T cell receptor beta chain lacking the large solvent-exposed Cbeta FG loop supports normal alpha/beta T cell development and function in transgenic mice.

The striking and unique structural feature of the T cell receptor (TCR) beta chain is the bulky solvent-exposed FG loop on the Cbeta domain, the size of almost half an immunoglobulin domain. The location and size of this loop suggested immediately that it could be a crucial structural link between the invariant CD3 subunits and antigen-recognizing alpha/beta chains during TCR signaling. However, functional analysis does not support the above notion, since transgene coding for TCR beta chain lacking the complete FG loop supports normal alpha/beta T cell development and function.     

Differential effects of the early and late intrauterine environment on corticotrophic cell development

The developing embryo and fetus respond to a range of intrauterine stressors, but the effect of chronic intrauterine stress on the programmed development of pituitary corticotrophs has not been investigated. We have used a pregnant sheep model in which the embryonic environment at conception has been surgically perturbed by uterine carunclectomy. This procedure results in the development of fetuses that either are placentally restricted and chronically hypoxemic or that demonstrate compensatory placental growth and maintain normoxemia throughout late gestation. We found that uterine carunclectomy resulted in the emergence of a population of non-corticotrophin-releasing hormone (non-CRH) target cells that secreted high amounts of adrenocorticotrophic hormone (ACTH) in the fetal pituitary. This change in corticotroph development was independent of late-gestation hypoxemia. However, chronic hypoxemia during late gestation (in either carunclectomized or non-carunclectomized uterine environments) resulted in a reduction in the proportion of ACTH stored in CRH-target. Thus, the early and late intrauterine environments differentially program the development of specific corticotrophic cell types in the fetal pituitary. These patterns of altered corticotroph development are important given the central roles of the hypothalamo-pituitary-adrenal axis in the fetal adaptive response to intrauterine stress and in the early programming of adult disease.     

Differential effects of growth hormone and prolactin on murine T cell development and function

We have previously shown that major heat-shock protein (hsp 70) protects WEHI-S tumor cells from cytotoxicity mediated by tumor necrosis factor alpha (TNF-alpha) and TNF-beta. In the present study, the effect of altered expression of hsp70 and low molecular weight heat- shock protein, hsp27, on tumor cell sensitivity to monocytes and lymphokine-activated killer (LAK) cells was studied. Constitutive and stable expression of transfected human hsp70 rendered cells almost completely resistant to monocytes. Conversely, inhibition of endogenous hsp70 by expression of antisense hsp70 RNA enhanced the sensitivity of cells to monocyte-mediated killing. Surprisingly, overexpression of human hsp27, which does not protect WEHI-S cells from TNF killing, conferred partial resistance to monocytes. Only approximately 60% of monocyte-mediated killing of WEHI-S cells could be blocked by neutralizing TNF-alpha antibody or immunoglobulin G-TNF receptor chimeric protein, suggesting the presence of both TNF-dependent and TNF- independent lytic mechanisms. As free radicals have been suggested to be mediators of monocyte cytotoxicity, we tested the sensitivity of transfected cells to oxidative stress. Overexpression of either hsp70 or hsp27 rendered cells partially resistant to hydrogen peroxide. No significant changes in the susceptibility of cell lines overexpressing hsp70 or hsp27 to cytotoxicity mediated by LAK cells were observed. Interestingly, monocytes but not LAK cells contained detectable levels of hsp27 and hsp70 in nonstressed conditions. Taken together, these data indicate that hsp70 protects tumor cells from TNF-mediated monocyte cytotoxicity and that both hsp27 and hsp70 confer resistance to TNF-independent, probably free radical-mediated lysis by monocytes. Moreover, hsp27 and hsp70 may provide monocytes with a protective mechanism against their own toxicity.     

Expression of two distinct T cell receptor alpha/beta heterodimers by an antigen-specific T cell clone

A cytochrome c-specific, MHC-restricted T cell clone with two complete rearrangements of the same V beta 1 gene element was shown to express two different TCR alpha/beta heterodimers. Antipeptide antisera specific for TCR C beta 1 and C beta 2 peptides each immunoprecipitated distinct disulfide-linked cell surface heterodimers. The clone was derived from immunized allogeneic chimeric mice, and displayed multiple Ia specificities, including the ability to recognize antigen in association with both I-Ek and I-Es Ia molecules, as well as alloreactivity to the I-Eb molecule. It will be important to determine whether each receptor contributes independently to the overall specificity of the clone.     

