Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

Adoptive Transfer and Bone Marrow Chimera Models to Analyze Treg Function and Differentiation.

Cellular adoptive transfer and mixed bone marrow chimera are cornerstone experimental tools for immuno-biology. Here we describe protocols for adoptive transfer and bone marrow chimera to address the effect of a specific mutation on T regulatory cell (Treg) function and differentiation, respectively. Treg function can be quantitatively measured by analyzing the expansion of conventional CD4 T cells and their differentiation into helper cells. The quantitative measure of Treg differentiation is addressed by analyzing the number and phenotype of Foxp3-expressing cells. The use of congenic markers is instrumental for these approaches.

Differentiation of Peripheral Treg.

This chapter shows protocols for the differentiation of peripheral Treg (pTreg) from polyclonal and monoclonal CD4+ T cells. Polyclonal naïve CD4+ T cells can differentiate into pTreg upon adoptive transfer into Foxp3-diphtheria toxin receptor transgenic recipient mice in which endogenous Tregs are transiently depleted by administration of diphtheria toxin before adoptive transfer. Differentiation of monoclonal pTreg is induced through oral delivery of ovalbumin into RAG-deficient DO11.10 mice, in which T cells are ovalbumin specific. We show the isolation of naïve CD4+ T cells by flow cytometry, the administration of ovalbumin in drinking water, and the analysis tools, including an optional protocol for the enrichment of analysis samples in CD4+ T cells using a magnetic purification.

Effect of Smoking on Treatment Efficacy and Toxicity in Patients with Cancer: A Systematic Review and Meta-Analysis.

AIM: The aim of the present systematic review and meta-analysis was to summarize the current evidence on the potential impact of smoking during cancer treatment on treatment efficacy and toxicity irrespective of cancer type. METHODS: A systematic literature search was performed using two electronic databases for potentially eligible studies. Only studies based on multivariable analysis for the association between smoking, compared to non-smokers (never or former), and treatment efficacy or toxicity were included. Pooled Hazard Ratios (HRs) or Odds Ratios (ORs) and corresponding 95% Confidence Intervals (CIs) were estimated through random-effects meta-analyses. RESULTS: In total, 97 eligible studies were identified, of which 79 were eligible for the pooled analyses. Smoking during radiation therapy, with or without chemotherapy, was associated with an increased risk of locoregional recurrence (pooled HR: 1.56; 95% CI: 1.28-1.91 for radiation therapy; pooled HR: 4.28; 95% CI: 2.06-8.90 for chemoradiotherapy) and worse disease-free survival (pooled HR: 1.88; 95% CI: 1.21-2.90 for radiation therapy; pooled HR: 1.92; 95% CI: 1.41-2.62 for chemoradiotherapy) as well as a higher risk for radiation-induced toxicity (pooled OR: 1.84; 95% CI: 1.32-2.56 for radiation therapy; pooled OR: 2.43; 95% CI: 1.43-4.07 for chemoradiotherapy) with low-to-moderate certainty of evidence. Smoking during treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) in patients with lung cancer was associated with worse progression-free survival compared to non-smokers (pooled HR: 1.43; 95% CI: 1.14-1.80; moderate certainty of evidence), whereas smoking was associated with improved progression-free survival in patients treated with checkpoint inhibitors (HR: 0.70; 95% CI: 0.58-0.84; moderate certainty of evidence). No statistically significant associations were observed between smoking and treatment efficacy or toxicity to chemotherapy. CONCLUSION: The present meta-analysis confirms earlier evidence of the negative impact of smoking during radiation therapy, with or without chemotherapy, on treatment efficacy and radiation-induced toxicity as well as a negative impact of smoking on the efficacy of EGFR-TKIs and a positive impact on the efficacy of checkpoint inhibitors. The evidence is too weak to draw firm conclusions on the potential association between smoking and chemotherapy, whereas there is no evidence for pooled analyses regarding other types of systemic oncological therapy.

Population homogeneity for the antibody response to COVID-19 BNT162b2/Comirnaty vaccine is only reached after the second dose across all adult age ranges.

