Basis of CTLA-4 function in regulatory and conventional CD4(+) T cells

CTLA-4 proteins contribute to the suppressor function of regulatory T cells (Tregs), but the mechanism by which they do so remains incompletely understood. In the present study, we assessed CTLA-4 protein function in both Tregs and conventional (Tconv) CD4(+) T cells. We report that CTLA-4 proteins are responsible for all 3 characteristic Treg functions of suppression, TCR hyposignaling, and anergy. However, Treg suppression and anergy only required the external domain of CTLA-4, whereas TCR hyposignaling required its internal domain. Surprisingly, TCR hyposignaling was neither required for Treg suppression nor anergy because costimulatory blockade by the external domain of CTLA-4 was sufficient for both functions. We also report that CTLA-4 proteins were localized in Tregs in submembrane vesicles that rapidly recycled to/from the cell surface, whereas CTLA-4 proteins in naive Tconv cells were retained in Golgi vesicles away from the cell membrane and had no effect on Tconv cell function. However, TCR signaling of Tconv cells released CTLA-4 proteins from Golgi retention and caused activated Tconv cells to acquire suppressor function. Therefore, the results of this study demonstrate the importance of intracellular localization for CTLA-4 protein function and reveal that CTLA-4 protein externalization imparts suppressor function to both regulatory and conventional CD4(+) T cells.     

Lack of a role for transforming growth factor-beta in cytotoxic T lymphocyte antigen-4-mediated inhibition of T cell activation

Similarities in the phenotypes of mice deficient for cytotoxic T lymphocyte antigen-4 (CTLA-4) or transforming growth factor-beta1 (TGF-beta1) and other observations have led to speculation that CTLA-4 mediates its inhibitory effect on T cell activation via costimulation of TGF-beta production. Here, we examine the role of TGF-beta in CTLA-4-mediated inhibition of T cell activation and of CTLA-4 in the regulation of TGF-beta production. Activation of AND TCR transgenic mouse T cells with costimulatory receptor-specific antigen presenting cells results in efficient costimulation of proliferation by CD28 ligation and inhibition by CTLA-4 ligation. Neutralizing antibody to TGF-beta does not reverse CTLA-4-mediated inhibition. Also, CTLA-4 ligation equally inhibits proliferation of wild-type, TGF-beta1(-/-), and Smad3(-/-) T cells. Further, CTLA-4 engagement does not result in the increased production of either latent or active TGF-beta by CD4(+) T cells. These results indicate that CTLA-4 ligation does not regulate TGF-beta production and that CTLA-4-mediated inhibition can occur independently of TGF-beta. Collectively, these data demonstrate that CTLA-4 and TGF-beta represent distinct mechanisms for regulation of T cell responses.     

Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4+ T cells and is quickly expressed on the cell surface in an antigen-specific manner

CD40 ligand (CD40L) is an essential effector cytokine for macrophage activation, dendritic cell licensing, and T-cell-dependent antibody responses. Although CD40L is known to be made de novo following antigen recognition, several reports have described surface mobilization of preformed, intracellular CD40L in certain CD4(+) effector T cells. Here we show that rapid surface expression of preformed CD40L following antigen recognition is a general property of both effector and memory CD4(+) T cells, including in vitro and in vivo activated T-cell-receptor transgenic T cells, memory phenotype CD4(+) T cells from pathogen-free naive mice, and polyclonal virus-specific effector and memory T cells. Intracellular CD40L is stored in secretory lysosomes, and colocalizes more strongly with Fas ligand than with CTLA-4, two other molecules that are delivered to the cell surface following antigen recognition. Stimulated surface expression of preformed CD40L is found in memory CD4(+) T cells from CD40-deficient mice, indicating that it does not depend on CD40-induced internalization for delivery to the secretory compartment. We suggest that delivery of preformed CD40L to antigen-presenting cells (APCs) could enable antigen-specific activation of APCs in transient interactions that are too brief to permit de novo synthesis of CD40L.     

