Single-positive CD4 Research Articles

Gads‐deficient thymocytes are blocked at the transitional single positive CD4+ stage

Positive selection of T-cell precursors is the process by which a diverse T-cell repertoire is established. Positive selection begins at the CD4(+)CD8(+) double positive (DP) stage of development and involves at least two steps. First, DP thymocytes down-regulate CD8 to become transitional single positive (TSP) CD4(+) thymocytes. Then, cells are selected to become either mature single positive CD4(+) or mature single positive CD8(+) thymocytes. We sought to define the function of Gads during the two steps of positive selection by analyzing a Gads-deficient mouse line. In Gads(+/+) mice, most TSP CD4(+) thymocytes are TCR(hi)Bcl-2(hi)CD69(+), suggesting that essential steps in positive selection occurred in the DP stage. Despite that Gads(-/-) mice could readily generate TSP CD4(+) thymocytes, many Gads(-/-) TSP CD4(+) cells were TCR(lo)Bcl-2(lo)CD69(-), suggesting that Gads(-/-) cells proceeded to the TSP CD4(+) stage prior to being positively selected. These data suggest that positive selection is not a prerequisite for the differentiation of DP thymocytes into TSP CD4(+) thymocytes. We propose a model in which positive selection and differentiation into the TSP CD4(+) stage are separable events and Gads is only required for positive selection.

Notch signaling is required for proliferation but not for differentiation at a well-defined β-selection checkpoint during human T-cell development

AbstractNotch signaling is absolutely required for β-selection during mouse T-cell development, both for differentiation and proliferation. In this report, we investigated whether Notch has an equally important role during human T-cell development. We show that human CD34+ thymocytes can differentiate into CD4+CD8β+ double positive (DP) thymocytes in the absence of Notch signaling. While these DP cells phenotypically resemble human β-selected cells, they lack a T-cell receptor (TCR)–β chain. Therefore, we characterized the β-selection checkpoint in human T-cell development, using CD28 as a differential marker at the immature single positive CD4+CD3−CD8α− stage. Through intracellular TCR-β staining and gene expression analysis, we show that CD4+CD3−CD8α−CD28+ thymocytes have passed the β-selection checkpoint, in contrast to CD4+CD3−CD8α−CD28− cells. These CD4+CD3−CD8α−CD28+ thymocytes can efficiently differentiate into CD3+TCRαβ+ human T cells in the absence of Notch signaling. Importantly, preselection CD4+CD3−CD8α−CD28− thymocytes can also differentiate into CD3+TCRαβ+ human T cells without Notch activation when provided with a rearranged TCR-β chain. Proliferation of human thymocytes, however, is clearly Notch-dependent. Thus, we have characterized the β-selection checkpoint during human T-cell development and show that human thymocytes require Notch signaling for proliferation but not for differentiation at this stage of development.

Phenotypic and Functional Characterization of HIV-1-Specific CD4+CD8+ Double-Positive T Cells in Early and Chronic HIV-1 Infection

CD4+CD8+ double-positive (DP) T cells represent a poorly characterized population of effector T cells found at low frequencies in the peripheral blood. Virus-specific DP T cells have been identified in HIV-1-infected patients but their origin, relationship to conventional CD4+ and CD8+ single-positive (SP) T cells, and role in disease pathogenesis are unclear. In this study, peripheral blood T cells were analyzed for cytokine production, maturation, and cytolytic marker expression by polychromatic flow cytometry in subjects with both early (n = 27) and chronic (n = 21) HIV-1 infection. HIV-1-specific interferon gamma (IFN-g)-producing DP T cells were identified at a median frequency of 0.48% compared with 1.08% and 0.02% for CD8 and CD4 SP cells, respectively, in response to pooled HIV-1 peptides. HIV-1- specific DP T cells exhibited polyfunctionality with characteristics of both CD4 and CD8 SP T cells, including coproduction of IFN-gamma and IL-2 and expression of cytolytic-associated lysosomal-associated membrane protein. No differences in frequencies of unstimulated DP T cells were observed in early compared with chronic infection. However, chronic infection was associated with higher frequencies of HIV-specific, IFN-gamma-producing DP T cells and higher fractions of effector memory and lysosomal-associated membrane protein expression among these cells, suggesting an effect of cumulative viral antigen burden on DP T-cell function.

