MAIT cell development in mice and humans.

MAIT cells arise in the thymus following rearrangement of a T cell receptor (TCR) reactive against microbial vitamin B2-derived metabolites presented by the MHC-Ib molecule, MR1. Mechanisms that are conserved in mammals ensure the frequent production of MR1-restricted TCRs and the intra-thymic differentiation of MR1-restricted thymocytes into effector cells. Upon thymic egress and migration into non-lymphoid tissues, additional signals modulate MAIT cell functions according to each local tissue environment. Here, we review the recent progress made towards a better understanding of the establishment of this major immune cell subset.

On the organization of human T-cell receptor loci: log-periodic distribution of T-cell receptor gene segments.

The human T-cell repertoire is complex and is generated by the rearrangement of variable (V), diversity (D) and joining (J) segments on the T-cell receptor (TCR) loci. The T-cell repertoire demonstrates self-similarity in terms clonal frequencies when defined by V, D and J gene segment usage; therefore to determine whether the structural ordering of these gene segments on the TCR loci contributes to the observed clonal frequencies, the TCR loci were examined for self-similarity and periodicity in terms of gene segment organization. Logarithmic transformation of numeric sequence order demonstrated that the V and J gene segments for both T-cell receptor α (TRA) and ß (TRB) loci are arranged in a self-similar manner when the spacing between the adjacent segments was considered as a function of the size of the neighbouring gene segment, with an average fractal dimension of approximately 1.5. Accounting for the gene segments occurring on helical DNA molecules with a logarithmic distribution, sine and cosine functions of the log-transformed angular coordinates of the start and stop nucleotides of successive TCR gene segments showed an ordered progression from the 5' to the 3' end of the locus, supporting a log-periodic organization. T-cell clonal frequency estimates, based on V and J segment usage, from normal stem cell donors were plotted against the V and J segment on TRB locus and demonstrated a periodic distribution. We hypothesize that this quasi-periodic variation in gene-segment representation in the T-cell clonal repertoire may be influenced by the location of the gene segments on the periodic-logarithmically scaled TCR loci. Interactions between the two strands of DNA in the double helix may influence the probability of gene segment usage by means of either constructive or destructive interference resulting from the superposition of the two helices.

Blood T-cell receptor diversity decreases during the course of HIV infection, but the potential for a diverse repertoire persists.

HIV infection results in a decrease in circulating CD4(+) T-cell and naive T-cell numbers. If such losses were associated with an erosion of T-cell receptor (TCR) repertoire diversity in the peripheral T-cell pool, this might exacerbate the state of persistent immunodeficiency. Existing methods for the analysis of the TCR repertoire have demonstrated skewed distributions of TCR genes in HIV-infected subjects but cannot directly measure TCR diversity. Here we used AmpliCot, a quantitative assay based on DNA hybridization kinetics, to measure TCR diversity in a cross-sectional comparison of 19 HIV-infected persons to 18 HIV-uninfected controls. HIV-infected persons had a 10-fold decrease in total TCR repertoire diversity in 1.5 mL of blood compared with uninfected controls, with decreased diversity correlating most closely with a lower CD4(+) T-cell percentage. Nonetheless, the TCR repertoire diversity of sort-purified T-cell subpopulations in HIV-infected and HIV-uninfected subjects was comparable. These observations suggest that the TCR repertoire diversity changes in whole blood during HIV disease progression are primarily the result of changes in the number and proportion of T-cell subpopulations and that most HIV-infected persons may retain a sufficiently diverse TCR repertoire to permit immune reconstitution with antiretroviral therapy alone, without thymopoiesis.

Expression of CD133 on leukemia-initiating cells in childhood ALL.

