TCR Alpha Beta Research Articles

Cytotoxic protein expression in natural killer cell lymphomas and in alpha beta and gamma delta peripheral T-cell lymphomas.

Lymphomas with T-cell phenotype represent a heterogeneous group of diseases differing in histopathology, tumour site, and cell origin. They include peripheral T-cell lymphomas (PTCLs) derived from alpha beta cells, but also some recently recognized entities such as gamma delta, hepatosplenic lymphomas and natural killer (NK) cell lymphomas. Only a few studies have investigated the possibility that at least some PTCLs could be derived from lymphocytes with cytotoxic potential. In order to investigate this possibility, 60 cases of PTCL, including 27 cases expressing the alpha beta T-cell receptor (TCR alpha beta), 15 TCR gamma delta cases and 18 cases expressing neither TCR (TCR silent), as well as 14 cases of NK-cell lymphomas, were studied by immunohistochemistry for the expression of TIA-1, perforin, and granzyme B proteins. Expression of TIA-1 is characteristic of cytotoxic cells regardless of their activation status, whereas expression of perforin and granzymes is highly increased in activated cytotoxic cells and correlates with the induction of cytolytic activity. All NK-cell lymphomas (11 sinonasal, three systemic cases) expressed TIA-1, perforin, and granzyme B in most tumour cells. All gamma delta PTCLs (15 cases) expressed TIA-1 protein in most tumour cells, with a different cytotoxic antigen profile in hepatosplenic gamma delta PTCL (TIA-1+, perforin-, granzyme B-) and in non-hepatosplenic gamma delta PTCLs (three nasal, one skin, one lung), the latter expressing the three cytotoxic proteins. Of the 45 cases of alpha beta and TCR silent PTCL, 15 (33 per cent) were considered to be derived from cytotoxic lymphocytes with expression of at least one cytotoxic protein (TIA-1, 15/45; perforin, 10/41; granzyme B, 14/38) in tumour cells. This cytotoxic protein expression appeared to be related to the site of localization, since 7/13 (54 per cent) extranodal and only 8/32 (25 per cent) nodal alpha beta and TCR silent PTCLs expressed TIA-1, and to histology, since this pattern was observed in a proportion of anaplastic (6/8, 75 per cent) and pleomorphic (8/17, 47 per cent) lymphomas, but not in AILD-type NHL (0/16). Taken together, our data suggest that NK-cell lymphomas and non-hepatosplenic gamma delta PTCLs represent tumours of activated cytotoxic NK cells and gamma delta T cells, respectively; that hepatosplenic gamma delta PTCLs represent tumours of non-activated cytotoxic gamma delta T cells; and that a small proportion of alpha beta and TCR silent PTCLs, mostly extranodal cases, or nodal anaplastic lymphomas, represent tumours of cytotoxic T cells.

Appropriate developmental expression of human CD8 beta in transgenic mice.

The human CD8 glycoprotein is expressed either as an alpha beta heterodimer or as an alpha alpha homodimer on thymocytes, mature T cells, and subpopulations of intestinal intraepithelial lymphocytes (IELs). The homodimeric form of CD8 is exclusively expressed on TCR gamma delta IELs and on subsets of NK cells and TCR alpha beta IELs. To understand the molecular mechanisms by which these genes are regulated, we created transgenic mice with a 95-kb human genomic DNA fragment containing the entire CD8 beta gene as well as a cluster of tissue-specific DNase I-hypersensitive sites 7 to 10 kb upstream of the gene. These sites were present in CD8 alpha beta+- but not CD8 alpha beta- T cell lines nor in a B cell line. We found that transgenic mice had correct developmental expression of human CD8 beta on thymocytes and mature CD8+ cells and no expression on mature CD4+ T cells or B cells. Interestingly, the percentage of mouse CD8 alpha+ cells that were human CD8 beta+ varied, depending on the founder line, from 4 to 88%, whereas the percentage among siblings was similar, indicative of a variegated phenotype resulting from site of integration effects. Expression was also observed on intestinal IELs, but only on those expressing the TCR alpha beta receptor and not the TCR gamma delta cells, which exclusively express CD8 alpha alpha. Of the TCR alpha beta+ cells, the transgene was expressed in both the CD8 alpha alpha and alpha beta subpopulations. These results indicate that this 95-kb fragment affords developmentally correct expression of the human CD8 beta gene on thymus-derived T cells in transgenic animals. Therefore, CD8 lineage-specific regulatory sequences must be located within the fragment.

