Single-positive CD4 Research Articles

Analysis of the human thymic perivascular space during aging.

The perivascular space (PVS) of human thymus increases in volume during aging as thymopoiesis declines. Understanding the composition of the PVS is therefore vital to understanding mechanisms of thymic atrophy. We have analyzed 87 normal and 31 myasthenia gravis (MG) thymus tissues from patients ranging in age from newborn to 78 years, using immunohistologic and molecular assays. We confirmed that although thymic epithelial space (TES) volume decreases progressively with age, thymopoiesis with active T-cell receptor gene rearrangement continued normally within the TES into late life. Hematopoietic cells present in the adult PVS include T cells, B cells, and monocytes. Eosinophils are prominent in PVS of infants 2 years of age or younger. In the normal adult and the MG thymus, the PVS includes mature single-positive (CD1a(-) and CD4(+) or CD8(+)) T lymphocytes that express CD45RO, and contains clusters of T cells expressing the TIA-1 cytotoxic granule antigen, suggesting a peripheral origin. PBMCs bind in vitro to MECA-79(+) high endothelial venules present in the PVS, suggesting a mechanism for the recruitment of peripheral cells to thymic PVS. Therefore, in both normal subjects and MG patients, thymic PVS may be a compartment of the peripheral immune system that is not directly involved in thymopoiesis.

The final maturation of at least some single-positive CD4(hi) thymocytes does not require T cell receptor-major histocompatibility complex contact.

The majority ( approximately 70%) of postselection CD4(+) single-positive (SP) thymocytes are CD8(lo)CD4(hi). These cells express very low levels of CD8, undetectable by flow cytofluorimetric (FCM) analysis, but sufficiently high to allow purification by panning. Unlike the fully mature CD8(-)CD4(hi) thymocytes, which account for the remaining approximately 30% of the SP CD4(+) thymocytes, CD8(lo)CD4(hi) cells are functionally immature and short-lived unless they receive an unidentified maturation signal from the thymus. In this study, we tested the hypothesis that this signal is provided by a T cell receptor (TCR)-major histocompatibility complex (MHC) class II interaction. Using intrathymic transfer, we show that the immature CD8(lo)CD4(hi) cells could complete their intrathymic maturation and populate the peripheral lymphoid organs in the absence of MHC class II (and class I) molecules. Furthermore, in mice devoid of class II (and class I) molecules, the progeny of CD8(lo)CD4(hi) cells was long-lived and functionally reactive to allogeneic class II molecules, although their numbers in the spleen and the mesenteric lymph node were approximately 40-50% lower than those in class II(+) mice 5 mo after transfer. Control experiments demonstrated that the surviving cells did not originate from the contaminating mature thymocytes. These results demonstrate that the final maturation, proliferation, and peripheral survival (up to 5 mo) of at least some postselection CD4(+) SP cells do not require the TCR-MHC class II interaction. They also indicate that the TCR-MHC class II interaction(s) required for the intrathymic development of long-lived CD4(+) SP cells occurs before the CD4(hi) SP stage of development.

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.

Fas Is Expressed Early in Human Thymocyte Development But Does Not Transmit an Apoptotic Signal

We investigated the expression and function of Fas on human thymocytes prepared from fetal and pediatric tissue specimens and from SCID-hu Thy/Liv grafts. Unlike mouse thymocytes, human thymocytes exhibited a pattern of Fas expression skewed to immature cells, in that the highest expression was seen on double negative thymocytes and on intrathymic T progenitor cells. Fas expression was intermediate on double positive human thymocytes, and low or negative on mature single positive CD4 and CD8 medullary thymocytes. In spite of this relatively abundant surface expression, cross-linking of Fas with agonist mAb was incapable of triggering an apoptotic signal in human thymocytes. Apoptotic signaling was not enhanced by treatment with cycloheximide, nor by restoring a cosignaling milieu by addition of thymic stromal cells. Mouse thymocytes were induced to apoptosis by cross-linked recombinant soluble human Fas ligand both in vitro and in vivo, though human thymocytes were also resistant to this mode of receptor ligation. Membrane-bound Fas ligand also induced apoptotic death in murine thymocytes but not in human thymocytes. Human thymocytes were as sensitive as Jurkat cells, however, to apoptosis induced by TNF-alpha, suggesting that these cells have a signaling defect before activation of the earliest caspases. These data demonstrate a durable and specific resistance of human thymocytes to apoptosis induced through Fas receptor engagement, and reveal significant species-specific differences in the biology of thymocyte-programmed cell death.

