Single-positive CD4 Research Articles

Interleukin-7 and infection itself by human immunodeficiency virus 1 favor virus persistence in mature CD4+CD8−CD3+ thymocytes through sustained induction of Bcl-2

The sequence of events and the mechanisms leading to the destruction of the thymus during human immunodeficiency virus (HIV) infection are still poorly characterized. Investigated here are the survival capacity on HIV-1 infection of the mature single-positive CD4(+)CD8(-)CD3(+) (SP CD4(+)) and the intermediate CD4(+) CD8(-)CD3(-) thymocytes previously shown to be able to replicate the virus in the thymic microenvironment. It is demonstrated that the mature SP CD4(+) thymocytes exhibit a high survival capacity despite the production of a high yield of viruses. Interleukin-7, reported to be a crucial cofactor of tumor necrosis factor (TNF) to promote HIV replication, is shown here to counteract the apoptotic activity of TNF. Resistance to apoptosis of SP CD4(+) cells is conferred by a high expression of the IL-7 receptor (IL-7R) associated with the capacity of IL-7 to permanently up-regulate Bcl-2. In addition, this high Bcl-2 level is further enhanced by infection itself. In contrast, intermediate thymocytes, which replicate the virus at a lower level, are more sensitive to apoptosis, and their differentiation into double-positive CD4(+)CD8(+)CD3(-) (DP CD3(-)) cells strongly increases their death rate on infection. This sensitivity is related to a lower expression of IL-7R and Bcl-2 in intermediate thymocytes, which further decreases at the DP CD3(-) stage. In addition, a decreased level of Bcl-2 is observed in this subset during infection. Altogether these data suggest that in vivo, HIV infection might create a persistent virus reservoir within the SP CD4(+) thymocytes, whereas the later infection of intermediate cells might lead to thymopoiesis failure.

A naturally processed mitochondrial self-peptide in complex with thymic MHC molecules functions as a selecting ligand for a viral-specific T cell receptor.

Peptide fragments of self-proteins bound to major histocompatibility complex molecules within the thymus are important for positively selecting T cell receptor (TCR)-bearing CD4(+)CD8(+) double positive (DP) thymocytes for further maturation. The relationship between naturally processed thymic self-peptides and TCR-specific cognate peptides is unknown. Here we employ HPLC purification of peptides released from H-2K(b) molecules of the C57BL/6 thymus in conjunction with mass spectrometry (MS) and functional profiling to identify a naturally processed K(b)-bound peptide positively selecting the N15 TCR specific for the vesicular stomatitis virus octapeptide (VSV8) bound to K(b). The selecting peptide was identified in 1 of 80 HPLC fractions and shown by tandem MS (MS/MS) sequencing to correspond to residues 68-75 of the MLRQ subunit of the widely expressed mitochondrial NADH ubiquinone oxidoreductase (NUbO(68-75)). Of note, the peptide differs at six of its eight residues from the cognate peptide VSV8 and functions as a weak agonist for mature CD8 single positive (SP) N15 T cells, with activity 10,000-fold less than VSV8. In N15 transgenic (tg) recombinase activating gene 2(-/)- transporter associated with antigen processing 1(-/)- fetal thymic organ culture, NUbO(68-75) induces phenotypic and functional differentiation of N15 TCR bearing CD8 SP thymocytes. Failure of NUbO(68-75) to support differentiation of a second K(b)-restricted TCR indicates that its inductive effects are not general.

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.

Thymic lesions in cats infected with a pathogenic molecular clone or an ORF-A/2-deficient molecular clone of feline immunodeficiency virus.

