Thymus and immune reconstitution after allogeneic hematopoietic stem cell transplantation in humans: never say never again

Assessment of the host immune status is becoming a key issue in allogeneic hematopoietic stem cell transplantation (allo-HSCT). In the long-term follow-up of these patients, severe post-transplant infections, relapse or secondary malignancies may be directly related to persistent immune defects. In allo-HSCT, T-cell differentiation of donor progenitors within the recipient thymus is required to generate naive recent T-cell emigrants (RTE). These cells account for a durable T-cell reconstitution, generating a diverse T-cell receptor (TCR) repertoire and robust response to infections. It is now possible to quantify the production of RTE by measuring thymic T-cell receptor excision circles or 'TREC' which are small circular DNA produced during the recombination of the genomic segments encoding the TCR alpha chain. Here we discuss the role of thymic function in allo-HSCT. The pre-transplant recipient thymic function correlates with clinical outcome in terms of survival and occurrence of severe infections. Post-transplant, TREC analysis showed that the thymus is a sensitive target to the allogeneic acute graft-versus-host disease (GvHD) reaction but is also prone to recovery in young adult patients. In all, thymus is a key player for the quality of immune reconstitution and clinical outcome after allo-HSCT. Thymic tissue is plastic and it is a future challenge to halt or reverse thymic GVHD therapeutically by acting at the level of T-cell progenitors generation, thymic homing and/or epithelial thymic tissue preservation.     

Pathways of T-cell regeneration in mice and humans: implications for bone marrow transplantation and immmunotherapy

Summary: Much of our understanding of the immunobiology of bone marrow transplantation (BMT) has come from studies in young adult mice reconstituted with T-cell-depleted bone marrow after lethal irradiation. Recent evidence indicates, however, that the applicability of conclusions drawn from this model to human BMT may be limited. While mice retain essentially normal thymic function well past sexual maturity, humans show significant age-related declines in thymic function age-early in life. Therefore, thymic-deficient mice may provide a more accurate model for study of the immunobiology of BMT T-cell regeneration in thymic-deficient mice occurs primarily via antigen-driven expansion of mature peripheral T cells resulting m limited immune competence due to quantitative deficiencies in T-cell number and severe restriction in the diversity of the regenerated T-cell receptor (TCR) repertoire. Similarly, immune reconstitution in adult humans after BMT is marked by quantitative T-cell deficiencies, especially in the CD4⁺ subset, and loss of TCR diversity. Taken together, prevailing evidence suggests that thymic function is suboptimal in most BMT recipients, and that thymic-independent pathways of T-cell regeneration are generally limited in their ability to restore host immune competence. New strategies to enhance thymic function in man after BMT would hold great therapeutic potential.     

Surface T-cell antigen receptor expression and availability for long-term antigenic signaling

Summary:  It is important to understand how T-cell antigen receptor (TCR) engagement and signaling are regulated throughout an immune response. This review examines the dynamics of surface TCR expression and signaling capacity during thymic and effector T-cell development. Although the TCR can undergo vast changes in surface expression, T cells remain capable of sustaining TCR engagement for long periods of time. This may be achieved by a combination of mechanisms that involve (a) controlling the quantity of surface TCR available for ligand interaction and (b) controlling the quality of surface TCR expression during T-cell activation. TCR signaling itself appears to be one of the main quantitative modulators of surface TCR expression, and it can cause both downregulation and upregulation at different times of T-cell activation. Recent studies indicate that the degree of upregulation is tunable by the strength of antigenic stimulation. There is evidence that qualitatively distinct forms of the TCR exist, and their potential role in sustained antigenic signaling is also discussed. A goal of future studies will be to better characterize these modulations in surface TCR expression and to clarify their impact on the regulation of immune responses.     