A thymic hormone protects mice from enteropathy during acute graft-versus-host disease.

We have previously reported that a nonapeptide thymic hormone, facteur thymique serique (FTS), is involved in the differentiation and activation of intestinal intraepithelial lymphocytes (i-IEL) in mice. In this study, we examined the effect of FTS treatment on enteropathy in a murine model for acute graft-vs.-host disease (GVHD) induced by injection of parental C57BL/6 splenocytes into unirradiated (C57BL/6 x DBA/2) F1 hybrids. FTS treatment significantly protected mice from developing acute GVHD as assessed by mortality rate, splenomegaly and enteropathy. The infiltration of donor-derived TCR alpha beta i-IEL bearing CD8 alpha beta was significantly inhibited in the small intestine of FTS-treated mice, and the frequencies of apoptosis of crypt cells in the intestinal mucosa were decreased in these mice during acute GVHD. These results suggest that FTS treatment contributes to protection against enteropathy of acute GVHD. Thus, FTS may provide a useful approach to control acute GVHD after blood transfusion or bone marrow transplantation.

Nematode infection induces Th2 cell-associated immune responses in LEC mutant rats with helper T cell immunodeficiency.

Long-Evans Cinnamon (LEC) rats have maturational arrest of CD4+8- T cells from CD4+8+ cells in the thymus. Despite this, CD4+8- T cells are always present in peripheral lymphoid organs of LEC rats, suggesting that these CD4+8- T cells are generated by an uncommon pathway. We investigated the role of LEC rat peripheral CD4+8- T cells in Th2-associated responses to infection with the nematode Nippostrongylus brasiliensis. After infection, the numbers of CD4+8- TCR alpha beta + T cells significantly increased in mesenteric lymph nodes (MLN) and the spleen, while those in the thymus were still negligible. Infection also induced significant up-regulation of IL-4 gene expression in LEC rat MLN cells. Total serum IgE levels in LEC rats were markedly increased two weeks after infection. Mucosal mast cell responses in the gut and lungs of LEC rats were induced as prominently as in control Long-Evans Agouti (LEA) rats. Faecal egg count data indicated that LEC rats rejected nematodes faster than LEA rats. These results suggested that Th2-associated responses can be induced by nematode infection in LEC rats probably through the extrathymic recruitment and proliferation of CD4+8- TCR alpha beta + T cells.

Interleukin-13 secretion by normal and posttransplant T lymphocytes; in vitro studies of cellular immune responses in the presence of acute leukaemia blast cells.

T lymphocyte secretion of interleukin-13 (IL-13) in response to different activation signals was characterized in vitro. IL-13 release was investigated when virus transformed B lymphocytes or acute myelogenous leukaemia (AML) blasts were used as accessory cells during T cell activation. First, a majority of both CD4+ and CD8+ TCR alpha beta + T lymphocyte clones, derived from normal individuals and bone marrow transplant recipients, secreted IL-13 in response to a standardized mitogenic activation signal (phytohaemagglutinin + IL-2 + B lymphocyte accessory cells). The CD4+ cells showed significantly higher IL-13 levels than the CD8+ subsets. Second, when leukaemic accessory cells (more than 95% AML blasts) were used during T cell activation, IL-13 was released both during alloactivation of normal T lymphocytes and during mitogen activation of posttransplant T cells. Third, when normal T lymphocytes were stimulated with allogeneic AML blasts, addition of IL-13-neutralizing monoclonal antibodies decreased interferon gamma levels. Although addition of IL-13-neutralizing antibodies did not alter granulocyte-colony-stimulating factor secretion by allostimulating AML blasts, altered blast proliferation was detected for certain patients. Thus, most T cell clones can release IL-13, and IL-13 can modulate cytokine responses during T cell recognition of allogeneic AML cells.

Gamma delta T cells of human early pregnancy decidua: evidence for local proliferation, phenotypic heterogeneity, and extrathymic differentiation.

