CD8 Single-positive Cells Research Articles

Overexpression of the Notch target genes Hes in vivo induces lymphoid and myeloid alterations.

To examine the effects of Notch signaling on hematopoiesis, we transplanted mice with progenitors transduced with a constitutively active form of Notch1 (Notch1IC) or the Notch1 target genes Hes. Notch1IC-transduced cells induce T cell tumors and cannot generate B lymphocytes in vivo. Hes-transplanted mice remained healthy but cells transduced with Hes1 or Hes5 were partially impaired in their ability to differentiate into B cells. Both Hes1 and Hes5 were upregulated in the BM of Notch1IC mice and their ability to interfere with the transcriptional activity of E2A in a reporter assay was comparable to that of Notch1IC. This suggests that the inhibition of B cell development in the Notch1IC-transduced cells could be mediated by the interference of HES1/HES5 proteins with E2A. Hes1-, Hes5- and Notch1IC-transduced bone marrow cells cultured ex vivo in a colony forming assay in the presence of cytokines that promote myeloid differentiation remained very immature, indicating that the myeloid potential of these bone marrow cells was altered. Thymocytes overexpressing Hes1, Hes5 or Notch1IC matured normally into CD4 and CD8 single positive cells in vivo. Altogether our data suggest that Notch1IC induces T cell tumors independently of Hes genes but that its interference with lymphoid B and myeloid maturation is partly mediated by Hes1 and Hes5. DOI:

A molecular marker for thymocyte-positive selection: selection of CD4 single-positive thymocytes with shorter TCRB CDR3 during T cell development.

The generation of the naive T cell repertoire is a direct result of maturation and selection events in the thymus. Although maturation events are judged predominantly on the expression of surface markers, molecular markers, more intimately involved in the selection process, can be informative. We have identified a molecular marker for selection in later stages of maturation in humans. Thymocytes are selected for the expression of TCR beta-chains with shorter CDR3 at the double-positive to single-positive (SP) transition. Here we extend these studies to the mouse and show that the selection phenotype is not related to alpha-chain pairing but is a function of the MHC haplotype. Interestingly, the selection is much more apparent in CD4 SP thymocytes than in CD8 SP cells. This is in contrast to human thymocytes, where the selection is equally apparent in both lineages. The involvement of MHC in the process argues that this is a positive selection stage. The difference in the extent of this selection between the two SP lineages may indicate a class difference in the nature of the TCR-MHC interaction, the role of coreceptors in the selection process, or both.

Overexpression of AML1 Transcription Factor Drives Thymocytes into the CD8 Single-Positive Lineage

To understand the gene regulation involved in the development of single-positive (SP) thymocytes, we generated transgenic mice in which the AML1 transcription factor is overexpressed. In these mice the number of CD8 SP thymocytes was greatly increased, and this continued to be true even when MHC class I was absent. This promotion to the CD8 SP lineage was not, however, observed when both class I and class II were absent. Furthermore, even thymocytes carrying MHC class II-restricted TCR differentiated into the CD8 SP lineage when AML1 was overexpressed. The selected CD8 SP cells were, however, unable to mature, as judged by the expression level of heat-stable Ag. Thus, overexpression of AML1 is able to skew class II-restricted thymocytes into the CD8 SP lineage, but not to drive the maturation of resulting selected CD8 SP cells.

Effect of phorbol ester and calcium ionophore on human thymocytes

Positive selection of immature thymocytes is a developmental process in which TCR ligation with low avidity induces generation of mature T cells. In mouse thymocytes, CD4 +8 + double-positive (DP) cells which were treated with a proper combination of calcium ionophore ionomycin and phorbol 12-myristate 13-acetate (PMA) have been reported to differentiate into CD4 single positive cells. However, in human thymocytes the effects of PMA and ionomycin have remained unclear. Here we report that DP cells that were treated with PMA and ionomycin up-regulated bcl-2 and down-regulated CD1 expression. However, CD3 expression remained low. This treatment induced prolonged CD4 down-regulation in DP cells which was an effect also seen in mature peripheral blood T cells. PMA/ionomycin-treated DP cells showed high cell proliferation and resistance to dexamethasone-induced apoptosis. These results indicate that PKC activation and calcium elevation may be part of the biochemical signals that induce positive selection of human DP cells and the system described in this paper may be a useful model to study the signals involved in the selection of human thymocytes.

