The TCR repertoires of regulatory and conventional T cells specific for the same foreign antigen are distinct.
ABSTRACT: The relationship between the TCR repertoires of natural regulatory T cells (nTregs) and conventional CD4(+) T cells (Tconv) capable of responding to the same antigenic epitope is unknown. In this study, we used TCR?-chain transgenic mice to generate polyclonal nTreg and Tconv populations specific for a foreign Ag. CD4(+) T cells from immunized 3.L2?(+/-) TCR?(+/-) Foxp3(EGFP) mice were restimulated in culture to yield nTregs (EGFP(+)) and Tconv (EGFP(-)) defined by their antigenic reactivity. Relative to Tconv, nTreg expansion was delayed, although a higher proportion of viable nTregs had divided after 72 h. Spectratype analysis revealed that both the nTreg and Tconv responses were different and characterized by skewed distributions of CDR3 lengths. CDR3 sequences from nTregs displayed a divergent pattern of J? usage, minimal CDR3 overlap (3.4%), and less diversity than did CDR3 sequences derived from Tconv. These data indicate that foreign Ag-specific nTregs and Tconv are clonally distinct and that foreign Ag-specific nTreg populations are constrained by a limited TCR repertoire.
Project description:The classical swine fever virus C-strain vaccine (C-strain vaccine) plays a vital role in preventing and controlling the spread of classical swine fever (CSF). However, the protective mechanisms of C-strain vaccine and cellular immunity conferred by T cell receptors (TCRs) are less well defined. We aimed to analyse the association between the complementarity determining region 3 (CDR3) spectratype of ??TCR in CD4+ T cells and C-strain vaccine; and to find conserved CDR3 amino acid motifs in specific TCR ?- and ?-chains. We found that the CDR3 spectratype showed dynamic changes correlating with C-strain vaccine immunisation and that TCR AV5S/8-3S/8-4S/14/38 and BV4S/6S/7S/15S/30 gene families showed clonal expansion in immunised pigs. The sequences of CDR3 from these clonally expanded T cells indicated a high frequency of the 'KLX' motif in the TCR ? chain and the 'GGX' motif in ? chain, and J?39, J?43, J?2.5 and J?2.3 genes were also found in high frequency. To the best of our knowledge, this is the first report describing the dynamic changes of ??TCRs and conserved CDR3 amino acid motifs in CD4+ T cells from C-strain vaccine-immunised pigs, which will provide a basis for the development of high-efficiency epitope vaccines.
Project description:We describe a simple iterative approach to augment TCR affinity, which we studied using a myelin oligodendrocyte glycoprotein-specific TCR. We hypothesized that single amino acid modifications in TCR CDR3 could enhance TCR sensitivity through focal interactions with antigenic peptide while minimizing the risk of cross-reactivity observed previously in TCR more broadly mutagenized using in vitro evolution techniques. We show that this iterative method can indeed generate TCR with Ag sensitivity 100-fold greater than the parental receptor and can endow TCR with coreceptor independence. However, we also find that single amino acid mutations in the CDR3 can alter TCR fine specificity, affecting recognition requirements for Ag residues over most of the length of the MHC binding groove. Furthermore, minimal changes in surface-exposed CDR3 amino acids, even the addition of a single hydroxyl group or conversion of a methyl or sulfhydryl moiety to a hydroxyl, can confer modified Ag-specific TCR with new self-reactivity. In vivo modeling of modified TCR through retroviral TCR gene transfer into Rag(-/-) mice confirmed the biological significance of these altered reactivities, although it also demonstrated the feasibility of producing Ag-specific, positively selecting, coreceptor-independent receptors with markedly increased Ag sensitivity. These results affirm the possibility of readily generating affinity-enhanced TCR for therapeutic purposes but demonstrate that minimal changes in TCR CDR3 structure can promote self reactivity and thereby emphasize the importance of caution in validating receptors with even subtle alterations before clinical application.