Exacerbated colitis associated with elevated levels of activated CD4+ T cells in TCRalpha chain transgenic mice

BACKGROUND AND AIMS: An unconventional CD4+ TCRalpha(-)beta(+) cell population mediates the development of colitis resembling ulcerative colitis in T-cell receptor alpha mutant (TCRalpha(-/-)) mice. However, the significance of such T cells in individuals with an intact TCRalpha locus remains unclear. Because a substantial proportion of naturally rearranged TCRalpha chains fails to pair with TCRbeta chains, the aim of this study was to analyze the development of CD4+ TCRalpha(-)beta(+) cells and the course of colitis in the presence of such a TCRalpha chain. METHODS: TCR chain transgenic TCRalpha(-/-) mice were generated and compared with wild-type and TCRalpha(-/-) mice by flow cytometric analysis of T lymphocytes with respect to their TCR expression and activation status and by histological analysis of colon tissue. The colitogenic potential of the unconventional CD4+ TCRalpha(-)beta(+) cells was assessed by adoptive transfer experiments. Furthermore, the half-life of TCRbeta chains was determined by pulse-chase labeling and immunoprecipitation. RESULTS: Transgenic expression of a TCR Valpha7.2 chain led to increased frequencies of CD4+ TCRalpha(-)beta(+) cells that caused rapid onset of colitis, reminiscent of, but even more severe than, that in TCRalpha(-/-) mice. This unconventional T-cell population displayed a constitutively activated phenotype in normal and transgenic TCRalpha(-/-) mice. An extended half-life of newly synthesized TCRbeta chains suggests a chaperone function of the TCR Valpha7.2 chain in TCRalpha(-/-) mice. CONCLUSIONS: Physiological TCRalpha rearrangement can promote the formation of chronically activated CD4+ TCRalpha(-)beta(+) T cells and may play a role in the etiology of UC.     

Competition for self ligands restrains homeostatic proliferation of naive CD4 T cells

T cell antigen receptor (TCR) diversity is a critical feature of adaptive immunity. However, restriction of TCR diversity is a potential risk during immune reconstitution by homeostatic proliferation. What peripheral mechanisms are in place to maintain TCR diversity during recovery from lymphopenia? Here, we examine competition between several monoclonal CD4 T cell populations in RAG(-/-) and TCR Tg RAG(-/-) environments. The results suggest that specific self ligands constitute a critical limiting resource essential for homeostatic proliferation of naive CD4 T cells. In addition, T cells ignore large numbers of competitors as long as their TCR specificity is different and other non-MHC resources are not limiting. Therefore, the numbers of self ligands expressed in the periphery set the limits on TCR diversity.     

CTLA-4 dysregulation of self/tumor-reactive CD8+ T-cell function is CD4+ T-cell dependent

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) maintains peripheral tolerance by suppressing T-cell activation and proliferation but its precise role in vivo remains unclear. We sought to elucidate the impact of CTLA-4 expression on self/tumor-reactive CD8(+) T cells by using the glycoprotein (gp) 100-specific T-cell receptor (TCR) transgenic mouse, pmel-1. pmel-1 CLTA-4(-/-) mice developed profound, accelerated autoimmune vitiligo. This enhanced autoimmunity was associated with a small but highly activated CD8(+) T-cell population and large numbers of CD4(+) T cells not expressing the transgenic TCR. Adoptive transfer of pmel-1 CLTA-4(-/-) CD8(+) T cells did not mediate superior antitumor immunity in the settings of either large established tumors or tumor challenge, suggesting that the mere absence of CTLA-4-mediated inhibition on CD8(+) T cells did not directly promote enhancement of their effector functions. Removal of CD4(+) T cells by crossing the pmel-1 CLTA-4(-/-) mouse onto a Rag-1(-/-) background resulted in the complete abrogation of CD8(+) T-cell activation and autoimmune manifestations. The effects of CD4(+) CLTA-4(-/-) T cells were dependent on the absence of CTLA-4 on CD8(+) T cells. These results indicated that CD8(+) CLTA-4(-/-) T-cell-mediated autoimmunity and tumor immunity required CD4(+) T cells in which the function was dysregulated by the absence of CTLA-4-mediated negative costimulation.     