Oligoclonality of Rat Intestinal Intraepithelial T Lymphocytes: Overlapping TCR β-Chain Repertoires in the CD4 Single-Positive and CD4/CD8 Double-Positive Subsets

AbstractPrevious studies in humans and mice have shown that gut intraepithelial lymphocytes (IELs) express oligoclonal TCR β-chain repertoires. These studies have either employed unseparated IEL preparations or focused on the CD8+ subsets. Here, we have analyzed the TCR β-chain repertoire of small intestinal IELs in PVG rats, in sorted CD4+ as well as CD8+ subpopulations, and important differences were noted. CD8αα and CD8αβ single-positive (SP) IELs used most Vβ genes, but relative Vβ usage as determined by quantitative PCR analysis differed markedly between the two subsets and among individual rats. By contrast, CD4+ IELs showed consistent skewing toward Vβ17 and Vβ19; these two genes accounted collectively for more than half the Vβ repertoire in the CD4/CD8 double-positive (DP) subset and were likewise predominant in CD4 SP IELs. Complementarity-determining region 3 length displays and TCR sequencing demonstrated oligoclonal expansions in both the CD4+ and CD8+ IEL subpopulations. These studies also revealed that the CD4 SP and CD4/CD8 DP IEL subsets expressed overlapping β-chain repertoires. In conclusion, our results show that rat TCR-αβ+ IELs of both the CD8+ and CD4+ subpopulations are oligoclonal. The limited Vβ usage and overlapping TCR repertoire expressed by CD4 SP and CD4/CD8 DP cells suggest that these two IEL populations recognize restricted intestinal ligands and are developmentally and functionally related.

Functional characteristics of human peripheral blood alpha/betaTCR+, CD4- and CD8- double-negative (DN) T cells.

The function of human peripheral blood alpha/betaTCR positive, CD4- and CD8- double-negative T lymphocytes (DN cells) in vivo is not completely understood. The response of immunomagnetically isolated DN cells to PHA and anti-CD3 was compared to the response of single-positive (SP) CD4 and CD8 subsets. Proliferation of DN cells in response to PHA was largely independent of APC. This suggests activation requirements for DN cells that are different from SP cells. Upon activation, HLA-DR was found to be upregulated early on DN cells, and IL-4 and IL-10 were detected in the supernatants of DN cells. These observations in vitro could correspond with an immunoregulatory role of human DN cells in vivo.

Thymosin-α1 stimulates maturation of CD34+ stem cells into CD3+4+ cells in an in vitro thymic epithelia organ coculture model

The effect of thymosin-α1 on thymopoiesis is largely unknown. Thymosin is found in the cortical and medullary thymic epithelia, as well as in nurse cells; thus, it is hypothesized that thymosin may affect both early and late stage of thymocyte maturation. In this study, the effect of thymosin-α1 on thymopoiesis was determined by coculturing in vitro CD34+ stem cells (SC) with allogeneic cultured thymic epithelia fragments (CTEF) for 1–4 weeks and analyzing T-cell maturation by flow cytometry.Thymosin-α1 significantly enhanced the cell number (e.g., proliferation) of mononuclear cells obtained at 2 and 4 weeks of the SC-CTEF cocultures (P < 0.01 and < 0.05, respectively). In particular, thymosin-α1 stimulated expression of CD3+ cells at 3 and 4 weeks (P < 0.05). The predominant subpopulation increased by thymosin stimulation was single positive mature CD4+ cells, which was confirmed to occur within the SC-CTEF thymic organ tissue by laser confocal immunofluorescence microscopy. Thymosin stimulation tended to enhance IL-7 synthesis, critical cytokine in the maturation of thymocytes.In summary, this is the first study to demonstrate that thymosin-α1 enhanced thymopoiesis of CD34+ stem cells in humans using an in vitro model of differentiation using stem cells and cultured thymic epithelial fragments cocultures. Furthermore, the thymosin significantly increased expression of CD3+4+ T cells.

