Protection against colitis by CD100-dependent modulation of intraepithelial γδ T lymphocyte function.

Intraepithelial γδ T lymphocytes (γδ IEL) have important roles in repair of tissue damage at epithelial sites, such as skin and intestine. Molecules that orchestrate these γδ T-cell functions are not well defined. Recently, interaction of the semaphorin CD100 on skin γδ T cells with plexin B2 on keratinocytes was shown to be important for effective γδ T-cell function in the epidermis, which raised the possibility that CD100 may exert similar functions in the intestinal tract. In this study, we find that CD100 is expressed on all IEL, and plexin B2 is present on all epithelial cells of the mouse colon. Using the dextran sulfate sodium (DSS) mouse model of colitis, disease severity is significantly exacerbated in CD100-deficient (CD100(-/-)) mice, with increased colon ulceration and mucosal infiltration with inflammatory cells. The severe colitis in CD100(-/-) mice is attributable to the failure of the colon epithelium to mount a proliferative response to damage. Unlike wild-type γδ IEL, γδ IEL from CD100(-/-) mice fail to produce keratinocyte growth factor-1 (KGF-1) in response to DSS treatment. Administration of recombinant KGF-1 to CD100(-/-) animals ameliorates disease and reverses colitis susceptibility. These results demonstrate that CD100-mediated signals are critical for effective activation of γδ IEL to produce growth factors, including KGF-1, that are required for healing of the colon epithelium during colitis.

Intraepithelial gamma delta T cells exposed by functional genomics.

Epithelial tissues house gammadelta T cells, which are important for the mucosal immune system and may be involved in controlling malignancies, infections and inflammation. Whole-genome gene-expression analysis provides a new way to study the signals required for the activation of gammadelta T cells, their mode of action and relationships among cells of the mucosal immune system.

CD81 and CD28 costimulate T cells through distinct pathways.

We have examined the role of CD81 in the activation of murine splenic alphabeta T cells. Expression of the CD81 molecule on T cells increases following activation, raising the possibility of a role for this molecule in progression of the activation process. Using an in vitro costimulation assay, we show that CD81 can function as a costimulatory molecule on both CD4+ and CD8+ T cells. This costimulation functions independently of CD28, and unlike costimulation through CD28, is susceptible to inhibition by cyclosporin A. Strikingly, the pattern of cytokine production elicited by costimulation via CD81 is unique. IL-2 production was not up-regulated, whereas both IFN-gamma and TNF-alpha expression significantly increased. Together our results demonstrate an alternate pathway for costimulation of T cell activation mediated by CD81.

A role for epithelial gammadelta T cells in tissue repair.

My colleagues and I have a long-term interest in interactions between intraepithelial gammadelta T cells and neighboring epithelial cells. We have focused our studies on interactions in the thymus, skin, and intestine, and are investigating the development, specificity, and function of these gammadelta T cells. Our results have defined unique properties of these cells and support a specialized role for epithelial gammadelta T cells in immune surveillance, wound repair, inflammation, and protection from malignancy.

Reduced gut intraepithelial lymphocytes in VLA1 null mice.

Very late antigen 1 (VLA1) is an integrin collagen receptor that is expressed by lymphocytes in several disease states. VLA1 blockade has been shown to ameliorate gut disease in experimental graft-versus-host disease. Here we show that in the VLA1 null mouse, which is generally healthy, there is a 50% reduction in gut intraepithelial lymphocytes (IELs) despite an otherwise normal lymphocyte distribution in peripheral blood and lymphoid organs. The gammadelta to alphabeta ratios of IELs are unchanged. We also find that IL2-stimulated splenocytes from VLA1 null animals show a deficiency in adhesion to fibrillar and basement membrane collagen as well as reduced proliferation in response to collagen substratum. These results suggest that some, but not all, intraepithelial lymphocytes require VLA1 to survive or proliferate within the gut epithelium or possibly to traverse the basement membrane.

Positive and negative regulation of V(D)J recombination by the E2A proteins.