RAG-dependent peripheral T cell receptor diversification in CD8+ T lymphocytes

Rearrangement of T cell receptor (TCR) genes is driven by transient expression of V(D)J recombination-activating genes (RAGs) during lymphocyte development. Immunological dogma holds that T cells irreversibly terminate RAG expression before exiting the thymus, and that all of the progeny arising from mature T cells express the parental TCRs. When single pancreatic islet-derived, NRP-A7 peptide-reactive CD8(+) T cells from nonobese diabetic (NOD) mice were repeatedly stimulated with peptide-pulsed dendritic cells, daughter T cells reexpressed RAGs, lost their ability to bind to NRP-A7K(d) tetramers, ceased to transcribe tetramer-specific TCR genes, and, instead, expressed a vast array of other TCR rearrangements. Pancreatic lymph node (PLN) CD8(+) T cells from animals expressing a transgenic NRP-A7-reactive TCR transcribed and translated RAGs in vivo and displayed endogenous TCRs on their surface. RAG reexpression also occurred in the PLN CD8(+) T cells of wild-type NOD mice and could be induced in the peripheral CD8(+) T cells of nondiabetes-prone TCR-transgenic B10.H2(g7) mice by stimulation with peptide-pulsed dendritic cells. In contrast, reexpression of RAGs could not be induced in the CD8(+) T cells of B6 mice expressing an ovalbumin-specific, K(b)-restricted TCR, or in the CD8(+) T cells of NOD mice expressing a lymphocytic choriomeningitis virus-specific, D(b)-restricted TCR. Extra-thymic reexpression of the V(D)J recombination machinery in certain CD8(+) T cell subpopulations, therefore, enables further diversification of the peripheral T cell repertoire.     

Stage-dependent reactivity of thymocytes to self-peptide--MHC complexes

In mice that express a transgene for the 2C T cell antigen-receptor (TCR) and lack a recombinase-activating gene (2C(+)RAG(-/-) mice) most of the peripheral T cells are CD8(+), a few are CD4(+), and a significant fraction are CD4(-)CD8(-) [double negative (DN)]. The DN 2C cells, like DN T cells that are abundant in various other alphabeta TCR-transgenic mice, appear to be derived directly from DN thymocytes that prematurely express the TCR transgene. The DN 2C cells are virtually absent in mice deficient in major histocompatibility complex class II (MHC-II) but more abundant in mice deficient in MHC-I, suggesting that the DN 2C thymocytes are positively selected by self-peptide-MHC-II (pMHC-II) complexes and negatively selected by self-pMHC-I complexes. The pMHC-I complexes, however, positively select CD8(+) 2C T cells in the same mice. The different effects of thymic pMHC-I on DN and CD8(+) thymocytes are consistent with the finding that DN 2C thymocytes are more sensitive than more mature CD4(+)CD8(+) [double positive (DP)] thymocytes to a weak pMHC-I agonist for the 2C TCR. Together with previous evidence that DP thymocytes respond more sensitively than T cells in the periphery to weak pMHC agonists, the findings suggest progressive decreases in responsiveness to self-pMHC-I complexes as thymocytes develop from DN to DP thymocytes and then to mature na?ve T cells in the periphery.     

