Influence of beta 2-microglobulin expression on gamma interferon secretion and target cell lysis by intraepithelial lymphocytes during intestinal Listeria monocytogenes infection

Numerous microbial pathogens, including Listeria monocytogenes, enter the host through the intestine. Although relatively little is known about the biological functions of intestinal intraepithelial lymphocytes (i-IEL), they are generally considered a first line of defense against intestinal infections. In the mouse, the vast majority of i-IEL express the CD8 coreceptor either as a CD8 alpha/alpha homodimer or as a CD8 alpha/beta heterodimer. The CD8 receptor of T-cell receptor TcR gamma/delta i-IEL is exclusively homodimeric, whereas the CD8-expressing TcR alpha/beta i-IEL segregate into equal fractions of CD8 alpha/alpha and CD8 alpha/beta cells. We infected beta 2-microglobulin (beta 2m)+/- mice (possessing all i-IEL populations) and beta 2m -/- mutant mice (lacking all CD8 alpha/beta + i-IEL and having few CD8 alpha/alpha + TcR alpha/beta i-IEL) with L. monocytogenes per os and determined their biological functions after TcR ligation with monoclonal antibodies. Cytolytic activities of TcR alpha/beta and TcR gamma/delta i-IEL from beta 2m +/- mice were not influenced by intestinal listeriosis. Cytolytic activities of TcR alpha/beta i-IEL were impaired in uninfected beta 2m -/- mice, but this reduction was reestablished as a consequence of intestinal listeriosis. Frequencies of gamma interferon (IFN-gamma)-producing TcR alpha/beta i-IEL in uninfected beta 2m -/- mice were reduced, compared with that in their heterozygous controls. Equally low frequencies of IFN-gamma-producing TcR gamma/delta i-IEL in beta 2M +/- and beta 2m-/- mutants were found. Listeriosis increased frequencies of INF-gamma-producing TcR alpha/beta and TcR gamma/delta i-IEL in both mouse strains. Most remarkably, the proportion of IFN-gamma-producing TcR gamma/delta i-IEL was elevated 10-fold in listeria-infected beta 2M -/- mice. Our findings show that the beta 2m-independent CD8 beta- i-IEL expressing either TcR alpha/beta or TcR gamma/delta are stimulated by intestinal listeriosis independent of regional beta 2m expression. We conclude that the three major CD8+ i-IEL populations are stimulated by intestinal listeriosis and that CD8 beta- i-IEL compensate for the total lack of CD8 beta+ i-IEL in beta 2M -/- mutant mice. Hence, in contrast to the peripheral immune system, which crucially depends on CD8 alpha/beta + TcR alpha/beta lymphocytes, the mucosal immune system can rely on additional lymphocytes expressing the CD8 alpha/alpha homodimer.     

Plasma levels and gene expression of granulocyte colony-stimulating factor, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-8, and soluble intercellular adhesion molecule-1 in neonatal early onset sepsis