Normal Thymic Selection of TCR Transgenic CD4 T Cells, but Impaired Survival in the Periphery Despite the Presence of Selecting MHC Molecules

In this paper, we investigate selection in the thymus and survival in the periphery of CD4 T cells, which carry a major histocompatibility class II-restricted transgenic TCR (A18 TCRtg) specific for a natural self Ag, the fifth component of complement (C5). A18 TCRtg thymocytes develop normal numbers of CD4 single-positive (SP) thymocytes, but do not show pronounced overselection as do some other TCR transgenic strains. CD4 SP cells are mature as judged by termination of CD8 synthesis, resistance to cortisone, and functional competence. The kinetics of positive selection, determined by BrdU labeling, are very fast. CD4 SP thymocytes are demonstrable within 2 days of labeling, and within 8 days after labeling a large proportion (20%) of lymph node T cells are recent thymic emigrants. The high number of recent thymic emigrants suggests rapid turnover of CD4 T cells in the periphery, which was confirmed by thymectomy and determination of CD4 T cell life spans. A18 TCRtg T cells have a t(1/2) of approximately 6 wk, despite the presence of selecting MHC molecules. This explains the failure to accumulate high numbers of peripheral T cells and suggests that the MHC-bound ligand(s) responsible for initiating survival signals is limiting for the selection and maintenance of A18 transgenic CD4 T cells.

In vitro positive selection and anergy induction of class II-restricted TCR transgenic thymocytes by a cortical thymic epithelial cell line.

Thymic epithelial cell lines isolated from hyperplastic thymi of transgenic mice over-expressing human papilloma viral oncogenes E6 and E7 constitutively displayed a phenotype consistent with a cortical origin. Exposure to IFN-gamma induced class II MHC and ICAM-1 expression, and up-regulated expression of VCAM-1 and class I MHC molecules. CD40 expression was maximally induced by a combination of IFN-gamma and IL-1, with lower levels of induction observed with a mixture of IFN-gamma and tumor necrosis factor (TNF)-alpha or TNF-alpha alone. B7-1 or B7-2 was not expressed constitutively or in response to cytokines. These stromal cells supported the development of CD4 single-positive (SP) cells in reaggregate co-cultures with CD4+ CD8+ thymocytes from TCR transgenic mice, but did not stimulate class II MHC-restricted, moth cytochrome c (MCC)-reactive T cells in vitro. The behavior of the culture system was consistent with positive selection, i.e. increased numbers of CD4 SP cells, gain of antigen responsiveness, and requirement for epithelial class II MHC products. Some variants of these stromal cell lines required exogenous MCC peptide in the reaggregation cultures (RC) for positive selection to occur. While a low concentration of MCC peptide (0.01-0.1 microM) significantly enhanced the accumulation of CD4 SP cells, higher concentrations of peptide (1-10 microM) resulted in recovery of predominantly CD4- CD8- and CD4(low) CD8- cells. Thymocytes recovered from RC containing low, but not high concentrations of peptide responded to MCC peptide in secondary cultures with splenic antigen-presenting cells.

Intestinal intraepithelial lymphocytes are primed for gamma interferon and MIP-1beta expression and display antiviral cytotoxic activity despite severe CD4(+) T-cell depletion in primary simian immunodeficiency virus infection.

Intraepithelial lymphocytes (IEL) are a critical effector component of the gut-associated lymphoid tissue (GALT) and play an important role in mucosal immunity as well as in the maintenance of the epithelial cell integrity and barrier function. The objective of this study was to determine whether simian immunodeficiency virus (SIV) infection of rhesus macaques would cause alterations in the immunophenotypic profiles of IEL and their mitogen-specific cytokine (gamma interferon [IFN-gamma] and MIP-1beta) responses (by flow cytometry) and virus-specific cytotoxic T-cell (CTL) activity (by the chromium release assay). Virally infected IEL were detected through the entire course of SIV infection by in situ hybridization. Severe depletion of CD4(+) single-positive and CD4(+)CD8(+) double-positive T cells occurred early in primary SIV infection, which was coincident with an increased prevalence of CD8(+) T cells. This was in contrast to a gradual depletion of CD4(+) T cells in peripheral blood. The CD8(+) IEL were the primary producers of IFN-gamma and MIP-1beta and were found to retain their potential to produce both IFN-gamma and MIP-1beta through the entire course of SIV infection. SIV-specific CTL activity was detected in primary IEL at 1, 2, and 4 weeks post-SIV infection. These results demonstrated that IEL may be involved in generating antiviral immune responses early in SIV infection and in suppressing viral infection thereafter. Alterations in homeostasis in epithelia due to severe CD4(+) T-cell depletion accompanied by changes in the cytokine and chemokine production by IEL may play a role in the enteropathogenesis of SIV infection.

