Regional Development Fund).

People with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) often report a high frequency of viral infections and flu-like symptoms during their disease course. Given that this reporting agrees with different immunological abnormalities and altered gene expression profiles observed in the disease, we aimed at answering whether the expression of the human angiotensin-converting enzyme 2 (ACE2), the major cell entry receptor for SARS-CoV-2, is also altered in these patients. In particular, a low expression of ACE2 could be indicative of a high risk of developing COVID-19. We then performed a meta-analysis of public data on CpG DNA methylation and gene expression of this enzyme and its homologous ACE protein in peripheral blood mononuclear cells and related subsets. We found that patients with ME/CFS have decreased methylation levels of four CpG probes in the ACE locus (cg09920557, cg19802564, cg21094739, and cg10468385) and of another probe in the promoter region of the ACE2 gene (cg08559914). We also found a decreased expression of ACE2 but not of ACE in patients when compared to healthy controls. Accordingly, in newly collected data, there was evidence for a significant higher proportion of samples with an ACE2 expression below the limit of detection in patients than healthy controls. Altogether, patients with ME/CFS can be at a higher COVID-19 risk and, if so, they should be considered a priority group for vaccination by public health authorities. To further support this conclusion, similar research is recommended for other human cell entry receptors and cell types, namely, those cells targeted by the virus., Myalgic encephalomyelitis/chronic fatigue syndrome, SARS-CoV-2, ACE2, Gene expression, DNA methylation

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.

The production of antibodies can constitute a powerful protective mechanism against infection, but antibodies can also participate in autoimmunity and allergic responses. Recent advances in the understanding of the regulation of germinal centres (GC), the sites where B cells acquire the ability to produce high-affinity antibodies, offered new prospects for the modulation of antibody production in autoimmunity and vaccination. The process of B cell affinity maturation and isotype switching requires signals from T follicular helper (Tfh) cells. In addition, Foxp3+ T follicular regulatory (Tfr) cells represent the regulatory counterpart of Tfh in the GC reaction. Tfr cells were identified one decade ago and since then it has become clear their role in controlling the emergence of autoreactive B cell clones following infection and immunization. At the same time, Tfr cells are essential for fine-tuning important features of the humoral response directed to foreign antigens that are critical in vaccination. However, this regulation is complex and several aspects of Tfr cell biology are yet to be disclosed. Here, we review the current knowledge about the regulation of antibody responses against self and foreign antigens by Tfr cells and its implications for the future rational design of safer and more effective vaccines.

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.

Patients affected by Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) show specific epigenetic and gene expression signatures of the disease. However, it is unknown whether these signatures include abnormal levels of the human angiotensin-converting enzymes, ACE and ACE2, the latter being the main receptor described for the host-cell invasion by SARS-CoV-2. To investigate that, we first re-analyzed available case-control epigenome-wide association studies based on DNA methylation data, and case-control gene expression studies based on microarray data. From these published studies, we found an association between ME/CFS and 4 potentially hypomethylated probes located in the ACE locus. We also found another disease association with one hypomethylated probe located in the transcription start site of ACE2. The same disease associations were obtained for women but not for men after performing sex-specific analyses. In contrast, a meta-analysis of gene expression levels could not provide evidence for a differentially expression of ACE and ACE2 in affected patients when compared to healthy controls. In line with this negative finding, the analysis of a new data set on the gene expression of ACE and ACE2 in peripheral blood mononuclear cells did not find any differences between a female cohort of 37 patients and 34 age-matched healthy controls. Future studies should be conducted to extend this investigation to other potential receptors used by SARS-CoV-2. These studies will help researchers and clinicians to improve the understanding of the health risk imposed by this virus when infecting patients affected by this debilitating disease.

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).

Research Areas: Immunology ; Research & Experimental Medicine Development of new immunogens eliciting broadly neutralizing antibodies (bNAbs) is a main priority for the HIV-1 vaccine field. Envelope glycoproteins from non-B-non-C HIV-1clades have not been fully explored as components of a vaccine. We produced Vaccinia viruses expressing a truncated version of gp120 (gp120t) from HIV-1 clades CRF02_AG, H, J, B, and C and examined their immunogenicity in mice and rabbits. Mice primed with the recombinant Vaccinia viruses and boosted with the homologous gp120t or C2V3C3 polypeptides developed antibodies that bind potently to homologous and heterologous envelope glycoproteins. Notably, a subset of mice immunized with the CRF02_AG-based envelope immunogens developed a cross-reactive neutralizing response against tier 2 HIV-1 Env-pseudoviruses and primary isolates. Rabbits vaccinated with the CRF02_AG-based envelope immunogens also generated potent binding antibodies, and one animal elicited antibodies that neutralized almost all (13 of 16, 81.3%) tier 2 HIV-1 isolates tested. Overall, the results suggest that the novel CRF02_AG-based envelope immunogens and prime-boost immunization strategy elicit the type of immune responses required for a preventive HIV-1 vaccine. info:eu-repo/semantics/publishedVersion

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.