Development of CD4 T cells from Human Hematopoietic stem Cells in a Lentiviral-engineered Stromal Culture (46.15)

Abstract Hematopoietic precursor/stem cells (HPCs) are progenitors to all functional immune cell lineages. We established a novel stromal cell line expressing (Delta-like 1) DL1, interleukin-7 (IL-7), and FMS-like tyrosine kinase 3 Ligand (Flt3-L) and demonstrated efficient differentiation and expansion of early T cells from adult CD34+ HPCs. However, continuous IL-7 signaling led to the production of immature CD8+ T lymphocytes at the expense of CD4+ T lymphocytes. It is known that the developing double positive T cells shut down IL-7 mediated signaling and require TCR signaling for survival and positive selection. We hypothesize that IL-7 inhibits the TCR signaling event required for positive selection. We found that by removal of IL-7, followed with CD3/CD28 stimulation, the early T cells were able to differentiate into mature single positive CD4 T cells expressing CD3, CD28, and TCRαβ. This can also be demonstrated by bypassing TCR signaling through stimulation with PMA/ionomycin. When further stimulated, these in vitro derived CD4+ T cells differentiated into various T helper subtypes and produced effector cytokines including IL-2, IFN-γ, and IL-17. This novel culture system can greatly facilitate the study of late T cell development and has potential for immunotherapeutic applications. Supported by Vectorite Biomedica Inc. and Yongling Foundation.

Glycerol-3-phosphate acyltransferase-1 Alters Thymic Development in Mice (85.16)

Abstract Our lab has recently shown that T-lymphocyte function ex vivo from glycerol-3-phosphate acyltransferase-1 (GPAT-1), first enzyme in de novo glycerophospholipid biosynthesis, knockout mice is similar to aged T-lymphocytes. In order to determine the mechanism(s) for this observation, we examined thymic development in GPAT-1 KO mice. Thymi were weighed then thymocytes isolated for determination of proliferation and thymic subset proportions. We found that the thymi from GPAT-1 KO mice were 30% smaller than the wild type controls and thymocyte proliferation was reduced. Furthermore, the thymic single positive CD4 and double negative subset proportions were increased while the double positive population was decerased. The changes in thymic weight and thymocyte subsets are most likely not linked to altered fetal thymic development since thymic weights from 3 week old mice were identical to that seen in wild type controls. Since T-lymphocyte GPAT-1 activity is reduced in aged T-lymphocytes these data give insight into a novel mechanism by which aging may alter thymic development and subsequent peripheral T-lymphocyte function. This work was supported by NIH AG20651 (CAJ).

Immune Ontogeny and Engraftment Receptivity in the Sheep Fetus

Objective: The biologic explanation for fetal receptivity to donor engraftment and subsequent long-term tolerance following transplantation early in gestation is not known. We investigated the role fetal immune ontogeny might play in fetal transplantation tolerance in sheep. Methods: Engraftmentof allogeneic and xenogeneicHSC was determined 60 days following transplantation at different time points in sheep fetal gestation. Parallel analysis of surface differentiation antigen expression on cells from lymphoid organs of timed gestational age fetal sheep was determined by flow cytometry using available reagents. Results: An engraftment window was identified after day 52 gestation lasting until day 71 (term gestation: 145 days). This period was associated with the expression of the leukocyte common antigen CD45 on all cells in the thymus. Double-positive and single-positive CD4 and CD8 cells began appearing in the thymus just prior (day 45 gestation) to the beginning of the engraftment window, while single-positive CD4 or CD8 cells do not begin appearing in peripheral organs until late in the engraftment period, suggesting deletional mechanisms may be operative. In concert, surface IgM-positive cells express CD45 in the thymus at day 45, with a comparable delay in the appearance of IgM/CD45 cells in the periphery until late in the engraftment window. Conclusions: These findings support a central role for the thymus in multilineage immune cell maturation during the period of fetal transplantation receptivity. Further, they suggest that fetal engraftment receptivity is due to gestational age-dependent deletional tolerance.