Partial Agonism Elicits an Enduring Phase of T-Cell-Mediated Antigen Presentation

Previous studies have shown that the anti-CD4 mAbW3/25 strongly enhances T cell APC (T-APC) activity.In this study, single positive CD4+and double negative(DN) (CD4−CD8−) T-helper cells specific for the 55–69 or 72–86 sequence of guinea pig (GP) myelin basic protein (GPMBP) were used to study CD4 regulation ofT-APC activity. Clones were cultured with irradiatedSPL and GPMBP or rat (R) MBP for 2–3 days, were propagated in IL-2 for another 1-3 days, were irradiated, andwere used as T-APC. DN T cells specific for GP55-69 effectively presented GPMBP and were superior APC compared to other CD4+T cells for presentation of this antigen. In contrast, DN T cells specific for the dominantencephalitogenic 72–86 determinant did not effectively present the agonist GPMBP but potently presented thepartial agonist RMBP. The heightened APC activity ofDN T cells reflected the lack of CD4 because the anti-CD4 mAb W3/25 promoted T-APC activity of CD4+T cells to those levels expressed by DN T cells. Overall, T cells with potent reactivity to GPMBP or RMBP were subsequently unable to present that antigen, whereas T cells exhibiting partial or low antigen reactivities were highly effective APC for presentation of that antigen. The unrelated antigen conalbumin was presented by MBP-specific clones only when added to culture with a specific partial agonist. Together, these data indicate that partially agonistic MHC ligands promote prolonged expression of T-APC activity and that DN T cells may be specialized to mediate postactivational antigen presentation.

PORCINE THYMUS SUPPORTS DEVELOPMENT OF FUNCTIONAL HUMAN T CELLS

40 Since immune responses toward xenogeneic organs tend to be greater than those to allografts, unacceptably high levels of immunosuppression would likely be required for their success. A state of donor-specific tolerance would obviate the risk of both acute and chronic rejection while eliminating the need for chronic immunosuppressive therapy, thereby permitting normal immunocompetence. We now demonstrate that fetal porcine thymus grafts can reconstitute a polyclonal human T cell repertoire when co-transplanted with fetal human liver under the kidney capsule of SCID mice (SW/HU). Single positive CD4 and CD8 human T cells repopulated the peripheral lymphoid organs of the grafted SCID mice. To determine the functional status of T cells developing n SW/HU SCID thymic grafts, human T cells were stimulated with Concavalin A and phytohematoagglutinin. Human lymphocytes in porcine thymus grafts or mouse spleens showed substantial proliferative responses to lectin stimulation (stimulation indices >50). Con A-stimulated thymocytes from HU/HU and SW/HU grafts showed similar upregulation of the early activation markers CD25 and CD69 at 24 hours. In order to further assess the function of human T cells that developed in thymic xenografts, we performed allogeneic anti-human mixed lymphocyte reactions (MLR). Human lymphocytes that developed in porcine thymic grafts mounted comparable MLR alloresponses to those elicited from human lymphocytes that developed in HU/HU grafts. No evidence of graft-versus-host disease, autoimmune disease, wasting syndrome or rejection of swine grafts was detected in any SW/HU SCID mice, presumably due to tolerance of human and swine lymphocytes towards mouse, swine and human antigens. Consistent with this possibility, porcine and murine class I+ and class II+ cells with a dendritic morphology were detected by immunohistochemistry, mainly in the corticomedullary junction and the medulla of SW/HU grafts. In summary, this study provides the first evidence that the swine thymus can support normal maturation and export to the periphery of functional polyclonal human lymphocytes in vivo. In addition to inducing xenogeneic transplantation tolerance, the xenogeneic thymus grafting approach could be utilized in the treatment of patients with HIV-associated thymic atrophy, given the HIV resistance of swine tissues.