We have previously reported that T lymphocytes proliferating in vitro to the hapten trinitrochlorobenzene (TNCB) exhibit a very restricted V beta gene usage and response to TNCB is limited to T-cell receptors (TCR) composed of V beta 8.2 in combination with V alpha 3.2, V alpha 8 and V alpha 10. This paper investigates the role played by T lymphocytes expressing the V beta 8.2 gene segment in the contact sensitivity (CS) reaction to TNCB in the intact mouse and in its passive transfer into naive recipient mice. Mice injected with monoclonal antibodies to V beta 8 are unable to develop CS upon immunization with TNCB and 4-day TNCB-immune lymph node cells from mice that had been depleted in vivo or in vitro of V beta 8+ T lymphocytes fail to transfer CS. However, when separated V beta 8+ and V beta 8- cells were used for passive transfer, it was found that V beta 8+ T lymphocytes failed to transfer CS when given alone to recipient mice and a V beta 8- population was absolutely required. Further analysis revealed that within the V beta 8- population, T lymphocytes expressing the gamma delta TCR were fundamental to allow transfer of the CS reaction. These gamma delta cells were found to be antigen non-specific, genetically unrestricted and to rearrange the V gamma 3 gene segment. This indicates that transfer of the CS reaction requires cross-talk between V beta 8+ and gamma delta+ T lymphocytes, thus confirming our previous results obtained using TNCB-specific T-cell lines. Time-course experiments showed that V beta 8+ lymphocytes taken 4-24 days after immunization with TNCB were able to proliferate and produce interleukin-2 (IL-2) in response to the specific antigen in vitro. Similar time-course experiments were then undertaken using the passive transfer of the CS reaction system. The results obtained confirm that TNCB-specific V beta 8+ T lymphocytes are present in the lymph nodes of immunized mice from day 4 to day 24, and reveal that gamma delta+ T lymphocytes are active for a very short period of time, i.e. days 4 and 5 after immunization. In fact, TNCB-specific V beta 8+ cells are able to transfer CS when taken 4-24 days after immunization, providing the accompanying V beta 8- or gamma delta+ T lymphocyte are obtained 4 days after immunization. In contrast, injection of V beta 8+ T lymphocytes together with V beta 8- or gamma delta+ T lymphocytes that had been taken 2 or 6 days after immunization, failed to transfer significant CS into recipient mice. Taken together, our results confirm that cross-talk between V beta 8+ and gamma delta+ T lymphocytes is necessary for full development of the CS reaction and may explain why the CS reaction in the intact mouse lasts up to 21 days after immunization while the ability of immune lymph node cells to transfer CS is limited to days 4 and 5 after immunization.     

Thymic medulla epithelial cells acquire specific markers by post-mitotic maturation

We recently showed that secretion of non-chimeric disulfide-linked human gamma delta TCR ('soluble' TCR, sTCR) comprising V gamma 9 and V delta 2 regions could be achieved by simply introducing translational termination codons upstream from the sequences encoding TCR transmembrane region. Here we extended these findings by demonstrating efficient secretion and heterodimerization of gamma delta sTCR comprising V gamma 8, V delta 1 and V delta 3 regions, obtained via the same strategy. After immunization against immunoaffinity-purified soluble TCR, several hundreds of TCR-specific monoclonal antibodies (mAb) were generated, which fell in at least seven groups. One set of mAb was directed against a V gamma 8-specific epitope. Strikingly, despite the high degree of sequence homology between V gamma 8 and other V gamma I domains, none of these mAb were crossreactive with other members of the V gamma I family. Three other sets of mAbs were shown to recognize delta chains comprising V delta 1, V delta 2 and V delta 3 regions respectively, regardless of their junctional sequence or of the gamma chain to which they were paired. Among the V delta 1-specific mAb, some specifically recognized V delta 1D delta J delta C delta chains while others reacted with both V delta 1 D delta J delta C delta and V delta 1J alpha C alpha chains, which suggested V domain conformational alterations induced by the C region. Moreover, reactivity of one V delta 1-specific mAb (#R6.11) was affected by a polymorphic residue located on the predicted CDR4 loop of the V delta region. Two delta chain-specific mAb (#178 and #515) showed a highly unusual reactivity, which was negatively affected by particular V delta and J delta sequences: (i) mAb #515 and #178 recognized all TCR delta chains except those comprising V delta 1 or V delta 2 regions, respectively and (ii) within TCR delta chains carrying 'permissive' V delta regions, none of those comprising the J delta 2 region were recognized by #515 and/or #178 mAbs, which suggested a highly specific conformation adopted by this particular J delta sequence. Apart from its usefulness in TCR structural studies, this novel set of mAb represents an important tool for the characterization and isolation of gamma delta T cells expressing particular combinations of V gamma/V delta regions and for analysis of V alpha/V delta usage by alpha beta T cells. Moreover, since our present data strongly suggest that gamma delta TCR are easier to obtain in a soluble form than alpha beta TCR, an efficient strategy for the generation of V alpha region-specific mAb might be to immunize with chimeric gamma delta sTCR comprising particular V alpha regions.     

