Delineation of chicken thymocytes by CD3-TCR complex, CD4 and CD8 antigen expression reveals phylogenically conserved and novel thymocyte subsets.

To further define the relationship between thymocyte subsets and their developmental sequence, multi-parameter flow cytometry was used to determine the distribution of the CD3-TCR complex and the accessory molecules CD4 and CD8 on chicken thymocytes. As in mammals, adult thymocytes could be subdivided into CD3-, CD3lo, and CD3hi staining populations. CD4 and CD8 distribution on such populations revealed the presence of CD3-CD4+CD8- and CD3-CD4-CD8+ thymocytes, putative precursors to CD4+CD8+ cells, detectable in the adult and at high frequency during ontogeny. Of particular interest was the existence of CD3lo expression on CD4+CD8- and CD4-CD8+, and in some instances, on CD4-CD8- thymocytes. Such phenotypes are not easily detectable in the mammalian thymus but were readily observed in both adult and embryonic chicken thymus from 16 days of embryogenesis. Further analysis of the TCR lineage of these CD3lo cells revealed that they were essentially all of the alpha beta TCR type. Mature CD3hi thymocytes were found within the CD4+CD8+ and CD4+CD8- and CD4-CD8+ subsets. Both alpha beta and gamma delta TCR lineage thymocytes were detected within all CD4- and CD8-defined subsets, thus identifying novel thymocyte subsets in the chicken thymus, namely alpha beta TCR+CD4-CD8- and gamma delta TCR+ CD4+CD8- cells. Hence, this analysis of chicken thymocytes, while confirming the phylogenically conserved nature of the thymus, has revealed novel T cell subsets, providing further insight into the complexity of mainstream thymocyte maturation pathways.     

Decreases in alpha beta T cell receptor negative T cells and CD8 cells, and an increase in CD4+ CD8+ cells in active Hashimoto''s disease and subacute thyroiditis.

We examined peripheral lymphocyte subsets in patients with autoimmune thyroid disease, or subacute thyroiditis, in the active stage when possible. During destructive thyrotoxicosis arising from alpha beta T cell receptor (TCR) negative T (WT31-CD3+) cells and CD8 (CD4-CD8+) cells decreased and those of CD4+CD8+ cells increased slightly, resulting in proportional increases in CD4 (CD4+CD8-) cells, non-T, non-B (CD5-CD19-) cells, and the CD4/CD8 cell ratio. Changes were similar in active subacute thyroiditis. During stimulative thyrotoxicosis in active Graves' disease, the numbers of such T lymphocyte subsets were not changed, but only the number of CD5+ B (CD5+CD19+) cells increased markedly, resulting in proportional decreases in total T (CD3+) cells, alpha beta+ TCR T (WT31+CD3+) cells, CD8 cells, and non-T, non-B cells. A serial study of some of the patients showed opposite changes in alpha beta TCR- T cells, the CD4/CD8 cell ratio, and CD5+ B cells between the active stages of Graves' and Hashimoto's diseases. alpha beta TCR- T cells were mostly gamma delta TCR+ T (IIF2+ CD3+) cells in these patients. These data suggest that alpha beta TCR-T (gamma delta TCR+ T), CD8, and CD4+ CD8+ cells are important in thyroid destruction in Hashimoto's disease and subacute thyroiditis, and that CD5+ B cells are important in thyroid stimulation in Graves' disease.     

Analysis of clones derived from human CD7+CD4-CD8-CD3- thymocytes.