PORCINE THYMUS SUPPORTS DEVELOPMENT OF FUNCTIONAL HUMANT T CELLS

40 Since immune responses toward xenogeneic organs tend to be greater than those to allografts, unacceptably high levels of immunosuppression would likely be required for their success. A state of donor-specific tolerance would obviate the risk of both acute and chronic rejection while eliminating the need for chronic immunosuppressive therapy, thereby permitting normal immunocompetence. We now demonstrate that fetal porcine thymus grafts can reconstitute a polyclonal human T cell repertoire when co-transplanted with fetal human liver under the kidney capsule of SCID mice (SW/HU). Single positive CD4 and CD8 human T cells repopulated the peripheral lymphoid organs of the grafted SCID mice. To determine the functional status of T cells developing in SW/HU SCID thymic grafts, human T cells were stimulated with Concavalin A and phytohematoagglutinin. Human lymphocytes in porcine thymus grafts or mouse spleens showed substantial proliferative responses to lectin stimulation (stimulation indices >50). Con A-stimulated thymocytes from HU/HU and SW/HU grafts showed similar up-regulation of the early activation markers CD25 and CD69 at 24 hours. In order to further asses the function of human T cells that developed in thymic xenografts, we performed allogeneic anti-human mixed lymphocyte reactions (MLR). Human lymphocytes that developed in porcine thymic grafts mounted comparable MLR alloresponses to those elicited from human lymphocytes that developed in HU/HU grafts. No evidence of graft-versus-host disease, autoimmune disease, wasting syndrome or rejection of swine grafts was detected in any SW/HU SCID mice, presumably due to tolerance of human and swine lymphocytes towards mouse, swine and human antigens. Consistent with this possibility, porcine and murine class I+ and class II+ cells with a dendritic morphology were detected by immunohistochemistry, mainly in the corticomedullary junction and the medulla of SW/HU grafts. In summary, this study provides the first evidence that the swine thymus can support normal maturation and export to the periphery of functional polyclonal human lymphocytes in vivo. In addition to inducing xenogeneic transplantation tolerance, the xenogeneic thymus grafting approach could be utilized in the treatment of patients with HIV-associated thymic atrophy, given the HIV resistance of swine tissues.

Mouse NK1.1 + cytotoxic T cells can be generated by IL-2 exposure from lymphocytes which express an intermediate level of T cell receptor

NK-like T cells which express the NK1.1 molecule and CD3 (or TCR) of intermediate level (CD3 int or TCR int cells) were recently demonstrated to be present in various immune organs, and to have NK-like cytotoxic activity against NK target cells. In this study, we investigated whether NK1.1 − T cells could express NK1.1. We found that NK1.1 + TCR int cells were much more abundant in the liver (20%) than in the spleen (2%). When hepatic and splenic mononuclear cells (MNCs) were cultured either in the absence of IL-2 or in the presence of CD3/TCR cross-linking, the original NK1.1 + TCR int cells disappeared. However, when they were cultured in the presence of a high dose of IL-2 for 4 days, a new type of NK1.1 + T cell was formed to the extent of approximately 15–20%, and the liver and spleen contained similar percentages of this new type of NK1.1 + T cells. The phenotypes of the original and the new type of NK1.1 + T cells were clearly distinct. The freshly obtained NK1.1 + TCR int cells consisted of double-negative (DN) CD4 −CD8 − cells and single-positive (SP) CD4 + cells, whereas the new type of NK1.1 + T cells predominantly consisted of DN CD4 −CD8 − cells and SP CD8 + cells and expressed a high level of CD3 (CD3 high or TCR high cells). When NK1.1 − cells or IL-2 receptor β-chain (IL-2R β) − cells were isolated from the liver and spleen, and cultured in the presence of IL-2 for 4 days, NK1.1 + T cells were generated from NK1.1 − cells, but not from IL-2R β − cells. Our results suggested that the NK1.1 − cells, but not IL-2R β − cells, contained the precursor of IL-2-stimulated NK1.1 + TCR high cells. When purified NK1.1 − IL-2R β + TCR int cells were cultured in the presence of IL-2 for 4 days, approximately 10% of the cells became NK1.1 + TCR high cells. Approximately 60% of the purified NK1.1 + TCR int cells lost NK1.1 expression. The IL-2-stimulated NK1.1 + TCR high cells that had arisen from NK1.1 − TCR int cells exerted an NK cell-like cytotoxic activity similar to that of the original NK1.1 + T cells. Thus, NK1.1 − TCR int cells could express NK1.1 and exert NK-like cytotoxic activity regardless of their origin. It appears that NK1.1 + TCR high cells can only be induced through an IL-2-stimulation pathway but not via CD3/TCR cross-linking.