Enforced expression of GATA-3 during T cell development inhibits maturation of CD8 single-positive cells and induces thymic lymphoma in transgenic mice.

The zinc finger transcription factor GATA-3 is of critical importance for early T cell development and commitment of Th2 cells. To study the role of GATA-3 in early T cell development, we analyzed and modified GATA-3 expression in vivo. In mice carrying a targeted insertion of a lacZ reporter on one allele, we found that GATA-3 transcription in CD4(+)CD8(+) double-positive thymocytes correlated with the onset of positive selection events, i.e., TCRalphabeta up-regulation and CD69 expression. LacZ expression remained high ( approximately 80% of cells) during maturation of CD4 single-positive (SP) cells in the thymus, but in developing CD8 SP cells the fraction of lacZ-expressing cells decreased to <20%. We modified this pattern by enforced GATA-3 expression driven by the CD2 locus control region, which provides transcription of GATA-3 throughout T cell development. In two independent CD2-GATA3-transgenic lines, approximately 50% of the mice developed thymic lymphoblastoid tumors that were CD4(+)CD8(+/low) and mostly CD3(+). In tumor-free CD2-GATA3-transgenic mice, the total numbers of CD8 SP cells in the thymus were within normal ranges, but their maturation was hampered, as indicated by increased apoptosis of CD8 SP cells and a selective deficiency of mature CD69(low)HSA(low) CD8 SP cells. In the spleen and lymph nodes, the numbers of CD8(+) T cells were significantly reduced. These findings indicate that GATA-3 supports development of the CD4 lineage and inhibits maturation of CD8 SP cells in the thymus.

The DNA binding activity of TAL-1 is not required to induce leukemia/lymphoma in mice.

Activation of the basic helix-loop-helix (bHLH) gene TAL-1 (or SCL) is the most frequent gain-of-function mutation in pediatric T cell acute lymphoblastic leukemia (T-ALL). Similarly, mis-expression of tal-1 in the thymus of transgenic mice results in the development of clonal T cell lymphoblastic leukemia. To determine the mechanism(s) of tal-1-induced leukemogenesis, we created transgenic mice expressing a DNA binding mutant of tal-1. Surprisingly, these mice develop disease, demonstrating that the DNA binding properties of tal-1 are not required to induce leukemia/lymphoma in mice. However, wild type tal-1 and the DNA binding mutant both form stable complexes with E2A proteins. In addition, tal-1 stimulates differentiation of CD8-single positive thymocytes but inhibits the development of CD4-single positive cells: effects also observed in E2A-deficient mice. Our study suggests that the bHLH protein tal-1 contributes to leukemia by interfering with E2A protein function(s).

Determination of three isoforms of the receptor activator of nuclear factor-kappaB ligand and their differential expression in bone and thymus.

The receptor activator of nuclear factor (NF)-kappaB ligand [RANKL; also known as tumor necrosis factor-related activation-induced cytokine, osteoprotegerin ligand, and osteoclast differentiation factor] is known to bind with the receptor activator of NF-kappaB (RANK) and act not only as a key factor for osteoclastogenesis but also as a regulator of lymphocyte development. In this study, we found two additional isoforms of RANKL. RANKL 2 has a shorter intracellular domain than the original RANKL (RANKL 1), and RANKL 3 lacks a transmembrane domain and was thought to act as a soluble form. In the bone marrow stromal cell line ST2 and preosteoblastic cell line MC3T3-E1, all three RANKL isoforms were detected, but the expression of RANKL 2 was preferentially suppressed by treatment with 1alpha,25-dihydroxyvitamin D(3) and dexamethasone. In young adult thymus, CD4(-)CD8(-) double-negative cells were positive for all three isoforms, CD4(+)CD8(+) double-positive cells were positive for RANKL 1 and RANKL 3 but negative for RANKL 2, and CD4(+)CD8(-) and CD4(-)CD8(+) single-positive cells were positive for all three isoforms. Immunofluorescence analyses of NIH3T3 cells transfected with each RANKL isoform indicated that the three RANKL isoforms were translated, and RANKL 2 protein predominantly stayed in the endoplasmic reticulum and Golgi networks. These results indicate that there are three kinds of RANKL-RANK pathways. The presence of multiple RANKL-RANK pathways suggests a more complicated RANKL-RANK system for osteoclastogenesis or T cell differentiation than previously thought.