Intestinal double-positive CD4+CD8+ T cells of neonatal rhesus macaques are proliferating, activated memory cells and primary targets for SIVMAC251 infection

AbstractPeripheral blood and thymic double-positive (DP) CD4+CD8+ T cells from neonates have been described earlier, but the function and immunophenotypic characteristics of other tissue-derived DP T cells are not clearly understood. Here, we demonstrate the functional and immunophenotypic characteristics of DP cells in 6 different tissues, including thymus from normal neonatal rhesus macaques (Macaca mulatta) between 0 and 21 days of age. In general, intestinal DP T cells of neonates have higher percentages of memory markers (CD28+CD95+CD45RAlowCD62Llow) and proliferation compared with single-positive (SP) CD4+ and CD8+ T cells. In addition, percentages of DP T cells increase and CD62L expression decreases as animals mature, suggesting that DP cells mature and proliferate with maturity and/or antigen exposure. Consistent with this, intestinal DP T cells in neonates express higher levels of CCR5 and are the primary targets in simian immunodeficiency virus (SIV) infection. Finally, DP T cells produce higher levels of cytokine in response to mitogen stimulation compared with SP CD4+ or CD8+ T cells. Collectively, these findings demonstrate that intestinal DP T cells of neonates are proliferating, activated memory cells and are likely involved in regulating immune responses, in contrast to immature DP T cells in the thymus.

Immune Ontogeny and Engraftment Receptivity in the Sheep Fetus

The therapeutic application of in utero hematopoietic stem cell (HSC) transplantation (IUHSCT) is theoretically attractive for definitive treatment of congenital disease states. Investigating this technique in sheep, we have previously shown long-term engraftment and expression of both allogeneic and xenogeneic donor cells without cytoablation and, under appropriate conditions, without GVHD. The theoretical basis for IUHSCT is the well-recognized immune receptivity of the fetus to engraftment of donor cells. Engraftment and long-term expression of donor human and allogeneic sheep HSC reliably occur in the fetal sheep model if the IUHSCT is performed prior to day 71 of gestation (term: 145 days), during the period of presumed immuno-naïveté. Investigations using alternate animals have also noted that gestational age at transplantation is critical to achieving long-term engraftment, presumably as a result of inducing durable immune tolerance to the donor. Despite this presumption, however, the biologic explanation for fetal receptivity to donor engraftment and subsequent long-term tolerance following transplantation early in gestation is not known. In the present studies, we investigated the role fetal immune ontogeny plays in the induction of tolerance following IUHSCT in sheep. To this end, we performed parallel experiments examining engraftment receptivity of fetal sheep to allogeneic and xenogeneic HSC and the appearance of immune phenotypes in fetal sheep lymphoid organs at varying gestational ages (days 39 to birth), attempting to draw correlations between the appearance/absence of specific immune cells and the ability to achieve durable engraftment and immune tolerance. Engraftment receptivity was determined 60 days post-transplantation at different time points in sheep fetal gestation, while immune phenotypes were determined by flow cytometry using commercially available antibodies to immune cell surface markers. Our results indicate that the fetus is largely non-receptive to engraftment of both allogeneic and xenogeneic donor HSC prior to day 52 gestation and possesses a peak in engraftment receptivity between days 64–71 of gestation, which rapidly declines thereafter. With respect to the developing fetal immune system, the period of peak engraftment receptivity was associated with the expression of CD45 on all cells in the thymus. Double-positive and single-positive CD4 and CD8 cells began appearing in the thymus just prior (day 45 of gestation) to the beginning of the engraftment window, while single-positive CD4 or CD8 cells did not begin appearing in peripheral organs until late in the engraftment period, suggesting deletional mechanisms predominate during this time. In a similar fashion, surface IgM (sIgM)+ cells in the thymus were the first to express CD45, commencing expression around day 45 of gestation, with a comparable delay in the appearance of IgM+/CD45+ cells in the peripheral blood and spleen until late in the engraftment window. These findings support a central role for the thymus in multilineage immune cell maturation during the period of fetal transplantation receptivity. Further, they suggest that fetal engraftment receptivity/long-term engraftment and expression following IUHSCT is due to gestational age-dependent deletional tolerance. Further, our findings suggest that IUHSCT in humans may be more successful if performed during the comparable period in human gestation.