Optimization of therapy for childhood acute lymphoblastic leukemia (ALL) requires a greater understanding of the cells that proliferate to maintain this malignancy because a significant number of cases relapse, resulting from failure to eradicate the disease. Putative ALL stem cells may be resistant to therapy and subsequent relapses may arise from these cells. We investigated expression of CD133, CD19, and CD38 in pediatric B-ALL. Cytogenetic and molecular analyses demonstrated that karyotypically aberrant cells were present in both CD133(+)/CD19(+) and CD133(+)/CD19(-) subfractions, as were most of the antigen receptor gene rearrangements. However, ALL cells capable of long-term proliferation in vitro and in vivo were derived from the CD133(+)/CD19(-) subfraction. Moreover, these CD133(+)/CD19(-) cells could self-renew to engraft serial nonobese diabetic-severe combined immunodeficient recipients and differentiate in vivo to produce leukemias with similar immunophenotypes and karyotypes to the diagnostic samples. Furthermore, these CD133(+)/CD19(-) ALL cells were more resistant to treatment with dexamethasone and vincristine, key components in childhood ALL therapy, than the bulk leukemia population. Similar results were obtained using cells sorted for CD133 and CD38, with only the CD133(+)/CD38(-) subfraction demonstrating xenograft repopulating capacity. These data suggest that leukemia-initiating cells in childhood B-ALL have a primitive CD133(+)/CD19(-) and CD38(-) phenotype.

An early decrease in Notch activation is required for human TCR-alphabeta lineage differentiation at the expense of TCR-gammadelta T cells.

Although well characterized in the mouse, the role of Notch signaling in the human T-cell receptor alphabeta (TCR-alphabeta) versus TCR-gammadelta lineage decision is still unclear. Although it is clear in the mouse that TCR-gammadelta development is less Notch dependent compared with TCR-alphabeta differentiation, retroviral overexpression studies in human have suggested an opposing role for Notch during human T-cell development. Using the OP9-coculture system, we demonstrate that changes in Notch activation are differentially required during human T-cell development. High Notch activation promotes the generation of T-lineage precursors and gammadelta T cells but inhibits differentiation toward the alphabeta lineage. Reducing the amount of Notch activation rescues alphabeta-lineage differentiation, also at the single-cell level. Gene expression analysis suggests that this is mediated by differential sensitivities of Notch target genes in response to changes in Notch activation. High Notch activity increases DTX1, NRARP, and RUNX3 expression, genes that are down-regulated during alphabeta-lineage differentiation. Furthermore, increased interleukin-7 levels cannot compensate for the Notch dependent TCR-gammadelta development. Our results reveal stage-dependent molecular changes in Notch signaling that are critical for normal human T-cell development and reveal fundamental molecular differences between mouse and human.

Continuous improvement in the immune system of HIV-infected children on prolonged antiretroviral therapy.

BACKGROUND: The goal of HAART is to promote reconstitution of CD4+ T cells and other immune responses. We evaluated the extent and the kinetics of immune reconstitution in HIV-infected children over 144 weeks of successful HAART. METHODS: Thirty-seven children receiving their first HAART regimen had plasma HIV RNA; T cells and subpopulations; T-cell rearrangement excision circles (TREC) DNA; candida, HIVCD4 and HIVCD8 enzyme-linked immunospot measured at regular intervals. RESULTS: Plasma HIV RNA became undetectable in 81% of patients at 24 weeks and remained undetectable in 77% at 144 weeks. In contrast, CD4+% continuously increased. Distribution of T-cell subpopulations changed rapidly during the first 48 weeks of HAART and more slowly thereafter. At 144 weeks, total, naive and activated CD4+% and naive CD8+% of HIV-infected children were not significantly different from those of healthy age-matched controls, whereas total and activated CD8+% remained elevated. CD4 and CD8 TREC content increased only during the first 48 weeks of HAART. They positively correlated with each other and with total CD4+%, naive CD4+% and naive CD8+%. Candida and HIVCD4 enzyme-linked immunospot increased over time reaching peak values at 48 weeks and 144 weeks, respectively. HIVCD8 enzyme-linked immunospot decreased in magnitude over 144 weeks of HAART but retained its breadth. Baseline CD4+% positively correlated with CD4+% and with functional immune reconstitution at week 144, whereas baseline TREC correlated with TREC at week 144. CONCLUSION: HIV-infected children acquired normal distribution of CD4 T cells and other subpopulations and recovered CD4-mediated HIV immunity after 144 weeks of HAART.

Recovering probabilities for nucleotide trimming processes for T cell receptor TRA and TRG V-J junctions analyzed with IMGT tools.