Diminution of the AML1 transcription factor function causes differential effects on the fates of CD4 and CD8 single-positive T cells.

In the thymic cortex, T lymphocytes are positively selected to survive and committed either to the CD4 single-positive (SP) or the CD8 SP lineage. The SP cells then pass through a step of maturation in the medulla and are delivered to peripheral lymphoid tissues. We examined the role of AML1, the gene encoding a transcription factor, in the above processes by using the transgenic mice expressing a dominant interfering form of AML1 as well as mice targeted heterozygously for AML1. One phenotypic change seen in the AML1-diminished mice was the reduction in the numbers of both CD4 SP and CD8 SP thymocytes, reflecting the partial impairment of the transition from the double-positive to SP stage. In addition, distinct from the above abnormality, perturbed were several aspects of SP cells, including the maturation of SP thymocytes, the recent thymic emigration, and the proliferative responsiveness of peripheral T cells to TCR stimulation. Interestingly, the AML1 diminution caused inhibitory and enhancing effects on the CD4 SP and CD8 SP cells, respectively. These differential effects are most likely related to the reduction in the peripheral CD4 SP/CD8 SP ratio observed in the AML1-diminished mice. The AML1 transcription factor thus maintains the homeostasis of each SP subset by functioning at the later stages of T lymphocyte differentiation.

Extrathymic human t lymphocyte development and regulation in immune deficient mice

Abstract We previously studied extrathymic human T lymphocyte development from stem/progenitor subsets in the bone marrow of bnx mice. Current studies examined human T cell development from purified CD34 + CD3 − stem cells in a new strain that we developed, nude/NOD/SCID mice. Human T cells isolated from bnx mice contained double CD4 + /CD8 + and single CD3 + /CD4 + or CD3 + /CD8 + cells, but they were anergic to activation by PMA plus ionomycin (P+1) and to CD3 and CD28 crosslinking. In nude/NOD/SCID mice, there was more variability in human T cell development in the marrow. Double positive CD4/CD8 and CD4 + /cytoCD3 + populations, which expressed CD3 mRNA, were found in some of the mice. But no CD8 single positive cells were found in any mice. In some mice, only myeloid progenitor cells and B cells were found, much like the NOD/SCID strain. The differences may be due to different microenvironments or to the types of human cells engrafted in the starting populations. Studying the influence of implanting human pediatric thymus slices into nude/NOD/SCID on the development of human T cells is in progress. Thymus grafts get well vascularized in the peritoneal cavity of mice. Early studies suggest that the presence of the thymus makes the human T cells less hyporesponsive. Future studies will detemine whether the human cells are being selected in the thymus or whether thymic stromal factors are influencing development and/or activation of human T cells.

The quantity of TCR signal determines positive selection and lineage commitment of T cells.

It is generally accepted that the avidity of TCR for self Ag/MHC determines the fate of immature thymocytes. However, the contribution of the quantity of TCR signal to T cell selection has not been well established, particularly in vivo. To address this issue, we analyzed DO-TCR transgenic CD3zeta-deficient (DO-Tg/zetaKO) mice in which T cells have a reduced TCR on the cell surface. In DO-Tg/zetaKO mice, very few CD4 single positive (SP) thymocytes developed, indicating that the decrease in TCR signaling resulted in a failure of positive selection of DO-Tg thymocytes. Administration of the peptide Ag to DO-Tg/zetaKO mice resulted in the generation of functional CD4 SP mature thymocytes in a dose-dependent manner, and, unexpectedly, DO-Tg CD8 SP cells emerged at lower doses of Ag. TCR signal-dependent, sequential commitment from CD8(+) SP to CD4(+) SP was also shown in a class I-restricted TCR-Tg system. These in vivo analyses demonstrate that the quantity of TCR signal directly determines positive and negative selection, and further suggest that weak signal directs positively selected T cells to CD8 lineage and stronger signal to CD4 lineage.