Previous studies using feline immunodeficiency virus (FIV) molecular clones lacking the putative transactivator gene (ORF-A/2) failed to address the issue of thymus pathogenesis or investigate the levels of viral replication in separate lymphoid compartments (Y. Inoshima, et al., J. Virol. 70:8518-8526, 1996; E. E. Sparger, et al., Virology 205:546-553, 1994). Using a highly pathogenic molecular clone of FIV, JSY3, and an ORF-A/2-deficient mutant, JSY3DeltaORF-A/2, we compared viral replication and the extent of thymic dysfunction as measured by the formation of lymphoid follicles and alteration of the thymocyte subsets. Viral replication was reduced in JSY3DeltaORF-A/2-infected cats as measured by lymphocyte coculture, immunohistochemistry, and quantitative PCR. Cell-associated viral load measured by lymphocyte coculture varied in a tissue-dependent manner with replication highest in lymphocytes isolated from the thymus, lower in those from the peripheral blood, and lowest in those from lymph node. Thymic proviral load and the number of viral p24 Gag-positive cells within the thymus detected by immunohistochemistry were also reduced. In addition, the onset of a reduced peripheral blood CD4/CD8 ratio was delayed in JSY3DeltaORF-A/2-infected cats. The formation and extent of thymic lymphoid follicular hyperplasia were similar in JSY3 and JSY3DeltaORF-A/2-infected cats as measured by anticytokeratin immunohistochemistry and flow cytometry for percent pan T-negative, immunoglobulin G-positive cells within the thymus. In contrast, comparison of thymocyte subpopulations demonstrated a reduced expansion of single-positive CD4(-) CD8(+) thymocytes in JSY3DeltaORF-A/2-infected cats. Level of viral replication, therefore, may not correlate with the formation of thymic lymphoid follicles but may correlate with the expansion of the single-positive CD4(-) CD8(+) thymocyte subpopulation.

Role of hypothalamic-pituitary axis in morphine-induced alteration in thymic cell distribution using mu-opioid receptor knockout mice

Mu-opioid receptor knockout mice (MORKO), were used to address two questions: (1) if morphine induced decrease in thymic weight and cell distribution is mediated by the mu-opioid receptor and (2) the role of corticosteroids in morphine mediated alteration in thymic cell distribution. Our result show that morphine mediated increase in plasma corticosterone is mediated by the mu-opioid receptor since morphine at doses as high as 25 mg/kg-body weight does not increase plasma corticosterone levels in the MORKO. In addition, we have also shown that morphine treatment results in the differentiation of CD4+CD8+ (double positive cells) to single positive CD4+ cells while dexamethasone treatment results in the deletion of CD4+CD8+ (double positive) cells.

Expression of CCR9 β-chemokine receptor is modulated in thymocyte differentiation and is selectively maintained in CD8+ T cells from secondary lymphoid organs

AbstractChemokines appear to have an important role in the seeding of lymphoid progenitors in the thymus, the regulation of the coordinated movements of the maturing T cells within this organ, and the egress of the resulting naive T cells to secondary lymphoid organs. CCR9, the specific receptor for the β-chemokine TECK/CCL25, is selectively expressed in thymus, lymph node, and spleen. Using a specific anti-CCR9 polyclonal antibody, K629, and a semiquantitative reverse transcriptase–polymerase chain reaction procedure, a detailed study of CCR9 expression in the thymus and secondary lymphoid organs was performed. The results show that CD4+CD8+ double-positive thymocytes have the highest CCR9 expression in thymus. Single-positive CD8+ thymocytes continue to express this receptor after abandoning the thymus as mature naive T cells, as suggested by the existence of a CD8+CD69lowCD62LhighCCR9+ cell subset. Consistent with this, CD8+lymphocytes from lymph nodes, spleen, and Peyer patches express a functional CCR9, as its expression correlates with migration in response to CCL25. Conversely, CD4+ thymocytes lose CCR9 before abandoning the thymus, and CD4+ T cells from secondary lymphoid organs also lack CCR9 expression. Analysis of CCR9 expression in thymocytes from mice of different ages showed that CCR9 levels are affected by age, as this receptor is more abundant, and its response to CCL25 is more potent in newborn animals. Collectively, these results suggest that CCR9 has a role in thymocyte development throughout murine life, with clear differences between the CD4+ and CD8+ lineages.

C-C chemokine receptor 4 expression defines a major subset of circulating nonintestinal memory T cells of both Th1 and Th2 potential.

