Activation and peripheral expansion of murine T-cell receptor gamma delta intraepithelial lymphocytes

BACKGROUND & AIMS: The intestinal epithelial compartment is populated by CD8(+) alpha beta and gamma delta intraepithelial lymphocytes (IELs), which monitor the integrity of the epithelial barrier. alpha beta IELs are activated by peptide antigens presented by class I major histocompatibility complex (MHC) molecules, but it is unclear how gamma delta IELs are activated. METHODS: G8 T-cell receptor (TCR) gamma delta transgenic (Tg) mice (specific for the class I MHC alloantigen, T22/10(b)) were crossed to class I MHC-deficient beta2-microglobulin-knockout (beta2m degrees) mice, and Tg+ IELs were examined for relative yields and surface and functional phenotype. RESULTS: Evidence for class I MHC-induced activation of Tg+ IELs was supported by the detection of 4-fold greater numbers of Tg+ IELs in G8 x beta2m+ mice that proliferated at 15-fold higher levels than IELs from G8 x beta2m degrees mice. However, expression of CD69, production of cytokine (interleukin 2 and interferon gamma), and detection of cytolytic function for IELs in G8 x beta2m degrees mice suggested that class I MHC was not required for gamma delta IEL development or maturation. CONCLUSIONS: These results suggest that CD8(+) TCR gamma delta IELs do not require class I MHC for development but support the notion that antigens presented by class I MHC molecules are involved in the peripheral expansion and differentiation of this subset.     

A potential role for interleukin-15 in the regulation of human natural killer cell survival

Resting lymphocyte survival is dependent upon the expression of Bcl-2, yet the factors responsible for maintaining lymphocyte Bcl-2 protein expression in vivo are largely unknown. Natural killer (NK) cells are bone marrow-derived lymphocytes that constitutively express the beta and common gamma(c) subunits of the IL-2 receptor (R) as a heterodimer with intermediate affinity for IL-2. IL-15 also binds to IL-2Rbeta gamma(c) and is much more abundant in normal tissues than IL-2. Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2R gamma(c) do not have NK cells. Further, treatment of mice with an antibody directed against IL-2Rbeta results in a loss of the NK cell compartment. These data suggest that a cytokine other than IL-2, which binds to IL-2Rbeta gamma(c), is important for NK cell development and survival in vivo. In the current report, we show that the recently described IL-15R(alpha) subunit cooperates with IL-2Rbeta gamma(c) to transduce an intracellular signal at picomolar concentrations of IL-15. We demonstrate that resting human NK cells express IL-15R(alpha) mRNA and further, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 d in the absence of serum. NK cell survival was not sustained by other monocyte-derived factors (i.e., TNF-alpha, IL-1beta, IL-10, IL-12) nor by cytokines known to use gamma(c) for signaling (i.e., IL-4, IL-7, IL-9, IL- 13). One mechanism by which IL-15 promotes NK cell survival may involve the maintenance of Bcl-2 protein expression. Considering these functional properties of IL-15 and the fact that it is produced by bone marrow stromal cells and activated monocytes, we propose that IL-15 may function as an NK cell survival factor in vivo.     

The relation between arterial oxygen tension and cerebral blood flow during cardiopulmonary bypass

BACKGROUND: The precise mechanisms involved in islet xenograft rejection remain unknown. The purpose of the present study was to determine cellular mechanisms responsible for islet xenograft rejection in the liver to facilitate finding a procedure for prevention of immune rejection. METHODS: Hepatic mononuclear cells (MNC) as well as splenocytes, peripheral blood MNC, and thymocytes from streptozotocin-induced diabetic mice (BALB/c) rejecting the intrahepatic rat (Lewis) islet xenografts were isolated and examined by two-color FACS analysis. RESULTS: The characteristic finding of the hepatic MNC from the mice rejecting islet xenografts compared with mice receiving isografts was a significant increase in the yield as well as in the percentage of the cells expressing CD3+ interleukin-2 receptor (IL-2R) alpha- beta+, CD3+ CD8alpha+ beta+, and T cell receptor (TCR) alphabeta+ lymphocyte function-associated antigen-1+. The expression of CD3 and TCR alphabeta of these T cells was found to be of intermediate intensity (TCR(int) cells). The expansion of these TCR(int) cells occurred predominantly in the liver. There was no significant difference in the cells expressing CD3+ IL-2R alpha+, CD3+ CD4+, CD3+ TCRgammadelta+, CD3- IL-2Rbeta+ (natural killer cells), and B220+ (B cells). In vivo administration of anti-IL-2Rbeta monoclonal antibody directed to the expanded cells produced a prevention of rejection. CONCLUSIONS: These findings suggest that islet xenograft rejection in the liver from rat to mouse is an event for which the TCR(int) cells are responsible.     

