CD8 Alpha Beta Research Articles

Spontaneous generation of human CD8+ TCRαβ+ cells derived from precursors within the double negative compartment

Flow cytometric analysis demonstrated that fresh human thymocytes contain only a low level of mature CD8+ TCR alpha beta + or CD8+ TCR gamma delta + cells and they consist consist of approximately 70% double positive (DP) and approximately 10% double negative (DN) cells. These unfractionated thymocytes could be selectively expanded in vitro by stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) and PHA in the presence of IL-2. The majority of the cells expanded from unfractionated thymocytes expressed CD3, TCR alpha beta and CD8 molecules after long-term culture (18 days). When highly purified DN thymocytes were expanded over a period of 18 days in the presence of DP cells, they also co-expressed CD3, TCR alpha beta and CD8 molecules on their surface. However, when purified DN thymocytes were expanded alone, that is, in the absence of DP cells for 18 days, they expressed CD3-associated TCR gamma delta, but not CD8 or TCR alpha beta. Despite the expression of measurable levels of IL-2 alpha and beta receptors, as well as a significant level of TCR alpha beta, purified DP cells failed to proliferate. These findings provide the first evidence, in humans, that the progression of precursor cells in the DN compartment to a later stage of differentiation can be induced outside the thymus and that DP cells can affect the development of TCR expression in proliferating DN thymocytes.

Regional specialization of intraepithelial T cells in the murine small and large intestine.

We investigated intraepithelial T cells from the small intestine, SI (jejunum, ileum) and the large intestine, LI (colon) of euthymic (BALB/c, H-2d; C.B-17+/+, H-2d; C57BL/6, H-2b) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/xid) mice. From individual euthymic and athymic mice, 7 x 10(6) intraepithelial lymphocytes (IEL) per mouse were isolated from the SI. Ten-fold lower numbers of IEL were obtained from the LI epithelium (4 x 10(5) IEL per mouse). Thymus-dependent and -independent T cells represented > 80% of SI-IEL but the fraction of T cells was reduced from 20% to 40% in LI-IEL. In euthymic mice, alpha beta T cells predominated in SI-IEL and in particular in LI-IEL populations, while SI-IEL and LI-IEL populations of athymic mice contained predominantly gamma delta T cells. The intraepithelial T cell subset distribution was different in SI versus LI: mainly CD8+ T cells were present in the SI, but a large CD4+ T cell subset was present in the LI. 'Double positive' CD4+ CD8 alpha+ T cells were present mainly in the SI epithelium but were rare in the LI epithelium. In euthymic as well as athymic mice, T cells expressing the homodimeric CD8 alpha alpha isoform predominated in the SI epithelium, while T cells expressing the heterodimeric CD8 alpha beta isoform predominated in the LI epithelium. LI-derived TCR alpha beta+ IEL displayed the CD2+ CD28+ LPAM-1/2- M290+ phenotype, and a fraction of them expressed the L-selectin LECAM-1. In contrast, a large fraction of TCR alpha beta+ SI-IEL was CD2- CD28- LPAM-1/2- M290+ and LECAM-1-. RAG-1/2 expression was detectable by RT-PCR in IEL from the SI but not the LI. Striking differences in phenotype were thus apparent between thymus-dependent and thymus-independent T cells in the epithelial layer of the jejunum/ileum and the colon of the mouse.

Composition of TCR-CD3 complex in human intestinal intraepithelial lymphocytes: lack of FcεRlγ chain