Distinct NFAT Family Proteins Are Involved in the Nuclear NFAT-DNA Binding Complexes from Human Thymocyte Subsets

The nuclear factor of activated T cells (NFAT) is involved in the transcriptional induction of cytokine and other immunoregulatory genes during an immune response. Among four distinct NFAT family members identified to date, mRNAs of NFAT1, NFATc, and NFATx are expressed in the thymus. Here, we report the distribution of these three NFAT family members in human fetal thymocyte subsets and in peripheral mature T cells. We show that NFATx mRNA was expressed in all T lymphocyte subsets tested and was highest in CD4+CD8+ double positive (DP) thymocytes. Conversely, NFAT1 mRNA was preferentially expressed in the mature CD4+ single positive (SP) populations. NFATc mRNA was present at low levels in all subsets but strongly induced upon treatment with phorbol ester and calcium ionophore. Interestingly, we detected NFAT-DNA binding complexes in DP thymocytes, albeit at lower levels than in CD4 SP cells. Corresponding to the mRNA expression, we observed that NFATx was responsible for the NFAT-DNA binding in DP thymocytes. Moreover, this DNA binding was inhibited by cyclosporin A, indicating that NFATx nuclear translocation was regulated by the calcineurin phosphatase in DP thymocytes. For the CD4 SP populations, NFAT1 and NFATc, and to some extent NFATx, were responsible for the NFAT-DNA binding complexes. These results indicate that NFAT family members are differentially regulated during the development of T cells, and that NFATx may play a distinct role in calcineurin-dependent signaling in DP thymocytes.

Cypermethrin-induced alteration of thymocyte distribution and functions in prenatally-exposed rats

The synthetic pyrethroid insecticide, cypermethrin (50 mg/kg) was given during gestation to pregnant rats by gavage in corn oil. Prenatal cypermethrin exposure induced a significant decrease in the absolute number of all thymocyte subsets during the first 30 days after birth, being the double negative CD4 −CD8 −, single positive CD4 and CD8 T cells preferentially affected. Later on day 60 and 90 double positive CD4 +CD8 + and single positive thymocytes gradually recovered, while the total number of CD4 −CD8 − cells was increased. Moreover, thymocytes from rats prenatally exposed to cypermethrin showed an impaired ability to proliferate in response to different doses of Concanavalin A (ConA) and human recombinant interleukin-2 (hrIL-2) and to produce and/or release IL-2. Overall, our results indicate that cypermethrin administered during prenatal period can affect multiple steps in thymocyte differentiation pathways resulting in an altered cell subset distribution and an impairment of thymocyte functions.

Clonal deletion of some V beta+ T cells in peripheral lymphocytes from C57BL/6 mice infected with MHV3.

Mouse hepatitis virus type 3 infection is generally accompanied by a severe immune dysfunction involving thymic or splenic T cell subpopulations. We postulate that the peripheral lymphoid cell depletions were caused by a selective deletion of some V beta subsets of mature T cells, as observed with superantigens. We have examined the expression of V beta 6, V beta 8 and V beta 14 in T cell subpopulations from the spleen and lymph nodes of pathogenic L2-MHV3-infected C57BL/6 mice. Cytofluorometric study showed decreases in splenic V beta 8+, V beta 6+, and V beta 14+ T cell subpopulations at 72 hrs post-infection. Single positive CD4+ T cells were diminushed but not the CD8+ cells. In contrast, the various V beta splenic cell populations were not modified in mice infected with a non- pathogenic YAC-MHV3 variant. However, the V beta 8/CD4 ratio increased in splenic cells but decreased in lymphocytes from lymph nodes. The V beta 14/CD4 ratio decreased only in splenic cells while V beta 6/CD4 ratios were not modified. These results suggest that alterations in V beta cell populations may play a role in the L2-MHV3-induced immunodeficiency.

Intrahepatic alpha beta-TcRintermediate LFA-1high T cells are stimulated during mouse hepatitis viral infection.