Human epidermal T cells predominantly belong to the lineage expressing alpha/beta T cell receptor

The epidermis of clinically normal-appearing human skin harbors a phenotypically heterogeneous population of T lymphocytes (TCs), the majority of which are CD2+/CD3+/CD5+ "memory" cells, but in an unactivated state, and express the TCR-alpha/beta. In contrast to murine skin, only a very minor subpopulation of CD3+ cells in the human epidermis bears the TCR-gamma/delta. Epidermal TCs primarily are distributed along the rete ridges in the basal keratinocyte layer and are often in close apposition to Langerhans cells (LCs). These TCs were propagated from epidermal cell suspensions after stimulation with TC activating agents (Con A, rIL-1, rIL-2), then evaluated for phenotypic features and TCR diversity. Similar to the in situ situation, most were CD4-/CD8+/TCR-alpha/beta+. In addition, two cultures contained TCR- gamma/delta+ cells; one of these determined to be an adherent CD4-/CD8+ population. Epidermal TCs were significantly (p less than 0.0001) more abundant in the sole than in the other body regions examined (i.e., 40 vs. 7 CD3+ cells/linear centimeter of epidermis) and seemed to have a particular affinity for the acrosyringial epithelium of eccrine sweat ducts. Moreover, the sole usually contained a greater number of CD8+ relative to CD4+ TCs, whereas the epidermal CD4/CD8 ratio in the trunk and extremities was quite variable, although the trend also was towards a slightly larger percentage of CD8+ cells. Collectively, our data suggest that the volar epidermis has a unique microenvironment which is responsible for both the higher density of TCs, preferentially CD8+, and lower number of LCs. This study has not only provided evidence for significant regional variability in the human epidermal TC population of normal skin, but also strengthens the concept for skin-associated lymphoid tissues (SALT), whereby memory TCs recirculate back to the epidermis and interact with resident antigen-presenting cells (i.e., LC).     

The V beta repertoire of mouse gut homodimeric alpha CD8+ intraepithelial T cell receptor alpha/beta + lymphocytes reveals a major extrathymic pathway of T cell differentiation

Gut intraepithelial lymphocytes (IEL) contain two independent T cell receptor alpha/beta + T cell populations, with different V beta repertoires. In DBA/2 mice (Mlsa, IE+), the CD4+ and heterodimeric alpha/beta CD8+ thymodependent T cell pool shows the same deletion of V beta 6, 8.1, and 11+ cells as found in peripheral lymphoid organs. In contrast, such deletions are not observed in the pool of IEL bearing homodimeric alpha CD8+ chains, in which these V beta families are frequently observed in high amounts. The size of this gut homodimeric alpha CD8+ IEL pool and its different V beta repertoire selection demonstrate the existence of a major extrathymic pathway of T cell differentiation with a gut-restricted localization. The large amount of the thymo-independent, homodimeric alpha CD8+ IEL found in the small bowel may contribute to a first line of defense against exogenous superantigens.     

Qa-2-dependent selection of CD8alpha/alpha T cell receptor alpha/beta(+) cells in murine intestinal intraepithelial lymphocytes

Murine intestinal intraepithelial lymphocytes (iIELs) are made up of a heterogeneous mix of T cells with unique phenotypes. Whereas CD8(+) T cells in peripheral lymphoid organs use CD8alpha/beta and are selected on MHC class Ia molecules, a majority of iIELs use CD8alpha/alpha. Here, we report that the presence of CD8alpha/alpha TCR-alpha/beta cells in iIELs is independent of classical MHC class I molecules K(b) and D(b), as illustrated by their presence in K(b)/D(b) double-knockout mice and in mice lacking a nonclassical MHC class I molecule, CD1d. Most strikingly, their presence is decreased by approximately 70% in mice lacking transporter associated with antigen processing (TAP). The TAP-dependent nonclassical MHC class I molecule Qa-2 is strongly implicated in the presence of these cells, as inferred from the low numbers of CD8alpha/alpha TCR-alpha/beta T cells in mice deficient in Qa-2 genes. Second, a Qa-2-transgenic mouse made in a Qa-2(-) strain showed an increase in the numbers of CD8alpha/alpha cells among its iIELs. Thus, the presence of CD8alpha/alpha TCR-alpha/beta cells in iIELs is mainly dependent on the nonclassical MHC class I molecule Qa-2.     