Human intestinal intraepithelial lymphocytes (iIEL) are a unique population of predominantly CD8 alpha beta+, TCR alpha beta+ lymphocytes and, to a lesser extent, TCP gamma delta+ lymphocytes that proliferate poorly to anti-CD3 mitogenic signals but display significant cytolytic activity. Studies in mouse model systems have shown that the gamma chain of the high-affinity receptor for IgE (Fc epsilon RI gamma) may substitute for the zeta chain in the TCR-CD3 complex of iIEL. This has suggested that the functional properties of these cells may be associated with an altered composition of the TCR-CD3 complex. We therefore analyzed the TCR-CD3 complex of normal human iIEL. One- and two-dimensional non-reducing/reducing SDS-PAGE analysis of CD3 gamma, CD3 delta, CD3 epsilon, zeta and Fc epsilon RI gamma chain immunoprecipitates of cell surface radiolabeled proteins with subunit-specific antibodies revealed a TCR-CD3 complex without associated Fc epsilon RI gamma chains. Thus, normal human iIEL contain a TCR-CD3 complex that consists predominantly of zeta homodimers in association with the alpha beta TCR and CD3 gamma, delta and epsilon, similar to the majority of peripheral lymphocytes. This indicates that the distinct properties of human iIEL are not associated with substitutions of the Fc epsilon RI gamma chain in the TCR-CD3 complex.

T Cell Development in CD3-ζ Mutant Mice

Increasing evidence points to multiple pathways of T lymphocyte development. The well characterized thymus-dependent pathway gives rise to T cells bearing TCR alpha beta heterodimers and either CD4 or CD8 alpha beta co-receptors. T cells of this lineage populate peripheral lymphoid compartments including lymph nodes, spleen, skin, and Peyer's patches. By comparison, factors which govern extrathymic T cell development are poorly understood. A variety of experiments have shown that intestinal intraepithelial lymphocytes (IELs) develop outside of the thymic environment, e.g., in the gut of nude, SCID, and beta 2m-/- mutant mice, and after transplanting bone marrow or fetal liver cells into irradiated thymectomized adult mice. This review focuses on the role of the CD3-zeta subunit in the development of both thymically and extrathymically derived T cells as determined by gene-targeting experiments in mice. Data from these and other T cell-related mutations continue to define crucial stages in thymocyte differentiation. Most interestingly, CD3-zeta mutant mice contain a unique population of intestinal IELs that develops independently of thymic selective processes and expresses a novel TCR/CD3 complex.

Cellular and molecular aspects of organotin-induced thymus atrophy.

1. Organotin compounds, di-n-butyltin dichloride (DBTC) in particular, have been shown to cause thymus atrophy in the rat. 2. DBTC-induced thymus atrophy results from a depletion of small CD4+CD8+ thymocytes which is caused by a diminished production of immature CD4-CD8+ and CD4+CD8+ thymoblasts. 3. DBTC inhibits the activation, but not the differentiation of immature CD4-CD8+ thymocytes in vitro and in vivo suggesting a selective antiproliferative activity of DBTC. 4. DBTC inhibits the adhesion molecule-mediated binding of thymocytes to thymic epithelial cells. 5. DBTC enhances the Ca2+ release elicited by cross-linking of the T cell receptor complex (TcR alpha beta-CD3) on thymocytes and moreover delays cap formation of the TcR alpha beta-CD3 receptor. 6. It is concluded that DBTC possibly interferes with the functioning of the cytoskeleton. The relation of the in vitro findings to the inhibition of immature CD4-CD8+ thymocyte activation and the induction of thymus atrophy is unknown as yet.

Oligoclonal repertoire of the CD8 alpha alpha and the CD8 alpha beta TCR-alpha/beta murine intestinal intraepithelial T lymphocytes: evidence for the random emergence of T cells.

The epithelium of the small intestine in normal euthymic mice contains a large number of intraepithelial lymphocytes (IEL), some of which bear a T cell receptor alpha/beta (TCR-alpha/beta). About half of these TCR-alpha/beta IEL display the CD8 alpha alpha phenotype and the remaining have the CD8 alpha beta or the CD4 phenotypes. To examine whether TCR-alpha/beta IEL have a TCR-beta chain repertoire as diverse as that of TCR-alpha/beta lymph node lymphocytes (LNL), we used a recently described PCR technique that allows a global analysis of the TCR-beta chain repertoire. Within any given mouse, the repertoires expressed in both CD8 alpha alpha and CD8 alpha beta TCR-alpha/beta IEL populations are oligoclonal and nonoverlapping between the two subsets. The clones are largely conserved through the length of the small intestine of the same individual. However, genetically identical individuals raised under indistinguishable environmental conditions display distinct oligoclonal repertoires. Those findings indicate that few cells of CD8 alpha alpha or of the CD8 alpha beta phenotype are responsible for the repopulation of the intestinal epithelium.