OP9-DL1 Cell Line Supports the Development of Phenotypically and Functionally Mature Tcrαβ And Tcrγδ T Cells, through Both Conventional and Aberrant Developmental Pathways

In vitro generation of mature T cells from human hematopoietic stem and progenitor cells (HSPC) could fulfill two existing needs. First, it could enhance and quicken T cell immune reconstitution after stem cell transplantation, which is very slow and generates a skewed TCR repertoire. Second, by generation of tumour antigen specific T cells it could provide an efficient therapy for numerous malignancies and could enhance GVT effect in the context of allogeneic SCT, without aggravating GVHD. T cells can be generated from human HSPC by culturing them on the murine stromal cell line OP9-transduced with the Notch ligand Delta-like-1 (OP9-DL1). Notch receptor activation is essential for T cell development. However, it is unclear whether Notch activation is sufficient for end maturation into functionally and phenotypically mature TCR positive cells. It was shown that human CD34+ cells cultured on OP9-DL1 differentiate to T cells which can proliferate and produce interferon-g upon polyclonal stimulation. The nature of the mature cells generated in these cultures, however, has not been well studied. CD34+ HSPC from postnatal thymus (PNT) or cord blood were cocultured with OP9-DL1, in the presence of the cytokines Flt-3L (5 ng/ml), SCF (2.5 ng/ml) and IL-7 (5 ng/ml). Every 3–5 days cells were harvested and transferred to fresh OP9-DL1 cells. At repetitive timepoints, an aliquot of the cells was analysed phenotypically. In some experiments, IL-15 was added to the culture. For some experiments, cells harvested from OP9-DL1 at the timepoint mature T cells were observed (usually about d 40 of culture), were transferred to feeder cells, consisting of JY cell line (5.104 cells/ml irradiated with 50 Gy and PBMC (5.105/ml irradiated with 40 Gy), in the presence of PHA (1 mg/ml). After 7 days, IL-2 (50 IU/ml) was added to the culture. Every 14 days, cells were restimulated with new feeders (irradiated JY and PBMC) and new addition of PHA. After 3 weeks of stimulation cells were stimulated overnight with 15 ng/ml PMA and 1500 ng/ml ionomycin, and 18 hours later cells were checked for intracellular presence of cytokines. We investigated whether the T cell population generated in these cultures contains mature cells with the characteristics of TCRγδ cells and of positively selected CD8 or CD4 single positive (SP) TCRαβ cells as observed in the human thymus. We found that under the described conditions, HSPC mature into CD1-CD27+ phenotypically mature T cells, with the TCRγδ fraction maturing faster and more efficiently compared to the TCRαβ fraction. Consistent with a mature phenotype, TCRγδ cells were mostly CD8αα or double negative (DN). No mature CD4 SP TCRαβ cells were observed and the mature CD8 SP cells co-expressed variable ratios of CD8αβ and CD8αα dimers, suggesting that these cells are not conventional positively selected TCRαβ cells. In support of this hypothesis, both mature CD1- TCRαβ and TCRγδ cells expressed the IL2Rβ receptor consitutively and both populations proliferated on IL-15 without prior antigen stimulation, CD8αα (TCRαβ and TCRγδ) cells being the most IL-15 responsive. Mature activated T cells secreted IFN-γ and TNFα, little or no IL-2 and IL-4, with no difference observed between TCRαβ and TCRγδ cells. These data suggest that CD8 TCRαβ cells generated in these cultures are unconventional CD8 cells possibly maturated through agonist selection. However, when cells harvested after 40 days of culture on OP9-DL1 were stimulated with PHA and IL-2 for 3 weeks, conventional appearing CD8αβ cells emerged, with a cytokine production profile similar to that of thymic CD8αβ TCRαβ T cells, with the majority of cells secreting IFN-γ and IL-2. We can conclude from these data that OP9-DL1 supports the development of both unconventional and conventional CD8+ TCRαβ cells, of which the generation and selection process are currently being investigated. Also the in vitro anti-tumor capacities of both populations need to be addressed.

Prevention of Autoimmunity and Control of Recall Response to Exogenous Antigen by Fas Death Receptor Ligand Expression on T Cells

Mice with mutations in the gene encoding Fas ligand (FasL) develop lymphoproliferation and systemic autoimmune diseases. However, the cellular subset responsible for the prevention of autoimmunity in FasL-deficient mice remains undetermined. Here, we show that mice with FasL loss on either B or T cells had identical life span as littermates, and both genotypes developed signs of autoimmunity. In addition, we show that T cell-dependent death was vital for the elimination of aberrant T cells and for controlling the numbers of B cells and dendritic cells that dampen autoimmune responses. Furthermore, we show that the loss of FasL on T cells affected the follicular dentritic cell network in the germinal centers, leading to an impaired recall response to exogenous antigen. These results disclose the distinct roles of cellular subsets in FasL-dependent control of autoimmunity and provide further insight into the role of FasL in humoral immunity.