Murine gamma/delta-expressing T cells affect alloengraftment via the recognition of nonclassical major histocompatibility complex class Ib antigens

T cells with antidonor specificities have been isolated from human recipients experiencing graft rejection after allogeneic bone marrow transplantation (BMT). Partial T-cell depletion of unrelated BM grafts with an anti- T-cell receptor (TCR) monoclonal antibody (MoAb) directed against the TCR alpha/beta heterodimer have shown that the incidence of graft-versus-host disease is low and that the incidence of durable engraftment is high. These studies suggest either that the number of residual TCR alpha/beta+ cells was sufficient to permit alloengraftment or that the preservation of cells other than TCR alpha/beta+ cells was beneficial for engraftment. With respect to the latter, one such candidate cell is the TCR gamma/delta+ T cell. Because no studies have specifically examined whether TCR gamma/delta+ cells might be capable of eliminating BM-derived hematopoietic cells, we established a new graft rejection model system in which transgenic (Tg) H-2d mice (termed G8), known to express gamma/delta heterodimers on high proportion of peripheral T cells, were used as BMT recipients. These Tg TCR gamma/delta+ cells respond vigorously to target cells that express the nonclassical major histocompatibility complex (MHC) class lb region gene products encoded in H-2T region of H-2T(b)+ strains. G8 Tg mice were used as recipients for C57BL/6 (B6: H-2(b); H-2T(b)) T-cell-depleted (TCD) donor BM. We show that G8 Tg (H-2(d), H-2T(d)) mice are potent mediators of B6 BM graft rejection and that the rejection process was inhibited by anti-TCR gamma/delta MoAbs. In contrast, BM from a B6 congenic strain that expresses the H-2T(a) allele, B6.A-Tl(a)/BoyEg, was readily accepted, suggesting that H-2T antigens on repopulating donor BM cells are the targets of host graft rejecting T cells that express the TCR gamma/delta heterodimer. PB chimerism studies were performed at > or = 1.5 months post-BMT using TCD BM from severe combined immunodeficient allogeneic donors, which is highly susceptible to rejection by the host. The addition of donor G8 TCR gamma/delta+ cells to TCD donor BM was shown to significantly increase alloengraftment in B6 recipients. These results show that (1) host TCR gamma/delta+ cells can reject repopulating donor cells, presumably by responding to nonclassical MHC class lb gene products expressed on BM-derived hematopoietic progenitor cells; and (2) donor TCR gamma/delta+ cells can facilitate the alloengraftment of rigorously TCD donor BM.     

Preferential V beta3 usage by hepatic T lymphocytes in patients with primary sclerosing cholangitis

BACKGROUND/AIMS: Primary sclerosing cholangitis and primary biliary cirrhosis are two biliary destructive disorders characterized by prominent T lymphocyte infiltrates in areas of portal destruction. The specificity of the T cell is determined by the T cell receptor for antigens. The aim of this study was to investigate the preference by which certain V alpha and V beta gene segments are expressed by peripheral and hepatic T cells in primary sclerosing cholangitis and primary biliary cirrhosis. METHODS: The usage of the alpha/beta T cell receptor (TcR) V gene of liver infiltrating lymphocytes and peripheral blood lymphocytes from 12 primary sclerosing cholangitis patients, 10 primary biliary cirrhosis patients and healthy controls was investigated, using alpha/beta TcR V gene product-specific monoclonal antibodies. HLA class II antigen typing with genomic typing technique was done in 11/12 primary sclerosing cholangitis patients. RESULTS: A significant difference between the studied groups of patients was an increase in the expression of V beta3+ T cells in liver tissue from patients with primary sclerosing cholangitis compared to patients with primary biliary cirrhosis and healthy controls (p<0.01). No significant differences were found in the peripheral blood between the three groups. Furthermore, no relation between the different TcR V alpha/beta cells and histological staging and class II antigen association was observed. CONCLUSIONS: Predominant TcR V beta3 gene usage in liver tissue in primary sclerosing cholangitis may indicate the presence of a specific antigen in this tissue with the capacity of selectively driving T cells, utilizing the V beta3 gene segment product, in primary sclerosing cholangitis patients.     