The uterine mucosa in pregnancy, the decidua, allows placenta formation and survival of the fetus despite the fact that it is semiallogeneic. Decidua contains large numbers of lymphocytes, of which CD56+ cells dominate, followed by T cells expressing either alpha beta or gamma delta TCR. We have investigated the developmental relationship between the CD56- and TCR gamma delta-expressing cells in early pregnancy decidua using dual labeling immunoelectron microscopy, immunoflow cytometry, and cell fractionation. Lymphocyte subpopulations were, in addition, analyzed for expression of the cytokine receptor for IL-7 and c-kit and for mRNA expression of recombinase-activating genes 1 and 2. Four different cell populations could be distinguished: CD56+bright, CD56+dim/TCR gamma delta+low, CD56+dim/TCR gamma delta+high, and TCR gamma delta+low. Recombinase-activating genes 1 and 2 were expressed in the CD56+bright cells and to a limited degree in CD56+dim/TCR gamma delta+low cells. c-kit was preferentially expressed on the CD56+bright cells, while IL-7R was preferentially expressed on CD56+dim/TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells. The CD56+dim TCR gamma delta+low and CD56+dim/TCR gamma delta+high cells displayed the characteristic morphology of large granular lymphocytes, while single positive TCR gamma delta+low cells were usually smaller and did not contain cytoplasmic granules. The gamma delta 1 gene segment was almost exclusively used in the TCR. Gamma delta T cells in mitosis were seen. We suggest that human early pregnancy decidua is a transient site for extrathymic maturation and that the progenitors of TCR gamma delta+ cells are bone marrow-derived immature cells expressing the CD56 (neural cell adhesion molecule) homing receptor.

Pre-TCR signaling components trigger transcriptional activation of a rearranged TCR alpha gene locus and silencing of the pre-TCR alpha locus: implications for intrathymic differentiation.

A rearranged TCR alpha transgene remains transcriptionally inactive in rag-2-/- thymocytes but can be induced by CD3-mediated signals with concomitant maturation of double-negative (DN) thymocytes to the CD4+CD8+ double-positive (DP) stage. Reciprocally, the same signals silence pre-TCR alpha (pT alpha) expression. In normal C57BL/6 thymocytes, TCR alpha expression is not detected in DN thymocytes while, in contrast, TCR beta expression is initiated at the most immature c-kit+CD44+CD25- stage and continues throughout thymocyte development. pT alpha expression is first detected at the intermediate c-kit +/- CD44+CD25+ DN stage, increases during transition to the more mature c-kit-CD44-CD25+ stage and is lost at the DP stage. Thus, although TCR beta and pT alpha expression are independent, the pre-TCR complex mediates signals controlling the appearance of alpha beta TCR through selective regulation of TCR alpha and pT alpha genes.

Role of IL-2 in rat fetal thymocyte development.

Early during rat thymus ontogeny, an important proportion of thymocytes expresses IL-2R and contains IL-2 mRNA. To investigate the role of the IL-2-IL-2R complex in rat T cell maturation, we supplied either recombinant rat IL-2 or blocking anti-CD25 mAb to rat fetal thymus organ cultures (FTOC) under several experimental conditions. The IL-2 treatment initially stimulated the growth of thymocytes and, as a result, induced T cell differentiation, but the continuous addition of IL-2 to rat FTOC, as well as the anti-CD25 administration, resulted in cell number decrease and inhibition of thymocyte maturation. These results indicate that immature rat thymocytes bear functional high-affinity IL-2R and that IL-2 promotes T cell differentiation as a consequence of its capacity to stimulate cell proliferation. Modifications in TCR alpha beta repertoire and increased numbers of NKR-P1+ cells, largely NK cells, were also observed in IL-2-treated FTOC. Furthermore, IL-2-responsiveness of different thymocyte subsets changed throughout thymic ontogeny. Immature CD4-CD8-cells responded to IL-2 in two stages, early in thymus development and around birth, in correlation with the maturation of two distinct waves of thymic cell progenitors. Mature CD8+ thymocytes maximally responded to IL-2 around birth, supporting a role for IL-2 in the increased proliferation of mature thymocytes observed in vivo in the perinatal period. Taken together, these findings support a role for IL-2 in rat T cell development.

HEL-Flu: an influenza virus containing the hen egg lysozyme epitope recognized by CD4+ T cells from mice transgenic for an alphabeta TCR.