An invariant T cell receptor alpha chain defines a novel TAP-independent major histocompatibility complex class Ib-restricted alpha/beta T cell subpopulation in mammals.

We describe here a new subset of T cells, found in humans, mice, and cattle. These cells bear a canonical T cell receptor (TCR) alpha chain containing hAV7S2 and AJ33 in humans and the homologous AV19-AJ33 in mice and cattle with a CDR3 of constant length. These T cells are CD4(-)CD8(-) double-negative (DN) T cells in the three species and also CD8alphaalpha in humans. In humans, their frequency was approximately 1/10 in DN, 1/50 in CD8alpha+, and 1/6,000 in CD4(+) lymphocytes, and they display an activated/memory phenotype (CD45RAloCD45RO+). They preferentially use hBV2S1 and hBV13 segments and have an oligoclonal Vbeta repertoire suggesting peripheral expansions. These cells were present in major histocompatibility complex (MHC) class II- and transporter associated with antigen processing (TAP)-deficient humans and mice and also in classical MHC class I- and CD1-deficient mice but were absent from beta2-microglobulin-deficient mice, indicating their probable selection by a nonclassical MHC class Ib molecule distinct from CD1. The conservation between mammalian species, the abundance, and the unique selection pattern suggest an important role for cells using this novel canonical TCR alpha chain.     

Most alpha/beta T cell receptor diversity is due to terminal deoxynucleotidyl transferase.

The contribution of template-independent nucleotide addition to antigen receptor diversity is unknown. We therefore determined the size of the T cell receptor (TCR)alpha/beta repertoire in mice bearing a null mutation on both alleles of the terminal deoxynucleotidyl transferase (Tdt) gene. We used a method based upon polymerase chain reaction amplification and exhaustive sequencing of various AV-AJ and BV-BJ combinations. In both wild-type and Tdt degrees / degrees mice, TCRAV diversity is one order of magnitude lower than the TCRBV diversity. In Tdt degrees / degrees animals, TCRBV chain diversity is reduced 10-fold compared with wild-type mice. In addition, in Tdt degrees / degrees mice, one BV chain can associate with three to four AV chains as in wild-type mice. The alpha/beta repertoire size in Tdt degrees / degrees mice is estimated to be 10(5) distinct receptors, approximately 5-10% of that calculated for wild-type mice. Thus, while Tdt activity is not involved in the combinatorial diversity resulting from alpha/beta pairing, it contributes to at least 90% of TCRalpha/beta diversity.     

Depletion of alpha/beta T cells by a monoclonal antibody against the alpha/beta T cell receptor suppresses established adjuvant arthritis, but not established collagen-induced arthritis in rats

The effects of treatment with a monoclonal antibody (R73 mAb) against T cell receptor alpha/beta (TCR-alpha/beta) on both established adjuvant arthritis (EAA) and established collagen-induced arthritis (ECIA) in rats have been investigated. Rats were treated with R73 mAb when arthritis reached a peak. Treatment with the anti-TCR-alpha/beta mAb markedly suppressed EAA, whereas ECIA was not affected by the mAb treatment. Histologically, R73 mAb-treated rats with EAA showed mild hyperplasia of synovial tissues, sparse infiltration of inflammatory cells, and minimal erosion of cartilage, whereas arthritic rats treated with PBS and an irrelevant control mAb against Giardia had marked hyperplasia of synovium with pannus, massive inflammatory cell infiltrate, and severe destruction of cartilage and subchondral bone. R73 mAb-treated rats with ECIA exhibited pronounced formation of pannus containing many inflammatory cells and marked cartilage and subchondral damage similar to those in arthritic rats that received the control treatments. Treatment with R73 mAb depleted markedly alpha/beta+ T cells in both peripheral blood and synovial tissues of rats with EAA and ECIA. R73 mAb treatment was associated with marked reduction in arthritogen-specific delayed-type hypersensitivity responses in both EAA and ECIA. The titers of antibodies against type II collagen produced in rats with ECIA were not affected by the mAb. Thus, alpha/beta+ T cells appear to have a central role in EAA, but not in chronic ECIA.     