It has long been known that CD4 T cells are necessary to provide help to B cells, triggering a germinal centre (GC) reaction where affinity maturation and isotype switching occur. However, the nature of the dedicated CD4 helper T cells, known as T follicular helper (Tfh), was only recently described. Here, we review the biology and function of the recently described T follicular regulatory (Tfr) cells, another CD4 T-cell population also found within GCs but with regulatory function and characteristics. Tfr cells have been identified in mice and humans as simultaneously presenting characteristics of T follicular cells (namely CXCR5 expression) and regulatory T cells (including Foxp3 expression). These Tfr cells have been implicated in the regulation of the magnitude of the GC reaction, as well as in protection from immune-mediated pathology.

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.

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.

Selection from a phage display library derived from human Interleukin-2 (IL-2) yielded mutated variants with greatly enhanced display levels of the functional cytokine on filamentous phages. Introduction of a single amino acid replacement selected that way (K35E) increased the secretion levels of IL-2-containing fusion proteins from human transfected host cells up to 20-fold. Super-secreted (K35E) IL-2/Fc is biologically active in vitro and in vivo, has anti-tumor activity and exhibits a remarkable reduction in its aggregation propensity- the major manufacturability issue limiting IL-2 usefulness up to now. Improvement of secretion was also shown for a panel of IL-2-engineered variants with altered receptor binding properties, including a selective agonist and a super agonist that kept their unique properties. Our findings will improve developability of the growing family of IL-2-derived immunotherapeutic agents and could have a broader impact on the engineering of structurally related four-alpha-helix bundle cytokines.

© 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

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

Costimulation blockade has been one of the most studied strategies to achieve immune tolerance, particularly in transplantation. Yet, in spite of the robust nature of the tolerance-inducing potential of costimulation blockade, a comprehensive understanding of the molecular and cellular mechanisms underlying tolerance induction is still missing. Nevertheless, progress has been continuously made. In this issue of the European Journal of Immunology, Chai et al. [Eur. J. Immunol. 2015. 45: 2017-2027] show that transplantation tolerance induced with an anti-CD154 monoclonal antibody relies on the coexistence of several tolerogenic mechanisms rather than one simple regulatory mechanism. These observations highlight the importance of concerted actions involving multiple pathways, namely apoptosis, acquisition of regulatory cells, or inhibition of proliferation, all of which contribute to the induction and maintenance of robust immune tolerance. A better understanding of these distinct tolerogenic pathways may lead to the development of better tolerance-inducing therapeutics.

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.

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. Here, we found that blood Tfr cells are increased in Sjögren syndrome, especially in patients with high autoantibody titres, as well as in healthy subjects upon influenza vaccination. While, 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. Here, we found these cells were generated in peripheral lymphoid tissues prior to 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 competent Tfr cells.

© The Author(s) 2017. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/, Immunization leads to the formation of germinal centres (GCs) that contain both T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. Whether T-cell receptor (TCR) specificity defines the differential functions of Tfh and Tfr cells is unclear. Here we show that antigen-specific T cells after immunization are preferentially recruited to the GC to become Tfh cells, but not Tfr cells. Tfh cells, but not Tfr cells, also proliferate efficiently on restimulation with the same immunizing antigen in vitro. Ex vivo TCR repertoire analysis shows that immunization induces oligoclonal expansion of Tfh cells. By contrast, the Tfr pool has a TCR repertoire that more closely resembles that of regulatory T (Treg) cells. Our data thus indicate that the GC Tfh and Tfr pools are generated from distinct TCR repertoires, with Tfh cells expressing antigen-responsive TCRs to promote antibody responses, and Tfr cells expressing potentially autoreactive TCRs to suppress autoimmunity., The research was funded by Fundação para Ciência e Tecnologia (FCT) grants PTDC/SAU-IMU/120225/2010, HMSP-ICT/0034/2013 and FCT-FAPESP/19906/2014 (to L.G.). A.R.M. and S.C.P.A. are funded by FCT scholarships SFRH/BD/88030/2012 and SFRH/BDP/81391/2011, respectively. The work by D.K., A.S., E.M.-F., W.C. and F.J. has been funded by Assistance Publique-Hôpitaux de Paris, Université Pierre and Marie Curie (Paris VI), LabEx Transimmunom (ANR-11-IDEX-0004-02) and ERC Advanced Grant TRiPoD (322856). The work by J.F. has been supported by PIRSES-GA-2012-317893 (7th FP, EU) and BIOCAPS (FP7/REGPOT-2012-2013.1, EC) under grant agreement no. 316265.