The structure of ClpP at 2.3 A resolution suggests a model for ATP-dependent proteolysis

Previous studies in congenitally athymic nude rats have suggested that the thymus is important for the development of intestinal T cells. Here we have examined the effect of the nude mutation on intraepithelial lymphocyte (IEL) development from the perinatal period. By immunohistochemistry it was shown that CD3(-)CD8 alpha alpha + putative IEL precursors colonized the epithelium of both normal and athymic neonatal rats. Mature T cells, however, did not develop in athymic neonates. In normal rats, gamma delta T cells were present at birth and alpha beta T cells appeared within 8 days of postnatal life. At this age, the composition and relative number of intraepithelial T cells were similar to that in normal adult rats, with the exception that most neonatal T-cell receptor-gamma delta + and -alpha beta + IEL expressed CD8 beta. By contrast, extrathymic T-cell maturation in the gut of congenitally athymic rats occurred slowly, as CD3+ IEL did not appear until 4-6 months of age. These intraepithelial T cells displayed variable phenotypes and appeared to be induced by environmental antigens as they were not found in isolator-kept old nudes. In conclusion, the present results indicate that the major colonization of the gut epithelium with gamma delta and alpha beta T cells expressing CD8 alpha beta takes place perinatally and requires the presence of the thymus. The developmental relationship between these neonatal T cells and more immature CD3- CD8 alpha alpha +/- IEL remains elusive.     

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.     

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.     

TCR-gamma delta cells in CD3 zeta-deficient mice contain Fc epsilon RI gamma in the receptor complex but are specifically unresponsive to antigen

Unlike TCR-alpha beta cells, TCR-gamma delta cells express a distinct member of the zeta family, the gamma-chain of Fc epsilon RI (Fc epsilon RI gamma) within the TCR complex. To study the role of the Fc epsilon RI gamma-chain in TCR-gamma delta cells, a TCR-gamma delta transgenic mouse (G8) has been crossed with CD3 zeta-chain-deficient mice (G8.zeta-/-). Thy-1+ spleen and lymph node cells of these animals expressed low levels of CD3/TCR. These results suggested that the zeta-chain is required for effective TCR transport to the cell surface. In contrast, intraepithelial TCR-gamma delta cells of G8.zeta-/- mice expressed high levels of TCR. Immunoprecipitation with anti-CD3 showed that Fc epsilon RI gamma-chains were associated with the TCR complex in T cells isolated from zeta-deficient mice. Although the Fc epsilon RI gamma-expressing T cells proliferated in response to stimulation by TCR-specific Abs including anti-CD3 epsilon, anti-pan gamma delta, and anti-V gamma 2 mAb, the G8.zeta-/- T cells did not respond to the G8-specific Ag (T10b), anti-Thy-1 mAb, or Con A. The unresponsiveness to the Ag was not due to the reduced TCR expression, because intraepithelial TCR-gamma delta cells from the zeta-deficient mice did not respond to Ag. The inability of the G8.zeta-/- T cells to respond to Ag could not be overcome by providing an anti-CD28 costimulatory signal or by adding exogenous rIL-2. Taken together, our data suggest that the Fc epsilon RI gamma-chain associates with the TCR-gamma delta complex in the absence of the zeta-chain, but it is not able to substitute for the zeta-chain for effective transport of TCR to the cell surface or functional responses to Ag.     

CD8 dimer usage on alpha beta and gama delta T lymphocytes from equine lymphoid tissues

Eight murine monoclonal antibodies (mAb) were used to identify the equine CD8 alpha or CD8 beta chains and to define the expression of these chains on lymphocytes from various lymphoid tissues. CD8 alpha was a 39 kDa protein and CD8 beta was a 32 kDa protein. Both chains were expressed on most of the CD8+ T lymphocytes in the peripheral blood, spleen, thymus, mesenteric lymph nodes and ileal intraepithelial lymphocytes (IEL), however, in each lymphoid compartment a percentage of lymphocytes expressed only the CD8 alpha chain. The largest percentage of CD8 alpha alpha expressing T lymphocytes was 37.7% of the IELs. Purified T lymphocytes from the ileum expressing CD8 alpha beta co-expressed the alpha beta T cell receptor (TCR). In contrast, purified CD8+ T lymphocytes from the PBMC co-expressed either the alpha beta or gamma delta TCR by RT-PCR. Use of pooled anti-CD8 alpha mAb of the murine IgG2a isotype and rabbit complement resulted in lysis of the entire CD8 expressing population in peripheral blood mononuclear cells (PBMC). These results indicated that CD8 dimer usage by equine T lymphocytes is similar to other species and that the mAb described can be further used to separate equine CD8+ T lymphocyte subsets from the lymphoid tissues to define their function in protection against viral and other infections.     