NF-kappa B is required for the positive selection of CD8+ thymocytes.

To examine the role of NF-kappaB in T cell development, we analyzed thymocyte ontogeny in transgenic (mutant I-kappaBalpha (mI-kappaBalpha)) mice that express a superinhibitory form of the NF-kappaB inhibitory protein, I-kappaBalpha (I-kappaBalpha(A32/36)), under the control of the T cell-specific CD2 promoter and enhancer. Thymi from mI-kappaBalpha mice contained increased numbers of double-positive (DP) and decreased numbers of both CD4(+) and CD8(+) single-positive cells, consistent with a block in DP thymocyte maturation. In addition, expression of CD69, a marker of positive selection, was decreased on DP thymocytes from the mI-kappaBalpha mice. To test directly whether NF-kappaB was required for positive or negative selection, we generated mI-kappaBalpha mice expressing the H-Y or 2C alphabeta TCR transgenes. Expression of the I-kappaBalpha(A32/36) transgene caused a block in the positive selection of CD8(+) single-positive cells in both strains of TCR transgenic animals. In contrast, negative selection was unaffected by expression of the I-kappaBalpha(A32/36) transgene. Taken together, these results identified a NF-kappaB-dependent transcriptional pathway that is selectively required for the positive selection of CD8(+) thymocytes.

Phenotypic analysis of peripheral CD4+ CD8+ T cells in the rat.

Among peripheral T cells, the expression of CD4 and CD8 is almost mutually exclusive. However, here we show, using flow cytometric analysis, that ex vivo approximately 6% of rat T cells stained for both CD4 and CD8. These double positive cells were also detected by confocal microscopy. Only around 50% of double positive cells expressed the CD8beta chain, the remaining cells expressed the CD8alpha chain alone. Double positive cells were blast-like with a phenotype, distinct from that of either CD4 or CD8 single positive cells, suggestive of an activated state. Previous reports of double positive T cells have also suggested that coexpression of CD4 and CD8 is linked to the activation state of the cell. There was an indication that priming animals with a hapten-carrier complex increased the ratio of CD8alphaalpha : alphabeta expressing double positive T cells, although we did not detect an increase in the frequency of double positive T cells following priming. We also show that the frequency of double positive cells was reduced following thymectomy and with age. In conclusion, these studies show that peripheral T cells expressing both CD4 and CD8 can be detected in the rat and that they are phenotypically distinct from CD4 and CD8 single positive T cells.

Differential Effects of Diethylstilbestrol and 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Thymocyte Differentiation, Proliferation, and Apoptosis in bcl-2 Transgenic Mouse Fetal Thymus Organ Culture

Both the estrogenic drug diethylstilbestrol (DES) and the pervasive environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inhibit thymocyte development. The mechanisms by which either agent induces thymic atrophy are still undetermined. We previously found that TCDD and DES inhibited C57BL/6 murine fetal thymocyte organ cultures (FTOC) at different stages of development. Now, using bcl-2 transgenic (TG) mice, we have further investigated their effects on FTOC proliferation, differentiation, maturation, and apoptosis. As with C57BL/6 mice, thymocyte development in C3H/bcl-2 FTOCs was inhibited by either TCDD (10 nM) or DES (20 μM) in both bcl-2 TG− and TG+ littermates. However, the percentage reduction of cell number induced by DES in bcl-2 TG+ FTOCs was significantly less than the level of inhibition in TG− FTOCs. There was no difference in the level of reduction from TCDD-exposed TG+ or TG− FTOC. Whereas TCDD increased production of mature CD8 cells in either strain, DES mainly yielded cells in the CD4−CD8− (DN) stage in TG− mice. The anti-apoptotic bcl-2 transgene overcame some DES blocking of DN thymocyte development, allowing more cells to differentiate into CD4 single-positive cells. Analysis of cell cycle showed that TCDD inhibited entry into S phase, whereas DES blocked cell cycling in the G2/M phase. TCDD did not induce detectable apoptosis in FTOC. However, unlike the effects of 17β-estradiol (E2) in vivo, DES induced apoptosis in the TG− FTOC, and these apoptotic cells were mainly in the DN subpopulation. This apoptosis could be prevented by the overexpression of bcl-2 in the TG+ mice. Our results demonstrate that, in addition to inhibition of fetal thymocytes at different stages of development by TCDD and DES, DES also induces thymic atrophy by both bcl-2-inhibitable apoptosis and by inducing cell cycle arrest in G2/M in the latest stage in the stem cell compartment. TCDD, on the other hand, does not induce apoptosis, but inhibits entry into cell cycle in the earliest stage in the stem cell compartment.