CCR4, a chemokine receptor for macrophage-derived chemokine (MDC) and thymus and activation-regulated chemokine (TARC), has been implicated as a preferential marker for Th2 lymphocytes. Following in vitro polarization protocols, most Th2 lymphocytes express CCR4 and respond to its ligands TARC and MDC, whereas Th1 lymphocytes express CXC chemokine receptor 3 and CCR5 (but not CCR4). We show in this study that CCR4 is a major receptor for MDC and TARC on T lymphocytes, as anti-CCR4 mAbs significantly inhibit the migration of these cells to MDC and TARC. CCR4 is also highly expressed in most single-positive CD4(+) thymocytes and on a major fraction of blood nonintestinal (alpha(4)beta(7)(-)) memory CD4 lymphocytes, including almost all skin memory CD4(+) cells expressing the cutaneous lymphocyte Ag (CLA), but weakly or not expressed in other subsets in thymus and blood. Interestingly, major fractions of circulating CCR4(+) memory CD4 lymphocytes coexpress the Th1-associated receptors CXC chemokine receptor 3 and CCR5, suggesting a potential problem in using these markers for Th1 vs Th2 lymphocyte cells. Moreover, although production of Th2 cytokines in blood T cells is associated with CCR4(+) CD4 lymphocytes, significant numbers of freshly isolated circulating CCR4(+) memory CD4 lymphocytes (including both CLA(+) and CLA(-) fractions) readily express the Th1 cytokine IFN-gamma after short-term stimulation. Our results are consistent with a role for CCR4 as a major trafficking receptor for systemic memory T cells, and indicate that the patterns and regulation of chemokine receptor expression in vivo are more complex than indicated by current in vitro models of Th1 vs Th2 cell generation.

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.

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.

CD8+ thymic lymphocytes express reduced levels of CD8beta and increased interferon gamma in cats perinatally infected with the JSY3 molecular clone of feline immunodeficiency virus.

Biological isolates of feline immunodeficiency virus (FIV) cause a relative expansion of activated single-positive CD8(+) (SP CD8(+)) lymphocytes within the thymus of infected cats. In this study, thymic SP CD8(+) lymphocytes were analyzed from cats inoculated as neonates with a pathogenic molecular clone of FIV, JSY3, which was previously derived from the wild-type biological isolate FIV(NCSU-1) (NCSU-1). Four cats were inoculated intraperitoneally with NCSU-1 and compared with 11 cats inoculated with JSY3. Five control cats matched in litter and age were administered an intraperitoneal sham inoculum. Between 12 and 16 weeks postinoculation, interferon-gamma (IFN-gamma) mRNA was quantified by RT-PCR in freshly isolated thymocytes and peripheral blood mononuclear cells (PBMCs). The quantity of IFN-gamma mRNA was increased more than 10-fold in thymocytes and PBMCs of 13 of 13 FIV-inoculated cats as compared with the sham-inoculated controls. IFN-gamma mRNA coenriched with magnetically sorted CD8(+) PBMCs and single-positive (SP) CD8(+) thymocytes. Cells expressing IFN-gamma mRNA were located within the thymic perivascular zone, along the corticomedullary junction, and adjacent to lymphoid follicles. The expansion of thymic SP CD8(+) cells was associated with an increase in CD8alpha(+)/beta(neg) and CD8alpha(+)/beta(lo) phenotypes, the latter population resembling a previously reported memory/effector peripheral blood cell with FIV suppressor activity. From these data we conclude that JSY3 and NCSU-1 induce similar phenotypic changes in thymic and peripheral blood CD8(+) cells. Thus, JSY3 is pathogenic for the thymus in vivo and will be useful for defining determinants of the CD8(+) cell response in this pediatric AIDS model.

Effect of murine thymic epithelial cell line (MTEC1) on the functional expression of CD4+CD8– thymocyte subgroups