While mRNA vaccines are administrated worldwide in an effort to contain the COVID-19 pandemic, the heterogeneity of the humoral immune response they induce at the population scale remains unclear. Here, in a prospective, longitudinal, cohort-study, including 1245 hospital care workers and 146 nursing home residents scheduled for BNT162b2 vaccination, together covering adult ages from 19 to 99 years, we analyse seroconversion to SARS-CoV-2 spike protein and amount of spike-specific IgG, IgM and IgA before vaccination, and 3-5 weeks after each dose. We show that immunogenicity after a single vaccine dose is biased to IgG, heterogeneous and reduced with increasing age. The second vaccine dose normalizes IgG seroconversion in all age strata. These findings indicate two dose mRNA vaccines is required to reach population scale humoral immunity. The results advocate for the interval between the two doses not to be extended, and for serological monitoring of elderly and immunosuppressed vaccinees.

Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies.

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.

Production of high-quality SARS-CoV-2 antigens: Impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance.

Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.

Interruption of Thymic Activity in Adult Mice Improves Responses to Tumor Immunotherapy.

The thymus produces precursors of both conventional T cells (Tconv; also known as effector T cells) and regulatory T cells (Treg) whose interactions prevent autoimmunity while allowing efficient protective immune responses. Tumors express a composite of self-antigens and tumor-specific Ags and engage both Tconv and Treg. Along the aging process, the thymus involutes, and tumor prevalence increases, a correlation proposed previously to result from effector cell decline. In this work, we directly tested whether interruption of thymic activity in adult mice affects Foxp3-expressing Treg composition and function and alters tumor immune surveillance. Young adult mice, on two different genetic backgrounds, were surgically thymectomized (TxT) and analyzed or challenged 2 mo later. Cellular analysis revealed a 10-fold decrease in both Tconv and Treg numbers and a bias for activated cells. The persisting Treg displayed reduced stability of Foxp3 expression and, as a population, showed a compromised return to homeostasis upon induced perturbations. We next tested the growth of three tumor models from different tissue origins and/or presenting distinct degrees of spontaneous immunogenicity. In none of these conditions, adult TxT facilitated tumor growth. Rather, TxT enhanced the efficacy of antitumor immunotherapies targeting Treg and/or the immune checkpoint CTLA4, as evidenced by the increased frequency of responder mice and decreased intratumoral Treg to CD8+IFN-γ+ cell ratio. Together, our findings point to a scenario in which abrogation of thymic activities affects preferentially the regulatory over the ridding arm of the immune activities elicited by tumors and argues that higher prevalence of tumors with age cannot be solely attributed to thymic output decline.

Longitudinal Analysis of Antibody Responses to the mRNA BNT162b2 Vaccine in Patients Undergoing Maintenance Hemodialysis: A 6-Month Follow-Up.

Background: Patients on hemodialysis (HD) are at higher risk for COVID-19, overall are poor responders to vaccines, and were prioritized in the Portuguese vaccination campaign. Objective: This work aimed at evaluating in HD patients the immunogenicity of BTN162b2 after the two doses induction phase, the persistence of specific antibodies along time, and factors predicting these outcomes. Methods: We performed a prospective, 6-month long longitudinal cohort analysis of 156 HD patients scheduled to receive BTN162b2. ELISA quantified anti-spike IgG, IgM, and IgA levels in sera were collected every 3 weeks during the induction phase (t0 before vaccine; t1, d21 post first dose; and t2 d21 post second dose), and every 3-4 months during the waning phase (t3, d140, and t4, d180 post first dose). The age-matched control cohort was similarly analyzed from t0 to t2. Results: Upon exclusion of participants identified as previously exposed to SARS-CoV-2, seroconversion at t1 was lower in patients than controls (29 and 50%, respectively, p = 0.0014), while the second vaccine dose served as a boost in both cohorts (91 and 95% positivity, respectively, at t2, p = 0.2463). Lower response in patients than controls at t1 was a singularity of the participants ≤ 70 years (p = 2.01 × 10-05), associated with immunosuppressive therapies (p = 0.013), but not with lack of responsiveness to hepatitis B. Anti-spike IgG, IgM, and IgA levels decreased at t3, with IgG levels further waning at t4 and resulting in >30% seronegativity. Anti-spike IgG levels at t1 and t4 were correlated (ρ = 0.65, p < 2.2 × 10-16). Conclusions: While most HD patients seroconvert upon 2 doses of BNT162b2 vaccination, anti-spike antibodies levels wane over the following 4 months, leading to early seroreversion in a sizeable fraction of the patients. These findings warrant close monitoring of COVID-19 infection in vaccinated HD patients, and advocate for further studies following reinforced vaccination schedules.

