A tumor-suppressor function for Fas (CD95) revealed in T cell-deficient mice.

Fas (CD95) and its ligand are central regulatory molecules in hematopoietic cells. Previous studies have suggested a role for Fas in the regulation of tumor progression, but Fas has not yet been conclusively identified as a tumor suppressor. Fas-deficient individuals lack malignant tumors, perhaps because of regulation by T cells. To investigate such a possibility, mice deficient in both T cells and Fas were generated, and they were found to develop severe B cell dysregulation characterized by malignant, lethal B cell lymphoma. Lymphoma arose from a monoclonal B220+CD19-CD5-CD23- B cell secreting immunoglobulin M, kappa rheumatoid factor. In contrast, animals containing alpha beta T cells, gamma delta T cells, and/or functional Fas suppressed the development of lymphoma. These data indicate that Fas functions as a tumor suppressor, and identifies roles for both alpha beta T cells and gamma delta T cells in Fas-independent tumor regulation.

Gamma delta T cells from tolerized alpha beta T cell receptor (TCR)-deficient mice inhibit contact sensitivity-effector T cells in vivo, and their interferon-gamma production in vitro.

Contact sensitivity (CS) responses to reactive hapten Ag, such as picryl chloride (PCl) or oxazolone (OX), are classical examples of T cell-mediated immune responses in vivo that are clearly subject to multifaceted regulation. There is abundant evidence that downregulation of CS may be mediated by T cells exposed to high doses of Ag. This is termed high dose Ag tolerance. To clarify the T cell types that effect CS responses and mediate their downregulation, we have undertaken studies of CS in mice congenitally deficient in specific subsets of lymphocytes. The first such studies, using alpha beta T cell-deficient (TCR alpha -/-) mice, are presented here. The results clearly show that TCR alpha -/- mice cannot mount CS, implicating alpha beta T cells as the critical CS-effector cells. However, TCR alpha -/- mice can, after high dose tolerance, downregulate alpha +/+ CS-effector T cells adoptively transferred into them. By mixing ex vivo and then adoptive cell transfers in vivo, the active downregulatory cells in tolerized alpha -/- mice are shown to include gamma delta TCR+ cells that also can downregulate interferon-gamma production by the targeted CS-effector cells in vitro. Downregulation by gamma delta cells showed specificity for hapten, but was not restricted by the MHC. Together, these findings establish that gamma delta T cells cannot fulfill CS-effector functions performed by alpha beta T cells, but may fulfill an Ag-specific downregulatory role that may be directly comparable to reports of Ag-specific downregulation of IgE antibody responses by gamma delta T cells. Comparisons are likewise considered with downregulation by gamma delta T cells occurring in immune responses to pathogens, tumors, and allografts, and in systemic autoimmunity.

The biological activity of natural and mutant pTalpha alleles.

beta selection is a major checkpoint in early thymocyte differentiation, mediated by successful expression of the pre-T cell receptor (TCR) comprising the TCRbeta chain, CD3 proteins, and a surrogate TCRalpha chain, pTalpha. The mechanism of action of the pre-TCR is unresolved. In humans and mice, the pTalpha gene encodes two RNAs, pTalpha(a), and a substantially truncated form, pTalpha(b). This study shows that both are biologically active in their capacity to rescue multiple thymocyte defects in pTalpha(-/-) mice. Further active alleles of pTalpha include one that lacks both the major ectodomain and much of the long cytoplasmic tail (which is unique among antigen receptor chains), and another in which the cytoplasmic tail is substituted with the short tail of TCR Calpha. Thus, very little of the pTalpha chain is required for function. These data support a hypothesis that the primary role of pTalpha is to stabilize the pre-TCR, and that much of the conserved structure of pTalpha probably plays a critical regulatory role.

Germinal center formation, immunoglobulin class switching, and autoantibody production driven by "non alpha/beta" T cells.