Lamina propria T cell subsets in the small and large intestine of euthymic and athymic mice

We investigated lamina propria T cells from the small intestine (jejunum/ileum) and the large intestine (colon) of euthymic (BALB/c, C.B-17, C57BL/6) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/xid) mice. CD3+ T cells represented about 40% of the lamina propria lymphocytes (LPL) from the small or the large intestine of euthymic mice, and 20-30% of the LPL populations from the small or large intestine of athymic mice. In the lamina propria T cell population of the small intestine, 85% were of the alpha beta lineage in euthymic mice, but only 40% were of the alpha beta lineage in athymic mice. T cells of the gamma delta lineage were thus more frequent than T cells of the alpha beta lineage in the intestinal lamina propria T cells of extrathymic origin. CD4+ T cells represented 40% of the lamina propria T cells in the small as well as in the large intestine of euthymic mice, and 20-30% of the T cells in the lamina propria of the nude mouse gut. In euthymic mice, 40% of the T cells in the small intestine lamina propria, and 30% of the T cells in the colonic lamina propria were CD8+. In intestinal lamina propria T cell populations of athymic mice, the CD8+ T cell population was expanded. Most (60-70%) CD8+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice expressed the homodimeric CD8 alpha + beta- form of the CD8 coreceptor. A fraction of 15-20% of all CD3+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice were 'double negative' CD4- CD8-. A large fraction of the TCR alpha beta + T cells in the colonic lamina propria (but not in the small intestine lamina propria) of euthymic mice expressed the CD2 and the CD28 costimulator molecules, the adhesion molecule LECAM-1 (CD62 L), and could be activated in vitro by CD3 ligation. These data reveal a considerable heterogeneity in the surface phenotype and the functional phenotype of murine lamina propria T cells.     

Sulcotrione soil metabolism in summer corn crops

The IL-15 receptor alpha subunit (IL-15Ralpha) mediates high-affinity binding of IL-15, a pleiotropic cytokine implicated in the development of innate immune cells. We have generated IL-15Ralpha null (IL-15Ralpha-/-) mice to understand the role of IL-15Ralpha in immune development and function. IL-15Ralpha-/- mice are markedly lymphopenic despite grossly normal T and B lymphocyte development. This lymphopenia is due to decreased proliferation and decreased homing of IL-15Ralpha-/- lymphocytes to peripheral lymph nodes. These mice are also deficient in natural killer cells, natural killer T cells, CD8+ T lymphocytes, and TCRgammadelta intraepithelial lymphocytes. In addition, memory phenotype CD8+ T cells are selectively reduced in number. Thus, IL-15Ralpha has pleiotropic roles in immune development and function, including the positive maintenance of lymphocyte homeostasis.     

Lineage relationships of the fetal thymocyte subset that expresses the beta chain of the interleukin-2 receptor

The beta chain (p75) of the interleukin-2 (IL-2) receptor (IL-2R) is expressed on up to 5-7% of fetal thymocytes on day 16 of gestation, declining thereafter to a minute proportion of less than 1% around birth, and of 1-2% of adult thymocytes. A significant part of fetal IL-2R beta+ thymocytes are gamma delta cells. The precursor-progeny relationships of fetal IL-2R beta+ thymocytes to the alpha beta T cell lineage have not been previously studied, nor has their position within the developmental sequence been determined. Here we show that IL-2R beta is expressed on a subset of very immature cells, along with high amounts of Pgp1 and Fc gamma RII/III, partially preceding the expression of intracellular CD3 epsilon. IL-2-R beta disappears before expression of IL-2R alpha. IL-2R beta+ cells, purified by sorting on day 15 of gestation, efficiently reconstituted fetal thymic lobes depleted of lymphoid cells by treatment with desoxyguanosine. They developed into T cell receptor (TCR) alpha beta+, TCR gamma delta+, and CD4/CD8 double- and single-positive cells in similar proportions as did sorted IL-2R alpha+ day 15 fetal thymocytes. These data suggest that IL-2R beta expression marks a short period of very early thymocyte development, perhaps immediately after entry into the thymus.     

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.     