Differential expression and regulation of CD6 on T-cell subsets revealed by monoclonal antibody (MAb) CH11

A monoclonal antibody (MAb), CH11, was developed by immunizing mice with CD4+ gammadelta T-cell receptor (TCR)+ cells. It recognized an antigen expressed in the surface membrane of T-cell lines, but not of U937, lymphoblastoid B cells (LBC), K562, Raji or Daudi cells, indicating selectivity for the T-cell lineage. In addition, it labelled 70-80% of normal peripheral blood mononuclear cells (PBMC), with high expression on the erythrocyte rosetting (E+) fraction, and low/absent expression on E- cells. However, CD4+ T cells expressed higher levels of reactivity than CD8+ or gammadelta+ T-cell receptor (TCR)+ lymphocytes in PB. Furthermore, in 7 of 10 individuals tested, 7.34+/-3.88% of unselected PBMC were CH11- CD3+ and were relatively enriched in CD8+ and in gammadelta TCR+-cells. In addition, thymic gammadelta T cells, and gammadelta lymphoproliferations from two patients were nonreactive or weakly reactive with the MAb. Activation of E+ cells with phorbol-12-myristate-13-acetate (PMA) enhanced CH11 expression uniformly, whereas activation with phytohemagglutinin (PHA) selectively down-regulated expression of the antigen on the CD8+ subset. In Western blots performed in nonreducing (NR) conditions, MAb CH11 detected a 100 kDa molecule in PBMC and Jurkat T-cell lysates. Preincubation of T cells with MAb CH11 specifically abrogated their subsequent reactivity with MAb to CD6, suggesting that MAb CH11 is recognizing an epitope of CD6. Given its function as a receptor for ligands on thymic epithelium, activated leukocytes and synoviocytes, this newly defined heterogeneity of expression and regulation of the CD6 molecule on subsets of T cells may help determine their functional repertoire in vivo.     

Dual-Receptor T Cells Expressing One Self-Restricted TCR

During thymic development, T cells rearrange and express genes encoding clonotypic T-cell receptors (TCR), and undergo selective events involving interactions between TCR and thymic major histocompatibility complex (MHC) molecules. Recent studies indicate that up to 30% of peripheral T cells may express two TCR, that both TCR on these cells are functional, and that dual-specific T cells can promote autoimmunity. Here we demonstrate that dual-receptor T cells are readily generated in class II-deficient (class II⁻) mice expressing the class II-restricted AND TCR transgene (TCR_tg). While this TCR_tg is unable to promote positive selection in class II⁻ mice, T cells arise in class II⁻TCR⁺_tg mice by co-expressing non-transgenic endogenous TCR that permit positive selection on class I molecules. Our findings indicate that when a rearranged TCR fails to promote positive selection on self MHC molecules, expression of a second receptor can rescue the developing T cell. Accordingly, many dual-receptor T cells may actually be mono-specific in vivo , possessing only one self MHC-restricted TCR, and therefore should not require unique regulatory controls to prevent autoreactivity.     

Preselection TCR repertoire predicts CD4+ and CD8+ T-cell differentiation state

T cells must display diversity regarding both the cell state and T-cell receptor (TCR) repertoire to provide effective immunity against pathogens; however, the generation and evolution of cellular T-cell heterogeneity in the adaptive immune system remains unclear. In the present study, a combination of multiplex PCR and immune repertoire sequencing (IR-seq) was used for a standardized analysis of the TCR β-chain repertoire of CD4⁺ naive, CD4⁺ memory, CD8⁺ naive and CD8⁺ memory T cells. We showed that the T-cell subsets could be distinguished from each another with regard to the TCR β-chain (TCR-β) diversity, CDR3 length distribution and TRBV usage, which could be observed both in the preselection and in the post-selection repertoire. Moreover, the Dβ-Jβ and Vβ-Dβ combination patterns at the initial recombination step, template-independent insertion of nucleotides and inter-subset overlap were consistent between the pre- and post-selection repertoires, with a remarkably positive correlation. Taken together, these results support differentiation of the CD4⁺ and CD8⁺ T-cell subsets prior to thymic selection, and these differences survived both positive and negative selection. In conclusion, these findings provide deeper insight into the generation and evolution of TCR repertoire generation.     