The differentiation of human thymocyte precursors was studied by analysis of clonal progeny of CD4-CD8-CD3- (triple negative or TN) thymocytes. Using a culture system of phytohemagglutinin, IL-2, and irradiated allogeneic lymphoid feeder cells, we found that 48% of clones (104 total) derived from TN thymocyte suspensions were TCR gamma delta cells, 12% of clones were TCR alpha beta cells, and 34% were CD16+CD3- cells. Importantly, 6% of clones were novel subsets of CD4+CD8-CD3- or CD4-CD8+CD3- thymocytes. The majority of TCR alpha beta, TCR gamma delta, and CD16+CD3- clones expressed low levels of CD4. Molecular analysis of freshly isolated TN- thymocytes prior to in vitro culture demonstrated that up to 40% of cells had TCR gamma, delta, and beta gene rearrangements, but were negative in indirect immunofluorescence assays for cytoplasmic TCR delta and beta. These data provide evidence at the clonal level for the presence of precursors of the TCR alpha beta and TCR gamma delta lineages in the human TN thymocyte pool. Moreover, a substantial proportion of freshly isolated human TN thymocytes had already undergone TCR gene rearrangement prior to in vitro culture. Whether these precursors of the TCR alpha beta and TCR gamma delta lineages mature from cells already containing TCR gene rearrangements into sTCR+ cells or differentiate in vitro from cells with TCR genes in germline configuration remains to be determined. Nonetheless, these data demonstrate that the predominant clone types that grow out of human TN thymocytes in vitro are TCR gamma delta and NK cells.     

Definition of unique traits of human CD4-CD8- alpha beta T cells.

We have studied the nature of human CD4-CD8- (double negative) alpha beta T cells to determine whether they possess unique characteristics which could further differentiate them from conventional CD4+ or CD8+ (single positive) T cells. We observed that double negative TCR alpha beta+ T cells differ from single positive T cells in the following respects: (i) their T cell receptor (TCR) repertoire is different, as revealed by the analysis of 47 clones derived from three individuals and by analysis of peripheral blood lymphocytes (PBL) prior to in vitro manipulation; (ii) their in vivo CD3:TCR expression is lower before in vitro manipulation and expansion; (iii) their direct proliferative response to IL-3, which is not mediated by secondary release of other T cell growth factors. These characteristics have also been recently ascribed to murine double negative alpha beta T cells, which develop extrathymically and are considered to be a distinct T cell lineage. Our data suggest that, like their murine counterparts, human double negative alpha beta T cells may represent a distinct T cell lineage which might develop extrathymically.     

Autoreactive T cells in normal mice: unrestricted recognition of self peptides on dendritic cell I-A molecules by CD4-CD8- T cell receptor alpha/beta+ T cell clones expressing V beta 8.1 gene segments

CD4-CD8- double-negative (DN) and CD4+CD8- T cell clones were derived from splenic precursors resistant to killing by anti-Thy-1, -CD5, -CD4 and -CD8 monoclonal antibodies and complement. Both DN and CD4+ clones express functional T cell receptor (TcR) alpha/beta and exhibit strong autoreactivity in vitro. DN cells can be induced to proliferate by dendritic cells (DC) of all haplotypes tested, although this activation is inhibited by antibodies specific for I-A determinants expressed on the stimulatory DC. In contrast, CD4+ clones only respond to syngeneic or I-Ad-compatible DC. Both DN and CD4+ autoreactive clones do not proliferate when cultured with class II+ H-2d normal or tumor macrophages and B cell lines or with class II-transfected L cells, suggesting that these cells recognize self peptides only present on the surface of DC. Despite their phenotype resembling that of immature thymocytes and their inability to interact directly with B lymphocytes, DN cloned T cells, like CD4+ T cells, exhibit nonspecific helper functions and can induce polyclonal B cell proliferation and differentiation. DN TcR alpha/beta+ peripheral T cells represent, like TcR gamma/delta+ lymphocytes, a new T cell subset physiological role whose remains to be defined.     

Stimulation of CD2-negative T cells by staphylococcal enterotoxin superantigens.