Inhibition of cytotoxic T cell development by transforming growth factor beta and reversal by recombinant tumor necrosis factor alpha

The immunoregulatory effects of transforming growth factor beta (TGF-beta) and recombinant murine tumor necrosis factor alpha (rMuTNF-alpha) on CTL generation and activity were examined. The results demonstrate that TGF-beta, in a dose-dependent manner, inhibited CTL generation but not CTL activity. The inhibitory effects were detected only when TGF-beta was added within the first 48 h of the MLC. Little activity was seen when it was added thereafter, including the addition of TGF-beta to the cytotoxicity assay. The production of TNF-alpha, which occurs during early phases of the MLC and which is inhibited in the presence of TGF-beta, appears to have an important regulatory role, as altering the levels of TNF-alpha in an MLC can significantly influence CTL development. The inhibitory effects of TGF-beta on the MLC can be significantly reversed by the addition of rMuTNF-alpha to the cultures. These results demonstrate that TGF-beta can inhibit MLC and subsequent CTL generation at early stages of the reaction, and such inhibition may involve the suppression of TNF-alpha production.     

Differential effects of three antibiotics on T helper cell cytokine expression

OBJECTIVES: To test whether ciprofloxacin, moxifloxacin and clarithromycin affect the expression of the T helper (Th) cell cytokines, interferon-gamma and interleukin-4. Quinolone and macrolide antibiotics are routinely used for the treatment of critically ill patients with sepsis. These antibiotics modulate some aspects of immune cell function. Alteration in the profile of Th cell cytokine expression will affect the T helper cell ratio and subsequent immune responses. METHODS: Following ethics committee approval and informed consent, mononuclear cells were isolated from 20 healthy volunteers using single density gradient centrifugation. Cells were incubated with ciprofloxacin (0--100 mg/L), moxifloxacin (0--50 mg/L) or clarithromycin (0--125 mg/L) and stimulated with phorbol myristate acetate and ionomycin. For flow cytometric analysis, cells were stained with antibodies to CD3 and CD4, prior to permeabilization with saponin and intracellular staining with anti-interleukin-4 and anti-interferon-gamma. RESULTS: Both moxifloxacin and ciprofloxacin dose-dependently decreased interferon-gamma and interleukin-4 expression by Th cells (both P<0.0001). Only interleukin-4 expression however, was affected by clarithromycin (P=0.04). There was no change in the Th1/Th2 ratio for moxifloxacin or ciprofloxacin, but the Th1/Th2 ratio increased with increasing concentrations of clarithromycin, from a median [range] of 5.3 [1.3--16.0] without antibiotic to 9.1 [1.8--18.8] at 125 mg/L (P=0.017). CONCLUSIONS: Moxifloxacin and ciprofloxacin had pronounced effects on both Th1 and Th2 cytokine expression, without altering Th1/Th2 ratios. However, clarithromycin decreased only interleukin-4 expression such that the Th1/Th2 ratio increased. Since a Th1 profile is considered favourable for resolution of infection, elucidation of immunomodulatory profiles of antibiotics may permit more rational antibiotic choice in future.     

Developmental arrest of NK1.1+ T cell antigen receptor (TCR)- alpha/beta+ T cells and expansion of NK1.1+ TCR-gamma/delta+ T cell development in CD3 zeta-deficient mice