Normal hematopoiesis is maintained by activated bone marrow CD4+ T cells

CD4(+) T cells produce hematopoietic-related cytokines and are essential for hematopoiesis stimulation during infection and hematologic recovery after bone marrow transplantation. However, it remains unclear if T cells are necessary to maintain normal hematopoiesis. We report here that, in T-cell-deficient mice, terminal differentiation of myeloid progenitors is defective, resulting in very low levels of granulocytes in the periphery. Hematopoiesis is restored after thymus graft or reconstitution with CD4(+) T cells but not CD8(+) T cells. Bone marrow CD4(+) T cells have an activated phenotype and produce cytokines, apparently, in the absence of exogenous stimulation. Transgenic mice carrying T-cell receptor specific for an ovalbumin peptide presented in the context of a specific class II molecule (I-A(d)) (DO11.10 RAG(-/-)) show the same hematopoietic deficiency as athymic mice. Their bone marrow CD4(+) T cells are not activated, suggesting that hematopoiesis maintenance requires the presence of cognate antigen in order to activate bone marrow T-helper cells. In fact, priming of transgenic mice with ovalbumin restores normal hematopoiesis. The data show that the current concept of "normal hematopoiesis" does not reflect a basal bone marrow activity, but it is an antigen-induced state maintained by constant activation of bone marrow CD4(+) T cells.     

Tpl2 and ERK transduce antiproliferative T cell receptor signals and inhibit transformation of chronically stimulated T cells

The protein kinase encoded by the Tpl2 protooncogene plays an obligatory role in the transduction of Toll-like receptor and death receptor signals in macrophages, B cells, mouse embryo fibroblasts, and epithelial cells in culture and promotes inflammatory responses in animals. To address its role in T cell activation, we crossed the T cell receptor (TCR) transgene 2C, which recognizes class I MHC presented peptides, into the Tpl2(-/-) genetic background. Surprisingly, the TCR2C(tg/tg)/Tpl2(-/-) mice developed T cell lymphomas with a latency of 4-6 months. The tumor cells were consistently TCR2C(+)CD8(+)CD4(-), suggesting that they were derived either from chronically stimulated mature T cells or from immature single positive (ISP) cells. Further studies showed that the population of CD8(+) ISP cells was not expanded in the thymus of TCR2C(tg/tg)/Tpl2(-/-) mice, making the latter hypothesis unlikely. Mature peripheral T cells of Tpl2(-/-) mice were defective in ERK activation and exhibited enhanced proliferation after TCR stimulation. The same cells were defective in the induction of CTLA4, a negative regulator of the T cell response, which is induced by TCR signals via ERK. These findings suggest that Tpl2 functions normally in a feedback loop that switches off the T cell response to TCR stimulation. As a result, Tpl2, a potent oncogene, functions as a tumor suppressor gene in chronically stimulated T cells.     

T helper type-2 cells induce ileal villus atrophy,goblet cell metaplasia,and wasting disease in T cell-deficient mice

BACKGROUND & AIMS: T helper (Th) 1 and Th2 cell subsets significantly influence the pathological features of inflammation in the gastrointestinal tract in a distinct manner. It is now established that the transfer of CD4(+)CD45RB(Hi) (RB(Hi)) T cells to either severe combined immunodeficient (SCID) or recombinase activation gene 2-deficient (RAG(-/-)) mice results in a severe granulomatous hypertrophic colitis mediated by Th1 cells. We have modified this approach to address the role of Th2 cells. METHODS: RB(Hi) T cells from wild-type (Wt) mice or mice genetically predisposed to Th2 responses (interferon-gamma-defective [IFN-gamma(-/-)]) with or without B cells were transferred to T cell receptor (TCR)-beta and delta-chain-defective (TCR(-/-)) or SCID mice. RESULTS: Transfer of Wt RB(Hi) T cells induced wasting disease with severe colitis in the TCR(-/-) mice. In contrast, IFN-gamma(-/-) RB(Hi) T cells induced severe weight loss and hypoalbuminemia without significant inflammation in the colon. The small intestine of these mice exhibited villus atrophy, a decrease in brush-border enzymes, reduced enterocyte proliferation, and an increased number of goblet cells. The presence of B cells was necessary for these changes, because SCID recipients required cotransfer of B cells, together with IFN-gamma(-/-) RB(Hi) T cells for ileal lesions to develop. Treatment of TCR(-/-) recipients of IFN-gamma(-/-) RB(Hi) T cells with anti-IL-4 mAb abrogated both the wasting disease and the villus atrophy. CONCLUSIONS: Dysregulated Th2 cells cause atrophic changes and goblet cell transformation in the small intestinal epithelium and wasting disease mediated by excess interleukin-4 and B cells.     