A key feature of B and T lymphocyte development is the generation of antigen receptors through the rearrangement and assembly of the germline variable (V), diversity (D), and joining (J) gene segments. However, the mechanisms responsible for regulating developmentally ordered gene rearrangements are largely unknown. Here we show that the E2A gene products are essential for the proper coordinated temporal regulation of V(D)J rearrangements within the T cell receptor (TCR) gamma and delta loci. Specifically, we show that E2A is required during adult thymocyte development to inhibit rearrangements to the gamma and delta V regions that normally recombine almost exclusively during fetal thymocyte development. The continued rearrangement of the fetal Vgamma3 gene segment in E2A-deficient adult thymocytes correlates with increased levels of Vgamma3 germline transcripts and increased levels of double-stranded DNA breaks at the recombination signal sequence bordering Vgamma3. Additionally, rearrangements to a number of Vgamma and Vdelta gene segments used predominantly during adult development are significantly reduced in E2A-deficient thymocytes. Interestingly, at distinct stages of T lineage development, both the increased and decreased rearrangement of particular Vdelta gene segments is highly sensitive to the dosage of the E2A gene products, suggesting that the concentration of the E2A proteins is rate limiting for the recombination reaction involving these Vdelta regions.

Gammadelta T cells in host defense and epithelial cell biology.

Recent studies have demonstrated increased numbers of gammadelta T cells in a variety of human infectious as well as noninfectious diseases. In some cases gammadelta T cells could be shown to destroy infected or transformed cells. Advances in the identification of ligands recognized by gammadelta T cells and the development of animal model systems to study these cells in vivo should overcome some of the major obstacles currently preventing a better understanding of gammadelta T cell function in immune responses. As we gain this knowledge it may become possible to design therapeutic strategies exploiting unique properties of gammadelta T cells to promote more effective immunity.

Integrin alpha 6A beta 1 induces CD81-dependent cell motility without engaging the extracellular matrix migration substrate.

It is well established that integrins and extracellular matrix (ECM) play key roles in cell migration, but the underlying mechanisms are poorly defined. We describe a novel mechanism whereby the integrin alpha 6 beta 1, a laminin receptor, can affect cell motility and induce migration onto ECM substrates with which it is not engaged. By using DNA-mediated gene transfer, we expressed the human integrin subunit alpha 6A in murine embryonic stem (ES) cells. ES cells expressing alpha 6A (ES6A) at the surface dimerized with endogenous beta 1, extended numerous filopodia and lamellipodia, and were intensely migratory in haptotactic assays on laminin (LN)-1. Transfected alpha 6A was responsible for these effects, because cells transfected with control vector or alpha 6B, a cytoplasmic domain alpha 6 isoform, displayed compact morphology and no migration, like wild-type ES cells. The ES6A migratory phenotype persisted on fibronectin (Fn) and Ln-5. Adhesion inhibition assays indicated that alpha 6 beta 1 did not contribute detectably to adhesion to these substrates in ES cells. However, anti-alpha 6 antibodies completely blocked migration of ES6A cells on Fn or Ln-5. Control experiments with monensin and anti-ECM antibodies indicated that this inhibition could not be explained by deposition of an alpha 6 beta 1 ligand (e.g., Ln-1) by ES cells. Cross-linking with secondary antibody overcame the inhibitory effect of anti-alpha 6 antibodies, restoring migration or filopodia extension on Fn and Ln-5. Thus, to induce migration in ES cells, alpha 6A beta 1 did not have to engage with an ECM ligand but likely participated in molecular interactions sensitive to anti-alpha 6 beta 1 antibody and mimicked by cross-linking. Antibodies to the tetraspanin CD81 inhibited alpha 6A beta 1-induced migration but had no effect on ES cell adhesion. It is known that CD81 is physically associated with alpha 6 beta 1, therefore our results suggest a mechanism by which interactions between alpha 6A beta 1 and CD81 may up-regulate cell motility, affecting migration mediated by other integrins.

Thymic cortical epithelium is sufficient for the development of mature T cells in relB-deficient mice.

Thymic development of T lymphocytes progresses as a consequence of both TCR-mediated and non-TCR-mediated interactions between thymocytes and stromal cells. As relB-deficient mice appear to lack thymic medullary epithelium and mature dendritic cells, we studied the effect of this "cortex-only" thymus on T cell development. Two major consequences were observed. First, in both relB mutant and TCR transgenic/relB mutant mice, positive selection of both TCR alpha beta and delta gamma T cells appeared to proceed normally, with export of fully functional T cells to the periphery, suggesting that the thymic medullary stromal cells are not required for full maturation of T cells nor is an organized medullary compartment required for accumulation of mature single positive CD4 and CD8 T cells. Second, thymic negative selection was impaired, as evidenced by significant autoreactive proliferative responses to normal spleen stimulators. Peripheral T cells in relB mutant mice showed an unusually high proportion of CD69+ and CD44high cells. While some of these cells may be autoreactive T cells, most of the cells appeared to be activated by cytokines produced by relB mutant nonlymphoid cells, as the effect is minimized in relB mutant bone marrow chimeras. In sum, while the TCR-mediated steps in T cell maturation require both thymic cortex and medulla (epithelium and dendritic cells) for normal positive and negative selection of the repertoire, non-TCR-mediated interactions in the thymic cortex alone are sufficient to generate mature functional T cells.