The production of class-switched antibodies, particularly immunoglobulin (Ig) G1 and IgE, occurs efficiently in T cell receptor (TCR) alpha-/- mice that are congenitally devoid of alpha/beta T cells. This finding runs counter to a wealth of data indicating that IgG1 and IgE synthesis are largely dependent on the collaboration between B and alpha/beta T cells. Furthermore, many of the antibodies synthesized in TCR alpha-/- mice are reactive to a similar spectrum of self-antigens as that targeted by autoantibodies characterizing human systemic lupus erythematosus (SLE). SLE, too, is most commonly regarded as an alpha/beta T cell-mediated condition. To distinguish whether the development of autoantibodies in TCR alpha-/- mice is due to an intrinsic de-regulation of B cells, or to a heretofore poorly characterized collaboration between B and "non-alpha/beta T" cells, the phenotype has been reconstituted by transfer of various populations of B and non-alpha/beta T cells including cloned gamma/delta T cells derived from TCR alpha-/- mice, to severe combined immunodeficient (SCID) mice. The results establish that the reproducible production of IgG1 (including autoantibodies) is a product of non-alpha/beta T cell help that can be provided by gamma/delta T cells. This type of B-T collaboration sustains the production of germinal centers, lymphoid follicles that ordinarily are anatomical signatures of alpha/beta T-B cell collaboration. Thus, non-alpha/beta T cell help may drive Ig synthesis and autoreactivity under various circumstances, especially in cases of alpha/beta T cell immunodeficiency.

In vivo manipulation of Vgamma9Vdelta2 T cells with zoledronate and low-dose interleukin-2 for immunotherapy of advanced breast cancer patients.

The potent anti-tumour activities of gammadelta T cells have prompted the development of protocols in which gammadelta-agonists are administered to cancer patients. Encouraging results from small Phase I trials have fuelled efforts to characterize more clearly the application of this approach to unmet clinical needs such as metastatic carcinoma. To examine this approach in breast cancer, a Phase I trial was conducted in which zoledronate, a Vgamma9Vdelta2 T cell agonist, plus low-dose interleukin (IL)-2 were administered to 10 therapeutically terminal, advanced metastatic breast cancer patients. Treatment was well tolerated and promoted the effector maturation of Vgamma9Vdelta2 T cells in all patients. However, a statistically significant correlation of clinical outcome with peripheral Vgamma9Vdelta2 T cell numbers emerged, as seven patients who failed to sustain Vgamma9Vdelta2 T cells showed progressive clinical deterioration, while three patients who sustained robust peripheral Vgamma9Vdelta2 cell populations showed declining CA15-3 levels and displayed one instance of partial remission and two of stable disease, respectively. In the context of an earlier trial in prostate cancer, these data emphasize the strong linkage of Vgamma9Vdelta2 T cell status to reduced carcinoma progression, and suggest that zoledronate plus low-dose IL-2 offers a novel, safe and feasible approach to enhance this in a subset of treatment-refractory patients with advanced breast cancer.

Conservation of T cell receptor conformation in epidermal gammadelta cells with disrupted primary Vgamma gene usage.

A feature that distinguishes gammadelta T cell subsets from most alphabeta T cells and B cells is the association of expression of single T cell receptor (TCR) gamma and delta variable (V) region gene segments with specific anatomic sites. Mice lacking the TCR Vgamma5 chain normally expressed by most dendritic epidermal T cells were shown to retain a conformational determinant (idiotype) ordinarily expressed exclusively by such Vgamma5+ cells. Conservation by shuffled gammadelta TCR chains of an idiotype associated with a specific anatomic site indicates that for TCRgammadelta, as for immunoglobulin, conformation is associated to a greater extent with the function or development of lymphocyte repertoires than is the use of particular gene segments.

Regulation of cutaneous malignancy by gammadelta T cells.

