A Distinctive γδ T Cell Repertoire in NOD Mice Weakens Immune Regulation and Favors Diabetic Disease.

Previous studies in mice and humans suggesting that γδ T cells play a role in the development of type 1 diabetes have been inconsistent and contradictory. We attempted to resolve this for the type 1 diabetes-prone NOD mice by characterizing their γδ T cell populations, and by investigating the functional contributions of particular γδ T cells subsets, using Vγ-gene targeted NOD mice. We found evidence that NOD Vγ4+ γδ T cells inhibit the development of diabetes, and that the process by which they do so involves IL-17 production and/or promotion of regulatory CD4+ αß T cells (Tregs) in the pancreatic lymph nodes. In contrast, the NOD Vγ1+ cells promote diabetes development. Enhanced Vγ1+ cell numbers in NOD mice, in particular those biased to produce IFNγ, appear to favor diabetic disease. Within NOD mice deficient in particular γδ T cell subsets, we noted that changes in the abundance of non-targeted T cell types also occurred, which varied depending upon the γδ T cells that were missing. Our results indicate that while certain γδ T cell subsets inhibit the development of spontaneous type 1 diabetes, others exacerbate it, and they may do so via mechanisms that include altering the levels of other T cells.

γδ T Cells Shape Preimmune Peripheral B Cell Populations.

We previously reported that selective ablation of certain γδ T cell subsets, rather than removal of all γδ T cells, strongly affects serum Ab levels in nonimmunized mice. This type of manipulation also changed T cells, including residual γδ T cells, revealing some interdependence of γδ T cell populations. For example, in mice lacking Vγ4(+) and Vγ6(+) γδ T cells (B6.TCR-Vγ4(-/-)/6(-/-)), we observed expanded Vγ1(+) cells, which changed in composition and activation and produced more IL-4 upon stimulation in vitro, increased IL-4 production by αß T cells as well as spontaneous germinal center formation in the spleen, and elevated serum Ig and autoantibodies. We therefore examined B cell populations in this and other γδ-deficient mouse strains. Whereas immature bone marrow B cells remained largely unchanged, peripheral B cells underwent several changes. Specifically, transitional and mature B cells in the spleen of B6.TCR-Vγ4(-/-)/6(-/-) mice and other peripheral B cell populations were diminished, most of all splenic marginal zone (MZ) B cells. However, relative frequencies and absolute numbers of Ab-producing cells, as well as serum levels of Abs, IL-4, and BAFF, were increased. Cell transfers confirmed that these changes are directly dependent on the altered γδ T cells in this strain and on their enhanced potential of producing IL-4. Further evidence suggests the possibility of direct interactions between γδ T cells and B cells in the splenic MZ. Taken together, these data demonstrate the capability of γδ T cells of modulating size and productivity of preimmune peripheral B cell populations.

γδ T cells affect IL-4 production and B-cell tolerance.

γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αß T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4-producing T cells (both residual γδ T cells and αß T cells) and in systemic IL-4 levels. Its B cells expressed IL-4-regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4-inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance.

γδ T cells recognize the insulin B:9-23 peptide antigen when it is dimerized through thiol oxidation.

The insulin peptide B:9-23 is a natural antigen in the non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). In addition to αß T cells and B cells, γδ T cells recognize the peptide and infiltrate the pancreatic islets where the peptide is produced within ß cells. The peptide contains a cysteine in position 19 (Cys19), which is required for the γδ but not the αß T cell response, and a tyrosine in position 16 (Tyr16), which is required for both. A peptide-specific mAb, tested along with the T cells, required neither of the two amino acids to bind the B:9-23 peptide. We found that γδ T cells require Cys19 because they recognize the peptide antigen in an oxidized state, in which the Cys19 thiols of two peptide molecules form a disulfide bond, creating a soluble homo-dimer. In contrast, αß T cells recognize the peptide antigen as a reduced monomer, in complex with the MHCII molecule I-A(g7). Unlike the unstructured monomeric B:9-23 peptide, the γδ-stimulatory homo-dimer adopts a distinct secondary structure in solution, which differs from the secondary structure of the corresponding portion of the native insulin molecule. Tyr16 is required for this adopted structure of the dimerized insulin peptide as well as for the γδ response to it. This observation is consistent with the notion that γδ T cell recognition depends on the secondary structure of the dimerized insulin B:9-23 antigen.

Antigen-specific regulation of IgE antibodies by non-antigen-specific γδ T cells.