The multifaceted Foxp3fgfp allele enhances spontaneous and therapeutic immune surveillance of cancer in mice.

It is well established that therapeutic impairment of Foxp3+ Treg in mice and humans favors immune rejection of solid tumors. Less explored is the impact Foxp3 allelic variants may have on tumor incidence, progression and therapy. In this work, we tested and demonstrate that the Foxp3fgfp reporter allele, found previously to either enhance or reduce Treg function in specific autoimmunity settings, confers increased anti-tumor immunity. Our conclusions stem out of the analysis of three tumor models of different tissue origin, in two murine genetic backgrounds. When compared to wild type animals, mice carrying the Foxp3fgfp allele spontaneously delay, reduce or prevent primary tumor growth, decrease metastasis growth, and potentiate the response to anti-CTLA4 monotherapy. These findings suggest allelic variances at the Foxp3 locus may serve as predictive indicators for personalized therapy and prognostics, and point at possible new therapeutic targets.

Remodelagem cardíaca adicional induzida pelo treinamento militar intenso em atletas de nível competitivo / Additional cardiac remodeling induced by intense military in competitive athletes

Fundamento: A remodelagem cardíaca depende da intensidade, da duração e do método de treinamento. Objetivo: Avaliar se o treinamento realizado em uma tropa especial portuguesa incrementa a remodelagem cardíaca em uma amostra de jovens que previamente praticavam esporte de competição. Métodos: Estudo prospectivo que incluiu 76 militares candidatos a tropas especiais, 45 dos quais praticavam previamente exercício físico de nível competitivo (> 10 horas por semana). Destes militares atletas, apenas 17 concluíram com sucesso o curso. A avaliação foi realizada com 6 meses de intervalo e incluiu história clínica completa, exame físico, sinais vitais, dados antropométricos e avaliação ecocardiográfica. Considerou-se significância estatística quando p < 0,05, com intervalo de confiança de 95%. Resultados: No final do curso, verificaram-se diminuição da porcentagem de massa gorda (19,1 ± 3,3% vs. 13,1 ± 3,5%; p < 0,01), aumento da porcentagem de massa muscular (41,3 ± 2,1% vs. 44,4 ± 1,8%; p < 0,01), e diminuição da pressão arterial sistólica, diastólica e frequência cardíaca. Em relação à remodelagem cardíaca, verificaram-se aumento do diâmetro diastólico do ventrículo esquerdo (49,7 ± 3,2 mm vs. 52,8 ± 3,4 mm; p < 0,01), aumento tendencial do volume do átrio esquerdo (27,3 ± 4,5 mL/m2 vs. 28,2 ± 4,1 mL/m2; p = 0,07) e aumento da massa do ventrículo esquerdo (93,1 ± 7,7g/m2 vs. 100,2 ± 11,4 g/m2; p < 0,01). As variáveis funcionais também sofreram alterações, com aumento do S' (15 (13-16) cm/s vs. 17 (16-18) cm/s; p < 0,01) e diminuição da fração de ejeção do ventrículo esquerdo (60 ± 6% vs. 54 ± 6%; p < 0,01). Conclusão: O treinamento físico militar intenso provocou remodelagem cardíaca adicional em atletas de nível competitivo, tanto estrutural como funcional