Flow cytometric analysis of lymphoid cells in thymic epithelial neoplasms✩

There have been conflicts concerning the criteria for diagnosing malignant epithelial neoplasms of thymic origin. To differentiate thymic carcinomas from thymomas, the maturation stage of T-lineage lymphoid cells infiltrating thymomas and thymic carcinomas was examined by flow cytometry to associate it with the degree of tumor malignancy. Multidimensional flow cytometric analysis was performed on the lymphoid cells extracted from 27 thymic epithelial neoplasms (14 encapsulated thymomas, ten invasive thymomas, and three thymic carcinomas) by using anti-CD3, -CD4, -CD8, -CD10, -CD20, -CD38, -CD45RA, and -CD45RO monoclonal antibodies. CD4 and CD8 were co-expressed on 76.8% of the lymphoid cells in encapsulated thymoma (N=14), 59.2% in invasive thymoma (N=10), and 6.7% in thymic cancer (N=3). The percentage of CD4- or CD8- single positive cells was 11.4% in encapsulated thymoma, 23.9% in invasive thymoma, and 77.7% in thymic cancer. The percentage of CD10-positive cells was 20.8% in encapsulated thymoma, 13.2% in invasive thymoma, and 6.0% in thymic cancer. The percentage of CD20-positive cell was 2.6% in encapsulated thymoma, 3.3% in invasive thymoma, and 31.6% in thymic cancer. There were significant statistical differences in the percentages of CD4/CD8 double positive cells, CD4- or CD8-single positive cells, CD10-positive cells and CD20-positive cells among the three groups. Two cases classified as invasive thymoma by pathohistological examination, however, showed the infiltration of mature lymphocytes like as thymic cancers. CD4+CD8+ or CD10+ T-lineage cells were the most reliable markers of the benignancy of thymic epithelial tumors. CD4- or CD8-single positive cells or CD20-positive cells were characteristic in thymic carcinoma. Flow cytometry on the maturity of lymphoid cells infiltrating thymic epithelial tumors was feasible for determining their degree of malignancy. Some invasive thymomas showed the intermediate characteristics with thymomatous epithelia and mature lymphoid cells.

Alterations of thymocyte development, thymic emigrants and peripheral T cell population in rats exposed to 2,3,7,8-tetrachlorodibenzo- p-dioxin

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) exerts diverse biological effects by activating the cytosolic transcription factor, arylhydrocarbon receptor (AhR), which translocates to nuclei by TCDD binding and induces gene expressions. Among the well known-adverse effects of TCDD is thymus atrophy. In thymus atrophy, TCDD alters the proliferation as well as the differentiation of immature thymocytes. Previous studies on the effects of TCDD on thymocyte development were primarily carried out with high doses of TCDD. The present study investigates the effects of lower doses of TCDD (1 or 2 μg TCDD/kg by gavage) on thymocyte development, and furthermore, their sequential consequences on the peripheral T cell repertoire. Seven days after treatment with 1 or 2 μg TCDD/kg, the expression of CYP1A1 mRNA, one of the sensitive responses caused by the binding of TCDD to AhR, was detected in the thymus of rats. Thymus weights and thymus cell numbers decreased in TCDD-treated rats in a dose-dependent manner. The ratios of CD4 single-positive (SP) cells/CD8 SP cells were significantly reduced by TCDD exposure, indicating that the maturation of CD4 +CD8 + double-positive (DP) cells was skewed toward CD8 SP cells. These changes in the thymus were parallel to those previously observed with high doses of TCDD exposure. However, the specific reduction of DP cells reported in previous studies with high doses of TCDD was not detected in the present study. On the other hand, the skewing of mature CD4/CD8 T cell ratio in thymocytes by TCDD was not reflected in mesenteric lymph node (LN) lymphocytes, where the proportion of CD8 T cells was rather lowered by TCDD with a significant difference at 1 μg TCDD/kg. In LN lymphocytes, the percentage of recent thymic emigrants (RTEs), defined by the surface markers of Thy1 +CD45RC −, was shown to be significantly reduced by exposure to 1 and 2 μg TCDD/kg. T cell supply from the thymus has a crucial role in keeping the diversity of the T cell repertoire. The results of the present study indicated that lower doses of TCDD affect thymocyte development, especially differentiation, and reduce the proportion of RTE in LN, which may cause immunosuppression by reducing the variety of the T cell receptor repertoire.