Studies with MHC-deficient knock-out mice reveal impact of both MHC I- and MHC II-dependent T cell responses on Listeria monocytogenes infection.

Mutant mice with a defined genetic defect in the beta 2-microglobulin (beta 2m) or the H2-I-A beta chain, which are virtually devoid of functional CD8 or CD4 alpha beta T cells, respectively, were employed for analyzing immune mechanisms involved in acquired resistance against Listeria monocytogenes. Although the lethal dose of L. monocytogenes was markedly lower for either mouse mutant as compared with their heterozygous control littermates, both beta m -/- and A beta -/- mutants were able to resolve low dose infection. However, in both mouse mutants, the course of disease was exacerbated and clearance was markedly delayed. Vaccine induced immunity against a secondary high dose infection lethal for naive animals was also impaired in beta 2m -/- and A beta -/- mice. However, both mutant mice were still capable of controlling secondary infection. Based on numbers of L. monocytogenes organisms in spleens, beta 2m -/- mutants suffered more dramatically from primary and secondary infection than A beta -/- mice. Ag-induced IFN-gamma secretion was impaired during the early phase of infection in beta 2m -/- mice and at later stages in A beta -/- mice. Modulation of gamma delta T cells by mAb treatment led to significant increase in bacterial load of spleens in both beta 2m -/- and A beta -/- mice. Finally, the development of granulomatous lesions was markedly affected in both mutants. In beta 2m -/- mutants, infiltrative lesions appeared and in A beta -/- mice few inflammatory islets with necrotic centers developed. These data demonstrate the importance of both MHC I- and MHC II-dependent immune mechanisms in acquired resistance to L. monocytogenes and point to the necessity of a coordinated interaction between CD8 and CD4 alpha beta T cells (and probably gamma delta T cells) in anti-L. monocytogenes resistance.

Role of coreceptors in positive selection and lineage commitment.

Recent experiments have re-awakened interest in a stochastic/selective model of positive selection of T lymphocytes. A revised version of the model has been proposed whereby commitment of double-positive thymocytes to either the CD4 or CD8 lineage requires two engagements with MHC molecules: the first, initiating the differentiation program, signals down-regulation of one or the other coreceptor, regardless of the T cell receptor's specificity for MHC class I or II molecules; the second, leading to terminal differentiation, screens the choice of coreceptor by permitting only those cells with matched receptors and coreceptors to proceed. Here we explore the role of coreceptors in the two stages of positive selection by manipulating CD8 expression in MHC class II-deficient mice, crossing them with either CD8-negative animals or animals carrying combinations of CD8 alpha and CD8 beta transgenes. We find that coreceptors are required at both stages of positive selection and that artificial expression of the down-modulated CD8 molecule can quite efficiently rescue cells that have made a 'mistake' in their choice of coreceptor. We also establish that commitment to the CD4 pathway and to the helper phenotype can be linked.

Regulation of IgE responses to inhaled antigen in mice by antigen-specific gamma delta T cells.

Indirect evidence implicates gamma delta T cells in the cross-regulation of CD4 alpha beta T cell responses. Adoptive transfer of small numbers of gamma delta T cells from ovalbumin (OVA)-tolerant mice selectively suppressed TH2-dependent immunoglobulin E (IgE) antibody production without affecting parallel IgG responses. Challenge of these gamma delta T cells in vitro with specific antigen resulted in production of high levels of interferon gamma. The effects of the gamma delta T cells may be mediated by direct inhibition of OVA-specific CD4+ TH2 cell proliferation or selection for specific CD4 TH2 cells.

Phenotype and suppressor activity of T-lymphocyte clones extracted from lesions of oral lichen planus.