BACKGROUND: Nucleotides are trimmed from the ends of variable (V), diversity (D) and joining (J) genes during immunoglobulin (IG) and T cell receptor (TR) rearrangements in B cells and T cells of the immune system. This trimming is followed by addition of nucleotides at random, forming the N regions (N for nucleotides) of the V-J and V-D-J junctions. These processes are crucial for creating diversity in the immune response since the number of trimmed nucleotides and the number of added nucleotides vary in each B or T cell. IMGT sequence analysis tools, IMGT/V-QUEST and IMGT/JunctionAnalysis, are able to provide detailed and accurate analysis of the final observed junction nucleotide sequences (tool "output"). However, as trimmed nucleotides can potentially be replaced by identical N region nucleotides during the process, the observed "output" represents a biased estimate of the "true trimming process." RESULTS: A probabilistic approach based on an analysis of the standardized tool "output" is proposed to infer the probability distribution of the "true trimmming process" and to provide plausible biological hypotheses explaining this process. We collated a benchmark dataset of TR alpha (TRA) and TR gamma (TRG) V-J rearranged sequences and junctions analysed with IMGT/V-QUEST and IMGT/JunctionAnalysis, the nucleotide sequence analysis tools from IMGT, the international ImMunoGeneTics information system, http://imgt.cines.fr. The standardized description of the tool output is based on the IMGT-ONTOLOGY axioms and concepts. We propose a simple first-order model that attempts to transform the observed "output" probability distribution into an estimate closer to the "true trimming process" probability distribution. We use this estimate to test the hypothesis that Poisson processes are involved in trimming. This hypothesis was not rejected at standard confidence levels for three of the four trimming processes: TRAV, TRAJ and TRGV. CONCLUSION: By using trimming of rearranged TR genes as a benchmark, we show that a probabilistic approach, applied to IMGT standardized tool "outputs" opens the way to plausible hypotheses on the events involved in the "true trimming process" and eventually to an exact quantification of trimming itself. With increasing high-throughput of standardized immunogenetics data, similar probabilistic approaches will improve understanding of processes so far only characterized by the "output" of standardized tools.

Thymic function-related markers within the thymus and peripheral blood: Are they comparable?

The thymus involutes with age and its functionality has traditionally been assumed to be limited early in life. However, some studies have demonstrated that thymic function persists in adults. In humans, since it is difficult to obtain thymic samples from healthy individuals, indirect parameters have been used to study the thymic function. The aim of this study was to compare thymic function parameters within both the thymus and peripheral blood mononuclear cells from thirty-three patients who underwent cardiac surgery, as well as to relate these parameters with aging. The proportion of peripheral naïve T cells and intrathymic T cell differentiation stages, as well as peripheral and intrathymic TREC levels were analysed. We demonstrated that thymopoyesis persists in the healthy elderly since all T cell differentiation stages were found within the thymus. Among the studied parameters, peripheral TREC levels are found to be a good thymic function marker since they correlated with age. In healthy individuals, peripheral TREC levels are a good reflect of thymic function as demonstrated by their correlation with intrathymic TREC values.

Human thymus contains multipotent progenitors with T/B lymphoid, myeloid, and erythroid lineage potential.

It is a longstanding question which bone marrow-derived cell seeds the thymus and to what level this cell is committed to the T-cell lineage. We sought to elucidate this issue by examining gene expression, lineage potential, and self-renewal capacity of the 2 most immature subsets in the human thymus, namely CD34+ CD1a- and CD34+ CD1a+ thymocytes. DNA microarrays revealed the presence of several myeloid and erythroid transcripts in CD34+ CD1a- thymocytes but not in CD34+ CD1a+ thymocytes. Lineage potential of both subpopulations was assessed using in vitro colony assays, bone marrow stroma cultures, and in vivo transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The CD34+ CD1a- subset contained progenitors with lymphoid (both T and B), myeloid, and erythroid lineage potential. Remarkably, development of CD34+ CD1a- thymocytes toward the T-cell lineage, as shown by T-cell receptor delta gene rearrangements, could be reversed into a myeloid-cell fate. In contrast, the CD34+ CD1a+ cells yielded only T-cell progenitors, demonstrating their irreversible commitment to the T-cell lineage. Both CD34+ CD1a- and CD34+ CD1a+ thymocytes failed to repopulate NOD/SCID mice. We conclude that the human thymus is seeded by multipotent progenitors with a much broader lineage potential than previously assumed. These cells resemble hematopoietic stem cells but, by analogy with murine thymocytes, apparently lack sufficient self-renewal capacity.