A minor fraction of CD3+ T cells lacks expression of the CD2 antigen, which is the target for an "alternative" T cell activation pathway. CD2-CD3+ T cells can be stimulated by anti-CD3 or anti-T cell receptor (TCR) antibodies, indicating that the CD3/TCR signal transduction pathway functions in the absence of cell surface CD2. In the present study we have analyzed whether CD2-CD3+ T cells also respond to antigen stimulation. We show here that cloned CD2-negative T cells expressing the alpha/beta TCR are activated by one or several staphylococcal enterotoxin "superantigens". Activation of CD2-CD3+ T cell clones by staphylococcal enterotoxins resulted in IL-2 production and/or proliferative activity, and was dependent on the presence of HLA class II-bearing feeder cells. These data demonstrate that T cells can recognize (and respond to) antigen in the absence of a functional CD2 molecule.     

Interleukin 7 preferentially supports the growth of gamma delta T cell receptor-bearing T cells from fetal thymocytes in vitro.

Murine fetal thymus cells were cultured with various interleukins (IL-1, 2, 3, 4, 5, 6, and 7) in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and it was found that only IL-4 and IL-7 induced a prominent proliferative response in the presence of PMA. A large proportion of cells grown in the cultures of fetal thymus cells (days 15 and 17 of gestation) stimulated with PMA plus IL-4 or with PMA plus IL-2 remained CD4-CD8-. In marked contrast, nearly 70% of the cells generated in the cultures of the same fetal thymocytes stimulated with PMA plus IL-7 expressed CD8 on their surface. Approximately 30% of these cells expressed TCR gamma, delta, whereas TCR alpha beta+ cells were virtually undetectable. The cells grown in cultures stimulated with PMA plus IL-7 comprised three populations: CD4-Lyt-2-3-, CD4-Lyt-2 + Lyt-3- and CD4-Lyt-2 + Lyt-3+, and that TCR gamma delta+ T cells were found in all three populations. It was also found that the addition of IL-7 in the culture of adult CD4-CD8- thymocytes on the monolayer of a thymic stromal cell line, which selectively promotes the generation of alpha beta T cells, resulted in the generation of gamma delta T cells. These results strongly suggest that IL-7 plays an important role in the development of gamma delta T cells.     

Proliferative properties of murine intestinal intraepithelial lymphocytes (IEL): IEL expressing TCR alpha beta or TCR tau delta are largely unresponsive to proliferative signals mediated via conventional stimulation of the CD3-TCR complex.

Murine intestinal intraepithelial lymphocytes (IEL) were studied for their capacity to proliferate in vitro following stimulation of the T cell receptor (TCR)-associated CD3 epsilon molecule, or upon direct stimulation of the TCR complex itself. Although IEL consisted primarily of CD3+ T cells which included activated cytotoxic T lymphocytes as demonstrated in CD3- and TCR-mediated redirected cytotoxic assays, IEL displayed minimal proliferative responses following stimulation with anti-CD3, anti-TCR alpha beta, or anti-TCR tau delta monoclonal antibodies under soluble conditions, or under conditions which effect membrane cross-linking, including the addition of accessory cells to IEL cultures. The lack of proliferation induction could not be overcome by stimulation of IEL in the presence of T cell-dependent cytokines, phorbol ester, or interleukin-4. Moreover, unlike splenic T cells, stimulation of IEL failed to result in expression of interleukin-2 receptor, further demonstrating an inability of IEL to respond to exogenous proliferative signals. This study is the first to examine the proliferative potential of murine IEL following direct CD3 or TCR stimulation. The findings described here: (i) identify an important functional distinction between intestinal IEL and other peripheral alpha beta or tau delta T cells which generally respond well to proliferative signals mediated through the CD3-TCR complex, and (ii) demonstrate that on murine IEL the CD3-TCR complex can discriminate signals of lytic activity from those of cell proliferation.     

Cytotoxic activity of V beta 8+ T cells in Crohn's disease: the role of bacterial superantigens.