T cells from epicutaneously immunized mice are prone to T cell receptor revision

Epicutaneous immunization of T cell receptor (TCR) transgenic (Tg) mice whose CD4(+) T cells are specific for the Ac1-11 fragment of myelin basic protein (MBP) with Ac1-11 elicits T cells with dominant suppressor/regulatory activity that confers protection against Ac1-11-induced experimental autoimmune encephalomyelitis. We now report that such disease-resistant MBP TCR Tg mice also harbor a sizeable fraction of peripheral CD4(+) T cells lacking surface expression of the Tg TCR beta chain and expressing diverse, endogenously rearranged TCR beta chains. Ex vivo incubation at physiological temperature caused the loss of neo-beta-chain expression and reversion to the MBP alphabeta TCR(+) phenotype. The presence of recombination activating gene 1 and 2 proteins in CD4(+) T cells with revised TCRs was consistent with effective V(D)J recombination activity. The emergence of these cells did not depend on the thymic compartment. We conclude that in mice epicutaneously immunized with an autoantigen, peripheral specific T cells are susceptible to multiple mechanisms of tolerance.     

CD4+ and CD8+ T cell receptor repertoire perturbations with normal levels of T cell receptor excision circles in HIV-infected,therapy-naive adolescents

Our objective was to determine whether treatment-naive HIV-infected adolescents manifest abnormalities in thymus function and peripheral T cell repertoire, and to assess relationships of these immunologic characteristics with each other, with plasma HIV virus load, and T cell surface markers. TCR Vbeta repertoire was determined by CDR3 length spectratyping in purified CD4(+) and CD8(+) T cells of high-risk, HIV-negative adolescents and of treatment-naive, HIV-infected adolescents. Thymus function was investigated by the simultaneous examination of T cell receptor excision circles (TRECs) in the CD4(+) and CD8(+) T cell subsets. HIV-infected adolescents exhibited significantly greater perturbations in their TCR Vbeta repertoire in comparison with HIV-negative subjects. Perturbations in the CD8(+) T cell compartment were more profound in comparison with CD4(+) T cells. The CD4(+) TCR Vbeta perturbations were negatively correlated with the total and phenotypically naive CD4(+) T cells, and with CD4(+) TRECs. CD8(+) TRECs, although not correlated with CD8(+) TCR Vbeta perturbations, showed negative correlation with memory and activated CD8(+) T cells. Interestingly, TRECs in CD4(+) and CD8(+) T cells were not significantly different between HIV-infected and uninfected adolescents. The TCR Vbeta repertoire in adolescents is profoundly perturbed even in early stages of HIV infection, when total CD4(+) cell counts in most subjects are within normal limits. The correlative analyses demonstrating negative association of CD4(+) cell TRECs with CD4(+) TCR Vbeta perturbations and of CD8(+) TRECs with CD8(+) cell activation markers provide evidence of the intense activation of the central and peripheral immune compartments in this study population.     

Class I MHC molecules on hematopoietic cells can support intrathymic positive selection of T cell receptor transgenic T cells.