The role of B7 costimulation in CD4/CD8 T cell homeostasis.

The effect of B7-mediated costimulation on T cell homeostasis was examined in studies of B7-1 (CD80) and B7-2 (CD86) transgenic as well as B7-deficient mice. B7 overexpression in transgenic mice resulted in marked polyclonal peripheral T cell hyperplasia accompanied by skewing toward an increased proportion of CD8 single-positive cells and a decreased proportion of CD4 single-positive cells in thymus and more markedly in peripheral T cells. B7-induced T cell expansion was dependent on both CD28 and TCR expression. Transgenic overexpression of B7-1 or B7-2 resulted in down-regulation of cell surface CD28 on thymocytes and peripheral T cells through a mechanism mediated by intercellular interaction. Mice deficient in B7-1 and B7-2 exhibited changes that were the reciprocal of those observed in B7-overexpressing transgenics: a marked increase in the CD4/CD8 ratio in peripheral T cells and an increase in cell surface CD28 in thymus and peripheral T cells. These reciprocal effects of genetically engineered increase or decrease in B7 expression indicate that B7 costimulation plays a physiological role in the regulation of CD4+ and CD8+ T cell homeostasis.

The selection of M3-restricted T cells is dependent on M3 expression and presentation of N-formylated peptides in the thymus.

The major histocompatibility complex (MHC) class Ib molecule H2-M3 binds N-formylated peptides from mitochondria and bacteria. To explore the role of M3 expression and peptide supply in positive and negative selection, we generated transgenic mice expressing an M3-restricted TCR-alpha/beta from a CD8(+) T cell hybridoma (D7) specific for a listerial peptide (LemA). Development of M3-restricted transgenic T cells is impaired in both beta2-microglobulin-deficient and transporter associated with antigen processing (TAP)-deficient mice, but is not diminished by changes in the H-2 haplotype. Maturation of M3/LemA-specific CD8(+) single positive cells in fetal thymic organ culture was sensitive to M3 expression levels as determined by antibody blocking and use of the castaneus mutant allele of M3. Positive selection was rescued in TAP(-/-) lobes by nonagonist mitochondrial and bacterial peptides, whereas LemA and a partial agonist variant caused negative selection. Thus, M3-restricted CD8(+) T cells are positively and negatively selected by M3, with no contribution from the more abundant class Ia molecules. These results demonstrate that class Ib molecules can function in thymic education like class Ia molecules, despite limited ligand diversity and low levels of expression.

Thymic transplantation in miniature swine. I. Development and function of the "thymokidney".

Previous studies in our laboratory have demonstrated the importance of the thymus for rapid and stable tolerance induction in an allotransplant model. The focus of the present study was to explore the feasibility of autologous thymic transplantation to produce a new transplantable organ (thymokidney) and to examine the function of subsequent vascularized thymokidney transplants in T cell development. Eight juvenile swine received autologous thymic grafts under the renal capsule. Thymic tissue was obtained through a partial (n=6) or complete (n=2) thymectomy, and growth of the autologous thymic graft was compared between partially and completely thymectomized animals. Two of the partially thymectomized animals received irradiated (1000 cGy) as well as non-irradiated autologous thymic grafts. Graft survival, growth and evidence of thymocyte development was determined by (a) macroscopic examination of the implanted tissue, (b) histological examination, and (c) flow cytometry. Naive CD4 SP T cells were identified by CD45RA-expression. Growth of transplanted thymic tissue was demonstrated in all thymic graft recipients. No difference was seen between partially and completely thymectomized animals. By POD 60, the thymic grafts exhibited normal macroscopic and microscopic structure, and normal thymocyte composition. Irradiated thymic tissue displayed a similar pattern of development, but growth was markedly delayed. To evaluate thymic function of the graft, a composite thymokidney was transplanted into a recipient which had previously been thymectomized, had few circulating CD4-single positive cells and had lost MLR reactivity. The number of CD4+/CD45RA+ cells in this animal increased steadily from POD 30 to POD 150, indicating that the thymus of the composite thymokidney allograft was functional; in addition, MLR assays demonstrated that the recipient recovered immunocompetence. The establishment of a thymokidney by thymic autografting to the renal subcapsular space results in normal thymic growth and function, and may provide a valuable tool for studying the role of the thymus in tolerance induction. As far as we are aware, we provide the first evidence of functional vascularized thymic graft reconstituting T cells and leading to a return of a immunocompetence in a large animal model.

