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.

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.

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.

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.

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.

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.

Biological insights into TCRgammadelta+ and TCRalphabeta+ intraepithelial lymphocytes provided by serial analysis of gene expression (SAGE).

Intraepithelial lymphocytes (IELs) are abundant, evolutionarily conserved T cells, commonly enriched in T cell receptor (TCR) gammadelta expression. However, their primary functional potential and constitutive activation state are incompletely understood. To address this, serial analysis of gene expression (SAGE) was applied to murine TCRgammadelta+ and TCRalphabeta+ intestinal IELs directly ex vivo, identifying 15,574 unique transcripts that collectively portray an "activated yet resting," Th1-skewed, cytolytic, and immunoregulatory phenotype applicable to multiple subsets of gut IELs. Expression of granzymes, Fas ligand, RANTES, prothymosin beta4, junB, RGS1, Btg1, and related molecules is high, whereas expression of conventional cytokines and high-affinity cytokine receptors is low. Differentially expressed genes readily identify heterogeneity among TCRalphabeta+ IELs, whereas differences between resident TCRgammadelta+ IELs and TCRalphabeta+ IELs are less obvious.

Defining the specific physiological requirements for c-Myc in T cell development.

c-Myc is associated with cell growth and cycling in many tissues and its deregulated expression is causally implicated in cancer, particularly lymphomagenesis. However, the contribution of c-Myc to lymphocyte development is unresolved. We show here that the formation of normal lymphocytes by c-Myc-/- cells is selectively defective. c-Myc-/- cells are inefficient, in an age-dependent manner, at populating the thymus, and subsequent thymocyte maturation is ineffective: they fail to grow and proliferate normally at the late double-negative (DN) CD4-CD8- stage. Because N-Myc expression in thymocytes usually declines at the late DN stage, these results confirm that the nonredundant contributions of Myc family members to development are related to their distinct patterns of developmental gene expression.

NF-kappa B activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development.

Activation of the transcription factor NF-kappa B and pre-T cell receptor (pre-TCR) expression is tightly correlated during thymocyte development. Inhibition of NF-kappa B in isolated thymocytes in vitro results in spontaneous apoptosis of cells expressing the pre-TCR, whereas inhibition of NF-kappa B in transgenic mice through expression of a mutated, superrepressor form of I kappa B alpha leads to a loss of beta-selected thymocytes. In contrast, the forced activation of NF-kappa B through expression of a dominant-active I kappa B kinase allows differentiation to proceed to the CD4(+)CD8(+) stage in a Rag1(-/-) mouse that cannot assemble the pre-TCR. Therefore, signals emanating from the pre-TCR are mediated at least in part by NF-kappa B, which provides a selective survival signal for developing thymocytes with productive beta chain rearrangements.

An alphabeta T-cell-independent immunoprotective response towards gut coccidia is supported by gammadelta cells.

Although gammadelta cells are commonly hypothesized to provide a 'first line of defence', gammadelta-cell-deficient mice are generally only marginally more susceptible to pathogens. Because gammadelta cells are enriched within epithelia, it is important to resolve whether immunoprotective capacity towards epithelial-tropic pathogens is absent from the gammadelta-cell compartment, or whether such activity is present but simply redundant with that of alphabeta T cells. In this work, following infection of the intestinal epithelium of alphabeta T-cell-deficient mice with the coccidian parasite, Eimeria vermiformis, gammadelta cells were shown to support the rapid activation of other lymphoid cells and to confer a transferable antipathogen effect that could be eradicated by neutralization of interferon-gamma. However, unlike alphabeta T cells, these effects of gammadelta cells showed no evidence of functional immunological memory. These results are directly relevant to coccidiosis, an economically significant disease of livestock, and should have general relevance to infections involving alphabeta T-cell deficiencies, e.g. cryptosporidiosis in patients with acquired immune deficiency syndrome (AIDS).

gammadelta cells and the regulation of mucosal immune responses.

We are only now uncovering the potentially important contributions made to immune responses by gammadelta cells. These contributions are likely to be particularly important at mucosal sites, where gammadelta cells are disproportionately enriched. Indeed, gammadelta cells have proven biological activity in the lung. In addition, gammadelta cells are also enriched in young rather than adult animals. Studies of mutant mice have demonstrated that alphabeta T cells are seemingly essential for high-affinity, cognate immunological memory, whereas gammadelta cells contribute to the early stages of an immune response and to the regulation of alphabeta T cell- and B cell-mediated immunity. To explore further the role of gammadelta cells in immune responses, we have investigated whether their contribution is greater during the early period of life, when the cells are more abundant. In a natural system of coccidial infection of gut epithelial cells, we find that alphabeta T cell responses are less essential for immunoprotection during primary challenge of young mice than is true for adult animals. This "ineffectiveness" creates a "window of importance" for the immunoprotective capacity of gammadelta cells, which seem thereby to be more crucial in young compared with older animals. The relative ineffectiveness of alphabeta T cells in young mice may be attributable to a bias toward Th2 activity. We therefore hypothesize that gammadelta cell activity, elicited by infection early in life, may compensate for defects in Th1 activity and may actually accelerate the bias in alphabeta T cells away from Th2. This has obvious implications for susceptibility to Th2-type allergic responses.

Genetic dissection of primary and secondary responses to a widespread natural pathogen of the gut, Eimeria vermiformis.