Background: Cardiac remodeling depends on the intensity, duration, and training method. Objective: To evaluate if the training performed in a Portuguese military special operations troop increases cardiac remodeling in a sample of young individuals who previously practiced competitive sports. Methods: A prospective study involving 76 military candidates for military special operations, 45 of whom previously practiced at competitive level (> 10 hours per week). Of these military athletes, only 17 successfully completed the course. The evaluation was performed at 6 months intervals and included a complete clinical history, physical examination, vital signs, anthropometric data and echocardiographic evaluation. Statistical significance was considered when p < 0.05, with a 95% confidence interval. Results: At the end of the course, there was a decrease in the percentage of fat mass (19.1 ± 3.3% vs. 13.1 ± 3.5%; p < 0.01), an increase in the percentage of lean mass (41.3 ± 2.1% vs. 44.4 ± 1.8%; p < 0.01), and decreased systolic and diastolic blood pressure and heart rate. Regarding cardiac remodeling, there was an increase in left ventricular diastolic diameter (49.7 ± 3.2 mm vs. 52.8 ± 3.4 mm; p < 0.01), an increase trend in left atrial volume (27.3 ± 4.5 mL/m2 vs. 28.2 ± 4.1 mL/m2; p = 0.07) and increased left ventricular mass (93.1 ± 7.7 g/m2 vs.100.2 ± 11.4 g/m2 ; p < 0.01). Functional variables also changed, with an increase in S' (15 (13-16) cm/s vs. 17 (16-18) cm/s; p < 0,01) and a decrease in left ventricular ejection fraction (60 ± 6% vs. 54 ± 6%; p < 0.01). Conclusion: Intense military physical training resulted in additional cardiac remodeling in athletes of competitive level, both structural and functional

Exercise-induced cardiac remodeling in athletes and in special forces soldiers. / Remodelagem cardíaca induzida pelo exercício físico em atletas de nível competitivo e militares de forças especiais.

INTRODUCTION: Exercise-induced cardiac remodeling is frequent in athletes. This adaptation is structurally manifested by an increase in cardiac dimensions and mass. Soldiers are also subject to intense physical exercise, although with different characteristics. OBJECTIVE: To compare exercise-induced cardiac remodeling in competitive athletes and in soldiers on a special forces training course. METHODS: We studied 17 soldiers (all male and Caucasian, mean age 21±3 years) who completed a special forces course and 17 basketball players (47.3% male, 64.7% Caucasian, mean age 21±3 years). Assessment included a transthoracic echocardiogram and analysis of myocardial mechanics. This assessment was performed at the beginning and end of the military course and the sports season, respectively. RESULTS: Cardiac remodeling was observed in both groups. The soldiers presented a predominantly eccentric pattern, with increased left ventricular (LV) size (49.7±3.2 vs. 52.8±3.4 mm; p<0.01), increased LV mass (93.1±7.7 vs. 100.2±11.4 g/m2; p<0.01) and decreased relative wall thickness (0.40±0.1 vs. 0.36±0.1; p=0.05). The basketball players showed a concentric pattern, with decreased LV size (52.0±4.7 vs. 50.4±4.7 mm; p=0.05), and increased relative wall thickness (0.33±0.1 vs. 0.36±0.1; p=0.05). Although there was no significant difference in LV myocardial strain in the groups separately, when compared there was a significant decrease (-20.2±1.6% vs. -19.4±2.1%; p=0.03). CONCLUSION: Cardiac remodeling was frequent, with an eccentric pattern in soldiers and a concentric pattern in basketball players. Myocardial deformation may represent a physiological adaptation to physical exercise.

Tolerogenic insulin peptide therapy precipitates type 1 diabetes.

Daniel et al. (https://doi.org/10.1084/jem.20110574) have previously published in JEM a study on the preventive effect of tolerogenic vaccination with a strong agonist insulin mimetope in type 1 diabetes. Our study now challenges these results and shows that osmotic pump delivery of the modified insulin peptide R22E did not prevent hyperglycemia, accelerated disease onset, increased its incidence, and worsened insulitis.

Control of disease tolerance to malaria by nitric oxide and carbon monoxide.