V-D-J rearrangements at the T cell receptor delta locus in mouse thymocytes of the alpha beta lineage

The T cell receptor (TCR) delta locus lies within the TCR alpha locus and is excised from the chromosome by V alpha-J alpha rearrangement. We show here that delta sequences persist in a large fraction of the DNA from mature CD4+CD8- alpha beta+ mouse thymocytes. Virtually all delta loci in these cells are rearranged and present in extrachromosomal DNA. In immature alpha beta lineage thymocytes (CD3-/loCD4+CD8+) and in CD4+CD8- alpha beta+ thymocytes expressing a transgene-encoded alpha beta receptor, rearranged delta genes are present both in chromosomal and extrachromosomal DNA. Thus, contrary to earlier proposals, commitment to the alpha beta lineage does not require recombinational silencing of the delta locus or its deletion by a site-specific mechanism prior to V alpha-J alpha rearrangement.     

Restricted T cell receptor repertoire for acetylcholine receptor in murine myasthenia gravis

Immunization of C57BL/6 mice with AChR provokes symptoms similar to those seen in the disease myasthenia gravis. To elucidate the structural requirements for T cell recognition of AChR and to identify TcR features which might provide targets for immunotherapy, a panel of T cell hybridomas was generated after immunization of mice with the immunodominant peptide of the AChR alpha chain. The TcR genes expressed by these hybridomas were sequenced. TcR-V beta 6 was preferentially employed, but other V beta genes were also observed. A conserved acidic residue was present in all CDR3 regions, regardless of the V beta. The TcR-V alpha repertoire was somewhat skewed with three V alpha families accounting for 82% of the sequences. The utilization of multiple T cell receptor V beta genes may contribute to the inability to inhibit EAMG by elimination of V beta 6+ T cells.     

MHC-independent presentation of mycobacteria to human gamma delta T cells

The majority of human peripheral gamma delta T cells express antigen receptors using the V gamma 9 and V delta 2 gene products. Cells of this subset have been previously shown to uniformly recognize mycobacteria regardless of their V-(D)-J junctional sequences in an MHC-unrestricted manner. This reactivity superficially resembles activation of alpha beta cells by bacterial superantigens, which are thought to be presented by monomorphic regions of MHC class II molecules. It is not known whether presentation of the mycobacterial antigen to V gamma 9/V delta 2 T cells is also mediated by class II MHC molecules. In order to examine the similarity between presentation of bacterial superantigens to alpha beta T cells and the presentation of mycobacteria to gamma delta T cells we have studied the role of class II MHC molecules in presentation of the mycobacterial antigen AP-MT to V gamma 9/V delta 2 clones. Activation of gamma delta T cells by AP-MT required direct contact with antigen presenting cells, indicating that an interaction with cell surface molecules on antigen presenting cells is required. Class II MHC molecules were neither sufficient nor necessary for effective presentation of AP-MT to the gamma delta T cells, as transfectants expressing class II MHC molecules were unable to present, whereas cell lines lacking expression of MHC class II molecules could present this mycobacterial antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gamma delta T-cell help in responses to pathogens and in the development of systemic autoimmunity

Mice rendered deficient in alpha beta T-cells by single-gene knockout mutation show enhanced levels of autoantibody formation and even some symptoms of autoimmune disease. This is remarkable given that most experimental studies heretofore have indicated that the development of autoimmune disease is highly multigenic, requiring the complementary actions of multiple loci. The basis of the phenomenon in alpha beta T-cell-deficient mice appears to be the provision of help to B-cells by other cells, including gamma delta T-cells. Perhaps surprisingly, gamma delta T-cell help seems quite efficacious, particularly after infection, when it can culminate in the formation of germinal centers. Furthermore, two independent sets of studies reviewed here indicate that significant levels of self-reactive IgG can also be provoked by gamma delta T-cells independent of germinal center formation. The task ahead is to integrate this pathway into the physiologic immune responses to healthy individuals, immunocompromised individuals, and newborns.     