Because most pathogens initially challenge the body at epithelial surfaces, it is important to dissect the mechanisms that underlie T-cell responses to infected epithelial cells in vivo. The coccidian parasites of the genus Eimeria are protozoan gut pathogens that elicit a potent, protective immune response in a wide range of host species. CD4+ alpha beta T cells and gamma interferon (IFN-gamma) are centrally implicated in the primary immunoprotective response. To define any additional requirements for the primary response and to develop a comparison between the primary and the secondary response, we have studied Eimeria infections of a broad range of genetically altered mice. We find that a full-strength primary response depends on beta(2)-microglobulin (class I major histocompatibility complex [MHC] and class II MHC and on IFN-gamma and interleukin-6 (IL-6) but not on TAP1, perforin, IL-4, Fas ligand, or inducible nitric oxide synthetase. Indeed, MHC class II-deficient and IFN-gamma-deficient mice are as susceptible to primary infection as mice deficient in all alpha beta T cells. Strikingly, the requirements for a highly effective alpha beta-T-cell-driven memory response are less stringent, requiring neither IFN-gamma nor IL-6 nor class I MHC. The class II MHC dependence was also reduced, with adoptively transferable immunity developing in MHC class II(-/-) mice. Besides the improved depiction of an immune response to a natural gut pathogen, the finding that effective memory can be elicited in the absence of primary effector responses appears to create latitude in the design of vaccine strategies.

Constitutive activation of NF-kappaB and T-cell leukemia/lymphoma in Notch3 transgenic mice.

The multiplicity of Notch receptors raises the question of the contribution of specific isoforms to T-cell development. Notch3 is expressed in CD4(-)8(-) thymocytes and is down-regulated across the CD4(-)8(-) to CD4(+)8(+) transition, controlled by pre-T-cell receptor signaling. To determine the effects of Notch3 on thymocyte development, transgenic mice were generated, expressing lck promoter-driven intracellular Notch3. Thymuses of young transgenics showed an increased number of thymocytes, particularly late CD4(-)8(-) cells, a failure to down-regulate CD25 in post-CD4(-)8(-) subsets and sustained activity of NF-kappaB. Subsequently, aggressive multicentric T-cell lymphomas developed with high penetrance. Tumors sustained characteristics of immature thymocytes, including expression of CD25, pTalpha and activated NF-kappaB via IKKalpha-dependent degradation of IkappaBalpha and enhancement of NF-kappaB-dependent anti-apoptotic and proliferative pathways. Together, these data identify activated Notch3 as a link between signals leading to NF-kappaB activation and T-cell tumorigenesis. The phenotypes of pre-malignant thymocytes and of lymphomas indicate a novel and particular role for Notch3 in co-ordinating growth and differentiation of thymocytes, across the pre-T/T cell transition, consistent with the normal expression pattern of Notch3.

[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.

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.

Signals involved in gamma/delta T cell versus alpha/beta T cell lineage commitment.

In seeking an explanation for the complexity of tissue development, biologists are obliged to explain lineage commitment, the events that dictate whether or not a progenitor cell will differentiate into one cell type or another. Such explanations have been sought across a broad spectrum of biological systems, although in no case has a full under- standing been developed. For immunologists, attention has been focused on the lineage commitment of a T cell progenitor to becoming either a gammadelta T cell or an alphabeta T cell. In this review, we compare the signals that thymocytes may receive from the pre T cell receptor (preTCR) with signalling from TCRgammadelta. These signals may determine, co-determine, facilitate, or cement the alphabeta/gammadelta lineage decision, in concert with signals from additional molecules, such as Notch and cytokine receptors. Elucidating the pleiotropic signalling events, particularly those elicited by the preTCR, may in the near future contribute to a molecular definition of lineage commitment.

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.

Widespread expression of an autoantigen-GAD65 transgene does not tolerize non-obese diabetic mice and can exacerbate disease.

Glutamic acid decarboxylase (GAD)65 is a pancreatic beta cell autoantigen implicated as a target of T cells that initiate and sustain insulin-dependent diabetes mellitus (IDDM) in humans and in non-obese diabetic (NOD) mice. In an attempt to establish immunological tolerance toward GAD65 in NOD mice, and thereby to test the importance of GAD in IDDM, we generated three lines transgenic for murine GAD65 driven by a major histocompatibility complex class I promoter. However, despite widespread transgene expression in both newborn and adult mice, T cell tolerance was not induced. Mononuclear cell infiltration of the islets (insulitis) and diabetes were at least as bad in transgenic mice as in nontransgenic NOD mice, and in mice with the highest level of GAD65 expression, disease was exacerbated. In contrast, the same transgene introduced into mouse strain, FvB, induced neither insulitis nor diabetes, and T cells were tolerant to GAD. Thus, the failure of NOD mice to develop tolerance toward GAD65 reflects at minimum a basic defect in central tolerance, not seen in animals not predisposed to IDDM. Hence, it may not be possible experimentally to induce full tolerance toward GAD65 in prediabetic individuals. Additionally, the fact that autoimmune infiltration in GAD65 transgenic NOD mice remained largely restricted to the pancreas, indicates that the organ-specificity of autoimmune disease is dictated by tissue-specific factors in addition to those directing autoantigen expression.

The expression in vivo of a second isoform of pT alpha: implications for the mechanism of pT alpha action.

A second isoform of pT alpha, "pT alpha(b)," is derived from the pT alpha locus by tissue-specific, alternative splicing. pT alpha(b) is coexpressed in the thymus with the previously characterized form of pT alpha (which we term pT alpha(a)) and is also expressed in peripheral cells without pT alpha(a). While pT alpha(a) acts to retain most TCR beta-chains intracellularly, pT alpha(b) permits higher levels of cell surface TCR beta expression and facilitates signaling from a CD3-TCR beta complex.