The identity of cells that mediate positive selection of CD8(+) T cells was investigated in two T cell receptor (TCR) transgenic systems. Irradiated beta(2)-microglobulin mutant mice or mice with mutations in both the K(b) and D(b) genes were repopulated with fetal liver cells from class I(+) TCR transgenic mice. In the case of the 2C TCR, mature transgene-expressing CD8(+) T cells appeared in the thymuses of the chimeras and in larger numbers in the peripheral lymphoid organs. These CD8(+) T cells were functional, exhibited a naive, resting phenotype, and were mostly thymus-dependent. Their development depended on donor cell class I expression. These results establish that thymic hematopoietic cells can direct positive selection of CD8(+) T cells expressing a conventional TCR. In contrast, no significant development of HY (male antigen)-TCR(+) CD8(+) T cells was observed in class I(+) into class I-deficient chimeras. These data suggest that successful positive selection directed by hematopoietic cells depends on specific properties of the TCR or its thymic ligands. The possibility that hematopoietic cell-induced, positive selection occurs only with TCRs that exhibit relatively high avidity interactions with selecting ligands in the thymus is discussed.     

Foxp3+ CD25- CD4 T cells constitute a reservoir of committed regulatory cells that regain CD25 expression upon homeostatic expansion

Expression of the IL-2 receptor alpha chain (CD25) by peripheral CD4 T cells follows cellular activation. However, CD25 expression by CD4 cells is widely used as a marker to identify regulatory T cells (T(R)), although cells with regulatory properties are also found in the CD4+CD25- subset. By using in vivo functional assays and Foxp3 expression as a faithful marker of T(R) differentiation, we have evaluated the requirements for CD25 expression by peripheral T(R). We first show that in vivo depletion of CD25+ cells prevents the development of spontaneous encephalomyelitis in recombination-activating gene (RAG)-deficient anti-myelin basic protein T cell antigen receptor (TCR) transgenic mice, and allows disease induction in otherwise healthy RAG-competent transgenic mice. Similar treatment in normal thymectomized animals is followed by the fast recovery of a normal number of CD25+ T(R). Consistently, Foxp3-expressing T(R) encompassed in the CD25- cell population convert to CD25+ after homeostatic expansion and are selectable by IL-2 in vitro. Surface expression of CD25 on T(R) is controlled by the activity of conventional CD4 cells and is fully labile because it can be lost and regained without affecting the functional potential of the cells. These findings reveal that Foxp3-expressing CD25- cells constitute a peripheral reservoir of differentiated T(R), recruited to the CD25+ pool upon homeostatic expansion and/or activation. This analysis, together with the notion that physiological commitment of T(R) takes place exclusively in the thymus should help for the interpretation of experiments assessing peripheral T(R) differentiation from naive CD4 T cells, defined as CD25-.     

Analysis of T cells in paroxysmal nocturnal hemoglobinuria provides direct evidence that thymic T-cell production declines with age.

Peripheral blood T cells in patients with paroxysmal nocturnal hemoglobinuria (PNH) comprise a mixture of residual normal and glycosylphosphatidylinositol (GPI)-deficient PNH cells. Using multicolor flow cytometry, we demonstrated significant differences between the proportions of naive and memory cells within these populations. PNH T cells comprise mainly naive cells (CD45RA(+)CD45R0(-)), whereas normal T cells in the same patients were predominantly memory (CD45RA(-)CD45R0(+)) cells. Functional analyses showed that GPI-deficient CD45RA(+) T cells can convert to a CD45R0(+) phenotype. We present data from a PNH patient in remission for 20 years who still had significant numbers of GPI-deficient T cells; these showed a normal distribution of naive and memory components. The predominantly naive phenotype of GPI-deficient T cells seen in PNH patients with active disease likely reflects the phenotype of recent normal thymic emigrants. In patients where hematopoiesis was predominantly derived from the PNH stem cell, absolute numbers of both naive PNH CD4(+) cells and CD8(+) cells show an inverse correlation with patient age, implying this age-related decline in T-cell production is secondary to a decrease in thymic activity rather than a stem cell defect.     