Human CD8+ TCR-alpha beta(+) and TCR-gamma delta(+) cells modulate autologous autoreactive neuroantigen-specific CD4+ T-cells by different mechanisms

To investigate the regulatory interactions among autologous T-cells during the course of multiple sclerosis (MS), proteolipid protein peptide-specific CD4+ T-cell clones (TCCs) were irradiated and used as immunogens to stimulate purified populations of autologous CD8+ TCR-alpha beta+ and TCR-gamma delta+ T-cells isolated from the peripheral blood of MS patients, patients with other non-inflammatory neurological diseases, and healthy blood donors. The resulting blasts were expanded in the presence of hIL-2 and then cloned by limiting dilution. Two different groups of CD8+ TCCs were revealed. A first group of CD8+ TCCs recognized autologous CD4+ T-cells based in their TCRV beta structures (anti-idiotypic responsiveness). A second group of CD8+ TCCs recognized Ag activated autologous CD4+ TCCs irrespective of their Ag specificity or TCRV beta expression (anti-ergotypic responsiveness). Both groups showed MHC class I restricted cytotoxicity against CD4+ T-cells and were able to secrete IFN-gamma, TNF alpha/beta and TGF-beta. TCR-gamma delta+ TCCs isolated in response to stimulation with autologous peptide-specific CD4+ TCCs showed only anti-ergotypic cytotoxicity, which was not inhibited by anti-MHC class Ia monoclonal antibodies. Moreover, they were able to secrete IFN-gamma and TNF alpha/beta, but not TGF-beta. These data demonstrate that regulatory mechanisms among human autologous T-cells can be mediated by cytolytic interactions or by the release of specific cytokines. Furthermore, they provide evidence that CD8+ TCR-alpha beta+ and TCR-gamma delta+ cells differ in their patterns of recognition and in their abilities to modulate the immune response mediated by autologous autoreactive CD4+ T-cells.     

A TCR alpha chain transgene induces maturation of CD4- CD8- alpha beta+ T cells from gamma delta T cell precursors

The proportion of CD4- CD8- double-negative (DN) alpha beta T cells is increased both in the thymus and in peripheral lymphoid organs of TCR alpha chain-transgenic mice. In this report we have characterized this T cell population to elucidate its relationship to alpha beta and gamma delta T cells. We show that the transgenic DN cells are phenotypically similar to gamma delta T cells but distinct from DN NK T cells. The precursors of DN cells have neither rearranged endogenous TCR alpha genes nor been negatively selected by the MIsa antigen, suggesting that they originate from a differentiation stage before the onset of TCR alpha chain rearrangements and CD4/CD8 gene expression. Neither in-frame V delta D delta J delta nor V gamma J gamma rearrangements are over-represented in this population. However, since peripheral gamma delta T cells with functional TCR beta gene rearrangements have been depleted in the transgenics, we propose that the transgenic DN population, at least partially, originates from the precursors of those cells. The present data lend support to the view that maturation signals to gamma delta lineage-committed precursors can be delivered via TCR alpha beta heterodimers.     

High expression of V gamma 8 is a shared feature of human gamma delta T cells in the epithelium of the gut and in the inflamed synovial tissue