Stepwise development of committed progenitors in the bone marrow that generate functional T cells in the absence of the thymus.

We identified committed T cell progenitors (CTPs) in the mouse bone marrow that have not rearranged the TCRbeta gene; express a variety of genes associated with commitment to the T cell lineage, including GATA-3, T cell-specific factor-1, Cbeta, and Id2; and show a surface marker pattern (CD44+ CD25- CD24+ CD5-) that is similar to the earliest T cell progenitors in the thymus. More mature committed intermediate progenitors in the marrow have rearranged the TCR gene loci, express Valpha and Vbeta genes as well as CD3epsilon, but do not express surface TCR or CD3 receptors. CTPs, but not progenitors from the thymus, reconstituted the alphabeta T cells in the lymphoid tissues of athymic nu/nu mice. These reconstituted T cells vigorously secreted IFN-gamma after stimulation in vitro, and protected the mice against lethal infection with murine CMV. In conclusion, CTPs in wild-type bone marrow can generate functional T cells via an extrathymic pathway in athymic nu/nu mice.

The role of the non-homologous end-joining pathway in lymphocyte development.

One of the most toxic insults a cell can incur is a disruption of its linear DNA in the form of a double-strand break (DSB). Left unrepaired, or repaired improperly, these lesions can result in cell death or neoplastic transformation. Despite these dangers, lymphoid cells purposely introduce DSBs into their genome to maximize the diversity and effector functions of their antigen receptor genes. While the generation of breaks requires distinct lymphoid-specific factors, their resolution requires various ubiquitously expressed DNA-repair proteins, known collectively as the non-homologous end-joining pathway. In this review, we discuss the factors that constitute this pathway as well as the evidence of their involvement in two lymphoid-specific DNA recombination events.

Sibling rivalry: competition between Pol X family members in V(D)J recombination and general double strand break repair.

The nonhomologous end-joining pathway is a major means for repairing double-strand breaks (DSBs) in all mitotic cell types. This repair pathway is also the only efficient means for resolving DSB intermediates in V(D)J recombination, a lymphocyte-specific genome rearrangement required for assembly of antigen receptors. A role for polymerases in end-joining has been well established. They are a major factor in determining the character of repair junctions but, in contrast to 'core' end-joining factors, typically appear to have a subtle impact on the efficiency of end-joining. Recent work implicates several members of the Pol X family in end-joining and suggests surprising complexity in the control of how these different polymerases are employed in this pathway.

Artemis sheds new light on V(D)J recombination.

V(D)J recombination represents one of the three mechanisms that contribute to the diversity of the immune repertoire of B lymphocytes and T lymphocytes. It also constitutes a major checkpoint during the development of the immune system. Indeed, any V(D)J recombination deficiency leads to a block of B-cell and T-cell maturation in humans and animal models, leading to severe combined immunodeficiency (T-B-SCID). Nine factors have been identified so far to participate in V(D)J recombination. The discovery of Artemis, mutated in a subset of T-B-SCID, provided some new information regarding one of the missing V(D)J recombinase activities: hairpin opening at coding ends prior to DNA repair of the recombination activating genes 1/2-generated DNA double-strand break. New conditions of immune deficiency in humans are now under investigations and should lead to the identification of additional V(D)J recombination/DNA repair factors.

Aging, autoimmunity and arthritis: T-cell senescence and contraction of T-cell repertoire diversity - catalysts of autoimmunity and chronic inflammation.

Rheumatoid arthritis (RA), like many other autoimmune syndromes, is a disease of adults, with the highest incidence rates reported in the elderly. The immune system undergoes profound changes with advancing age that are beginning to be understood and that need to be incorporated into the pathogenetic models of RA. The age-related decline in thymic function causes extensive remodeling of the T-cell system. Age-dependent changes in T-cell homeostasis are accelerated in patients with RA. The repertoire of naive and memory T cells is less diverse, possibly as a result of thymic insufficiency, and it is biased towards autoreactive cells. Presenescent T cells emerge that are resistant to apoptosis and that often expand to large clonal populations. These cells are under the regulatory control of nonconventional costimulatory molecules, display potent effector functions, and appear to be critical in the synovial and extra-articular manifestations of RA.