The relationship between the structure of the T cell antigen receptor (TCR)-CD3 complex and development of NK1.1+ T cells was investigated. The TCR complex of freshly isolated NK1.1+ TCR-alpha/beta+ thymocytes contained CD3 zeta homodimers and CD zeta-FcR gamma heterodimers, whereas that of the majority of NK1.1- T cells did not contain FcR gamma. The function of CD3 zeta and FcR gamma in the development of NK1.1+ T cells was determined by analyzing CD3 zeta- and FcR gamma- deficient mice. The NK1.1+ T cells from wild-type and CD3 zeta- deficient mice had equal levels of CD3 expression. However, the development of NK1.1+ TCR-alpha/beta+ T cells was almost completely disrupted in thymus and spleen in CD3 zeta-deficient mice, whereas no alteration was observed in FcR gamma-deficient mice. In contrast, the number of novel NK1.1+ TCR-gamma/delta+ thymocytes expressing a surface phenotype similar to NK1.1+ TCR-alpha/beta+ thymocytes increased approximately six times in CD3 zeta-deficient mice. These findings establish the distinct roles of the CD3 zeta chain in the development of the following different thymic T cell compartments: NK1.1- TCR+, NK1.1+ TCR-alpha/beta+, and NK1.1+ TCR-gamma/delta+ thymocytes, which cannot be replaced by CD3 eta or FcR gamma chains.     

Thymus-independent development and negative selection of T cells expressing T cell receptor alpha/beta in the intestinal epithelium: evidence for distinct circulation patterns of gut- and thymus-derived T lymphocytes.

We demonstrate that mouse intestinal intraepithelial lymphocytes (IEL) can be divided into subsets based on the differential expression of functional T cell receptor alpha/beta (TCR-alpha/beta) signaling complexes. Two subsets, CD4+ 8 alpha + beta - and CD8 alpha + beta -, are refractory to stimulation with anti-TCR-alpha/beta and contain high frequencies of potentially self-reactive cells. In contrast, the CD4+ and CD8 alpha + beta + IEL subsets are responsive to anti-TCR-alpha/beta and depleted of potentially self-reactive cells. The analysis of fetal liver radiation chimeras using adult thymectomized recipients demonstrates that the four TCR-alpha/beta + IEL subsets are generated in normal numbers in the absence of the thymus. Moreover, expression of the major histocompatibility complex class II-encoded I-E molecule and Mls1a in the gut of the athymic host results in the negative selection of potentially self-reactive T cells expressing V beta 11 and V beta 6, respectively, from those IEL subsets that express functional TCR-alpha/beta signaling complexes. Neither the spleen nor the Peyer's patches of athymic recipients contain T cells of donor origin. In contrast, normal numbers of phenotypically and functionally mature CD4+ and CD8 alpha + beta + T cells of donor origin are found in the lamina propria of chimeric animals. The phenotypic analysis of lymphocytes obtained from Ly5 congenic parabionts reveals that peripheral T cells migrate rapidly to the Peyer's patches and lamina propria, but not to the intestinal epithelium. Taken together, these results demonstrate that the intestinal epithelium is a thymus-independent site of T lymphopoiesis, where selection of the T cell repertoire involves the deletion of potentially self-reactive cells in situ. Moreover, the appearance of donor-derived, phenotypically mature T cells, exclusively in the lamina propria of athymic radiation chimeras, suggests that mature IEL expressing functional TCR-alpha/beta migrate to this site.     

Retroviral transformation in vitro of chicken T cells expressing either alpha/beta or gamma/delta T cell receptors by reticuloendotheliosis virus strain T

Exposure of normal juvenile chicken bone marrow cells to the replication defective avian reticuloendotheliosis virus strain T (REV- T) (chicken syncytial virus [CSV]) in vitro resulted in the generation of transformed cell lines containing T cells. The transformed T cells derived from bone marrow included cells expressing either alpha/beta or gamma/delta T cell receptors (TCRs) in proportions roughly equivalent to the proportions of TCR-alpha/beta and TCR-gamma/delta T cells found in the normal bone marrow in vivo. Essentially all TCR-alpha/beta- expressing transformed bone marrow-derived T cells expressed CD8, whereas few, if any, expressed CD4. In contrast, among TCR-gamma/delta T cells, both CD8+ and CD8- cells were derived, all of which were CD4-. Exposure of ex vivo spleen cells to REV-T(CSV) yielded transformed polyclonal cell lines containing > 99% B cells. However, REV-T(CSV) infection of mitogen-activated spleen cells in vitro resulted in transformed populations containing predominantly T cells. This may be explained at least in part by in vitro activation resulting in dramatically increased levels of T cell REV-T(CSV) receptor expression. In contrast to REV-T(CSV)-transformed lines derived from normal bone marrow, transformed lines derived from activated spleen cells contained substantial numbers of CD4+ cells, all of which expressed TCR- alpha/beta. While transformed T cells derived from bone marrow were stable for extended periods of in vitro culture and were cloned from single cells, transformed T cells from activated spleen were not stable and could not be cloned. We have therefore dissociated the initial transformation of T cells with REV-T(CSV) from the requirements for long-term growth. These results provide the first demonstration of efficient in vitro transformation of chicken T lineage cells by REV- T(CSV). Since productive infection with REV-T(CSV) is not sufficient to promote long-term growth of transformed cells, these results further suggest that immortalization depends not only upon expression of the v- rel oncogene but also on intracellular factor(s) whose expression varies according to the state of T cell physiology and/or activation.     