Reverse genetics was used to modify the influenza virus genome by inserting the p46-63 sequence of hen egg lysozyme (HEL) into the neuraminidase stalk of the virus. The resulting virus, HEL-Flu, contained the epitopes recognized by CD4+ T cells from 3A9-TCR transgenic mice (C3HTg). Here, we show that HEL-Flu was infectious in the respiratory tract of both C3H and C3HTg mice, the latter animals showing an early, transient morbidity. Splenic dendritic cells and certain cloned populations of splenic macrophages and brain microglia constitutively presented infectious and inactivated HEL-Flu to the T cells in an Ag-specific and MHC class II-restricted manner. These results demonstrate the utility of HEL-Flu in assessing the APC activity for naive T cells; they also extend the previous studies showing that discrete populations of macrophages and microglia constitutively process and present Ag to naive T cells.

Differential requirement of MHC class II molecules expressed on hematopoietic cells for positive selection of CD4+ thymocytes in TCR alpha beta and TCR beta transgenic mice.

To address whether expression of the relevant MHC molecules on thymic epithelial cells (TEC) is sufficient for positive selection, the fate of CD4+ T cells directed by the DR alpha E beta b molecule was compared between two lines of HLA-DRA transgenic mice, DR alpha-24 expressing DR alpha E beta b on TEC and hematopoietic cells, and DR alpha-30 expressing the molecule on TEC but not on other cell lineages. CD4+ T cells were selected to mature on DR alpha E beta b molecules in both lines deficient for the endogenous MHC class I and class II molecules, whereas CD4+ T cell differentiation in DR alpha-30, as compared with that in DR alpha-24, was severely impaired when these mice were crossed with mice transgenic for the DR alpha E beta b-restricted alpha beta TCR. In contrast, CD4+ T cells expressing the same TCR alpha beta as the transgenes were selected to mature in DR alpha-30 crossed with the single TCR beta transgenic mice. Taken together, these results indicate that the restricted expression of DR alpha E beta b on TEC is sufficient for positive selection in the TCR beta and non-TCR transgenic mice, but the expression on both TEC and hematopoietic cells is required for that in the TCR alpha beta transgenic mice. In the light of the difference in the developmental stage at which alpha beta TCR are expressed between TCR alpha beta and TCR beta transgenic mice, the interaction of the TCR alpha beta with DR alpha E beta b molecules expressed on hematopoietic cells required for positive selection in TCR alpha beta transgenic mice could be mapped to the early stage of T cell differentiation and might mimic 'undefined' early signaling of thymocytes in physiological T cell differentiation.

Isolation and characterization of human papillomavirus type 6-specific T cells infiltrating genital warts.

The potential role of T cells in the control of human papillomavirus type 6 (HPV-6) infections is an appealing premise, but their actual role has been sparsely investigated. Since HPV-6 infections are confined to the epithelium, such an investigation should focus on the T cells present at the site of infection (i.e., the warts). Therefore, we isolated wart-infiltrating lymphocytes (WIL) from patients with clinically diagnosed anogenital warts. These WIL were characterized by their phenotype and their specificity for E7 and L1 proteins of HPV-6. The phenotype of WIL varied drastically from patient to patient, as determined by their expression of CD4, CD8, T-cell receptor alpha/beta chain (TCR alpha beta), and TCR gamma delta. Despite this heterogeneity in phenotype, HPV-6 E7 and/or L1-specific WIL, as determined by lymphoproliferation, could be isolated from more than 75% of the patients studied. Among all L1 peptides recognized by WIL, peptides 311-330 and 411-430 were the most consistently detected, with seven of nine patients for whom L1 peptide reactivity was observed responding to at least one of them. Moreover, the HPV-6 epitopic peptides recognized by WIL differed to some extent from those recognized by peripheral T cells.

BCL-XL-regulated apoptosis in T cell development.

Thymocyte differentiation progresses through well-defined stages in which apoptosis is central to the selection of a functional TCR repertoire. In the present study, we explored the developmental effects of BCL-XL, a repressor of apoptosis. We found that endogenous BCL-XL is down-regulated by both positive and negative selection signals at the CD4+ CD8+ stage of thymocyte development. We examined the role of BCL-XL regulatable apoptosis in T cell development in the context of an alpha beta TCR transgene, Rag-1 deficiency and the scid model. We found that BCL-XL expression promoted accumulation of CD8 single-positive thymocytes even in MHC class II-restricted TCR transgenic mice. However, the apoptotic resistance conferred by BCL-XL could not fully substitute for TCR-mediated positive selection signals nor did it prevent negative selection. Overexpression of BCL-XL promoted partial maturation of CD4-CD8- thymocytes to CD4+CD8+ cells in a Rag-deficient, but not a scid background. Thus, TCR-mediated signals mediate the regulation of endogenous BCL-XL during thymocyte development, indicating that the principal protection of double-positive thymocytes by BCL-XL occurs prior to selection. The impact of BCL-XL on the CD8 but not CD4 lineage supports the asymmetric model of lineage commitment. Moreover, several critical control points in T cell development could be distinguished as BCL-XL responsive or unresponsive.