Alteration of T-cell receptor repertoires during thymic T-cell development

The majority of thymocytes die in the thymus, whereas small populations of T cells that are able to specifically recognize an antigen are considered to survive. Although the thymic selection is thought to have a profound effect on T-cell receptor (TCR) repertoire, little is known how TCR repertoire is formed during the thymocyte developmental process. We examined TCRalpha- and beta-chain variable regions (TCRAV and TCRBV) repertoire in thymic T-cell subpopulations from mice bearing different major histocompatibility (MHC) haplotypes. In Balb/c mice, but not C57BL/6, remarkable alterations of the TCR repertoire were observed in mature T-cell subpopulations as previously reported. In contrast, there were no significant differences of TCRBV repertoire between DN3 (CD25(+)CD44(-)) and DN4 (CD25(-)CD44(-)), and between DN4 and DP. These results suggest that (1) TCR repertoire of mature T cells was formed mainly under the influence of endogenous superantigens, while MHC haplotypes played the least role; (2) the 'beta-selection' process during immature stages had little impact on TCRBV repertoire formation; and (3) TCR repertoire in immature T-cell subpopulations was extremely similar between different strains of mice. We thus consider that pre-selection TCR repertoire in immature T cells could be determined by some genetic factors conserved among different strains.     

Post-Natal Ontogenesis of the T-Cell Receptor CD4 and CD8 Vβ Repertoire and Immune Function in Children with DiGeorge Syndrome

DiGeorge syndrome (DGS) is a congenital disorder characterized by typical facial features, hypoparatyroidism, conotruncal cardiac defects and thymic hypoplasia. Although there are some reports addressing lymphocytes counts and function in DGS children over time, few data have been reported on the T-cell receptor Vβ (TCRBV) repertoire in relation to disease progression. The aim of this study was to evaluate the degree and nature of immunodeficiency and to investigate a possible correlation to clinical findings.We used third complementary region (CDR3) size spectratyping as a tool for monitoring T-cell repertoire diversity in 7 DGS’s children. The rate of thymic output, the phenotype and function of peripheral T-cells and the humoral immunity were also investigated. At baseline a profound alteration of the TCR repertoire was noted, mainly in the CD8+ T-cells, in DGS patients when compared to a control group. Furthermore, analysis of thymic output showed a significant decrease in TCR rearrangement excision circles (TRECs) levels in the patient group. Immunoglobulin abnormalities were also detected. The observed TCR repertoire alterations, although not statistically significant, may suggest an increased susceptibility to infections. A parallel increase in the TCR repertoire diversity and clinical improvement occurred during the follow-up. Our results confirm that the extent of immunodeficiency is highly variable and could improve through childhood, and indicate that TCR repertoire may be a useful marker to clinically monitor thymic function in this primary immunodeficiency.C. Cancrini and M.L. Romiti contributed equally to this work.     

Fas (CD95/APO-1) limits the expansion of T lymphocytes in an environment of limited T-cell antigen receptor/MHC contacts

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.     

Immune constitution monitoring after PBMC transplantation in complete DiGeorge syndrome: an eight-year follow-up

A young boy with a confirmed complete DiGeorge Syndrome (cDGS) underwent a peripheral blood mononuclear cell transplantation (PBMCT) from his HLA-identical sister at 4.5 years of age, without a conditioning regimen. Eight years later, he is healthy with good immunological functions in the presence of a stable mixed T-cell chimerism. Absence of recent thymic emigrants is confirmed. We observe an inverted CD4+/CD8+ ratio, related to the CD8 subset expansion, a skewing of the TCR repertoire, especially on the CD8+ subset and a telomere loss on the CD8+ cells compared to the donor. However, these anomalies do not seem to have an impact on functional immunity. PBMCT in cDGS using an HLA-matched sibling donor provides good long-lasting immunity and is an easy alternative to bone marrow transplantation and to thymic transplantation.     

Role of TCR-restricting MHC density and thymic environment on selection and survival of cells expressing low-affinity T cell receptors

H-Y-specific T cell receptor (TCR) transgenic mice express an MHC class I H-2D(b)-restricted TCR specific for a male antigen-derived peptide (H-Y). Selection and survival of cells expressing this low-affinity TCR were analyzed under optimal and sub-optimal conditions. Optimal conditions were provided by a thymic environment with high H-2D(b) density. Sub-optimal conditions were provided by a thymic environment with decreased H-2D(b) density or by an athymic environment with either high or low H-2D(b) density. Whereas negative selection was still guaranteed under sub-optimal conditions, selection and survival efficiency of cells expressing the transgenic TCR were strongly dependent on optimal conditions. These results indicated that both a thymic microenvironment and a high density of TCR-restricting MHC molecules were needed to ensure selection and maintenance of cells expressing TCR with low affinity and hence a more diverse T cell repertoire.     