We have analyzed the V-gene usage in gamma delta T cells of the human gut and joint by using a new mAb (B18) specific for V gamma 8 of human TCR-gamma delta+ T cells. The B18+ population constituted a minor subset of the gamma delta T cells in peripheral blood (PB) of healthy persons (6 +/- 5%) and only 1 of 35 gamma delta T cell clones analyzed was positive. In contrast, the B18+ subset was a dominant gamma delta T cell population among intraepithelial lymphocytes (IEL) derived from the human intestine (74 +/- 29, p < 0.002), and two of three IEL clones from patients with coeliac disease were B18+. Interestingly, a higher proportion of B18+ gamma delta T cells was found in the synovial fluid of patients with rheumatoid arthritis (RA) (21 +/- 18%, 0.02 < p < 0.05) compared with normal PB. Furthermore, the B18+ subset was more frequent among IL-2-expanded gamma delta T cells (42 +/- 20%) derived from synovial tissue than among IL-2-expanded cells derived from synovial fluid (p < 0.002) and PB from RA patients (p < 0.02) as well as normal PB (p < 0.002). The V-gene usage of 13 gamma delta T cell clones from the synovial fluid of arthritic patients was analyzed. All B18+ clones (n = 7) expressed mRNA for V gamma 8 together with mRNA for V delta 1 (n = 5) or mRNA for V delta 3 (n = 2). None of the B18- clones expressed V gamma 8 (n = 6). We conclude that the gamma delta T cell that expresses V gamma 8, together with mainly V delta 1, is a major gamma delta T cell subset among the IEL of the gut and a highly frequent subset in the synovial tissue of patients with RA. This subset may correspond to the mouse V gamma 7+ IEL, which has a high degree of amino acid sequence homology with the human V gamma 8 protein.     

Diminution of the number of gamma delta T lymphocytes in hepatocellular carcinoma patients treated with transcatheter arterial embolization

gamma delta T lymphocytes, which are CD3+ lymphocytes that express gamma delta chains of the T-cell antigen receptor (TCR) on their surface, are functionally distinct from alpha beta T lymphocytes, which express alpha beta chains of the TCR. gamma delta T lymphocytes are thought to differentiate in mouse hepatic sinusoids, to play a role in antitumor action, and to act as natural killer cells. The purpose of this study was to examine whether gamma delta T lymphocytes in the peripheral blood are suppressed when hepatic sinusoids are damaged during transcatheter arterial embolization (TAE). The numbers of alpha beta T lymphocytes and gamma delta T lymphocytes in the peripheral blood were examined with monoclonal antibodies and flow cytometry before and after TAE in 32 patients (from 46 to 78 years of age) with liver cirrhosis and hepatocellular carcinoma. The number of alpha beta T lymphocytes before and after TAE was unchanged. However, the number of gamma delta T lymphocytes and the ratio of gamma delta T lymphocytes to CD3+ lymphocytes were significantly decreased for 3 weeks after TAE treatment. This decrease suggests that TAE suppresses the supply of gamma delta T lymphocytes to the peripheral blood. In addition, TAE may weaken a patient's antitumor immunity, because gamma delta T lymphocytes that have antitumor activity decrease after TAE.     

Involvement of the Fas/Fas ligand pathway in activation-induced cell death of mycobacteria-reactive human gamma delta T cells: a mechanism for the loss of gamma delta T cells in patients with pulmonary tuberculosis

Although the identity of T cells involved in the protection against Mycobacterium tuberculosis (Mtb) in humans remain unknown, patients with pulmonary tuberculosis (TB) have reduced numbers of Mtb-reactive, V gamma 9+/V delta 2+ T cells in their blood and lungs. Here we have determined whether this gamma deltaT loss is a consequence of Mtb Ag-mediated activation-induced cell death (AICD). Using a DNA polymerase-mediated dUTP nick translation labeling assay, 5% or less of freshly isolated CD4+ alpha beta or gamma delta T cells from normal healthy individuals and TB patients were apoptotic. However, during culture Mtb Ags induced apoptosis in a large proportion of V gamma 9+V delta 2+ peripheral blood T cells from healthy subjects (30-45%) and TB patients (55-68%); this was increased further in the presence of IL-2. By contrast, anti-CD3 did not induce any significant level of apoptosis in gamma delta T cells from healthy subjects or TB patients. Mtb Ag stimulation rapidly induced Fas and Fas ligand (FasL) expression by gamma delta T cells, and in the presence of metalloproteinase-inhibitors >70% of gamma delta T cells were FasL+. Blockade of Fas-FasL interactions reduced the level of Mtb-mediated gamma delta T cell apoptosis by 75 to 80%. Collectively, these findings demonstrate that Mtb-reactive gamma delta T cells are more susceptible to AICD and that the Fas-FasL pathways of apoptosis is involved. AICD of gamma delta T cells, therefore, provides an explanation for the loss of Mtb-reactive T cells during mycobacterial infection.     