An interleukin-7 internet for intestinal intraepithelial T cell development: knockout of ligand or receptor reveal differences in the immunodeficient state.

Both interleukin-7 (IL-7) and IL-7 receptor (R) gene knockout (IL-7-/- and IL-7R-/-) mice were employed in order to directly investigate the importance of the IL-7 and IL-7R signaling pathway for the development of intestinal intraepithelial lymphocytes (IEL). Loss of the IL-7R-specific gene resulted in complete deficiency of the gamma delta T cell lineage with lack of V gamma 4- and V gamma 7-specific messages in the epithelium of the gastrointestinal (GI) tract in comparison to control mice of the same genetic background (approximately 40%). Disruption of the IL-7-specific gene resulted in marked, but not complete depletion of gamma delta T cells (2-3%) in IEL. Furthermore, mRNA for both V gamma 4 and V gamma 7 genes were detected in the gamma delta IEL subset of IL-7-/- mice. The subtle differences between IL-7-/- and IL-7R-/- mice suggest that although IL-7 controls most of the expansion and/or development of gamma delta IEL, another ligand binding to the IL-7R also plays a discernable role. Furthermore, alpha beta IEL developed more slowly in IL-7R-/- mice when compared with ligand knockouts; however, the frequency of IEL T cells subsequently increased with age and normal levels of CD3+ T cells expressing the alpha beta TCR were detected by 2 and 3 months of age in IL-7-/- and IL-7R-/- mice, respectively. The direct comparison of IL-7-/- and IL-7R-/- mice clearly supports the hypothesis that both IL-7 and another IL-7R binding molecule can influence the development of gamma delta T cells in the intestinal epithelium.

P40 molecule regulates NK cell activation mediated by NK receptors for HLA class I antigens and TCR-mediated triggering of T lymphocytes.

p40 was previously described as a regulatory molecule capable of inhibiting both the natural and the CD16-mediated cytotoxicity of NK cells. In this study, we analyze the effect of p40 molecule engagement on the NK cell triggering induced by activating HLA class I-specific NK receptors (NKR) or on TCR alpha beta-mediated T cell activation. CD3-CD16+ NK cell clones expressing activating NKR (either CD94 or p50) were analyzed in a redirected killing assay using P815 target cells and appropriate mAb. A strong target cell lysis was detected in the presence of anti-NKR or anti-CD16 mAb alone. Addition of anti-p40 mAb resulted in a strong inhibition of both anti-NKR or anti-CD16 mAb-induced cytolysis. mAb specific for either CD45 or lymphocyte function associated antigen-1 did not exert any inhibitory effect in the same experimental system. Free intracellular calcium ([Ca2+]i) increase induced by mAb cross-linking of activating CD94 or p50 was inhibited by simultaneous engagement of p40 molecules, but not of other NK surface molecules including CD44 and CD56. In addition, cross-linking of p40 molecules strongly inhibited the CD94-induced tumor necrosis factor-alpha and IFN-gamma production. Analysis of TCR alpha beta or gamma delta T cell clones revealed that the engagement of p40 molecules, using specific mAb, induced some degree of inhibition only on anti-V beta (but not anti-V delta or anti-CD3) mAb-induced cytotoxicity. On the other hand, the p40 molecule engagement prevented T cell proliferation induced by either anti-V beta 8 or anti-V delta 2 mAb. A similar inhibitory effect was found on the IL-2-induced NK cell proliferation. Taken together, our present findings suggest that p40 may play a role in the regulation of NK and T lymphocyte activation and proliferation.

Recombinant soluble alphabeta T cell receptors protect T cells from immune suppression: requirement for aggregated multimeric, disulfide-linked alphabeta heterodimers.