The localization of gammadelta T cells within epithelia suggests that these cells may contribute to the down-regulation of epithelial malignancies. We report that mice lacking gammadelta cells are highly susceptible to multiple regimens of cutaneous carcinogenesis. After exposure to carcinogens, skin cells expressed Rae-1 and H60, major histocompatibility complex-related molecules structurally resembling human MICA. Each of these is a ligand for NKG2d, a receptor expressed by cytolytic T cells and natural killer (NK) cells. In vitro, skin-associated NKG2d+ gammadelta cells killed skin carcinoma cells by a mechanism that was sensitive to blocking NKG2d engagement. Thus, local T cells may use evolutionarily conserved proteins to negatively regulate malignancy.

Lymphoid development in mice congenitally lacking T cell receptor alpha beta-expressing cells.

Vertebrate T cells express either an alpha beta or gamma delta T cell receptor (TCR). The developmental relatedness of the two cell types is unresolved. alpha beta + T cells respond to specific pathogens by collaborating with immunoglobulin-producing B cells in distinct lymphoid organs such as the spleen and Peyer's patches. The precise influence of alpha beta + T cells on B cell development is poorly understood. To investigate the developmental effects of alpha beta + T cells on B cells and gamma delta + T cells, mice homozygous for a disrupted TCR alpha gene were generated. The homozygotes showed elimination of alpha beta + T cells and the loss of thymic medullae. Despite this, gamma delta + T cells developed in normal numbers, and there was an increase in splenic B cells.

Establishing humanized mice using stem cells: maximizing the potential.

Studies on physiology and pathology as they relate to the immune system draw heavily upon rodent models. With the increasing impetus provided by initiatives in translational medicine, the demand for ever more sophisticated, 'humanized' murine models is greater than ever. However, the design and implementation of studies in such mice is far from trivial. Here we provide a technical perspective on the increasing interest in developing humanized mice. We give examples of primary data starting with the routine procurement of human donor material, through CD34(+) cell purification prior to engraftment to injection into immunocompromised mice. Our goal is to provide practical advice to the many investigators who may be commencing or considering such studies.

Gamma(delta) T cells: non-classical ligands for non-classical cells.

A recent study describes direct binding between a gammadelta T-cell receptor and its ligand, T22, a non-classical class I major histocompatibility complex (MHC) molecule. A companion study, solving the crystal structure of T22, highlights the differences between this interaction and those of classical MHC molecules and alphabeta T cells.

Alpha beta and gamma delta T cells can share a late common precursor.

BACKGROUND: The subdivision of T cells into alpha beta and gamma delta subtypes is conserved throughout vertebrate development. The respective alpha beta and gamma delta T-cell receptors (TCRs) are encoded by somatically rearranged genes. There has been broad speculation as to whether an individual thymocyte can become either a gamma delta T cell or an alpha beta T cell as a result of stochastic gene rearrangement processes, or whether the two types of T cell are derived from separate lineages. Many of the experimental findings are apparently conflicting, however, and the issue--a basic one in immunology and development--remains unresolved. RESULTS: To address this issue, we have used the recently developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, which allows us to examine quantitatively the status of TCR gamma and delta genes in postnatal alpha beta T cells and their progenitors. Interestingly, such cells are depleted of productively rearranged delta and gamma genes, which can encode delta and gamma TCR polypeptide chains. However, in mice that can rearrange TCR delta gene segments, but in which the TCR delta gene is non-functional in other respects, no such depletion of productive rearrangements is seen. CONCLUSION: The quantitative data that we have obtained fulfill the predictions of the stochastic hypothesis: that is, a progenitor T cell first attempts to become a gamma delta T cell and, if unsuccessful, then attempts to become an alpha beta T cell. Thus, alpha beta and gamma delta T cells can derive from a common precursor thymocyte. In the simplest case, therefore, lineage-determining factors are the successful rearrangement of both gamma and delta genes before TCR alpha gene rearrangements occur, which lead to deletion of the TCR delta locus and thereby preclude further gamma delta T-cell differentiation. In contrast, successful rearrangement of the TCR beta locus remains compatible with cells becoming either gamma delta or alpha beta T cells.