Malnutrition alters the rates of apoptosis in splenocytes and thymocyte subpopulations of rats

Malnutrition continues to be a major public health problem throughout the developing world. Nutritional deficiencies may be the most common cause of secondary immunodeficiency states in humans. It has been suggested that nutritional imbalances can induce apoptosis in a variety of cell types. The purpose of this study was to examine the effect of severe malnutrition on cell subsets and the frequency of spontaneous and/or dexamethasone-induced cell death in vivo in the thymus and spleen from severely malnourished, lactating rats. Apoptosis frequency was estimated by flow cytometry using annexin-V and terminal transferase-mediated dUTP nick-end labelling assay assays. The results obtained in the present study indicate that malnutrition is associated with a significant increase of spontaneously apoptotic cells in the thymus (9.8-fold) and spleen (2.4-fold). Increase in apoptosis was associated largely with CD4(+)CD8(+) double-positive thymocytes. Unexpectedly, similar frequencies of spontaneous apoptosis of these cells were found in both well-nourished and malnourished rats. In contrast, consistent increases in the apoptosis of CD4(-)CD8(-) double-negative thymocytes were observed in malnourished rats. In addition, single-positive CD8(+) and single-positive CD4(+) thymocytes had higher frequencies of apoptosis in malnourished rats. The frequency of total dexamethasone-induced apoptosis was found to be similar in both groups of animals. Nevertheless, in malnourished dexamethasone-treated animals, the percentage of apoptotic double-negative thymocytes was significantly higher than in well-nourished animals, while the rate of apoptosis was lower among double-positive cells. In general, the thymus appears more sensitive to the effects of malnutrition and dexamethasone than the spleen. Furthermore, double-negative thymocytes appear to be the most affected.

2,3,7,8-Tetrachlorodibenzo- p-dioxin modulates the expression of cKrox and Runx3, transcription regulatory factors controlling the lineage commitment of CD4 +CD8 + into CD4 and CD8 thymocytes, respectively

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) was reported to skew the lineage commitment of thymocytes toward CD4 −CD8 + T (CD8 T) cells. However, the underlying mechanisms are not known. In the present study, we first demonstrated that the expression of transcription regulatory factors such as cKrox and Runx3, which have been shown to be intimately associated with the commitment of CD4 +CD8 + double-positive (DP) to CD4 or CD8 single-positive (SP) thymocyes, was down-regulated by TCDD in CD4 SP thymocytes, but up-regulated in DP, CD4 +CD8 + double-negative (DN), and CD8 SP thymocytes. Then, we found that TCDD inhibited the differentiation of DPK cells, an immature CD4 +CD8 + lymphoma cell line, into CD4 +CD8 − T cells, as well as the expression of cKrox and Runx3 upon antigen stimulation. Co-treatment with the AhR antagonist α-naphthoflavone did not completely block the inhibitory action of TCDD on DPK differentiation and the expression of cKrox and Runx3 in DPK cells, suggesting that the immunomodulatory abilities of TCDD are produced, at least in part, independently of the AhR pathway in DPK cells. Our findings could help in understanding the regulatory mechanisms of TCDD on thymocyte development, in particular on the skewed differentiation of DP into CD8 SP thymocytes.

B- and T-cell Development Both Involve Activity of the Unfolded Protein Response Pathway

The unfolded protein response (UPR) signaling pathway regulates the functional capacity of the endoplasmic reticulum for protein folding. Beyond a role for UPR signaling during terminal differentiation of mature B cells to antibody-secreting plasma cells, the status or importance of UPR signaling during hematopoiesis has not been explored, due in part to difficulties in isolating sufficient quantities of cells at developmentally intermediate stages required for biochemical analysis. Following reconstitution of irradiated mice with hematopoietic cells carrying a fluorescent UPR reporter construct, we found that IRE1 nuclease activity for XBP1 splicing is active at early stages of T- and B-lymphocyte differentiation: in bone marrow pro-B cells and in CD4(+)CD8(+) double positive thymic T cells. IRE1 was not active in B cells at later stages. In T cells, IRE activity was not detected in the more mature CD4(+) T-cell population but was active in the CD8(+) cytotoxic T-cell population. Multiple signals are likely to be involved in activating IRE1 during lymphocyte differentiation, including rearrangement of antigen receptor genes. Our results show that reporter-transduced hematopoietic stem cells provide a quick and easy means to identify UPR signaling component activation in physiological settings.