Recombinant soluble T cell receptors (sTCR) protected contact sensitivity (CS) effector T cells from down-regulation or immunosuppression. CS-protecting sTCR were released enzymatically from the surface of thymoma cells transfected with cDNAs encoding TCR-alpha and -beta extracellular domains that were expressed with a phosphatidylinositol linkage. sTCR affinity purified on anti-TCR-alpha and anti-TCR-beta mAb columns had identical CS-protective activity, as did sTCR from a CD4+ Th2 clone or from a CD8+ cytotoxic clone. Reduced sTCR alpha- and beta-chains had no CS-protective activity, but this was restored when the TCR chains were rejoined into disulfide-linked alphabeta heterodimers. sTCR CS protection was Ag nonspecific, MHC unrestricted, and not influenced by the relevant synthetic peptide specific for the TCR complexed with appropriate MHC. CS protection may have resided in the sTCR constant region. When heated at 62 degrees C for 30 min, sTCR formed a CS-protecting aggregate, with a molecular mass of 481 +/- 37 kDa, corresponding to an alphabeta TCR pentamer. HPLC gel filtration essentially confirmed the molecular mass at 516 kDa for the multimer, while the monomer, which was an alphabeta TCR heterodimer, had an expected molecular mass of approximately 104 kDa and no bioactivity. In summary, the pentameric sTCR may bind to and activate lymphoid cells, perhaps via constant domains, resulting in protection of CS effector T cells from down-regulation. The ability of sTCR to protect CS effector T cells from down-regulation/suppression, if generalized, could overcome immunosuppression accompanying infectious diseases, particularly AIDS, or in tumors.

Distinct methylation states of the CD8 beta gene in peripheral T cells and intraepithelial lymphocytes.

The CD8 coreceptor is expressed on both immature and mature T cells as either an alphabeta heterodimer or an alpha alpha homodimer. Thymocytes and peripheral T cells express CD8 alphabeta, whereas TCR alphabeta+ intraepithelial lymphocytes (IEL) express CD8 alpha alpha or CD8 alphabeta, and the majority of TCR gammadelta+ IEL bear CD8 alpha alpha. The presence of CD8 beta enhances the signaling and adhesion properties of the CD8 alphabeta coreceptor and is necessary for efficient T cell development in the thymus, but is not required for the extrathymic maturation of CD8 alpha alpha+ IEL. To address whether CD8 alpha alpha+ IEL express CD8 beta during their development, we examined the methylation state of cytosines in the CD8 beta gene 5' regulatory region to identify those for which the methylation state inversely correlates with expression of the CD8 beta protein. We identified four such cytosines that were demethylated in CD8 beta-expressing thymocytes and T cells. Interestingly, these cytosines were also demethylated in CD4+ lymph node T cells that had transiently expressed CD8 beta during their development. The methylation state of these cytosines was examined in DNA purified from TCR alphabeta+ CD8 alpha alpha+ and TCR alphabeta+ CD8 alphabeta+ IEL, as well as from TCR gammadelta+ CD8 alpha alpha+ and CD3- CD8 alpha alpha+ IEL. The methylation pattern for TCR alphabeta+ CD8 alpha alpha+ IEL DNA was distinct from that seen for DNA from CD4+ lymph node cells, suggesting that TCR alphabeta+ CD8 alpha alpha+ IEL have not previously expressed CD8 beta. Analysis of DNA from CD3- CD8 alpha alpha+ IEL indicated that the unique methylation pattern of the CD8 beta gene in TCR alphabeta+ CD8 alpha alpha+ IEL DNA was not due to transcription of the CD8 alpha gene or the influence of the gut microenvironment.

IL-2 and IL-7 differentially induce CD4-CD8- alpha beta TCR+NK1.1+ large granular lymphocytes and IL-4-producing cells from CD4-CD8- alpha beta TCR+NK1.1- cells: implications for the regulation of Th1- and Th2-type responses.

Effector functions of CD4-CD8- double negative (DN) alpha beta TCR+ cells were examined. Among mouse DN alpha beta TCR+ thymocytes, NK1.1+ cells expressing a canonical V alpha 14/J alpha 281 TCR but not NK1.1- cells produce IL-4 upon TCR cross-linking and IFN-gamma upon cross-linking of NK1.1 as well as TCR. Production of IL-4 but not IFN-gamma from DN alpha beta TCR+NK1.1+ cells was markedly suppressed by IL-2. Whereas V alpha 14/J alpha 281 TCR+ cells express NK1.1+, these cells are not the precursor of DN alpha beta TCR+NK1.1+CD16+B220+ large granular lymphocytes (LGL). IL-2 induces rapid proliferation and generation of NK1.1+ LGL from DN alpha beta TCR+NK1.1- but not from DN alpha beta TCR+NK1.1+ cells. LGL cells exhibit NK activity and produce IFN-gamma but not IL-4 upon cross-linking of surface TCR or NK1.1 molecules. In contrast to IL-2, IL-7 does not induce LGL cells or NK activity from DN alpha beta TCR+NK1.1- cells but induces the ability to produce high levels of IL-4 upon TCR cross-linking. Our results show that DN alpha beta TCR+ T cells have several distinct subpopulations, and that IL-2 and IL-7 differentially regulate the functions of DN alpha beta TCR+ T cells by inducing different types of effector cells.