In Crohn's disease, disease-related stimuli could alter the T cell receptor (TCR) repertoire. To examine the possibility that changes in function may occur in T cell subsets without obvious changes in expression of TCR, we analysed the TCR repertoire of cytotoxic T lymphocytes in Crohn's disease peripheral blood. Furthermore, we examined the effect of bacterial superantigens, staphylococcal enterotoxin B (SEB) and E (SEE) on the cytotoxic function of T cell subsets bearing different TCR V genes using MoAbs specific for CD3 and TCR V gene products in a redirected cytotoxicity assay. There was no difference between patients and controls in the cytotoxicity measured in concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) with anti-CD3 or with six of seven anti-TCR V gene MoAbs. However, the cytotoxicity of V beta 8 T cells was decreased in Crohn's disease patients. This was not due to a decrease in total or CD8+ T cells expressing V beta 8. Furthermore, in normal subjects, PBMC stimulation with SEE and SEB selectively expanded and increased the cytotoxicity of V beta 8 and V beta 12 T cells, respectively. In Crohn's disease, although SEB stimulation increased the number and cytolytic function of the V beta 12 subset, SEE stimulation failed to increase cytolytic activity of V beta 8+ T cells in spite of the expansion of V beta 8+ T cells. These results suggest that the changes in cytotoxic function observed in V beta 8 T cells in Crohn's patients may reflect previous exposure to a V beta 8-selective superantigen.     

Functionally distinct subsets of human gamma/delta T cells.

To determine if effector subsets exist among human gamma/delta T cells, we examined the cytokine production and cytotoxic activity of gamma/delta T cell clones with different accessory molecule phenotypes, V delta and V gamma gene expression, and J gamma rearrangements. T cell clones bearing gamma/delta T cell receptor produce an array of cytokines like alpha/beta T cell clones. Individual gamma/delta T cell clones produced a characteristic array of cytokines without correlation with V delta or V gamma gene expression. However, when phenotypic subsets were considered, CD4+ gamma/delta clones produced significantly higher levels of interleukin 2 and granulocyte-monocyte colony-stimulating factor compared with CD4-CD8- and CD8+ gamma/delta clones. Similarly, when cytotoxic potential was assessed, CD4+ gamma/delta clones exhibited minimal activity when compared with CD4-CD8- and CD8+ adult peripheral blood gamma/delta clones. We conclude that functionally distinct gamma/delta T cell subsets exist and suggest that these subsets may correlate with expression of the CD4 accessory molecule.     

Lymphokine requirements for the development of specific cytotoxic T cells from single precursors

A high cloning efficiency, filler cell-free culture system was developed for the growth of single murine cytotoxic T lymphocyte precursors (CTLp) and their differentiation into cytotoxic T lymphocytes (CTL). The system used nonspecific stimulation with phorbol ester and calcium ionophore in the presence of recombinant lymphokines. The optimal lymphokine combination was interleukin 2 throughout, together with interferon-gamma during the first 6 days and interleukin 6 during the last 2 days of culture. Under these conditions half of all CD4-CD8+ T cells became CTL clones. The CTL were CD4-CD8+CD3+ TcR alpha/beta+ and were derived from CD4-CD8+Pgp-1- precursors.     

The cytotoxic T lymphocyte response in reticuloendotheliosis virus-infected chickens is mediated by alpha beta and not by gamma delta T cells.

We induced a virus-specific cytotoxic T lymphocyte (CTL) response in B2 chickens by i.v. inoculation with 100 TCID50 of the reticuloendotheliosis virus (REV). Chickens were sacrificed 7 days after the infection and cytotoxic activity of the spleen cells against various target cells was assayed in a 4 h 51Cr-release assay at an effector to target ratio of 100:1. In addition, T cell receptor (TCR) alpha beta and TCR gamma delta cells were negatively selected from the REV-immune spleen cells and used as effector cells against REV-infected B2 target cells. (On average 40% of spleen T cells express TCR gamma delta in the chicken.) By inhibition of the cytotoxic activity of the immune spleen cells against REV-infected syngeneic target cells with monoclonal antibodies specific for chicken CD3 and CD8 molecules, the effector cells could be identified as CD8+ T cells. The cytotoxic activity was MHC-restricted, as only syngeneic but not allogeneic REV-infected target cells were lysed by REV-immune spleen cells, and virus-specific, as no cytotoxic activity could be found using uninfected syngeneic target cells. When assaying the activity of the negatively selected, > 98% pure alpha beta and gamma delta T cells, it was found that alpha beta T cells exerted virus-specific CTL activity ranging from 26 to 62% specific 51Cr-release, while gamma delta T cells showed only 2-4% 51Cr-release. These data indicate that REV-specific CTL response is mediated by alpha beta T cells and that gamma delta T cells are not involved in virus-specific CTL activity in the spleen of REV-infected chickens.     