Phenotypes and invariant alpha beta TCR expression of peripheral V alpha 14+ NK T cells

A novel subset of peripheral T cells, peripheral NK T cells, is found to be a major population comprising 5% of splenic T and 40% of bone marrow T cells. The majority of peripheral NK T cells are characterized by the expression of an invariant TCR-alpha encoded by V alpha 14/J alpha 281 with a one nucleotide N region. Moreover, a specific reduction of V alpha 14+ NK T cells has been demonstrated to be tightly associated with various autoimmune diseases, indicating their decisive role in autoimmune disease development. In this study, we investigated the phenotypes of peripheral V alpha 14+ NK T cells and their TCR-beta repertoire. Peripheral V alpha 14+ NK T cells, comprise two populations, i.e., small and large sized cells, at an equal frequency, belonged to the CD4- CD8- fraction, and are heat stable antigen(bright), macrophage-1bright, B220bright, CD45RBdim, and Mel-14dim, but CD5-, distinct from thymic NK T cells. TCR-beta analysis clearly showed that peripheral V alpha 14+ NK T cells utilized two to three dominant invariant TCR-beta, such as V beta 8.2 D beta J beta 2.5/V beta 7 D beta J beta 2.1 in the spleen and liver, V beta 8.2 D beta J beta 2.5/V beta 8.3 D beta J beta 2.2/V beta 7 D beta J beta 2.6 in the bone marrow, and V beta 7 D beta J beta 2.1/V beta 3 D beta J beta 1.2 in intestinal intraepithelial lymphocytes. Judging from the unusual surface phenotypes, such as heat stable antigen, macrophage-1, B220, CD45RBdim, and Mel-14dim, which are known to be T cell activation markers, peripheral V alpha 14+ NK T cells may always be activated under physiologic conditions, resulting in the oligoclonal expansion of V alpha 14+ NK T cells with different invariant TCR-beta in different peripheral organs. The unique features of V alpha 14+ NK T cells are discussed.     

Requirement of IL-4 and liver NK1+ T cells for concanavalin A-induced hepatic injury in mice

Con A-induced hepatic injury of mice accompanied by elevated transaminase was inhibited after in vivo depletion of liver NK cells and NK1+ T cells with intermediate TCR by anti-NK1 Ab or anti-IL-2Rbeta Ab. However, depletion of liver NK cells alone by anti-asialo-GM1 Ab did not inhibit hepatic injury. Although depletion of NK1+ T cells inhibited Con A-induced IL-2R expression of CD4+ high TCR (TCRhigh) cells and IL-4 mRNA expression of hepatic mononuclear cells, exogenous IL-4 engendered Con A-induced hepatic injury and endowed the expression of IL-2R of CD4+ TCRhigh cells. It was also found that in vivo treatment with anti-IL-4 Ab before Con A administration inhibited Con A-induced hepatic injury. In addition, although Con A did not induce hepatic injury in MHC class I-deficient mice, exogenous IL-4 again engendered severe hepatic injury in these mice. Further, while serum TNF-alpha levels induced by Con A were greatly decreased in NK1+ T cell-depleted mice and class I-deficient mice, TNF-alpha levels were recovered by exogenous IL-4. These findings reveal that although CD4+ TCRhigh cells in the liver and their production of TNF-alpha are the direct effectors of Con A-induced hepatic injury, liver NK1+ T cells also play an important role in this hepatitis model. Con A hepatitis may serve as an experimental model for human autoimmune hepatitis.     

Expression of the mucosal homing receptor alpha4beta7 correlates with the ability of CD8+ memory T cells to clear rotavirus infection

The integrin alpha4beta7 plays an important role in lymphocyte homing to mucosal lymphoid tissues and has been shown to define a subpopulation of memory T cells capable of homing to intestinal sites. Here we have used a well-characterized intestinal virus, murine rotavirus, to investigate whether memory/effector function for an intestinal pathogen is associated with alpha4beta7 expression. Alpha4beta7(hi) memory phenotype (CD44hi), alpha4beta7- memory phenotype, and presumptively naive (CD44(lo)) CD8+ T lymphocytes from rotavirus-infected mice were sorted and transferred into Rag-2 (T- and B-cell-deficient) recipients that were chronically infected with murine rotavirus. Alpha4beta7(hi) memory phenotype CD8+ cells were highly efficient at clearing rotavirus infection, alpha4beta7- memory cells were inefficient or ineffective, depending on the cell numbers transferred, and CD44(lo) cells were completely unable to clear chronic rotavirus infection. These data demonstrate that functional memory for rotavirus resides primarily in memory phenotype cells that display the mucosal homing receptor alpha4beta7.     