Poly(lactic acid) (PLA) is one of the most successful and versatile polymers explored for controlled delivery of bioactive molecules. Its attractive properties of biodegradability and biocompatibility in vivo have contributed in a meaningful way to the approval of different products by the FDA and EMA for a wide range of biomedical and pharmaceutical applications, in the past two decades. This polymer has been widely used for the preparation of particles as delivery systems of several therapeutic molecules, including vaccines. These PLA vaccine carriers have shown to induce a sustained and targeted release of different bacterial, viral and tumor-associated antigens and adjuvants in vivo, triggering distinct immune responses. The present review intends to highlight and discuss the major advantages of PLA as a promising polymer for the development of potent vaccine delivery systems against pathogens and cancer. It aims to provide a critical discussion based on preclinical data to better understand the major effect of PLA-based carrier properties on their interaction with immune cells and thus their role in the modulation of host immunity. Statement of Significance During the last decades, vaccination has had a great impact on global health with the control of many severe diseases. Polymeric nanosystems have emerged as promising strategies to stabilize vaccine antigens, promoting their controlled release to phagocytic cells, thus avoiding the need for multiple administrations. One of the most promising polymers are the aliphatic polyesters, which include the poly(lactic acid). This is a highly versatile biodegradable and biocompatible polymer. Products containing this polymer have already been approved for all food and some biomedical applications. Despite all favorable characteristics presented above, PLA has been less intensively discussed than other polymers, such as its copolymer PLGA, including regarding its application in vaccination and particularly in tumor immunotherapy. The present review discusses the major advantages of poly(lactic acid) for the development of potent vaccine delivery systems, providing a critical view on the main properties that determine their effect on the modulation of immune cells.

Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the fourth cause of cancer death worldwide. It is respossible for approximately 7 00 000 deaths per year. This work focused on the development of a combinatorial multivalent nanoplatform for CRC immunotherapy and immunomodulation based on the design of polymeric nanoparticles (NP) able to deliver a combination of CRC-associated antigen, adjuvants and gene regulators according to targeted cells, dendritic cell (DC) and CRC cells. Material and methods Poly(lactic-co-glycolic) (PLGA)-based NP were prepared by the double emulsion (w/o/w) solvent evaporation method. NP were physicochemically characterised in terms of size, zeta potential and surface morphology. CRC antigen loadings were quantified by fluorescence. Immature DC (ATCC ® CRL-11904 TM ) were used to evaluate the in vitro NP cytotoxicity by the propidium iodide assay, as well as NP cellular uptake profile by flow cytometry. In vivo biodistribution assay of plain NP was also performed using the IVIS Lumina ® Bioimaging system. NP uptake in vivo by myeloid antigen presenting cells and the expression of maturation and co-stimulatory molecules at the surface of these cells sorted within draining lymph nodes, were also evaluated by flow cytometry. Results and discussions PLGA-based NP presented a mean size diameter close to 200 nm, with low polydispersity index (PdI) (≤0.200), a surface charge close to neutrality, as well as, a spherical shape and smooth surface. These multivalent delivery systems presented high loadings for antigen and adjuvants. No cytotoxic effect was observed on immature DC up to 48 hour of incubation. NP were extensively internalised by immature DC in vitro after 48 hour incubation, and by migratory DC in vivo 17 hour after animal immunisation. In vivo real-time monitoring of NP accumulation in mice whole bodies and dissected organs showed a fluorescent signal at 17 hour close to the site of immunisation and in the lymph nodes. No significant differences in the expression of the co-stimulatory CD80, CD86 and MHC class I markers on CD11b + CD11c + MHCII + population at lymph nodes were observed among different polymeric combinations upon mice immunisation with NP carrying CRC antigen and adjuvant. Conclusion According to NP physicochemical characteristics, internalisation and biodistribution patterns, this innovative nanoplatform can lead to a safe multivalent nanomedicine able to modulate dendritic cell activity and T cell expansion against tumour cells expressing entrapped antigens.

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).