Lymphocytes were extracted from six biopsy specimens of oral lichen planus. T-lymphocyte lines were expanded in culture with phytohaemagglutinin and interleukin 2, and cloned by limiting dilution. Fifteen T-cell clones were isolated with a probability of clonality of 96.3%. The majority of clones (n = 13) expressed the alpha beta T-cell receptor, and of these, 11 were CD8+ and two were CD4+. Two clones were CD4- and CD8-, and expressed the gamma delta T-cell receptor. The ability of these clones (effectors) to suppress concanavalin-A-stimulated proliferation of autologous lesional T-cell lines (responders) was assessed. Maximum suppressor activity ranged from 17 to 100%. The majority of clones (n = 12), including a CD3+ CD4+ CD8-alpha beta+ clone, displayed suppressor activity which was proportional to the effector to responder ratio. A CD3+CD4+CD8-alpha beta+ clone and a CD3+CD4-CD8-gamma delta+ clone displayed substantial helper activity at higher effector to responder ratios. These results demonstrate differential helper and suppressor activity of T-lymphocyte clones extracted from oral lichen planus lesions. The balance between help and suppression may be a fundamental determinant of immunological activity within the lymphocytic infiltrate of oral lichen planus, and hence may dictate the clinical behaviour of the disease.

Development of intestinal intraepithelial T lymphocytes is independent of Peyer's patches and lymph nodes in aly mutant mice.

We have previously reported a new spontaneous recessive mutation that induces a generalized lack of lymph nodes (LNs) and Peyer's patches (PPs) accompanied by immunodeficiency in mice (gene symbol, aly). In this study, we have analyzed gut-associated lymphatic tissues of the mutant aly/aly mice and have compared the intestinal intraepithelial T lymphocytes (IEL) in aly/aly and normal aly/+ littermate mice. Immunohistochemical studies revealed that colonization of IEL and lamina propria T cells takes place in the absence of PPs and IgA-producing B cells in the lamina propria. Absolute numbers of Thy-1- IEL-alpha beta and -gamma delta are not altered in aly/aly mutant mice, whereas absolute numbers of Thy-1+ IEL-alpha beta and -gamma delta in aly/aly mice are about half of those in aly/+ mice. In IEL-alpha beta from aly/aly mice, the major CD8 alpha alpha+ and CD8 alpha beta+ subsets are maintained, whereas CD4+ and CD4+, CD8+ subsets are reduced. Although the population size of major CD8 alpha alpha+ and CD4-, CD8- IEL-gamma delta subsets is slightly reduced, the use of TCR-gamma- and -delta-chain variable gene segments by IEL-gamma delta remains almost the same in aly/aly mice. The constitutive cytolytic activity of IEL-alpha beta and -gamma delta is attenuated sharply in the aly/aly condition. This activity is, however, augmented significantly after in vitro stimulation with anti-CD3 mAb. These results indicate that most of the IEL subpopulations develop independently of passage through PPs and mesenteric LNs and that the aly mutation interrupts cytotoxic IEL development during relatively late differentiation steps that convert cytotoxic precursors to the constitutively cytolytic state.

Reduced thymic maturation but normal effector function of CD8+ T cells in CD8 beta gene-targeted mice.

CD8 is a cell surface glycoprotein on major histocompatibility complex class I-restricted T cells. Thymocytes and most peripheral T cells express CD8 as heterodimers of CD8 alpha and CD8 beta. The intestinal intraepithelial lymphocytes (IEL), which have been suggested to be generated extrathymically, express CD8 predominantly as homodimers of CD8 alpha. We have generated CD8 beta gene-targeted mice. CD8 alpha+ T cell population in the thymus and in most peripheral lymphoid organs was reduced to 20-30% of that in wild-type littermates. CD8 alpha expression on thymocytes and peripheral T cells also decreased to 44 and 53% of the normal levels, respectively. In contrast, neither the population size nor the CD8 alpha expression level of CD8 alpha+ IEL was reduced. This finding indicates that CD8 beta is important only for thymic-derived CD8+ T cells. The lack of CD8 beta reduces but does not completely abolish thymic maturation of CD8+ T cells. Our result also reveals the role of CD8 beta in regulating CD8 alpha expression on thymic derived T cells. Peripheral T cells in these mice were efficient in cytotoxic activity against lymphocytic choriomeningitis virus and vesicular stomatitis virus, suggesting that CD8 beta is not essential for the effector function of CD8+ T cells.