Project description:Thymic regulatory T cells (tTreg) are critical in the maintenance of normal T cell immunity and tolerance. The role of TCR in tTreg selection remains incompletely understood. In this study, we assessed TCR? and TCR? sequences of mouse tTreg and thymic conventional CD4<sup>+</sup> T cells (Tconv) by high-throughput sequencing. We identified ?? TCR sequences that were unique to either tTreg or Tconv and found that these were distinct as recognized by machine learning algorithm and by preferentially used amino acid trimers in ?? CDR3 of tTreg. In addition, a proportion of ?? TCR sequences expressed by tTreg were also found in Tconv, and machine learning classified the great majority of these shared ?? TCR sequences as characteristic of Tconv and not tTreg. These findings identify two populations of tTreg, one in which the regulatory T cell fate is associated with unique properties of the TCR and another with TCR properties characteristic of Tconv for which tTreg fate is determined by factors beyond TCR sequence.
Project description:Regulatory T lymphocytes (Tregs) expressing the Foxp3 transcription factor are critical modulators of autoimmunity. Foxp3(+) Tregs may develop in the thymus as a population distinct from conventional Foxp3(-) ?? T cells (Tconvs). Alternatively, plasticity in Foxp3 expression may allow for the interconversion of mature Tregs and Tconvs. We examined >160,000 TCR sequences from Foxp3(+) or Foxp3(-) populations in the spleens or CNS of wild-type mice with experimental allergic encephalomyelitis to determine their relatedness and identify distinguishing TCR features. Our results indicate that the CNS-infiltrating Tregs and Tconvs arise predominantly from distinct sources. The repertoires of CNS Treg or Tconv TCRs showed limited overlap with heterologous populations in both the CNS and the spleen, indicating that they are largely unrelated. Indeed, Treg and Tconv TCRs in the CNS were significantly less related than those populations in the spleen. In contrast, CNS Treg and Tconv repertoires strongly intersected those of the homologous cell type in the spleen. High-frequency sequences more likely to be disease associated showed similar results, and some public TCRs demonstrated Treg- or Tconv-specific motifs. Different charge characteristics and amino acid use preferences were identified in the CDR3? of Tregs and Tconvs infiltrating the CNS, further indicating that their repertoires are qualitatively distinct. Therefore, discrete populations of Tregs and Tconvs that do not substantially interconvert respond during experimental allergic encephalomyelitis. Differences in sequence and physical characteristics distinguish Treg and Tconv TCRs and imply dissimilar Ag recognition properties.
Project description:The source, specificity, and plasticity of the forkhead box transcription factor 3 (Foxp3)(+) regulatory T (Treg) and conventional T (Tconv) cell populations active at sites of autoimmune pathology are not well characterized. To evaluate this, we combined global repertoire analyses and functional assessments of isolated T cell receptors (TCR) from TCRalpha retrogenic mice with autoimmune encephalomyelitis. Treg and Tconv cell TCR repertoires were distinct, and autoantigen-specific Treg and Tconv cells were enriched in diseased tissue. Autoantigen sensitivity and fine specificity of these cells intersected, implying that differences in responsiveness were not responsible for lineage specification. Notably, autoreactive Treg and Tconv cells could be fully distinguished by an acidic versus aliphatic variation at a single TCR CDR3 residue. Our results imply that ontogenically distinct Treg and Tconv cell repertoires with convergent specificities for autoantigen respond during autoimmunity and argue against more than limited plasticity between Treg and Tconv cells during autoimmune inflammation.
Project description:We evaluated immune reconstitution in 58 adults who received hematopoietic SCTs from allogeneic siblings (allosib), matched unrelated donors (MUD) or cord blood (CB) at 90-day intervals for 1 year post transplant. CB recipients had a higher incidence of infections in the first 100 days compared with allosib and MUD recipients. The number of circulating T cells was lower in CB recipients compared with MUD recipients at 90 days and compared with allosib recipients at 180 days. Spectratype analysis of the TCR V? complementarity determining region 3 (CDR3) of patient lymphocytes revealed that the TCR repertoire remained poorly diversified even at 360 days in nearly all patients. In contrast, the number of circulating B cells was significantly elevated in CB recipients compared with allosib recipients throughout the first year post transplant and compared with MUD recipients at 9-12 months. Spectratype analysis of the B-cell receptor V(H) CDR3 showed that the B-cell repertoire was diversified in most patients by 90 days. CD5(pos) B cells from assayed CB recipients expressed intracellular IL-10 early post transplant. Our data suggest that B cells, in addition to T cells, may have a role in impaired immune responses in CB transplant patients.