Greater CD8+ TCR heterogeneity and functional flexibility in HIV-2 compared to HIV-1 infection.

Virus-specific CD8(+) T cells are known to play an important role in the control of HIV infection. In this study we investigated whether there may be qualitative differences in the CD8(+) T cell response in HIV-1- and HIV-2-infected individuals that contribute to the relatively efficient control of the latter infection. A molecular comparison of global TCR heterogeneity showed a more oligoclonal pattern of CD8 cells in HIV-1- than HIV-2-infected patients. This was reflected in restricted and conserved TCR usage by CD8(+) T cells recognizing individual HLA-A2- and HLA-B57-restricted viral epitopes in HIV-1, with limited plasticity in their response to amino acid substitutions within these epitopes. The more diverse TCR usage observed for HIV-2-specific CD8(+) T cells was associated with an enhanced potential for CD8 expansion and IFN-gamma production on cross-recognition of variant epitopes. Our data suggest a mechanism that could account for any possible cross-protection that may be mediated by HIV-2-specific CD8(+) T cells against HIV-1 infection. Furthermore, they have implications for HIV vaccine development, demonstrating an association between a polyclonal, virus-specific CD8(+) T cell response and an enhanced capacity to tolerate substitutions within T cell epitopes.

Allelic exclusion at the TCRbeta locus.

Assembly of TCRbeta chain variable-region genes is regulated in the context of allelic exclusion. Differential epigenetic modifications of the two TCRbeta alleles established early in embryonic development may be important for permitting allelic exclusion by ordering rearrangement of the two alleles in double-negative thymocytes. Expression of a TCRbeta chain, as part of the pre-TCR complex, activates signaling pathways that enforce allelic exclusion in double-positive thymocytes. These signaling pathways, which utilize p56(lck) and SLP-76, may be distinct from those used to promote other processes initiated by pre-TCR expression. In double-positive thymocytes allelic exclusion is enforced, in part, by changes in Vbeta gene segment accessibility promoted by cis-acting elements that may be distinct from those regulating accessibility of D/Jbeta gene segments.

Somatic immunoglobulin hypermutation.

Immunoglobulin hypermutation provides the structural correlate for the affinity maturation of the antibody response. Characteristic modalities of this mechanism include a preponderance of point-mutations with prevalence of transitions over transversions, and the mutational hotspot RGYW sequence. Recent evidence suggests a mechanism whereby DNA-breaks induce error-prone DNA synthesis in immunoglobulin V(D)J regions by error-prone DNA polymerases. The nature of the targeting mechanism and the trans-factors effecting such breaks and their repair remain to be determined.

Quantitation of T-cell neogenesis in vivo after allogeneic bone marrow transplantation in adults.

Following myeloablative therapy, it is unknown to what extent age-dependent thymic involution limits the generation of new T cells with a diverse repertoire. Normal T-cell receptor gene rearrangement in T-cell progenitors results in the generation of T-cell receptor rearrangement excision circles (TRECs). In this study, a quantitative assay for TRECs was used to measure T-cell neogenesis in adult patients with leukemia who received myeloablative therapy followed by transplantation of allogeneic hematopoietic stem cells. Although phenotypically mature T cells had recovered by 1 to 2 months after bone marrow transplantation (BMT), TREC levels remained low for 3 months after BMT. T-cell neogenesis became evident by 6 months, and normal levels of adult thymic function were restored at 6 to 12 months after BMT. Subsequent leukemia relapse in some patients was associated with reduced TREC levels, but infusion of mature donor CD4(+) T cells resulted in rapid restoration of thymic function. These studies demonstrate that T-cell neogenesis contributes to immune reconstitution in adult patients and suggest that thymic function can be manipulated in vivo. (Blood. 2001;98:1116-1121)

Which T cells cause psoriasis?

Today, T cells appear to be the main protagonists in the pathogenesis of psoriasis vulgaris. This article summarizes how T cells might contribute to the generation of psoriatic skin lesions. It discusses the preferential T cell receptor usage and the putative mode of T cell activation in psoriatic skin lesions, and how streptococcal throat infections could be involved in disease manifestations. The results are integrated into a pathogenetic concept which considers psoriasis as a T-cell mediated autoimmune disorder.