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.

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.

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.

Defective induction of CTLA-4 in the NOD mouse is controlled by the NOD allele of Idd3/IL-2 and a novel locus (Ctex) telomeric on chromosome 1.

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), or CD152, is a negative regulator of T-cell activation and has been shown to be associated with autoimmune diseases. Previous work has demonstrated a defect in the expression of this molecule in nonobese diabetic (NOD) mice upon anti-CD3 stimulation in vitro. Using a genetic approach we here demonstrate that a novel locus (Ctex) telomeric on chromosome 1 together with the Idd3 (Il-2) gene confers optimal CTLA-4 expression upon CD3 activation of T-cells. Based on these data, we provide a model for how gene interaction between Idd3 (IL-2), Ctex, and Idd5.1 (Ctla-4) could confer susceptibility to autoimmune diabetes in the NOD mouse. Additionally, we showed that the Ctex and the Idd3 regions do not influence inducible T-cell costimulator (ICOS) protein expression in NOD mice. Instead, as previously shown, higher ICOS levels in NOD mice appear to be controlled by gene(s) in the Idd5.1 region, possibly a polymorphism in the Icos gene itself.

The enlarged population of marginal zone/CD1d(high) B lymphocytes in nonobese diabetic mice maps to diabetes susceptibility region Idd11.

The NOD mouse is an important experimental model for human type 1 diabetes. T cells are central to NOD pathogenesis, and their function in the autoimmune process of diabetes has been well studied. In contrast, although recognized as important players in disease induction, the role of B cells is not clearly understood. In this study we characterize different subpopulations of B cells and demonstrate that marginal zone (MZ) B cells are expanded 2- to 3-fold in NOD mice compared with nondiabetic C57BL/6 (B6) mice. The NOD MZ B cells displayed a normal surface marker profile and localized to the MZ region in the NOD spleen. Moreover, the MZ B cell population developed early during the ontogeny of NOD mice. By 3 wk of age, around the time when autoreactive T cells are first activated, a significant MZ B cell population of adult phenotype was found in NOD, but not B6, mice. Using an F2(B6 x NOD) cross in a genome-wide scan, we map the control of this trait to a region on chromosome 4 (logarithm of odds score, 4.4) which includes the Idd11 and Idd9 diabetes susceptibility loci, supporting the hypothesis that this B cell trait is related to the development of diabetes in the NOD mouse.

Prevention of diabetes in nonobese diabetic mice mediated by CD1d-restricted nonclassical NKT cells.

A role for regulatory lymphocytes has been demonstrated in the pathogenesis of type 1 diabetes in the NOD mouse but the nature of these cells is debated. CD1d-restricted NKT lymphocytes have been implicated in this process. Previous reports of reduced diabetes incidence in NOD mice in which the numbers of NKT cells are artificially increased have been attributed to the enhanced production of IL-4 by these cells and a role for classical NKT cells, using the Valpha14-Jalpha18 rearrangement. We now show that overexpression in NOD mice of CD1d-restricted TCR Valpha3.2(+)Vbeta9(+) NKT cells producing high levels of IFN-gamma but low amounts of IL-4 leads to prevention of type 1 diabetes, demonstrating a role for nonclassical CD1d-restricted NKT cells in the regulation of autoimmune diabetes.