Cloning of a novel cell type from human fetal liver expressing cytoplasmic CD3 delta and epsilon but not membrane CD3.

Seventeen-week human fetal liver cells cultured with a feeder cell mixture of irradiated PBL, irradiated JY cells (an EBV-transformed B cell line) and PHA contained a subpopulation of CD3- cells in addition to a major population of T cells with the mature phenotype. After 12 days in culture, CD3- CD16- cells were sorted and cloned by limiting dilution. Two representative clones, FL121 and FL125, were expanded and characterized. They shared the phenotype of CD2+CD3-CD4-CD5-CD6+CD16-CD56+. FL121 did not express CD8 whereas FL125 expressed CD8 alpha but not CD8 beta. Both clones were found to express cytoplasmic CD3 delta and CD3 epsilon Ag while CD3- NK clones isolated from PBL were negative for them. These results indicate that FL121 and FL125 were committed to the T-cell lineage. Southern blot analysis showed that the TCR beta genes and the TCR gamma genes of these clones were in the germ-line configuration. The establishment of FL121 and FL125 may provide a novel insight into the earliest stage of T-cell development in man.     

Intrathyroidal lymphocyte subsets, including unusual CD4+ CD8+ cells and CD3loTCR alpha beta lo/-CD4-CD8- cells, in autoimmune thyroid disease.

Intrathyroidal lymphocyte subsets were analysed in 13 euthyroid patients with autoimmune thyroid disease by two-colour flow cytometry and compared with subsets in peripheral blood. In both Graves' and Hashimoto's diseases, proportions of intrathyroidal CD5- B cells were higher than in peripheral blood. The numbers of such cells were correlated with serum levels of anti-thyroid microsomal antibodies. Proportions of T cells bearing alpha beta chains of T cell receptors (TCR alpha beta+ T; T alpha beta) and CD16+CD57+ natural killer (NK) cells were lower in the thyroid, but proportions of CD3hiTCR alpha beta-TCR gamma delta+ (T gamma delta) cells were not different. Proportions of CD4+Leu-8- helper T cells and CD4+CD57+ germinal centre T cells were higher and proportions of CD4+Leu-8+ suppressor-inducer T cells and CD8+CD57+ or CD8+CD11b+ suppressor T cells were lower than in the blood in both diseases. Proportions of CD5+ B cells were high in Graves' disease, and proportions of CD8+CD11b- cytotoxic T cells were high in Hashimoto's disease. Unexpectedly, CD4+CD8+ cells and CD3loTCR alpha beta lo/-CD4-CD8- cells were present in thyroid tissues of both diseases. These findings suggest that: (i) an imbalance in the numbers of regulatory T cells and of NK cells that had appeared in the thyroid resulted in the proliferation of CD5- B cells, which were related to thyroid autoantibody production; (ii) CD5+ B cells and cytotoxic T cells are important for the different pathological features in Graves' and Hashimoto's diseases, respectively; and (iii) intrathyroidal CD4+CD8+ cells and CD3loTCR alpha beta lo/-CD4-CD8- cells may be related to the pathogenesis of autoimmune thyroid disease.     

The amplifier role of T cells in the human in vitro B cell response to type 4 pneumococcal polysaccharide.