To determine the effect of thymic stromal cells on the functional maturation of CD4 single-positive (SP) thymocytes, the functional status of isolated CD4 SP thymocyte subgroups was investigated by means of cell proliferation and cytokine production in response to concanavalin A (Con A) prior and after co-culturing with a murine thymic epithelial cell line (MTEC1). Mouse medullary CD4 SP thymocytes were phenotypically divided into seven discrete subgroups predicted to reflect the maturation pathway from newly emerging CD4 SP thymocytes to terminally differentiated cells. For functional analysis, six major subgroups (6C10(+)CD69(+), 6C10(-)CD69(+), 6C10(-)CD69(-)3G11(+)Qa-2(-), 6C10(-)CD69(-)3G11(+)Qa-2(+), 6C10(-)CD69(-)3G11(-)Qa-2(-) and 6C10(-)CD69(-)3G11(-)Qa-2(+)) cells were isolated and their functional status in response to Con A stimulation assessed. A functional hierarchy is revealed among these subgroups, consistent with their phenotypic maturation status, which may imply that these cells undergo a functional maturation process within thymic medulla. The function of cytokine production by CD4 SP thymocytes is acquired in a stepwise manner from a low to high level and characterized by T(h)0-type cytokines in the main stream of differentiation pathway. However, a minor subgroup that appeared at the late stage as 3G11(-)6C10(-) cells was biased to produce T(h)2-type cytokines. Nevertheless, the functional capacity of the final two Qa-2(+) subgroups of CD4 SP thymocytes was still significantly lower than that of spleen CD4(+) T cells. After co-cultivation with MTEC1 cells, four subgroups of TCRalphabeta(+)CD4(+)CD8(-) thymocytes exhibited significantly higher levels of proliferation capability and modulation in cytokine production capability. However, co-culturing with MTEC1 cells did not change the pattern of T(h)0- or T(h)2-like cytokine production by respectively medullary CD4 SP thymocyte subgroups nor could MTEC1 induce CD4 SP thymocytes to secrete T(h)1-type cytokines. The results suggest that MTEC1 can regulate the functional status of these thymocyte subgroups.

Peripheral blood extrathymic CD4(+)CD8(+) T cells with high cytotoxic activity are from the same lineage as CD4(+)CD8(-) T cells in cynomolgus monkeys.

We have previously reported that CD4/CD8 double-positive (DP) T cells with the resting memory phenotype are present in the periphery of healthy cynomolgus monkeys. In the present study, we performed functional studies on the T cells. The expression of CD4 and CD8 on DP, CD4 single-positive (SP) or CD8 SP T cells was stable in cultures with either mitogen or anti-CD3 antibody stimulation. In spite of lacking CD28 expression, DP T cells showed similar proliferative ability and apoptosis sensitivity to CD4 SP and CD8 SP T cells. DP T cells showed both helper and cytotoxic activities. Although the helper activity of DP T cells was lower than that of CD4 SP T cells, cytotoxic activity was comparable to that of CD8 SP T cells. Fresh DP T cells killed target cells mainly by the perforin-granzyme pathway. In addition, fresh DP T cells expressed a high level of mRNA for IFN-gamma and produced a high level of IFN-gamma when they were activated by anti-CD3 antibody ligation. On the other hand, several expanded DP T cell clones shared TCR V(beta) with expanded CD4 SP T cell clones, strongly suggesting that those two corresponding clones with DP and CD4 SP phenotypes might be derived from the same ancestor T cell. These results showed that the DP T cells are a novel T cell subset with functions overlapping with those of CD4 SP and CD8 SP T cells, and that they might play protective and regulatory roles in secondary immune response in cynomolgus monkeys.

Ligand-specific selection of MHC class II-restricted thymocytes in fetal thymic organ culture.

Positive and negative selection of thymocytes is determined by the specificity of the TCR and signaling through its associated molecules. We have studied selection of thymocytes bearing a MHC class II-restricted TCR using fetal thymic organ culture. This system allows the addition of peptides to the already diverse panoply of endogenous peptide ligands and is useful for analyzing ligand-specific negative selection of CD4 single positive (CD4SP) thymocytes. The data reveal that the ability of a given ligand to mediate negative selection is related to its dissociation rate from the TCR. We find that negative selection is very sensitive, and only the weakest ligand that we can identify fails to induce negative selection. None of the numerous peptides tested were able to induce an increase in CD4SP thymocytes. In addition, the ligands that induce negative selection of CD4SP thymocytes also cause an increase in numbers of CD8SP thymocytes bearing high levels of the class II-restricted TCR. Although these cells have a cell surface phenotype consistent with positive selection, they most likely represent cells in the process of negative selection. Further analysis reveals that these cells are not induced by these ligands in intact adult animals and that their induction is probably only revealed in the organ culture system.