High frequency of CD8αα homodimer‐bearing T cells in human fetal intestine

Immunohistochemistry on frozen sections was used to identify CD8 alpha alpha cells and CD8 alpha beta cells in human intestine. As observed previously, CD8 alpha beta cells predominate (> 95%) in tonsil and post-natal intestine. However in human fetal intestine (16-24 weeks gestation), almost half the CD8+ cells in the lamina propria are CD8 alpha alpha, and many CD8 alpha alpha cells can be identified in the epithelium. In contrast, in the T cell zones of the Peyer's patches, CD8 alpha beta cells are dominant. The CD8 alpha alpha cells are virtually all alpha beta T cell receptor positive. By analogy with the murine system, these CD8 alpha alpha cells in the fetal gut may be directly derived from the marrow, undergoing thymus-independent differentiation in the gut mucosa.

Decreased CD8‐p561ck Activity in Peripheral Blood T‐Lymphocytes from Patients with Hereditary Haemochromatosis

Hereditary haemochromatosis (HH) is an autosomal recessive disease linked to certain MHC class-I specificities. The disease is characterized by increased iron absorption and, in some patients, abnormally low numbers of CD8+ T cells in the periphery. We were interested in whether CD4- and CD8-associated p56lck kinase activities were altered in patients with HH. In a study of 18 patients with HH (with and without low numbers of CD8+ cells), the level of autophosphorylation of the CD8-associated p56lck as well as its phosphotransferase activity, as determined by phosphorylation of an exogenous substrate, was significantly reduced by two- to three-fold relative to a control population of 23 healthy blood donors (P < 6 x 10(-7). CD8-p56lck activity was decreased in 16 out of 18 patients (ranging from 1.5- to 10-fold decrease). By contrast, the level of CD4-p56lck activity did not show an overall decrease relative to controls. In addition to an occasional decrease in the amount of CD8-associated lck, HH patient-derived T cells showed a consistent decrease in the relative CD8-p56lck specific activity. Immunofluorescence staining showed further that the difference could not be accounted by a discrepancy in the expression of CD8 alpha alpha or CD8 alpha beta complexes or MHC class I molecules. Decreased CD8-p56lck activity was seen both in patients undergoing intensive phlebotomy treatment and in patients in maintenance therapy (i.e. patients who had reached normal levels of iron stores), indicating that this abnormality does not appear to be corrected by iron depletion. To our knowledge, this is the first demonstration of an abnormality in a src-like receptor associated kinase in a human disease state linked to MHC class-I antigens.

Requirement for CD8 beta chain in positive selection of CD8-lineage T cells.

CD8 is either an alpha alpha homodimer or an alpha beta heterodimer, although most peripheral CD8-lineage T cells express only the CD8 alpha beta heterodimer. The physiological function of CD8 beta was elucidated with mice that were chimeric for the homozygous disruption of the CD8 beta gene. The CD8 beta-1- T cells developed normally to CD4+CD8+ stage, but did not efficiently differentiate further, which resulted in few peripheral CD8+ T cells. The number of peripheral CD8+ T cells was restored by transfer of an exogenous CD8 beta gene into CD8 beta-deficient T cells. Thus, CD8 beta is necessary for the maturation of CD8+ T cells.

Thymus-independent positive and negative selection of T cells expressing a major histocompatibility complex class I restricted transgenic T cell receptor alpha/beta in the intestinal epithelium.