The human B cell response to T cell independent type 2 antigens is regulated by thymus-derived lymphocytes. We analyzed the role of T cells in the in vitro antibody response to type 4 pneumococcal polysaccharide (PS4). We here show that the amplifying effect of T cells, which has previously been shown to be radioresistant and confined to T cell preparations enriched for CD4+ cells, is MHC non-restricted as demonstrated in cultures carried out in the presence of allogeneic T cells. Also, T cell clones derived from non-related donors are able to enhance the B cell response to PS4. All TCR alpha beta +, CD 4+ T cell clones, but none of the TCR alpha beta +, CD 8+ T cell clones tested, enhanced the B cell response to PS4. Furthermore, 3 out of 6 TCR gamma delta+ T cell clones were capable of enhancing the anti-PS4 B cell response. Experiments using recombinant lymphokines and glutaraldehyde-fixed T cells indicated that both lymphokines and T-B cell interactions are required for an optimal antibody response to PS4.     

Identification of αβ and γδ T Cell Receptor-Positive Cells

Two lineages of T lymphocytes bearing the CD3 antigen can be defined on the basis of the nature of the heterodimeric receptor chain (alpha beta or gamma delta T cell receptor (TCR) expressed. Precise identification of alpha beta and gamma delta TCR+ cells is essential when studying the tissue distribution and function of these different T cells. In immunofluorescence studies gamma delta TCR+ cells have been identified as CD3+WT-31- or CD3+CD4-CD8- cells. However, this may not be the optimal procedure because gamma delta TCR+ cells are weakly WT-31+, and some are CD8+. The aim of this study was to evaluate a panel of monoclonal antibodies (MoAb) directed against different chains of the TCR-T3 complex for a more precise identification of alpha beta+ and gamma delta TCR+ cells in flow cytometric studies. We found that the MoAb anti-Ti-gamma A and delta-TCS-1, recognizing the TCR-gamma and the TCR-delta chain respectively, only reacted with a subpopulation of gamma delta TCR+ cells, whereas another TCR-delta chain recognizing MoAb anti-TCR-delta 1 reacted with all gamma delta TCR+ cells. All MoAb reported to belong to the CD3 group reacted with both alpha beta TCR+ and gamma delta TCR+ cells as expected. Our results indicate that all gamma delta TCR+ cells can be identified with the MoAb anti-TCR-delta 1. Because no MoAb recognizing the TCR-alpha or TCR-beta chains at the cell surface of intact cells are yet available, we suggest that alpha beta TCR+ cells could be identified as CD3+ anti-TCR-delta 1-cells.     

T-cell receptor beta gene rearrangements in clones derived from human CD4-8- cells expressing natural killer cell activity.

Clones derived from highly purified human peripheral blood Leu 19+ cells in the presence of phytohaemagglutinin (PHA) and interleukin-2 (IL-2) expressed cytotoxic activity against natural killer (NK)-resistant as well as NK-sensitive targets. All 66 clones analysed had a germ line configuration of T-cell receptor (TCR) beta genes and 38/40 also had unrearranged TCR gamma genes. The two exceptions were both CD3+ clones, but these did not have a cytotoxic repertoire noticeably different from CD3- clones without TCR gamma gene rearrangements. Clones were also obtained from highly purified CD4-8- cells, most of which were also cytotoxic for NK-resistant and NK-sensitive targets. About 90% of these clones were CD3+ but only around 50% remained negative for CD4 and CD8 while a significant number (12.7%) were positive for both CD4 and CD8. All clones analysed had rearranged TCR gamma genes and most had also rearranged TCR beta genes, including 20/25 of the clones which were CD3+4-8-. Many of the clones showed two rearrangements of TCR beta genes, and 3/4 CD3- clones had rearranged TCR beta as well as TCR gamma genes. There was no correlation between cytotoxic activity and TCR gene status or phenotype of these CD4-8- derived clones, except that clones which were Leu 19+ tended to have higher cytotoxic activity against NK-sensitive and NK-resistant targets than Leu 19-clones. The results strongly indicate that TCR beta and gamma gene products are not involved in the cytotoxicity mediated by these clones. They also suggest that some CD4-8- cells may be capable of limited differentiation in vitro.