Type I and Type II T Cell Profiles in Chronic Myelogenous Leukemia

T cell immunity is considered to play an important role in the control of cell growth in chronic myelogenous leukemia (CML) although information regarding the characteristics of T lymphocytes in CML patients is limited. Using flow cytometric analysis of intracellular cytokine expression at the single-cell level, we analyzed helper T and cytotoxic T subsets in 8 CML patients. The percentage of interferon-γ (IFN-γ) single-positive CD4 cells (Th1) and that of interleukin-4 (IL-4) single-positive CD4 cells (Th2) was markedly decreased in pretreated CML patients compared to normal controls. In addition, the percentage of IFN-γ/IL-4 double-positive CD4 cells (Th0) was also reduced. Consequently, the percentage of IFN-γ/IL-4 double-negative CD4 cells was markedly increased. Similarly, a reduction in IFN-γ single-positive CD8 cells (Tc1) and IFN-γ/IL-4 double-positive CD8 cells (Tc0) and an increase in IFN-γ/IL-4 double-negative CD8 cells were observed in pretreated CML patients. Imbalance of these parameters was markedly improved following cytoreduction therapy. Our findings directly indicate anergic states in CML patients. Determination of the factors that affect Th and Tc profiles may lead to further understanding of immunological states and the development of effective immunotherapy.

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.

MEK activity regulates negative selection of immature CD4+CD8+ thymocytes.

CD4+CD8+ thymocytes are either positively selected and subsequently mature to CD4 single positive (SP) or CD8 SP T cells, or they die by apoptosis due to neglect or negative selection. This clonal selection is essential for establishing a functional self-restricted T cell repertoire. Intracellular signals through the three known mitogen-activated protein (MAP) kinase pathways have been shown to selectively guide positive or negative selection. Whereas the c-Jun N-terminal kinase and p38 MAP kinase regulate negative selection of thymocytes, the extracellular signal-regulated kinase (ERK) pathway is required for positive selection and T cell lineage commitment. In this paper, we show that the MAP/ERK kinase (MEK)-ERK pathway is also involved in negative selection. Thymocytes from newborn TCR transgenic mice were cultured with TCR/CD3epsilon-specific Abs or TCR-specific agonist peptides to induce negative selection. In the presence of the MEK-specific pharmacological inhibitors PD98059 or UO126, cell recovery was enhanced and deletion of DP thymocytes was drastically reduced. Furthermore, development of CD4 SP T cells was blocked, but differentiation of mature CD8 SP T cells proceeded in the presence of agonist peptides when MEK activity was blocked. Thus, our data indicate that the outcome between positively and negatively selecting signals is critically dependent on MEK activity.

Impairment of rat thymocyte differentiation and functions by neonatal capsaicin treatment is associated with induction of apoptosis

The direct and indirect interaction between the nervous system and its transmitters with the immune system was evaluated in the rat by using the neurotoxin capsaicin (Caps). In the present study we investigated the effect of Caps administration to neonatal rats on thymocyte subpopulation distribution and functions at different times after treatment. Caps treatment results in a marked reduction of thymus weight and cellularity. As shown by immunofluorescence and FACS analysis, profound depletion of double negative (DN), double positive (DP), and single positive (SP) CD4 + cells was already evident at day 7 after treatment and persisted until day 28. Reduced numbers of SP CD8 + cells were observed only at later time points. Analysis of TCR phenotype indicates that CD5 + TCR γ/δ + are particularly sensitive to neonatal Caps treatment. Caps-induced thymocyte depletion was associated with reduced proliferation in response to T cell mitogens. Moreover, in situ TUNEL reaction and agarose gel electrophoresis indicate that neonatal Caps treatment induces apoptosis of thymus cells. Morphological analysis reveals the presence of apoptotic cells in the subcapsular thymus cortical region. Overall our results suggest that Caps when administered at birth, profoundly affects T cell differentiation, likely through its ability to activate apoptotic cell death program.

Janus kinase 3 (Jak3) is essential for common cytokine receptor gamma chain (gamma(c))-dependent signaling: comparative analysis of gamma(c), Jak3, and gamma(c) and Jak3 double-deficient mice.