Interleukin-4 and listeriosis.

Experimental infection of mice with Listeria monocytogenes (L. monocytogenes) has served as an appropriate model for analyzing Th1-cell-driven immune responses. Generally, Th2 responses are absent and IL-4 is not detectable. Here, we describe experimental settings under which IL-4 is detectable in listeriosis. Our data suggest that IL-4 is rapidly produced after infection. This prompt IL-4 burst seems to stimulate chemokine responses and, therefore, may participate in the regulation of the early antilisterial host response. Soon thereafter, IL-4 production wanes. At least partially this seems to be caused by downregulation of IL-4-producing CD4+ NK1+ TCR alpha beta int lymphocytes by IL-12. In the absence of IFN-gamma responsiveness, IL-4 production is demonstrable during acquired immunity against L monocytogenes, and this elevated IL-4 production apparently contributes to disease exacerbation. In conclusion, the data are consistent with a detrimental role of IL-4 in listeriosis and active control of IL-4 synthesis by the antilisterial immune response. The rapid, but transient, IL-4 burst in listeriosis probably contributes to host defense without impairing development of the acquired T-cell response because of its shortness.

Identification of the complete expressed human TCR V gamma repertoire by flow cytometry

Five of the six expressed human TCR V gamma regions (V gamma 2, 3, 4, 8 and 9) can be detected by available mAb. No mAb with specificity for the remaining V gamma 5 region has been described. In this study, we have characterized mAb 56.3, which was obtained after immunization with V gamma 2/3/4/9-negative thymic gamma delta cells. V gamma transcripts were analyzed by inverse PCR and RT-PCR in 56.3+ cell lines and clones derived from peripheral blood. mAb 56.3 recognized all cells that expressed in-frame V gamma 5 transcripts. In addition, mAb 56.3 recognized some but not all cells expressing V gamma 3, but did not react with a large panel of clones expressing V gamma 2, 4, 8 or 9. In combination with anti-V gamma 2/3/4 mAb 23D12, V gamma 5-expressing cells could be clearly identified as 56.3+23D12(-). In some donors, mAb 56.3 also recognized a small fraction of TCR alpha beta cells. At the clonal level, these cells expressed in-frame V gamma 5-J beta-C beta or V gamma 3-J beta-C beta trans-rearrangements. When mAb 56.3 was combined with V gamma 2/3/4-, V gamma 8- and V gamma 9-specific mAb, all peripheral blood gamma delta T cells were stained. Thus, mAb 56.3 supplements the panel of available TCR V gamma-specific mAb. It is now possible to analyze the complete expressed human V gamma repertoire by flow cytometry.

Reconstitution of the extrathymic intestinal T cell compartment in the absence of irradiation.

Extrathymic development of intestinal intraepithelial lymphocytes was studied using a reconstitution model that does not require irradiation. WBB6F1/J-Kit(W)/Kit(W-v) mice were reconstituted with normal fetal liver cells. In this system, reduced c-Kit activity in host hemopoietic progenitors imparts normal precursors with a growth advantage and, thus, chimerism can be established without irradiation. In control mice, TCR gammadelta and TCR alphabeta intraepithelial lymphocytes (IEL) developed efficiently from fetal liver cells, with a predominance of TCR alphabeta over TCR gammadelta IEL. In contrast, development in reconstituted thymectomized mice was heavily skewed toward TCR gammadelta IEL generation. In thymectomized mice, development of CD4+ 8- and CD4+ 8+ TCR alphabeta IEL did not occur, while TCR alphabeta CD8 alphabeta development was nearly absent. The results indicated that without irradiation the majority of TCR alphabeta IEL were thymus dependent, whereas TCR gammadelta IEL developed extrathymically. Thus, the discrepancies observed between different models of athymic development may be explained by the induction of T cell development as a result of irradiation.