Gamma delta T cell help of B cells is induced by repeated parasitic infection, in the absence of other T cells.

BACKGROUND: gamma delta T cells, like alpha beta T cells, are components of all well-studied vertebrate immune systems. Yet, the contribution of gamma delta T cells to immune responses is poorly characterized. In particular, it has not been resolved whether gamma delta cells, independent of any other T cells, can help B cells produce immunoglobulin and form germinal centers, anatomical foci of specialized T cell-B cell collaboration. RESULTS: TCR beta-/- mice, which lack all T cells except gamma delta T cells, routinely displayed higher levels of antibody than fully T cell-deficient mice. Repeated parasitic infection of TCR beta-/- mice, but not of T cell-deficient mice, increased antibody levels and induced germinal centers that contained B cells and monoclonal gamma delta cells in close juxtaposition. However, antibody specificities were more commonly against self than against the challenging pathogen. gamma delta T cell-B cell help was not induced by repeated inoculation of TCR beta-/- mice with mycobacterial antigens. CONCLUSIONS: In the absence of any other T cells, gamma delta T cell-B cell collaboration can be significantly enhanced by repeated infection. However, the lack of obvious enrichment for antibodies against the challenging pathogen distinguishes gamma delta T cell help from alpha beta T cell help induced under analogous circumstances. The increased production of generalized antibodies may be particularly relevant to the development of autoimmunity, which commonly occurs in patients suffering from alpha beta T cell deficiencies, such as AIDS.

Electric field-mediated DNA transfer: transient and stable gene expression in human and mouse lymphoid cells.

The technique of DNA transfer by electroporation was investigated in an effort to evaluate its utility for the identification of developmentally controlled regulatory sequences. Transient and stable gene expression was detected in a variety of lymphoid cell lines subjected to electroporation. No correlation existed between the levels of chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) expression and stable transfection frequency. In all lymphoid cell lines tested, the simian virus 40 early region was a better promoter than was the Rous sarcoma virus long terminal repeat.

Secondary structure and (1)H, (13)C and (15)N resonance assignments of Skint-1: a selecting ligand for a murine γδ T cell subset implicated in tumour suppression.

A study describing the (1)H, (13)C and (15)N backbone and side chain chemical shift assignments and secondary structure of Skint-1 a prototypic member of a family of mouse genes, of which Skint-1 is involved in the development of the dendritic epidermal T cell (DETC) subset of γδ T cells.

Heavy-metal modulation of the human intercellular adhesion molecule (ICAM-1) gene expression.

The intercellular adhesion molecule 1 (ICAM-1) can be induced on many different cell types by a set of various modulators (IL1 beta, TNF, LPS, IFN-gamma), which are released during the inflammatory process. We have investigated the possibility that other factors, related to the stress and biophysical perturbations of the inflammatory response, may also modulate ICAM-1. Here, we report that heavy metals, in particular zinc, can enhance the expression of the ICAM-1 gene on cells actively involved at different levels during inflammation. Kinetic studies of ICAM-1 gene expression shows a maximum level of induction 4 h after treatment with metals, followed by a rapid decrease to basal levels within 12 h. The effect on enhanced gene expression is mostly due to a rapid increase of the transcriptional rate as shown by nuclear run-on experiments. In B lymphoblastoid cells, but not in fibroblasts, the increase in RNA expression seems significantly greater that the subsequent increase in protein expression, suggesting that a further point of post-transcriptional regulation of ICAM-1 occurs and may be linked to the cellular specificity. may be linked to the cellular specificity.

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.

Genetic analysis of the essential components of the immunoprotective response to infection with Eimeria vermiformis.