An innate view of gamma delta T cells.

Findings made during the past few years demonstrate that gamma delta T cells apparently share with macrophages a propensity to recognize nonpeptidic molecules of the kind most commonly associated with microorganisms and stressed cells. In general, recognition of these antigens by gamma delta T cells involves the antigen receptor but does not require antigen presenting cells to express MHC gene products or to have a functional antigen processing machinery. Other recent advances continue to support the notion that gamma delta T cells can perform specialized functions related to the repair of tissue damage.

T cell receptor (TCR)-induced death of immature CD4+CD8+ thymocytes by two distinct mechanisms differing in their requirement for CD28 costimulation: implications for negative selection in the thymus.

Negative selection is the process by which the developing lymphocyte receptor repertoire rids itself of autoreactive specificities. One mechanism of negative selection in developing T cells is the induction of apoptosis in immature CD4+CD8+ (DP) thymocytes, referred to as clonal deletion. Clonal deletion is necessarily T cell receptor (TCR) specific, but TCR signals alone are not lethal to purified DP thymocytes. Here, we identify two distinct mechanisms by which TCR-specific death of DP thymocytes can be induced. One mechanism requires simultaneous TCR and costimulatory signals initiated by CD28. The other mechanism is initiated by TCR signals in the absence of simultaneous costimulatory signals and is mediated by subsequent interaction with antigen-presenting cells. We propose that these mechanisms represent two distinct clonal deletion strategies that are differentially implemented during development depending on whether immature thymocytes encounter antigen in the thymic cortex or thymic medulla.

Molecular and cellular biology of dendritic epidermal T cells.

The function and specificity of gamma delta T cells remain enigmatic. Dendritic epidermal T cells (DETC) expressing an invariant gamma delta TCR represent a well established model system to investigate these key issues. Accumulating evidence supports the initial observation that recognition of a keratinocyte self-antigen by DETC proceeds without a requirement for MHC gene products. In addition, recent data have identified bioactive polypeptides expressed by DETC but not by most other T cells. For example, keratinocyte growth factor appears to be exclusively produced by activated DETC and intestinal intraepithelial gamma delta cells. These findings suggest that DETC may recognize antigen in novel ways as well as perform specialized functions complementary to those normally attributed to T cells.

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.

A role for CD81 in early T cell development.

Early stages of T cell development are thought to include a series of coordinated interactions between thymocytes and other cells of the thymus. A monoclonal antibody specific for mouse CD81 was identified that blocked the appearance of alpha beta but not gamma delta T cells in fetal organ cultures initiated with day 14.5 thymus lobes. In reaggregation cultures with CD81-transfected fibroblasts, CD4-CD8- thymocytes differentiated into CD4+CD8+ T cells. Thus, interactions between immature thymocytes and stromal cells expressing CD81 are required and may be sufficient to induce early events associated with T cell development.

T-cell development. T-cell lineage commitment revisited.

Years of controversy about the lineage relationship between alpha beta and gamma delta T cells may at last have been resolved: it now appears that most T cells derive from an identical T-lineage committed precursor.

Modulation of epithelial cell growth by intraepithelial gamma delta T cells.

The role played in immune surveillance by gamma delta T cells residing in various epithelia has not been clear. It is shown here that activated gamma delta T cells obtained from skin and intestine express the epithelial cell mitogen keratinocyte growth factor (KGF). In contrast, intraepithelial alpha beta T cells, as well as all lymphoid alpha beta and gamma delta T cell populations tested, did not produce KGF or promote the growth of cultured epithelial cells. These results suggest that intraepithelial gamma delta T cells function in surveillance and in repair of damaged epithelial tissues.

Activation and function of gamma delta T cells.

T-cell receptor gene rearrangements in gamma delta T cells have been the subject of intense molecular investigations. This year, much has been learned about the mechanisms controlling this process. However, the specificity and function of gamma delta T cells still remains enigmatic. The application of molecular technology including the availability of mutant mice lacking defined T-cell populations and immunologically relevant surface proteins is beginning to provide answers as well as some surprises.