Predicting the spectrum of TCR repertoire sharing with a data-driven model of recombination

Despite the extreme diversity of T-cell repertoires, many identical T-cell receptor (TCR) sequences are found in a large number of individual mice and humans. These widely shared sequences, often referred to as “public,” have been suggested to be over-represented due to their potential immune functionality or their ease of generation by V(D)J recombination. Here, we show that even for large cohorts, the observed degree of sharing of TCR sequences between individuals is well predicted by a model accounting for the known quantitative statistical biases in the generation process, together with a simple model of thymic selection. Whether a sequence is shared by many individuals is predicted to depend on the number of queried individuals and the sampling depth, as well as on the sequence itself, in agreement with the data. We introduce the degree of publicness conditional on the queried cohort size and the size of the sampled repertoires. Based on these observations, we propose a public/private sequence classifier, “PUBLIC” (Public Universal Binary Likelihood Inference Classifier), based on the generation probability, which performs very well even for small cohort sizes.     

Evidence for down-regulation of highly expressed TCR by CD4 and CD45 on non-selected CD4+CD8+thymocytes

Immature CD4+CD8+ double-positive (DP) thymocytes are positivelyselected for further development if they express TCR reactingwith thymic ligands of low affinity. However, the majority ofDP thymocytes express low TCR levels. This low level of TCRmay be insufficient to recognize thymic ligands. To understandthe basis for the low expression of TCR on DP thymocytes, wedetermined the density of TCR expression at various stages oftheir development using TCR transgenic (TCR-Tg) mice. We foundthat TCR expression was high in the thymocytes that had recentlytransited into the DP stage but then gradually decreased onDP cells if they were not selected by TCR interaction with MHCmolecules. However, such TCR suppression was not observed inpositively selected DP cells and in the non-selected DP cellsobtained from CD45 deficient mice or from mice receiving anti-CD4mAb. These findings suggest that the once highly expressed TCRat the DP stage is suppressed by CD45 and/or CD4 on non-selectedthymocytes. Furthermore, TCR suppression is prevented by TCR-mediatedsignals. The maintenance of high TCR levels on positively selectedDP thymocytes may facilitate their selection.     

Frequency and cell specificity of T-cell receptor interlocus recombination in human cells

Immunoglobulin and T-cell receptor (TCR) genes are assembled by a site-specific rearrangement known as V(D)J [variable-(diversity)-joining] recombination. These rearrangements occur normally in pre-B- and pre-T-cells using signal sequences adjacent to coding exons for immunoglobulin and TCR genes, respectively. However, aberrant recombination may result in the generation of hybrid TCR genes by joining of TCR-β with TCR-γ specific sequences. Such hybrid TCR genes occur at a low frequency in peripheral blood lymphocytes (PBL) of healthy individuals, and can be detected by PCR amplification. We have determined the in vivo frequency of hybrid Vγ-Jβ1 TCR (hybrid TCR) genes in lymphocyte DNA from 12 healthy individuals. The average frequency was found to be 5.83 in 0.75 × 10⁶ PBL, with a threefold difference between the highest and lowest individual value. The presence of similar TCR gene rearrangements in individual samples suggests that T-cells with a hybrid TCR gene are capable of clonal expansion in vivo. The individual hybrid TCR gene frequency remained relatively constant during 72 hours of in vitro cultivation. In long-term culture, the frequency gradually decreased, and after 28 days no hybrid TCR genes were detectable in lymphocyte DNA. These results show that T-cells with a hybrid TCR gene are able to respond to mitogen stimulation in vitro, and may have a proliferative disadvantage or are selected against during prolonged in vitro cultivation. No hybrid TCR genes were detected in ten proliferating T-cell clones, indicating that the rate of hybrid TCR gene formation is <2.0 × 10⁻⁸ per cell per cell division. No hybrid TCR genes were detected in DNA from B-lymphocytes, sperm, granulocytes, fibroblasts, keratinocytes, and three B-lymphoblastoid ataxia telangiectasia cell lines. In agreement with previous reports, the frequency of hybrid TCR genes in peripheral blood DNA from two ataxia telangiectasia patients was found to be more than 15-fold higher than in lymphocytes from normal individuals. These data show that formation of hybrid TCR genes is restricted to T-cells in vivo, and occurs at a very low frequency, if at all, in proliferating T-cells in vitro, and with an increased frequency in patients with ataxia telangiectasia. Environ. Mol. Mutagen. 30:245–253, 1997 © 1997 Wiley-Liss, Inc.     