Homeostasis and Effector Function of Lymphopenia-Induced “Memory-Like” T Cells in Constitutively T Cell-Depleted Mice

Naive T lymphocytes acquire a phenotype similar to Ag-experienced memory T cells as a result of proliferation under lymphopenic conditions. Such "memory-like" T (T(ML)) cells constitute a large fraction of the peripheral T cell pool in patients recovering from T cell ablative therapies, HIV patients under highly active antiretroviral therapy, and in the elderly population. To generate a model that allows characterization of T(ML) cells without adoptive transfer, irradiation, or thymectomy, we developed genetically modified mice that express diphtheria toxin A under control of a loxP-flanked stop cassette (R-DTA mice). Crossing these mice to CD4Cre mice resulted in efficient ablation of CD4 single-positive thymocytes, whereas double-positive and CD8 single-positive thymocytes were only partially affected. In the periphery the pool of naive (CD44(low)CD62L(high)) T cells was depleted. However, some T cells were resistant to Cre activity, escaped deletion in the thymus, and underwent lymphopenia-induced proliferation resulting in a pool of T(ML) cells that was similar in size and turnover to the pool of CD44(high)CD62L(low) "memory phenotype" T cells in control mice. CD4Cre/R-DTA mice remained lymphopenic despite the large available immunological "space" and normal Ag-induced T cell proliferation. CD4Cre/R-DTA mice showed a biased TCR repertoire indicating oligoclonal T cell expansion. Infection with the helminth Nippostrongylus brasiliensis resulted in diminished effector cell recruitment and impaired worm expulsion, demonstrating that T(ML) cells are not sufficient to mediate an effective immune response.

Multiparameter flow cytometric analysis of GATA‐3 expression in T cell development and differentiation

GATA‐3 is a key zinc‐finger transcription factor essential for T cell development and differentiation. In this study, a monoclonal antibody (mAb) was raised against mouse and human GATA‐3. This mAb was used to analyze GATA‐3 expression in intrathymic T cell progenitors and polarized Th1 and Th2 cells by Western blotting, and multicolor immunofluorescent staining, and flow cytometric analysis. Our results confirm that GATA‐3 is expressed at every stage of intrathymic mouse T‐cell development and that its expression levels vary among the T cell progenitor subsets. The highest levels of GATA‐3 are expressed by a subset of double negative CD4−CD8− cells and by the single positive CD4+CD8− cell population. In addition, we found that mature T cells express relatively low levels of GATA‐3 upon in vitro CD3 and CD28 costimulation. Moreover, their levels of GATA‐3 remain unchanged or rise significantly if these cells are skewed towards the Th1 or Th2 phenotypes in the presence of exogenous IL‐12 or IL‐4, respectively. In summary, we show that this antibody is compatible with both saponin and methanol permeabilization methods. It can be used in combination with fluorescent antibodies specific for staining various cell surface markers, as well as other intracellular transcription factors or cytokines to elucidate the T cell signaling pathways utilized during development and differentiation.

Signal via IL‐12Rβ2 Negatively Controls Intrathymic Maturation

Mice lacking IL‐12 and its responsiveness exhibited more severe inflammatory responses and were more susceptible to autoimmune diseases than wild type mice, yet the mechanism underlying this phenomenon is not clear. An increased number of CD4+ T cells has been observed in the periphery of IL‐12Rβ2−/− mice compared to wild type mice, indicating that IL‐12 responsiveness is involved in either decreased T cell death and/or increased T cell generation. We report here that thymocytes in mice deficient in IL‐12Rβ2, thus lacking IL‐12 responsiveness, undergo dramatically accelerated maturation. This mainly occurs at the stage from double positive (DP) to single positive (SP), resulting in higher numbers of CD4 and CD8 SP cells in these mice. No difference was observed in percentage and absolute number of apoptotic cells between the two mouse trains. Accelerated maturation also occurs at the stage from double negative (DN) to DP, given the significantly lower CD24 and higher TCRαβ and IL‐7Rα expression in these subsets of IL‐12Rβ2‐deficient mice. This occurs not only in mice injected with anti‐CD3 to mimic pre‐T cell receptor stimulation, but also in mice immunized with autoantigen to induce EAE. Thus, signals through IL‐12Rβ2 negatively regulate thymocyte maturation and play a crucial immunoregulatory role in controlling autoimmunity.