Project description:?? T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short "foreign" peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.
Project description:Background: Treg cells represent important viral reservoirs during chronic HIV infection. CD39 is closely involved in Treg-mediated immunosuppressive effects. However, CD39 expression on nTregs and mTregs and a relationship with HIV DNA levels during HIV infection is still unclear. In this study, we analyzed the distribution of HIV DNA in Treg subsets and the association between HIV DNA and CD39 expression on Treg subsets. Methods: Sixty-two HIV-infected patients with different HIV stages and 14 uninfected individuals were enrolled. nTregs (CD4+CD25+CD127lowCD45RO-) and mTregs (CD4+CD25+CD127lowCD45RO+) were isolated by magnetic selection and flow cytometric sorting. HIV DNA was quantified by real-time polymerase chain reaction (PCR). CD39 expression on nTregs and mTregs was analyzed by flow cytometry. Results: Higher levels of HIV DNA were detected in mTregs than those in nTregs during chronic HIV infection. The frequency of CD39+ nTregs and HIV DNA levels in nTregs were increased in patients with advanced HIV infection. Furthermore, HIV DNA levels in nTregs correlated positively with CD39+ nTreg frequency. CD39+ nTreg frequency was also increased in immune non-responders. Conclusions: mTregs and nTregs are both important reservoirs of virus during chronic HIV infection and HIV DNA levels increase in nTregs in patients with advanced HIV infection. We observed increased frequency of CD39+ nTregs and HIV DNA levels in nTregs in patients with advanced HIV infection. HIV DNA levels in nTregs correlated positively with CD39+ nTreg frequency.
Project description:Thymus-derived Foxp3(+) natural regulatory CD4 T cells (nTregs) prevent autoimmunity through control of pathogenic, autoreactive T cells and other immune effector cells. Using T cell receptor (TCR) transgenic models, diversity within this lineage has been found to be similar to that of conventional CD4 T cells. To determine whether balanced TCR diversity may be perturbed in autoimmunity, we have analyzed receptor composition in C57BL/6 and autoimmune non-obese diabetic (NOD) mice. The natural regulatory and conventional CD4 repertoires of C57BL/6 had similar diversities. Despite the apparently normal thymic development of the NOD nTreg lineage, TCR diversity within the selected repertoire was markedly restricted. Detailed analysis of TCRalpha and -beta chain composition is consistent with positive selection into the natural regulatory lineage being under stringent audition for interaction with MHC class II/self-peptide. The NOD MHC region, including the unique H2-A(g7) class II molecule, partly accounts for the reduction in diversity, but additional NOD genetic contribution(s) are required for complete repertoire compaction. Mechanistic links between MHC, autoimmunity, and nTreg diversity identified in this study are discussed.
Project description:Introduction: Expansion of antigen (Ag)-specific natural occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloAg-specific nTregs. Methods: A highly enriched nTreg-fraction (CD4+CD25brightCD127- T cells) was alloAg-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDC) or peripheral blood mononuclear cells (PBMC). The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the TSDR of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4 and cytokines. In addition, the antigen-specific suppressive capacity of these expanded nTregs was tested. Results: Allogeneic mature moDC and skin-derived DC were superior in inducing nTreg-expansion compared to immature moDC or PBMC in an HLA-DR and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2, could facilitate optimal mature moDC-induced nTreg-expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloAg-induced proliferation without significant suppression of completely HLA-mismatched-Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the TSDR within the FOXP3 gene and highly expressed of FOXP3, HELIOS and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-g and TNF-a but very few IL-17 producing nTregs were found. Next generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Conclusions: Human allogeneic mature moDC are highly efficient stimulator cells, in presence of exogenous IL-15, for expansion of stable alloAg-specific nTregs with superior suppressive function. Four different batches of highly pure regulatory T cells (all from the same donor) were expanded in two different ways, and compared to non-expanded samples.