Chemokine expression by intraepithelial gamma delta T cells. Implications for the recruitment of inflammatory cells to damaged epithelia

T cells expressing gamma delta TCR may have evolved to recognize Ag in a different manner as well as perform a broader set of functions than T cells with alpha beta TCR. In this study, we tested the hypothesis that dendritic epidermal T cells (DETC) bearing the invariant V gamma 3V delta 1 TCR may be able to signal the migration of peripheral alpha beta T cells to the epidermis by secreting specific chemokines. Expression of macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, RANTES, and lymphotactin was inducible in DETC 7-17 cells, whereas mRNA for monocyte chemoattractant protein (MCP)-1 could not be detected. Strikingly, lymphotactin was the most abundant chemokine produced by activated DETC 7-17 cells. Activated primary DETC cultures also produced copious amounts of lymphotactin mRNA. Similarly, freshly isolated and activated intestinal intraepithelial T cells (i-IEL) with gamma delta TCR expressed high levels of lymphotactin mRNA. In contrast, lymphotactin mRNA was present in activated spleen gamma delta T cells at low basal levels. Migration of CD8+ T cells induced by culture supernatants from stimulated DETC 7-17 cells was strongly reduced in the presence of a neutralizing anti-lymphotactin antiserum and to a lesser extent by neutralizing anti-MIP-1 alpha, anti-MIP-1 beta, or anti-RANTES antiserum. The presence of lymphotactin in supernatants from activated DETC 7-17 cultures was directly demonstrated by Western blot analysis. These observations are consistent with a model in which gamma delta IEL play an active multi-faceted role in the maintenance of epithelia homeostasis.     

Clonal expansion of lung V delta 1+ T cells in pulmonary sarcoidosis

Sarcoidosis is a multisystem disease of unknown etiology characterized by the presence of noncaseating granulomas in involved tissues. To investigate a potential role for gamma/delta T cells in the pathogenesis of pulmonary sarcoidosis, we studied lung and blood T cells from patients for preferential expression of particular gamma/delta T cell receptors. An abnormally high percentage of gamma/delta cells was found in the blood of some patients. However, the increased percentage did not reflect an increase in absolute number, and appeared to be secondary to a decrease in T cells expressing alpha/beta receptors. Furthermore, as in normals, the circulating gamma/delta cells in patients predominantly expressed V gamma 9/V delta 2 receptors, a subset that was not enriched at the site of disease. In contrast, in the lung, an increased percentage of gamma/delta cells expressing V delta 1 was found in a subset of patients. Importantly, these cells demonstrated evidence of prior activation by selectively expanding in vitro in the presence of interleukin 2. Furthermore, an analysis of junctional region sequences revealed their clonal nature. These clonal expansions of V delta 1+ cells in pulmonary sarcoidosis provide evidence for a disease process that involves specific recognition of a local antigen by T cells, and contributes new information regarding the nature of the as yet undefined antigenic stimulus.     

Differential usage of T cell receptor V gene segments in CD4+ and CD8+ subsets of T lymphocytes in monozygotic twins