We re-examined the observation that γδ T cells, when transferred from mice tolerized to an inhaled conventional Ag, suppress the allergic IgE response to this Ag specifically. Using OVA and hen egg lysozyme in crisscross fashion, we confirmed the Ag-specific IgE-regulatory effect of the γδ T cells. Although only Vγ4(+) γδ T cells are regulators, the Ag specificity does not stem from specificity of their γδ TCRs. Instead, the Vγ4(+) γδ T cells failed to respond to either Ag, but rapidly acquired Ag-specific regulatory function in vivo following i.v. injection of non-T cells derived from the spleen of Ag-tolerized mice. This correlated with their in vivo Ag acquisition from i.v. injected Ag-loaded splenic non-T cells, and in vivo transfer of membrane label provided evidence for direct contact between the injected splenic non-T cells and the Vγ4(+) γδ T cells. Together, our data suggest that Ag itself, when acquired by γδ T cells, directs the specificity of their IgE suppression.

A canonical Vγ4Vδ4+ γδ T cell population with distinct stimulation requirements which promotes the Th17 response.

We previously reported a subset of γδ T cells in mice which preferentially responds following intradermal immunization with collagen in complete Freund's adjuvant (CFA). These cells express a nearly invariant "canonical" Vγ4Vδ4+ TCR. They are potent producers of IL-17A and promote the development of collagen-induced arthritis. In this study, we report that CFA emulsified with PBS alone (without collagen) is sufficient to induce a strong response of Vγ4Vδ4+ cells in the draining lymph nodes of DBA/1 and C57BL/6 mice and that the TCRs of the elicited Vγ4Vδ4+ cells in both strains heavily favor the canonical sequence. However, although both CFA and incomplete Freund's adjuvant (which lacks the killed mycobacteria present in CFA) induced Vγ4Vδ4+ γδ T cell to expand, only CFA stimulated them to express IL-17A. The route of immunization was also critical, since intraperitoneal CFA induced only a weak response by these cells, whereas intradermal or subcutaneous CFA strongly stimulated them, suggesting that the canonical CFA-elicited Vγ4Vδ4+ cells are recruited from Vγ4+ γδ T cells normally found in the dermis. Their IL-17A response requires the toll-like receptor adapter protein MyD88, and their activation is enhanced by IFNγ, although αß T cells need not be present. The CFA-elicited Vγ4Vδ4+ γδ T cells show a cytokine profile different from that of other previously described IL-17-producing γδ T cells. Finally, the Vγ4Vδ4+ subset appears to promote the Th17 αß T cell response, suggesting its importance in mounting an effective immune response against certain pathogens.

αß TCR⁺ T cells, but not B cells, promote autoimmune keratitis in b10 mice lacking γδ T cells.

PURPOSE: To investigate additional factors in the spontaneous development of keratitis previously reported in B10.TCRδ⁻/⁻ female mice. METHODS: The study tested whether susceptible B10.TCRδ⁻/⁻ mice have dry eyes compared with resistant B6.TCRδ⁻/⁻ females and also rederived the B10.TCRδ⁻/⁻ strain to test for the role of an infectious agent. Also assessed was whether adoptive transfer of αß T cells from autoimmune mice induced keratitis in resistant mice. In addition, a potential role was examined for B cells or autoantibodies by B-cell inactivation, and the role of female hormones was tested by ovariectomy. Finally, the study investigated whether adoptive transfer of Vγ1⁺ γδ T cells confers protection. RESULTS: Tear production in B10.TCRδ⁻/⁻ females was actually higher than in B6.TCRδ⁻/⁻ controls. Rederived B10.TCRδ⁻/⁻ mice still developed keratitis. Keratitis was induced in resistant mice after adoptive transfer of αß T cells from keratitic donors. Inactivation of B cells from susceptible mice had no effect on the development of keratitis. Ovariectomy did not significantly reduce disease in B10.TCRδ⁻/⁻ females. Adoptive transfer of Vγ1⁺ cells from wild-type donors reduced keratitis in B10.TCRδ⁻/⁻ females. CONCLUSIONS: Neither low tear levels nor ovarian hormones contribute to spontaneous keratitis in B10.TCRδ⁻/⁻ female mice, nor does it appear to depend on an infectious agent carried vertically in this strain. However, αß T cells from keratitic hosts are sufficient to induce disease in the resistant B10.TCRß⁻/⁻δ⁻/⁻ strain. Autoaggressive αß T cells in the absence of Vγ1⁺ T cells in B10.TCRδ⁻/⁻ mice may be insufficiently checked to prevent disease.

Peptide antigens for gamma/delta T cells.