A NUP98-HOXD13 Fusion Gene Impairs Differentiation of T and B Lymphocytes.

Chromosomal translocations leading to NUP98 gene fusions are associated with a wide range of hematologic malignancies, including acute myelogenous leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), and myelodysplastic syndrome (MDS). The NUP98-HOXD13 (NHD13) gene fusion was first identified in a patient with a MDS that progressed to AML. Recently, we used an NHD13 fusion gene to develop a mouse model of MDS that recapitulates all of the key findings of the human disease, including ineffective hematopoiesis leading to peripheral blood cytopenias, dysplasia, and progression to AML. In addition to the features noted above, we observed that the NHD13 mice were lymphopenic, and 10–30% of these mice, depending on the strain background, progressed to T-ALL. These findings prompted us to define the lymphocyte development of NHD13 mice. CBCs obtained from clinically healthy NHD13 mice showed lymphopenia (2.21 vs. 8.72 K/μL, p<0.01) compared with wild type (WT) littermate controls. This lymphopenia was due to a decrease in both T and B cells, as FACS analysis of peripheral blood (PB) from NHD13 mice revealed a marked decrease in CD4 single positive (SP) and B220+/IgM+ cells (p<0.05) compared to WT controls; similar findings were observed in the spleen (p<0.01). The percentage of CD8 SP cells was not different between the NHD13 and WT mice in either the PB or spleen. To investigate the cause for the B-cell lymphopenia, we determined the Hardy fractions of bone marrow B cells. Although the pro-B cell (B220+/CD43+, Hardy fractions A–C) populations showed no difference between NHD13 and WT BM, the NHD13 BM showed decreased pre-B and B cell (B220+/CD43−, Hardy fractions D–F) populations, suggesting impaired differentiation at the pro-B to pre-B stage. Thymi from NHD13 mice (n=7, median age=7 months) showed grossly decreased size and decreased total number of thymocytes (1.75×107 vs. 9.21×107, p<0.01). The CD4/CD8 DN population was markedly increased (p<0.001) and the CD4/CD8 DP population markedly decreased (p<0.001) in NHD13 compared to WT mice. In addition, there was a variable increase in the DN1 and DN2 population, as well as a decrease in the DN3 and DN4 population in thymi from the NHD13 thymus, suggesting a partial block at the DN2 to DN3 transition. To determine clonality of the NHD13 thymocyte population, we used degenerate RT-PCR to identify clonal TCRβ gene rearrangements. As expected, the WT thymi showed polyclonal TCRβ gene rearrangements. However, 5 of 6 NHD13 thymus samples showed clonal DJ rearrangements, with over half of the TCRβ rearrangements in the thymus showing an identical D-J junction, but distinct V-D junctions. This finding suggested that there was a massive clonal expansion of DN2 cells that had undergone a DJ rearrangement, but not completed a VDJ rearrangement, further supporting the contention of a partial block at the DN2 to DN3 transition. Interestingly, analysis of TCRβ gene rearrangements in NHD13 spleens showed no evidence of cells with clonal DJ rearrangements, suggesting that the thymocytes with clonal DJ rearrangements did not mature and migrate from the thymus. Finally, we noted that although there is a marked increase in DN thymocytes from the NHD13 mice, the T-ALL that developed in these mice were typically DP or CD4 SP, suggesting that a rare cell that “escapes” the T-cell differentiation block is susceptible to leukemic transformation. Taken together, these findings demonstrate that the NHD13 transgene inhibits lymphoid as well as myeloid and erythroid differentiation, and is oncogenic in lymphoid as well as myeloid cells.