Phenotypic identification of the subgroups of murine T-cell receptor alphabeta+ CD4+ CD8- thymocytes and its implication in the late stage of thymocyte development.

Phenotypic analysis of the medullary-type CD4+ CD8- [CD4 single-positive (SP)] thymocytes has revealed phenotypic heterogeneity within this cell population. The characteristic phenotype of mature peripheral T cells can be uniquely marked as Qa-2+ HSA- CD69-, whereas in the medullary-type CD4 SP thymocytes, the expression patterns of many markers were quite different. This suggests that there are many subgroups in the population, which reflects that medullary-type CD4 SP thymocytes may undergo phenotypic maturation. According to the results of two-colour flow cytometry, seven discrete phenotypes were identified by the expression capacity of Qa-2, HSA, CD69, 3G11 and 6C10 molecules. Consequently, the phenotypic precursor-progeny relationship can be envisaged as: 3G11- 6C10+ CD69+ HSAhi -->3G11+ 6C10+ CD69+ HSAhi --> 3G11+ 6C10- CD69+ HSAint -->3G11+ 6C10- CD69- HSAint Qa-2- -->3G11+ HSA-/lo Qa-2lo. At the stage of 3G11+ 6C10- CD69- HSAint Qa-2-, a branch pathway could be initiated, which gave rise to 3G11- HSAl degrees Qa-2- cells, which then, in turn, developed into 3G11- HSA-/loQa-2hi cells, a minor subgroup of the most mature CD4 SP cells. Consistent with this predicted pathway, experiments indicated that the first two subgroups were still cortisone sensitive, whereas the others were cortisone resistant. The cells in the last two Qa-2-positive subgroups are probably ready for emigration into the periphery.

Systemic and mucosal immune responses of pigs to parenteral immunization with a pepsin-digested Serpulina hyodysenteriae bacterin

Serpulina hyodysenteriae infection of pigs, swine dysentery, causes a mucohemorrhagic diarrhoea resulting in significant economic losses to swine producers. The pathogenesis of this disease is poorly understood. Regardless, commercial vaccines have been developed and are in use. Thus, the present study was designed to examine cellular immune responses induced by parenteral S. hyodysenteriae vaccination. Significant antigen-specific interferon-γ (IFN-γ) and blastogenic responses were detected from peripheral blood lymphocytes isolated from vaccinated pigs. However, poor IFN-γ responses were detected from colonic lymph node lymphocytes from these same pigs despite significant antigen-specific blastogenic responses. In addition, peripheral blood IFN-γ responses were diminished by either in vitro depletion of CD4 expressing cells or by in vitro treatment with porcine IL-10. Colonic lymph node IFN-γ responses were not inhibited by treatment with porcine IL-10. Vaccination also resulted in increased percentages of both mucosal and peripheral blood CD8 single positive cells with concurrent decreases in percentages of CD4 single positive cells as compared to percentages of these same populations from non-vaccinated pigs. In conclusion, these studies show that parenteral S. hyodysenteriae vaccination results in cellular immune responses detectable both peripherally (systemic immunity) as well as at the site of infection (mucosal immunity). However, it appears that regulatory mechanisms affecting IFN-γ production in response to S. hyodysenteriae antigen differ between peripheral and colonic compartments.