Mouse hepatitis virus type 3 (MHV3), a coronavirus, is an excellent animal model for the study of thymic and extrathymic T cell subpopulation disorders induced during the viral hepatitis. To understand local hepatic immune responses, the phenotypes of resident hepatic lymphocytes were determined and compared that of splenic and thymic T cell subpopulations during the acute viral hepatitis induced by MHV3 in susceptible C57BL/6 mice. Single positive (SP) CD4+ or CD8+ cells strongly increased in the liver. A specific cell population, the double positive (CD4+ C8+) cells, normally present in liver and thymic cell preparations, decreased in C57BL/6 mice following the viral infection. alpha beta-TcRintermediate T cells shifted toward alpha beta-TcRhigh T cells in the liver and thymus of infected mice, but not in their spleen. The specific alpha beta-TcRint or high lymphocytes occurring in the liver of MHV3-infected mice expressed higher levels of leukocyte function antigen-1 (LFA-1) and Pgp-1 (CD44) activation markers, suggesting that they were either activated or antigen-experienced during the viral infection. No significant changes in T cell subpopulations were detected in the spleen. These observations suggest that MHV3 infection could induce an early in situ stimulation of resident hepatic T cells, despite a peripheral immunodeficiency in the thymus and spleen.

Impaired thymopoiesis occurring during the thymic involution of tumor-bearing mice is associated with a down-regulation of the antiapoptotic proteins Bcl-XL and A1

The thymus is a central lymphoid organ in which T lymphocytes undergo differentiation and maturation without the need for antigenic stimulation. Apoptosis (programmed cell death), plays a critical role in shaping the T cell repertoire, deleting unproductive as well as potentially autoreactive T cells. Thymic atrophy has been observed in several model systems, including aging, graft-vs-host-disease and tumor development. However, the mechanisms involved in this phenomenon remain to be completely elucidated. We have previously shown that the progressive growth of D1-DMBA-3 mammary tumor leads to extreme thymic atrophy in the host. This thymic involution is associated with an early block in T cell maturation at the triple negative stage of differentiation. In the present study we have used our murine mammary tumor model to further analyze the specific T cell subpopulations present in the thymus of tumor-bearing animals as well as to characterize the alterations of the apoptotic process present during the impaired thymopoiesis associated to this thymic involution. Flow cytometric analysis revealed a moderate increase in the percentages of single positive CD4+ and CD8+ cells within the CD3 negative population in the thymuses of tumor-bearing mice. Moreover, we observed a prolonged increase in apoptosis among thymocytes from tumor-bearing mice compared with thymocytes from normal mice during tumor development. Lastly, we found a major decrease of Bcl-XL and A1, two crucial anti-apoptotic Bcl-2 family members that are developmentally regulated in T cells. Together, our data suggest that the severe thymic involution seen in mammary tumor bearers is due to an arrest in at least two steps of T cell differentiation and a down-regulation of important molecules that control programmed cell death.

Influence of Retinoic Acid on the Differentiation Pathway of T Cells in the Thymus

This study investigated the ability of retinoic acid (RA) to influence T cell differentiation. All-trans-RA had marked effects on T cell differentiation in murine fetal thymic organ cultures (FTOCs). The time course of the effect of all-trans-RA in FTOC of day 14 C57BL/6 embryos revealed a twofold increase in the frequency of CD4 single-positive (SP) cells and a high level of CD3-bearing cells (CD3highcells) at a later stage of T cell development. At an earlier stage, all-trans-RA induced a twofold increase in the frequency of CD4 SP cells, but significantly suppressed the upregulation of CD3 and TCR. Reverse transcription-PCR using RA receptor (RAR) subtype-specific primers showed that RAR α but not β and γ is expressed during T cell development in the thymus and that its expression was associated with the generation of CD4/CD8 double-positive (DP) cells. In FTOC of day 16 BALB/c embryos, the level of Vβ3highcells was greatly reduced (1.4% of the CD3highcells) in response to the mouse mammary tumor virus-6-encoded superantigen, but Vβ3-bearing cells were rescued from the deletion in the presence of all-trans-RA (5.6% of the CD3highcells). Further, the inhibitory effect of all-trans-RA on thymocyte deletion was observed when the deletion was induced by a low concentration of staphylococcal enterotoxin B in FTOC. Taken together, these data suggest that RA increases the frequency of mature and self-reactive T cells in the thymus, possibly by inhibiting the process of negative selection at the DP stage of T cell differentiation.