Regulatory CD4+CD25+ T Cells Prevent Thymic Dysfunction in Experimental Chronic Colitis

Chronic colitis in T-cell deficient Tg epsilon26 mice develops due to a dysfunction of the thymus which generates colitogenic T cells after bone marrow (BM) transplantation. Regulatory CD4+ CD25+ T cells have been shown to prevent colitis in this model by normalizing the peripheral T-cell pool. We tested the hypothesis that T-cell normalization takes place in the thymus. Tg epsilon26 mice were transplanted with BM (BM-->Tg epsilon26 mice) and consequently received either CD4+ CD25+ or CD4+ CD25- cells from syngenic wild type mice. Furthermore, untransplanted Tg epsilon26 mice received CD4+ CD25+ or CD4+ CD25- cells or complete mesenteric lymph node cells. Transfer of regulatory. CD4+ CD25+ cells normalized the total number of thymocytes and the percentage and number of double positive CD4+ CD8+ cells in transplanted mice while percentage of single positive CD4+ and CD8+ thymocytes in BM-->Tg epsilon26 mice was reduced upon CD4+ CD25+ transfer. Timing of CD4+ CD25+ cell injection was important as transfer later than 7 days after BM transplantation failed to prevent abnormal thymic T-cell distribution in BM-->Tg epsilon26 mice. Isolated CD4+ CD25+ cell transfer without preceding BM transplantation failed to reconstitute thymic architecture. Differences of thymic cell composition could not be exclusively explained by presence or absence of colitis, respectively, because 19 days after BM transplantation when both groups showed no histological signs of colitis, animals transferred with CD4+ CD25+ T cells had a significantly higher percentage and number of CD4+ CD25+ thymocytes and CD4+ Foxp3+ cells than BM-->Tg epsilon26 mice. In conclusion, early CD4+ CD25+ cotransfer prevents thymic dysfunction which underlies immune-mediated bowel inflammation in BM-->Tg epsilon26 mice.

Involvement of CCR9 at multiple stages of adult T lymphopoiesis

The chemokine CCL25 is constitutively expressed in the thymus, and its receptor CCR9 is expressed on subsets of developing thymocytes. Nevertheless, the function of CCL25/CCR9 in adult thymopoiesis remains unclear. Here, we demonstrate that purified CCR9(-/-) hematopoietic stem cells are deficient in their ability to generate all major thymocyte subsets including double-negative 1 (DN1) cells in competitive transfers. CCR9(-/-) bone marrow contained normal numbers of lineage(-) Sca-1+c-kit+, common lymphoid progenitors, and lymphoid-primed multipotent progenitors (LMPP), and CCR9(-/-) LMPP showed similar T cell potential as their wild-type (WT) counterparts when cultured on OP9-delta-like 1 stromal cells. In contrast, early thymic progenitor and DN2 thymocyte numbers were reduced in the thymus of adult CCR9(-/-) mice. In fetal thymic organ cultures (FTOC), CCR9(-/-) DN1 cells were as efficient as WT DN1 cells in generating double-positive (DP) thymocytes; however, under competitive FTOC, CCR9(-/-) DP cell numbers were reduced significantly. Similarly, following intrathymic injection into sublethally irradiated recipients, CCR9(-/-) DN cells were out-competed by WT DN cells in generating DP thymocytes. Finally, in competitive reaggregation thymic organ cultures, CCR9(-/-) preselection DP thymocytes were disadvantaged significantly in their ability to generate CD4 single-positive (SP) thymocytes, a finding that correlated with a reduced ability to form TCR-MHC-dependent conjugates with thymic epithelial cells. Together, these results highlight a role for CCR9 at several stages of adult thymopoiesis: in hematopoietic progenitor seeding of the thymus, in the DN-DP thymocyte transition, and in the generation of CD4 SP thymocytes.

A deletion mutation of the protein tyrosine phosphatase kappa (Ptprk) gene is responsible for T-helper immunodeficiency (thid) in the LEC rat

Bone marrow (BM)-derived T-cell progenitors differentiate into CD4 or CD8 single-positive (SP) cells in the thymus. We have previously reported that a single autosomal mutation, thid, causes a defect in the maturation of CD4 SP thymocytes and an abnormality of peripheral helper T cells in the LEC rat. In this study we attempted to identify a gene responsible for the thid mutation. We first performed genetic linkage analysis and mapped the thid locus between Myb and D1Rat392 on Chr 1. In this region we found an approximately 380-kb deletion from intron 3 of the Ptprk gene, which encodes a receptor-like protein tyrosine phosphatase type kappa (RPTPkappa) to intron 1 of the RGD1560849 predicted gene in the LEC rat genome. Reconstitution with syngenic BM cells transduced Ptprk but not the RGD1560849 predicted gene rescued development of CD4 SP cells in the LEC rat thymus. It is confirmed by this result that the Ptprk gene is responsible for the thid mutation in the LEC rat. Our results further suggest that RPTPkappa plays a critical role in the development of CD4 SP cells in the thymus.