Different role of Apaf-1 in positive selection,negative selection and death by neglect in foetal thymic organ culture

Apoptotic protease-activating factor 1 (Apaf-1) is a component of the apoptosome which is required for the activation of procaspase-9. As Apaf-1 knockout (KO) (Apaf-1-/-) mice die before birth, the role of Apaf-1 during thymic selection was investigated using 5 day foetal thymic organ culture (FTOC) of thymi obtained at gestational day 15. There was a lower ratio of CD4 single-positive (SP) to CD8 SP cells and decreased apoptosis of CD4+CD8+ (DP) thymocytes from Apaf-1-/- mice compared with wild-type. To determine if these defects resulted in increased production of neglected thymocytes, the Apaf-1-/- mice were crossed with the T-cell receptor (TCR)-alpha-chain KO mice. There was no difference in thymocyte development in the thymi of TCR-alpha-/-Apaf-1-/- and TCR-alpha-/-Apaf-1+/+ mice 5 days after FTOC. To determine if Apaf-1 is involved in apoptosis during death by negative or positive selection, FTOC of the thymus of Apaf-1-/- Db/HY TCR-alphabeta transgenic (Tg) mice was carried out. There was decreased apoptosis of the HY clonal-specific M33+ thymocytes and an increased percentage of the autoreactive CD8+M33+ thymocytes in male, but not female Apaf-1-/- Db/HY TCR Tg mice. Our data suggest that Apaf-1 is not involved in positive selection or death by neglect, but may have a partial role in negative selection during early thymic T-cell development.     

Towards an integrated view of thymopoiesis

One prediction from the complex series of steps in intrathymic T-cell differentiation is that to regulate it the stroma controlling the process must be equally complex: the attraction of precursors, commitment to the T-cell lineage, induction of T-cell receptor (TCR) gene rearrangement, accessory molecule expression, repertoire expansion, major histocompatibility complex (MHC) molecule-based selection (positive and negative), acquisition of functional maturity and migratory capacity must all be controlled. In this review, Richard Boyd and Patrice Hugo combine knowledge of T-cell differentiation with thymic stromal cell heterogeneity to offer an integrated view of thymopoiesis within the thymic microenvironment.     

Flexibility in T-cell receptor ligand repertoires depends on MHC and T-cell receptor clonotype.

T-cell receptors (TCR) recognize peptides complexed to self-major histocompatibility complex (MHC) molecules. Recognition of peptide/MHC ligands by the TCR is highly peptide specific. However, certain TCRs can also recognize sequence-related and -unrelated ('mimicry') epitopes presented by homologous MHC molecules. Using two human, human leucocyte antigen-DR1 (HLA-DR1)-restricted T-cell clones specific for HA p307-319, we identified several diverse combinations of peptide-MHC complexes that are functionally equivalent in their ability to trigger T-cell stimulation. These findings demonstrate that a single TCR can productively interact with different peptides complexed to self- as well as non-self-MHC molecules. This extended reactivity is human leucocyte antigen (HLA) allele and TCR clonotype dependent, as the peptide repertoire recognized depends on the presenting HLA-DR molecule and varies among different TCRs that both recognize the HA p307-319/DR1 complex. Importantly, certain peptide analogues can completely change the HLA-restriction pattern of the TCR: T-cell recognition of the wild-type peptide that was absent in the context of a non-self HLA-DR molecule, was restored by complementing substitutions in altered peptide ligands, that could not be presented by the original restriction element. This mechanism may play an important role in allorecognition.