Involvement of the interleukin 2 pathway in the rearrangement and expression of both alpha/beta and gamma/delta T cell receptor genes in human T cell precursors

In this report, we have undertaken the phenotypic, functional and molecular characterization of a minor (less than 5%) subpopulation of adult thymocytes regarded as the earliest intrathymic T-cell precursors. Pro-T cells were immunoselected and shown to express different hematopoietic cell markers (CD45, CD38, CD7, CD5) and some activation-related molecules (4F2, Tr, HLA class II), but lack conventional T cell antigens (CD2-1-3-4-8-). TCR-gamma RNA messages are already expressed at this early ontogenic stage, while alpha and beta chain TCR genes remain in germline configuration. In vitro analyses of the growth requirements of pro-T cells demonstrated the involvement of the IL-2 pathway in promoting their proliferation and differentiation into CD3+ CD4+ or CD8+ mature thymocytes. Moreover, during the IL-2-mediated maturation process rearrangements and expression of both alpha and beta chain TCR genes occurred, and resulted in the acquisition of alpha/beta as well as gamma/delta (either disulphide-linked or non-disulphide-linked) heterodimeric TCR among the pro-T cell progeny.     

Distinctive selection mechanisms govern the T cell receptor repertoire of peripheral CD4-CD8- alpha/beta T cells

The T cell receptor (TCR) repertoire of CD4+ and CD8+ alpha/beta T cells is heavily influenced by positive and negative selection events that occur during T cell development in the thymus. The coreceptors CD4 and CD8 appear to be essential for this selection to occur. To gain insight into whether T cells that express TCR alpha/beta but lack either coreceptor (CD4- CD8- TCR alpha/beta or alpha/beta double-negative [DN] cells) are also subject to positive and negative selection, and whether selection can occur in the absence of coreceptors, we have performed an extensive immunogenetic analysis of the TCR V beta repertoire of alpha/beta DN cells in lymph nodes of normal mice. Our results show that alpha/beta DN cells appear to be unaffected by clonal deletion of V beta 5 and V beta 11 in I-E-expressing mice, and do not undergo deletion of V beta 6- and V beta 8.1-expressing T cells in Mls-1a-positive mice. They are also unaffected by positive selection of V beta 17a+ T cells in the context of I-Aq. The results suggest that most selection events require the participation of CD4 and CD8, while alpha/beta DN cells are unselected. This argues that most alpha/beta DN cells probably have never expressed CD4 or CD8. However, a unique form of repertoire selection occurs: enrichment of V beta 17a+ alpha/beta DN cells in I-E+ mice. This could be an instance of coreceptor-independent selection.     

Requirement for tyrosine residues 315 and 319 within zeta chain-associated protein 70 for T cell development

Engagement of the T cell antigen receptor (TCR) induces the transphosphorylation of the zeta chain-associated protein of 70,000 Mr (ZAP-70) protein tyrosine kinase (PTK) by the CD4/8 coreceptor associated Lck PTK. Phosphorylation of Tyr 493 within ZAP-70's activation loop results in the enzymatic activation of ZAP-70. Additional tyrosines (Tyrs) within ZAP-70 are phosphorylated that play both positive and negative regulatory roles in TCR function. Phosphorylation of Tyr residues (Tyrs 315 and 319) within the Interdomain B region of the ZAP-70 PTK plays important roles in the generation of second messengers after TCR engagement. Here, we demonstrate that phosphorylation of these two Tyr residues also play important roles in mediating the positive and negative selection of T cells in the thymus.