We demonstrate that in the mouse intestinal epithelium the selection of T lymphocytes expressing a transgenic T cell receptor alpha/beta (TCR-alpha/beta) specific for male antigen (H-Y) in the context of H-2Db depends on the differential expression of H-Y and H-2Db in situ. In H-2Db transgenic males, there is no reduction in the number of intestinal intraepithelial lymphocytes (IEL), and the four main subsets of IEL expressing TCR-alpha/beta, defined by the differential expression of CD4, CD8 alpha, and CD8 beta, are present. Moreover, the level of expression of CD8 alpha and CD8 beta on CD8+ IEL subsets is unaltered. The frequency of CD8 alpha+ IEL expressing CD8 beta, in H-2Db male mice, however, is significantly decreased and these cells do not express the transgenic TCR. In contrast, virtually all CD8 alpha+beta- IEL in the same animals express the transgenic TCR. Still, these potentially autoreactive cells are refractile to H-Y/H-2Db stimulation in vitro. Both H-2Db and H-2Dd transgenic females contain high frequencies of cells expressing the transgenic TCR among CD8 alpha+beta- and CD8 alpha+beta+ IEL. However, two possibly related phenotypic features are peculiar to H-2Db female mice. The frequency of CD8 alpha+ IEL expressing CD8 beta is increased in these mice and, while in H-2Dd females the level of the transgenic TCR alpha chain expressed on CD8 alpha+beta+ IEL is uniformly low, some of the CD8 alpha+beta+ IEL in H-2Db females express a high level of both transgenic TCR chains. It is important to note, the ability of CD8 alpha+beta+ IEL to respond to H-Y/H-2Db stimulation in vitro is restricted to those coexpressing a high level of both transgenic TCR chains. The analysis of athymic radiation chimeras using adult thymectomized recipients of distinct H-Y/H-2 haplotypes, reconstituted with bone marrow from H-2Db transgenic females, demonstrates that all IEL subsets present in unmanipulated transgenic animals develop in the absence of a thymus. These IEL are phenotypically identical to those found in unmanipulated transgenic animals sharing the H-Y/H-2 haplotype of athymic recipients. Taken together, these results demonstrate that in the absence of male antigen, expression of H-2Db in the intestinal epithelium results in the positive selection of functional IEL specific for male antigen, in situ. When both H-Y and H-2Db are expressed in the intestinal epithelium, CD8 alpha+beta+ IEL expressing the transgenic TCR are negatively selected, while the frequency of nonfunctional CD8 alpha+beta- IEL expressing the transgenic CR is increased.(ABSTRACT TRUNCATED AT 400 WORDS)

Role of transmembrane domains in assembly and intracellular transport of the CD8 molecule.

Previous studies have shown that CD8 can be present at the cell surface either as a disulfide-linked homodimer of CD8 alpha or as a disulfide-linked heterodimer of CD8 alpha and CD8 beta. Here we analyzed the assembly and intracellular transport of CD8 with particular emphasis on the role of the transmembrane domains. A chimeric protein (alpha T alpha) made by replacing the transmembrane domain of CD8 alpha with that of the interleukin-2 receptor alpha chain (Tac) exhibited reduced ability to form homodimers, while a mutant of Tac containing the CD8 alpha transmembrane domain (T alpha alpha) dimerized efficiently. Contrary to CD8 alpha, CD8 beta expressed alone was retained in the endoplasmic reticulum (ER). Only a small amount of CD8 beta formed homodimers, and these also remained in the ER. A mutant of CD8 beta that dimerized efficiently was also retained in the ER, thus proving that ER retention of CD8 beta is not due to its poor homodimerization. Rather, the extracellular domain of CD8 beta requires interaction with that of CD8 alpha to exit the ER. The transmembrane domain of CD8 beta was also shown to participate in ER retention by preventing exit of monomeric CD8 beta out of the ER. These findings demonstrate the role of transmembrane domains in assembly and intracellular transport of the CD8 molecule.

Restoration of early thymocyte differentiation in T-cell receptor beta-chain-deficient mutant mice by transmembrane signaling through CD3 epsilon.