Nitric oxide (NO) and carbon monoxide (CO) are gasotransmitters that suppress the development of severe forms of malaria associated with Plasmodium infection. Here, we addressed the mechanism underlying their protective effect against experimental cerebral malaria (ECM), a severe form of malaria that develops in Plasmodium-infected mice, which resembles, in many aspects, human cerebral malaria (CM). NO suppresses the pathogenesis of ECM via a mechanism involving (1) the transcription factor nuclear factor erythroid 2-related factor 2 (NRF-2), (2) induction of heme oxygenase-1 (HO-1), and (3) CO production via heme catabolism by HO-1. The protection afforded by NO is associated with inhibition of CD4(+) T helper (TH) and CD8(+) cytotoxic (TC) T cell activation in response to Plasmodium infection via a mechanism involving HO-1 and CO. The protective effect of NO and CO is not associated with modulation of host pathogen load, suggesting that these gasotransmitters establish a crosstalk-conferring disease tolerance to Plasmodium infection.

The first steps of adaptation of Escherichia coli to the gut are dominated by soft sweeps.

The accumulation of adaptive mutations is essential for survival in novel environments. However, in clonal populations with a high mutational supply, the power of natural selection is expected to be limited. This is due to clonal interference--the competition of clones carrying different beneficial mutations--which leads to the loss of many small effect mutations and fixation of large effect ones. If interference is abundant, then mechanisms for horizontal transfer of genes, which allow the immediate combination of beneficial alleles in a single background, are expected to evolve. However, the relevance of interference in natural complex environments, such as the gut, is poorly known. To address this issue, we have developed an experimental system which allows to uncover the nature of the adaptive process as Escherichia coli adapts to the mouse gut. This system shows the invasion of beneficial mutations in the bacterial populations and demonstrates the pervasiveness of clonal interference. The observed dynamics of change in frequency of beneficial mutations are consistent with soft sweeps, where different adaptive mutations with similar phenotypes, arise repeatedly on different haplotypes without reaching fixation. Despite the complexity of this ecosystem, the genetic basis of the adaptive mutations revealed a striking parallelism in independently evolving populations. This was mainly characterized by the insertion of transposable elements in both coding and regulatory regions of a few genes. Interestingly, in most populations we observed a complete phenotypic sweep without loss of genetic variation. The intense clonal interference during adaptation to the gut environment, here demonstrated, may be important for our understanding of the levels of strain diversity of E. coli inhabiting the human gut microbiota and of its recombination rate.

Recent thymic emigrants are the preferential precursors of regulatory T cells differentiated in the periphery.

Most Forkhead box P3(+) (Foxp3(+)) CD4 regulatory T cell (Treg) precursors are newly formed thymocytes that acquire Foxp3 expression on antigen encounter in the thymus. Differentiation of Treg, however, can also occur in the periphery. What limits this second layer of self- and nonself-reactive Treg production in physiological conditions remains to be understood. In this work, we tested the hypothesis that, similarly to thymic Treg, the precursors of peripheral Treg are immature T cells. We show that CD4(+)CD8(-)Foxp3(-) thymocytes and recent thymic emigrants (RTEs), contrarily to peripheral naïve mature cells, efficiently differentiate into Treg on transfer into lymphopenic mice. By varying donor and recipient mice and conducting ex vivo assays, we document that the preferential conversion of newly formed T cells does not require intrathymic preactivation, is cell-intrinsic, and correlates with low and high sensitivity to natural inhibitors and inducers of Foxp3 expression, such as IL-6, T-cell receptor triggering, and TGF-ß. Finally, ex vivo analysis of human thymocytes and peripheral blood T cells revealed that human RTE and newly developed T cells share an increased potential to acquire a FOXP3(bright)CD25(high) Treg phenotype. Our findings indicating that RTEs are the precursors of Tregs differentiated in the periphery should guide the design of Treg-based therapies.

Cutting edge: Intrathymic differentiation of adaptive Foxp3+ regulatory T cells upon peripheral proinflammatory immunization.

Thymocytes differentiate into CD4(+) Foxp3(+) regulatory T cells (T(R)) upon interaction between their TCR and peptide-MHC II complexes locally expressed in the thymus. Conversion of naive CD4(+) T cells into T(R) can additionally take place in the periphery under noninflammatory conditions of Ag encounter. In this study, making use of TCR transgenic models naturally devoid of Foxp3(+) cells, we report de novo generation of T(R) upon a single footpad injection of Ag mixed with a classic proinflammatory adjuvant. Abrupt T(R) differentiation upon immunization occurred intrathymically and was essential for robust tolerance induction in a mouse model of spontaneous encephalomyelitis. This phenomenon could be attributed to a specific feature of thymocytes, which, in contrast to mature peripheral CD4(+) T cells, were insensitive to the inhibitory effects of IL-6 on the induction of Foxp3 expression. Our findings uncover a pathway for T(R) generation with major implications for immunity and tolerance induction.