γδ T cells express adaptive antigen receptors encoded by rearranging genes. Their diversity is highest in the small region of TCR V-J junctions, especially in the δ chain, which should enable the γδ TCRs to distinguish differences in small epitopes. Indeed, recognition of small molecules, and of an epitope on a larger protein has been reported. Responses to small non-peptides known as phospho-antigens are multi-clonal yet limited to a single γδ T cell subset in humans and non-human primates. Responses to small peptides are multi-clonal or oligo-clonal, include more than one subset of γδ T cells, and occur in rodents and primates. However, less effort has been devoted to investigate the peptide responses. To settle the questions of whether peptides can be ligands for the γδ TCRs, and whether responses to small peptides might occur normally, peptide binding will have to be demonstrated, and natural peptide ligands identified.

The influence of IgE-enhancing and IgE-suppressive gammadelta T cells changes with exposure to inhaled ovalbumin.

It has been reported that the IgE response to allergens is influenced by gammadelta T cells. Intrigued by a study showing that airway challenge of mice with OVA induces in the spleen the development of gammadelta T cells that suppress the primary IgE response to i.p.-injected OVA-alum, we investigated the gammadelta T cells involved. We found that the induced IgE suppressors are contained within the Vgamma4(+) subset of gammadelta T cells of the spleen, that they express Vdelta5 and CD8, and that they depend on IFN-gamma for their function. However, we also found that normal nonchallenged mice harbor IgE-enhancing gammadelta T cells, which are contained within the larger Vgamma1(+) subset of the spleen. In cell transfer experiments, airway challenge of the donors was required to induce the IgE suppressors among the Vgamma4(+) cells. Moreover, this challenge simultaneously turned off the IgE enhancers among the Vgamma1(+) cells. Thus, airway allergen challenge differentially affects two distinct subsets of gammadelta T cells with nonoverlapping functional potentials, and the outcome is IgE suppression.

Allergic airway hyperresponsiveness-enhancing gammadelta T cells develop in normal untreated mice and fail to produce IL-4/13, unlike Th2 and NKT cells.

Allergic airway hyperresponsiveness (AHR) in OVA-sensitized and challenged mice, mediated by allergen-specific Th2 cells and Th2-like invariant NKT (iNKT) cells, develops under the influence of enhancing and inhibitory gammadelta T cells. The AHR-enhancing cells belong to the Vgamma1(+) gammadelta T cell subset, cells that are capable of increasing IL-5 and IL-13 levels in the airways in a manner like Th2 cells. They also synergize with iNKT cells in mediating AHR. However, unlike Th2 cells, the AHR enhancers arise in untreated mice, and we show here that they exhibit their functional bias already as thymocytes, at an HSA(high) maturational stage. In further contrast to Th2 cells and also unlike iNKT cells, they could not be stimulated to produce IL-4 and IL-13, consistent with their synergistic dependence on iNKT cells in mediating AHR. Mice deficient in IFN-gamma, TNFRp75, or IL-4 did not produce these AHR-enhancing gammadelta T cells, but in the absence of IFN-gamma, spontaneous development of these cells was restored by adoptive transfer of IFN-gamma-competent dendritic cells from untreated donors. The i.p. injection of OVA/aluminum hydroxide restored development of the AHR enhancers in all of the mutant strains, indicating that the enhancers still can be induced when they fail to develop spontaneously, and that they themselves need not express TNFRp75, IFN-gamma, or IL-4 to exert their function. We conclude that both the development and the cytokine potential of the AHR-enhancing gammadelta T cells differs critically from that of Th2 cells and NKT cells, despite similar influences of these cell populations on AHR.

Protective role of gammadelta T cells in spontaneous ocular inflammation.

PURPOSE: A role for gammadelta T cells in immunoregulation has been shown in a number of studies, but in the absence of infection or induced disease, mice lacking gammadelta T cells generally appear to be healthy. That certain mice lacking gammadelta T cells often spontaneously develop keratitis, characterized by a progressive and destructive inflammation of the cornea is reported here. METHODS: The keratitis developing in these mice was characterized in terms of prevalence in males versus females, age of onset, and histologic features. Attempts were made to understand the underlying causes of the disease by removing alphabeta T cells, altering sex hormones, and reconstituting gammadelta T cells. RESULTS: The development of keratitis in these mice depended on the C57BL/10 genetic background, and was much more common among females than males. The incidence of the disease increased with age, exceeding 80% in females greater than 18 weeks old. Evidence that the keratitis in these mice is at least partly autoimmune in nature, and that despite its prevalence in females, male hormones do not protect against the disease is presented. CONCLUSIONS: These findings indicate an important role for gammadelta T cells in maintaining immune balance in the eye. The mice described in this study represent a potential new small animal model of keratitis.

Macrophages express multiple ligands for gammadelta TCRs.