Thymic repertoire selection requires the expression of the alpha beta CD3 T-cell receptor (TCR) together with the coreceptors CD4 and CD8. The appearance of CD4 and CD8 on thymocytes is the hallmark of a complex maturation step, accompanied by downregulation of the interleukin 2 receptor (IL-2R) alpha chain, arrest of rearrangement (i.e., allelic exclusion) of the TCR beta-chain locus, a burst of cell divisions, and reduction in cell size. This maturation step is inhibited in TCR beta-chain-deficient mouse strains and may depend on surface expression of an immature TCR complex containing CD3 and TCR beta chains but no TCR alpha chain. Here we show that the CD4+8+ double-positive (DP) stage can be induced by treatment of fetal thymic organ cultures with anti-CD3 epsilon monoclonal antibodies in several TCR beta-chain-deficient mouse strains: severe combined immunodeficient (scid) mice, mice carrying a mutation in the recombination activating gene 1 (Rag-1), or mice carrying a deletion in the TCR beta-chain locus itself. These findings suggest that CD3 epsilon is expressed on the thymocyte surface independent of and prior to the TCR beta chain. The data are consistent with the notion that in wild-type mice the DP stage is induced by transmembrane signaling through an immature CD3-TCR beta-chain complex, which can be bypassed by crosslinking of CD3 epsilon alone.

Cytolytic activity of intestinal intraepithelial lymphocytes in germ-free mice is strain dependent and determined by T cells expressing gamma delta T-cell antigen receptors.

We have compared the cytolytic activities and the cellular compositions of the intestinal intraepithelial lymphocyte (i-IEL) populations in three different combinations of conventional (CV) and germ-free (GF) mice. Cytolytic activity of i-IELs expressing gamma delta T-cell antigen receptors (TCRs) is strain dependent in CV mice (high vs. low), and this strain-dependent variability is unaltered in the GF condition. Although absolute numbers of gamma delta i-IELs are slightly decreased, the composition of CD8 alpha alpha+ and CD4-CD8- subsets and the usage of TCR gamma- and delta-chain variable gene segments by gamma delta i-IELs remain the same in GF mice. By contrast, cytolytic activity of alpha beta TCR-expressing i-IELs is uniformly high in CV mice but attenuated sharply in the GF condition. A conspicuous decrease in the total numbers of alpha beta i-IELs is also noted, and CD8 alpha beta+ and CD4+CD8+ subsets are reduced, whereas the CD8 alpha alpha+ subset is expanded in GF mice. These results indicate that microbial deprivation preferentially influences the alpha beta i-IEL population to decrease and become noncytolytic but has little effect on the pool size or characteristics of gamma delta i-IELs. Consequently, cytolytic activity of freshly isolated i-IELs from GF mice is determined by T cells expressing gamma delta TCRs and is found to be strain dependent.

Precursors of CD3+CD4+CD8+ cells in the human thymus are defined by expression of CD34. Delineation of early events in human thymic development.

Studies of the most immature T cell progenitors in the human thymus have been hampered by the lack of markers and assays that define these cells. In this report we used a novel human fetal thymic organ culture system to determine the potential of T cell precursors isolated from human postnatal thymus, to differentiate into CD3+ thymocytes, and to investigate early stages of human T cell development. It was found that thymocytes that lack the markers CD3, CD4, and CD8 (triple negative [TN]) can differentiate in an allogeneic organotypic thymic culture. The capacity of TN thymocytes to differentiate was exclusively confined to the CD34+ population. CD34- TN thymocytes failed to differentiate in this system. In contrast, cloned lines of CD3- thymocytes could only be established from CD34- TN thymocytes. Five subsets of CD3- thymocytes were found with the following phenotype: CD1-TN, CD1+TN, CD1+CD4+CD8-, CD1+CD4+CD8 alpha+ beta-, and CD1+CD4+CD8 alpha beta+. These subpopulations expressed decreasing levels of CD34. The CD1-CD3- population expressed the highest levels of CD34 supporting the notion that this population is the most immature T cell precursor in the thymus, whereas the CD1+CD4+CD8 alpha+ beta+ which did not express CD34 seems to be the most mature of these CD3- populations. This notion is supported by the observations that CD34+ cells isolated from fetal liver, which differentiated into T cells in a FTOC, developed into CD3+ cells via CD1- and CD4+CD8- intermediates. Based on these data, we present a model of early stages in human intrathymic development.