The common cytokine receptor gamma chain (gamma(c)) is an essential receptor component for IL-2, IL-4, IL-7, IL-9 and IL-15, and thereby gamma(c)-deficient mice exhibit impaired T cell and B cell development. The Janus family tyrosine kinase 3 (Jak3) is known to be associated with gamma(c), and the reported phenotypes of gamma(c)-deficient (gamma(c)(-)) and Jak3-deficient (Jak3(-)) mice are similar, indicating that Jak3 is an essential transducer of gamma(c)-dependent signals. Nevertheless, certain differences have been suggested related to the range of actions of gamma(c) and Jak3. To clarify whether gamma(c)-dependent cytokines can partially transduce their signals without Jak3, we compared lymphocyte development in gamma(c)(-), Jak3(-), and gamma(c) and Jak3 double-deficient (gamma(c)(-)Jak3(-)) mice in the same genetic background. With the exception that T and B cells in Jak3(-) mice express high levels of gamma(c), the defects in thymocyte and peripheral T cell and B cell development are indistinguishable among gamma(c)(-), Jak3(-) and gamma(c)(-)Jak3(-) mice. Interestingly, although Bcl-2 induction was previously suggested to be Jak3-independent, IL-7 cannot induce Bcl-2 expression in CD4 single-positive (SP) thymocytes in either gamma(c)(-) or Jak3(-) mice nor can IL-7 rescue CD4 SP thymocytes from dexamethasone-induced cell death in gamma(c)(-) or Jak3(-) mice. These results indicate that Jak3 is absolutely essential for gamma(c)-dependent T cell and B cell development, and for gamma(c)-dependent prevention of thymocyte apoptosis.

Human CD4 residue Phe 43 is critical for repertoire development and maturation of MHC class II restricted CD4 single-positive T lineage cells in vivo.

To determine the functional significance of structural alteration of CD4-MHC class II interaction in vivo, two human (h)CD4-transgenic (tg) mice were established on a murine (m)CD4(-/-) H-2(b) background. The MHC class II binding-competent hCD4 (R240AhCD4) rescues the number and helper activity of hCD4(+)CD8(-) single-positive (SP) mature T cells in mCD4(-/-) mice. In contrast, the MHC class II binding-deficient F43I hCD4 mutant cannot facilitate normal differentiation of double-positive thymocytes to CD4(+)CD8(-) SP thymocytes. Hence, only 20 - 25% of CD4(+)CD8(-) SP T cells found in wild-type or R240A hCD4tg mice are generated, with resultant diminished helper responses. Differentiation of F43I hCD4 SP T cells is MHC class II but not class I dependent as demonstrated by crossing F43I hCD4tg mice onto MHC-deficient mice. These cells show a different pattern of TCR Valpha and Vbeta gene usage relative to comparable R240A hCD4 SP T cells from R240 AhCD4tg animals. Expression of activation markers including CD25 and CD69 on F43I hCD4 SP T cells suggests that autoreactive specificites may not have been eliminated intrathymically. Collectively, the results show that CD4-MHC class II interaction significantly influences intrathymic repertoire selection.

Expression of the Bcl-2 family member A1 is developmentally regulated in T cells.

During T cell development, cells that fail to meet stringent selection criteria undergo programmed cell death. Thymocyte and peripheral T cell susceptibility to apoptosis is influenced by expression of Bcl-2 family members, some of which are expressed in a developmentally patterned manner. We previously showed developmentally regulated expression of A1, an anti-apoptotic Bcl-2 family member, among B cell developmental subsets. Here we show that cells of the T lineage also express A1 in a developmentally regulated manner. Both A1 mRNA and A1 protein are readily detectable in the thymus, and while present among DN cells, A1 mRNA is up-regulated to very high levels among double-positive (DP) thymocytes. It is then down-regulated to moderate levels among single-positive (SP) thymocytes, and finally expressed at approximately 25-fold lower levels among mature SP CD4(+) and CD8(+) lymph node T cells than among DP thymocytes. Furthermore, we find that in vitro TCR ligation up-regulates A1 expression among both DP and SP thymocytes. Together, these data show that A1 expression is developmentally regulated in T lymphocytes and is responsive to TCR signaling, suggesting that A1 may play a role in maintaining the viability of DP thymocytes.