Steroid treatments in mice do not alter the number and function of regulatory T cells, but amplify cyclophosphamide-induced autoimmune disease.

Corticosteroids are commonly used in the therapy of autoimmune disease (AID), although they are rarely, if ever, curative. This failure may result from their deleterious effects on regulatory T cells (Treg). In this work, we directly tested the effects of hydrocortisone (HC) administration on Treg number and function in established mouse models of multiple sclerosis and colitis. Treatment with pertussis toxin (Ptx) or Cyclophosphamide (Cyp), two compounds known to affect Treg function served as controls. We first show that contrarily to Ptx, HC administration to mice transgenic for a TCR specific to myelin basic protein induces a mild lymphopenia, without selective depletion of Treg, nor induction of experimental autoimmune encephalomyelitis (EAE). We next report that HC administration to normal mice has no effect on Treg suppressive function tested in vitro. Moreover, we document that Treg isolated from HC-treated animals maintain their capacity to prevent T cell-induced colitis. In contrast, the combined administration of HC and Cyp, as is frequently used in the therapy of severe AID, dramatically enhanced the deleterious effect of Cyp on Treg number and function. Our analysis indicates that while a short course of corticosteroids alone is not deleterious to immune regulation, combined therapies, notably with Cyp, should be avoided.

Natural Treg cells spontaneously differentiate into pathogenic helper cells in lymphopenic conditions.

Induction of Forkhead-box p3 (Foxp3) expression in developing T cells upon peptide-MHC encountering has been proposed to define a lineage of committed Treg cells. However, sustained expression of Foxp3 is required for Treg function and what maintains Foxp3 expression in peripheral Treg remains obscure. To address this issue, we monitored natural Treg phenotype and function upon adoptive transfer into lymphocyte-deficient mice. We first show that about 50% of Foxp3-GFP(+) Treg isolated from Foxp3(gfp) KI animals loose Foxp3 expression in severe lymphopenic conditions. We next evidence that the cytokine IL-2, either produced by co-transferred conventional T cells or administrated i.v. prevents Foxp3 downregulation. Moreover, we document that Treg that lost Foxp3 expression upon adoptive transfer produce IL-2 are not suppressive and promote tissue infiltration and damage upon secondary transfer into alymphoid mice. Our findings that Treg convert into pathogenic Th cells in absence of IL-2 provide new clues to the success of Treg-based immune therapies.

When three is not a crowd: a Crossregulation model of the dynamics and repertoire selection of regulatory CD4+ T cells.

Regulatory CD4(+) T cells, enriched in the CD25 pool of healthy individuals, mediate natural tolerance and prevent autoimmune diseases. Despite their fundamental and potential clinical significance, regulatory T (T(R)) cells have not yet been incorporated in a coherent theory of the immune system. This article reviews experimental evidence and theoretical arguments supporting a model of T(R) cell dynamics, uncovering some of its most relevant biological implications. According to this model, the persistence and expansion of T(R) cell populations depend strictly on specific interactions they make with antigen-presenting cells (APCs) and conventional effector T (T(E)) cells. This three-partner crossregulation imposes that T(R) cells feed on the specific autoimmune activities they suppress, with implications ranging from their interactions with other cells to their repertoire selection in the periphery and in the thymus, and to the relationship between these cells and the innate immune system. These implications stem from the basic prediction that the peripheral dynamics sort the CD4(+) T-cell repertoire into two subsets: a less diverse set of small clones of autoreactive effector and regulatory cells that regulate each other's growth, and a more diverse set of barely autoreactive T(E) cell clones, whose expansion is limited only by APC availability. It is argued that such partitioning of the repertoire sets the ground for self-non-self discrimination.