The TCR confers immunity by the specific recognition of foreign Ag peptides in the context of self-MHC molecules. The mechanisms controlling TCR selection and repertoire generation are not clearly understood and seem to occur in an apparently random, (self) Ag-driven manner. To address the question to what extent the TCR repertoire is randomly shaped or genetically predetermined, we have analyzed the alpha beta TCR repertoire of the CD4+ and CD8+ subsets of peripheral blood lymphocyte cultures of monozygotic twins by using the polymerase chain reaction technique with TCR V region gene family-specific oligonucleotide primers. Our studies demonstrate that there is high concordance in the overall patterns of V gene usage within a pair of twins, particularly in V beta usage (mean V beta CD4+ R2 = 0.869 and CD8+ R2 = 0.833) and to a lesser extent V alpha usage (mean V alpha CD4+ R2 = 0.621 and CD8+ R2 = 0.627); whereas the patterns between unrelated individuals show more variability. This study has also demonstrated that the V alpha and V beta genes are not randomly used within the CD4+ and CD8+ subsets. We observed significant preferential skewing of several V alpha or V beta gene families to either the CD4+ or CD8+ subset in the majority of individuals analyzed (p-value range = 0.0476 to < 0.001). In particular, V alpha 11, 17, 22, and V beta 3, 9, 12, 18 were skewed to the CD4+ subset; whereas V alpha 2, 6, 12, 15, 20 and V beta 7, 14, 17 were skewed to the CD8+ subset. Furthermore, a number of the V genes showed patterns of skewing consistent only within a pair of twins. In three pairs of twins, V beta 2 was skewed to the CD4+ subset, whereas the fourth pair used almost equal frequencies of V beta 2 in both subsets. This observation was made for the V beta 2, 4, 5, 6, 8, 19 and V alpha 7, 16, 18, 21 families. Finally, the ratio of the relative V gene usage frequency that could be observed within an individual was conserved within the sets of twins; for instance, the relative amount of V beta 2 to that of V beta 3 was higher in both individuals of one set of twins, whereas it was lower in all of the other three sets. Together these observations suggest that the predominant influence shaping the TCR repertoire is genetically predetermined, of which, HLA-predicted selection mechanisms exerted during thymic maturation might be contributing factors.     

An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus

Correlation studies between cytokines expressed in islets and autoimmune diabetes development in NOD mice and BB rats have demonstrated that beta-cell destructive insulitis is associated with increased expression of proinflammatory cytokines (IL-1, TNF alpha, and IFN alpha) and type 1 cytokines (IFN gamma, TNF beta, IL-2 and IL-12), whereas non-destructive (benign) insulitis is associated with increased expression of type 2 cytokines (IL-4 and IL-10) and the type 3 cytokine (TGF beta). Cytokines (IL-1, TNF alpha, TNF beta and IFN gamma) may be directly cytotoxic to beta-cells by inducing nitric oxide and oxygen free radicals in the beta-cells. In addition, cytokines may sensitize beta-cells to T-cell-mediated cytotoxicity in vivo by upregulating MHC class I expression on the beta-cells (an action of IFN gamma), and inducing Fas (CD95) expression on beta-cells (actions of IL-1, and possibly TNF alpha and IFN gamma). Transgenic expression of cytokines in beta-cells of non-diabetes-prone mice and NOD mice has suggested pathogenic roles for IFN alpha, IFN gamma, IL-2 and IL-10 in insulin-dependent diabetes mellitus (IDDM) development, and protective roles for IL-4, IL-6 and TNF alpha. Systemic administrations of a wide variety of cytokines can prevent IDDM development in NOD mice and/or BB rats; however, a given cytokine may retard or accelerate IDDM development, depending on the dose and frequency of administration, and the age and the diabetes-prone animal model studied (NOD mouse or BB rat). Islet-reactive CD4+ T-cell lines and clones that adoptively transfer IDDM into young NOD mice have a Th1 phenotype (IFN gamma-producing), but other islet-specific Th1 clones that produce TGF beta can adoptively transfer protection against IDDM in NOD mice. NOD mice with targeted deletions of IL-12 and IFN gamma genes still develop IDDM, albeit delayed and slightly less often. In contrast, post-natal deletions of IL-12 and IFN gamma, also IL-1, TNF alpha, IL-2, and IL-6--by systemic administrations of neutralizing antibodies, soluble receptors and receptor antagonists, and receptor-targeted cytotoxic drugs--significantly decrease IDDM incidence in NOD mice and/or BB rats. These cytokine deletion studies have provided the best evidence for pathologic roles for proinflammatory cytokines (IL-1, TNF alpha, and IL-6) and type 1 cytokines (IFN gamma, IL-2 and IL-12) in IDDM development.