The immune responses generated after infection with Eimeria spp. are complex, include both cellular and humoral components, and lead to protection against re-infection. To facilitate the rational development of the next generation of anticoccidial vaccines it is important that the nature of the immunoprotective response against infection with Eimeria spp. is determined. In this brief report we discuss results that were obtained using a combination of genetic and cellular approaches to dissect the essential immune effector components that operate against infection with Eimeria vermiformis. Mice rendered deficient of immune function by targeted gene disruption at a variety of immune loci represent an integral component of our studies and include those with targeted gene disruption at loci that encode the B- and T-cell receptors (BCR, TCR), antigen presentation molecules and immune-effector molecules. Our studies demonstrated that TCR-alpha-beta + T cells are essential for immunoprotection during both primary and secondary infection. Moreover, during primary infection the major effector cell type is a population of major histocompatibility complex class II-restricted, interferon-gamma-producing TCR-alpha-beta T cell consistent with a T helper 1 phenotype. In addition, there is a supplementary role for another class of cells (presumably T cells) that are restricted to either non-classical antigen presentation molecules or classical major histocompatibilty complex class I loaded via an atypical pathway. Mice with a deficiency in interleukin-6 were slightly more susceptible to primary infection than intact animals, consistent with the reported effects of interleukin-6 upon the generation of T helper 1-type responses in vivo. In terms of the host response to re-infection, TCR-alpha-beta T cells were essential for immunity, but the requirement for specific cell subsets and effector mechanisms was much less stringent. Mice deficient in gamma-delta T cells, classical major histocompatibility complex class I, non-classical antigen presentation pathways, the cytokines interferon-gamma, interleukin-4, interleukin-6 and the cytolytic effector molecules perforin or FasL were completely immune to secondary infection. Moreover, major histocompatibility complex class II-deficient I-A-beta-/- mice were capable of mounting a substantial response to secondary infection, manifest by a 95% reduction in oocyst output compared with primary infection. These data have important consequences for the development of immune intervention strategies and indicate that vaccine development may be targeted toward the generation of a wider range of effector mechanisms than those that operate during primary infection.

Suppression of airway inflammation by a natural acute infection of the intestinal epithelium.

Although chronic intestinal helminth infections may suppress allergen-induced airway pathology by inducing a combination of modified T-helper (Th) 2 and immunosuppressive cytokines, a similar capacity of natural acute intestinal infections has remained untested, despite their global prevalence. Here, we show that allergic airway phenotypes including eosinophilia, eotaxin mRNA, and Th2 cytokines are significantly suppressed in animals that were infected by and that have cleared the intestinal parasite Eimeria vermiformis. Unlike in helminth-infected animals, regulation requires temporal coincidence of infection with sensitization; depends on interferon-gamma; and is not associated with an enhanced antigen-specific immunoglobulin G1 response. Moreover, regulation was effective following allergen sensitization in different anatomical sites, and in young and adult mice. These data highlight a transient anatomical dissemination of "functional immunologic dominance" following infection of the gut mucosa. They strongly support the hypothesis that airway allergies are naturally suppressed by both acute and chronic mucosal pathogens, but by different mechanisms.

[gamma][delta] cells: a right time and a right place for a conserved third way of protection.

The tripartite subdivision of lymphocytes into B cells, alphabeta T cells, and gammadelta cells has been conserved seemingly since the emergence of jawed vertebrates, more than 450 million years ago. Yet, while we understand much about B cells and alphabeta T cells, we lack a compelling explanation for the evolutionary conservation of gammadelta cells. Such an explanation may soon be forthcoming as advances in unraveling the biochemistry of gammadelta cell interactions are reconciled with the abnormal phenotypes of gammadelta-deficient mice and with the striking differences in gammadelta cell activities in different strains and species. In this review, the properties of gammadelta cells form a basis for understanding gammadelta cell interactions with antigens and other cells that in turn form a basis for understanding immunoprotective and regulatory functions of gammadelta cells in vivo. We conclude by considering which gammadelta cell functions may be most critical.