The common cytokine receptor gamma chain controls survival of gamma/delta T cells

We have investigated the role of common gamma chain (gamma c)-signaling pathways for the development of T cell receptor for antigen (TCR)-gamma/delta T cells. TCR-gamma/delta-bearing cells were absent from the adult thymus, spleen, and skin of gamma c-deficient (gamma c-) mice, whereas small numbers of thymocytes expressing low levels of TCR-gamma/delta were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-gamma/delta development through induction of V-J (variable-joining) rearrangements at the TCR-gamma locus. In contrast, we detected clearly TCR-gamma rearrangements in fetal thymi from gamma c- mice (which fail to signal in response to IL-7) and reduced TCR-gamma rearrangements in adult gamma c thymi. No gross defects in TCR-delta or TCR-beta rearrangements were observed in gamma c- mice of any age. Introduction of productively rearranged TCR V gamma 1 or TCR V gamma 1/V delta 6 transgenes onto mice bearing the gamma c mutation did not restore TCR-gamma/delta development to normal levels suggesting that gamma c-dependent pathways provide additional signals to developing gamma/delta T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from gamma c- mice were dramatically reduced compared to gamma c+ transgenic littermates. We favor the concept that gamma c-dependent receptors are required for the maintenance of TCR-gamma/delta cells and contribute to the completion of TCR-gamma rearrangements primarily by promoting survival of cells committed to the TCR-gamma/delta lineage.     

Intraepithelial lymphocytes from villus tip and crypt portions of the murine small intestine show distinct characteristics

BACKGROUND & AIMS: Intraepithelial lymphocytes (IELs) are located between epithelial cells that are thought to display unique features and functions at the small intestinal villus tip and crypt levels. We have addressed whether the spatial differences in the intestinal epithelium extend to IELs and subsequent cross-talk between IELs and epithelial cells. METHODS: IELs were isolated from villus tip and crypt portions of mouse small intestine and then compared for spontaneous cytokine production and responsiveness to interleukin (IL)-2 and/or IL-7. RESULTS: No difference was observed between number of beta IELs in villus tips and crypts, whereas a trend toward increased frequencies of IELs bearing the gamma delta form of T-cell receptor was noted in villus tips. Interestingly, the number of beta IELs producing interferon gamma and IL-5 was significantly reduced in the cells from crypts compared with villus tips. Furthermore, villus tip beta IELs exhibited higher responses to stimulation signals provided by IL-2 and/or IL-7 than their crypt counterpart. Such functional differences were not observed with gamma delta IELs from the two intestinal sites. CONCLUSIONS: Distinct molecular cross-talk between IELs and epithelial cells occurs in intestinal villus tips and crypts.     

Immunohistochemical characterization of intraepithelial and subepithelial mononuclear cells of the upper airways

The upper airway is the first site of exposure to inhaled antigens and the site of initiation of mucosal immunity to certain antigens; however, the intraepithelial lymphoid populations of this region have not been well characterized. We studied 6-mu frozen tissue sections from tonsils, adenoids, and nasal mucosae using immunohistochemistry and a panel of antibodies to mononuclear antigens to determine whether nasal mucosa contained distinctive populations of mononuclear cells. Intraepithelial lymphocytes (IELs) of nasal mucosa were CD3+, CD8+, and mainly CD5+. Tonsil and adenoid both showed diffuse CD8+ IELs; clusters of CD4+ IELs were associated with B cells within the crypt epithelium. All nasal IELs were uniformly negative for Leu8 (homing receptor analog of Mel14). Scattered Leu8-positive cells were present within tonsil and adenoid crypt epithelium only. Nasal IELs rarely expressed HML1 and were often CD7-, whereas the majority of tonsillar and adenoidal IELs were HML1+ and variably CD7+. In nasal mucosa and in deep submucosa of tonsil and adenoid, 80 to 90% of T cell receptor expression was of alpha/beta type. There was a concentration of gamma/delta T cell receptor-positive cells in intraepithelial and subepithelial zones of tonsil and adenoid, with areas of up to 30% gamma/delta T cell receptor positivity. A population of intraepithelial dendritic cells was identified in all three tissues expressing mononuclear phagocyte system antigens CD14 and KiM1P, but lacking CD1a. Virtually no B cells and no organized subepithelial lymphoid tissue were identified in nasal mucosa. Nasal mucosal lymphoid tissue seems to differ from that of endodermally derived mucosae, tonsil, and adenoids to share similarities with both mucosa-associated lymphoid tissue and peripheral lymph nodes.     

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.