Allelic variant in CTLA4 alters T cell phosphorylation patterns

Little is known regarding the functional effects of common autoimmune susceptibility variants on human immune cells. The SNP CT60 (rs3087243; A/G) located in the 3' UTR of the CTLA4 gene has been associated with autoimmune diseases. We examined a cohort of healthy individuals stratified by genotypes at CTLA4 to gain insight into the functional effects of allelic variation on T cell signaling. Using phospho-site-specific mAbs, we tested the hypothesis that the CT60 genotype at CTLA4 is associated with altered T cell antigen receptor (TCR) signaling in naive and/or memory T cells. By normalizing for the extent of the initial TCR signaling event at CD3zeta, we observed that the relative responsiveness to TCR stimulation as assessed by phosphorylation levels of downstream signaling molecules was altered in naive (CD4(+)CD45RA(high)) and memory (CD4(+)CD45RA(low)) T cells obtained from individuals with the disease-susceptibility allele at CTLA4. Thus, allelic variation associated with autoimmune disease can alter the signaling threshold of CD4(+) T cells. These experiments provide a rational approach for the dissection of T cell-susceptibility genes in autoimmune diseases.     

Development of CD4+ T cells expressing a nominally MHC class I-restricted T cell receptor by two different mechanisms

Differences in T cell receptor (TCR) signaling initiated by interactions among TCRs, coreceptors, and self-peptide-MHC complexes determine the outcome of CD4 versus CD8 lineage of T cell differentiation. The H-2Ld and Kbm3 alloreactive 2C TCR is positively selected by MHC class I Kb and a yet-to-be identified nonclassical class I molecule to differentiate into CD8+ T cells. Here we describe two mechanisms by which CD4+ 2C T cells can be generated in 2C TCR-transgenic mice. In the RAG-/- background, development of CD4+ 2C T cells requires the expression of both I-Ab and the TAP genes, indicating that both MHC class I and II molecules are required for positive selection of these T cells. Notably, only some of the 2C+ RAG-/- mice (approximately 30%) develop CD4+ 2C T cells, with frequencies in individual mice varying from 0.5% to as high as approximately 50%. In the RAG+ background, where endogenous TCRalpha genes are rearranged and expressed, CD4+ 2C T cells are generated because these cells express the 2C TCR as well as additional TCRs, consisting of the 2C TCRbeta and endogenous TCRalpha chains. Similarly, T cells expressing the OT-1 TCR, which is nominally MHC class I-restricted, can also develop into CD4+ T cells through the same two mechanisms. Thus, expression of two TCRs by a single thymocyte, TCR recognition of multiple MHC molecules, and heterogeneity of TCR, coreceptors, and peptide-MHC interactions in the thymus all contribute to the outcome of CD4 versus CD8 lineage development.     

Adapted NOD/SCID model supports development of phenotypically and functionally mature T cells from human umbilical cord blood CD34(+) cells

The NOD-LtSZ scid/scid (NOD/SCID) repopulation assay is the criterion for the study of self-renewal and multilineage differentiation of human hematopoietic stem cells. An important shortcoming of this model is the reported absence of T-cell development. We studied this aspect of the model and investigated how it could be optimized to support T-cell development. Occasionally, low-grade thymic engraftment was observed in NOD/SCID mice or Rag2(-/-)gamma(c)(-/-) mice. In contrast, the treatment of NOD/SCID mice with a monoclonal antibody against the murine interleukin-2R beta, (IL-2R beta) known to decrease natural killer cell activity, resulted in human thymopoiesis in up to 60% of the mice. T-cell development was phenotypically normal and resulted in polyclonal, mature, and functional CD1(-) TCR alpha beta (+) CD4(+) or CD8(+) single-positive T cells. In mice with ongoing thymopoiesis, peripheral T cells were observed. TREC analysis showed that T cells with a naive phenotype (CD45RA(+)) emerged from the thymus. In approximately half of these mice, the peripheral T cells included a pauciclonal outgrowth of CD45RO(+) cells. These data suggest that all elements of a functional immune system were present in these animals.