As only a handful of ligands have been identified, the general nature of the ligands recognized by gammadelta T cells remains unresolved. In this study, soluble multimerized gammadelta T cell receptors (smTCRs) representing the TCRs of two gammadelta T cell subsets common in the mouse were used to detect and track their own ligands. Ligands for both subsets were found on resident peritoneal macrophages taken from untreated mice, and the expression of both was further induced by Listeria monocytogenes infection. Nevertheless, the two types of ligand differ from one another in abundance, in the kinetics of their induction following Listeria infection, and in their ability to be induced by in vitro culture with lipopolysaccharide (LPS). Surprisingly, because both are detectable on normal macrophages, these host-derived ligands are likely expressed constitutively, but are induced to higher levels of expression by stress or inflammation. In contrast to T22 and other known cell surface ligands for gammadelta T cells in mice and humans, expression of these smTCR-defined ligands does not depend on beta2-microglobulin, suggesting that they are not MHC class I or class I-like molecules.

Airway hyperresponsiveness through synergy of gammadelta} T cells and NKT cells.

Mice sensitized and challenged with OVA were used to investigate the role of innate T cells in the development of allergic airway hyperresponsiveness (AHR). AHR, but not eosinophilic airway inflammation, was induced in T cell-deficient mice by small numbers of cotransferred gammadelta T cells and invariant NKT cells, whereas either cell type alone was not effective. Only Vgamma1+Vdelta5+ gammadelta T cells enhanced AHR. Surprisingly, OVA-specific alphabeta T cells were not required, revealing a pathway of AHR development mediated entirely by innate T cells. The data suggest that lymphocytic synergism, which is key to the Ag-specific adaptive immune response, is also intrinsic to T cell-dependent innate responses.

gammadelta T lymphocytes-selectable cells within the innate system?

Lymphocytes expressing gammadelta T cell receptors (TCR) constitute an entire system of functionally specialized subsets that have been implicated in the regulation of immune responses, including responses to pathogens and allergens, and in tissue repair. The gammadelta TCRs share structural features with adaptive receptors and peripheral selection of gammadelta T cells occurs. Nevertheless, their specificities may be primarily directed at self-determinants, and the responses of gammadelta T cells exhibit innate characteristics. Continuous cross talk between gammadelta T cells and myeloid cells is evident in histological studies and in in vitro co-culture experiments, suggesting that gammadelta T cells play a functional role as an integral component of the innate immune system.

gammadelta T-cell receptors: functional correlations.

The gammadelta T-cell receptors (TCRs) are limited in their diversity, suggesting that their natural ligands may be few in number. Ligands for gammadeltaTCRs that have thus far been determined are predominantly of host rather than foreign origin. Correlations have been noted between the Vgamma and/or Vdelta genes a gammadelta T cell expresses and its functional role. The reason for these correlations is not yet known, but several different mechanisms are conceivable. One possibility is that interactions between particular TCR-V domains and ligands determine function or functional development. However, a recent study showed that at least for one ligand, receptor specificity is determined by the complementarity-determining region 3 (CDR3) component of the TCR-delta chain, regardless of the Vgamma and/or Vdelta. To determine what is required in the TCR for other specificities and to test whether recognition of certain ligands is connected to cell function, more gammadeltaTCR ligands must be defined. The use of recombinant soluble versions of gammadeltaTCRs appears to be a promising approach to finding new ligands, and recent results using this method are reviewed.

Distribution and leukocyte contacts of gammadelta T cells in the lung.

Pulmonary gammadelta T cells protect the lung and its functions, but little is known about their distribution in this organ and their relationship to other pulmonary cells. We now show that gammadelta and alphabeta T cells are distributed differently in the normal mouse lung. The gammadelta T cells have a bias for nonalveolar locations, with the exception of the airway mucosa. Subsets of gammadelta T cells exhibit further variation in their tissue localization. gammadelta and alphabeta T cells frequently contact other leukocytes, but they favor different cell-types. The gammadelta T cells show an intrinsic preference for F4/80+ and major histocompatibility complex class II+ leukocytes. Leukocytes expressing these markers include macrophages and dendritic cells, known to function as sentinels of airways and lung tissues. The continuous interaction of gammadelta T cells with these sentinels likely is related to their protective role.

Aerosolized anti-T-cell-receptor antibodies are effective against airway inflammation and hyperreactivity.

Aerosolized monoclonal antibodies (mAbs) specific for T-cell receptors (TCR) were used to manipulate T-cell function in airways of ovalbumin (OVA)-sensitized and -challenged mice with airway hyperresponsiveness (AHR). The inhaled mAbs were found to be effective at low doses, had little or no systemic effect and specifically abrogated both effector and regulatory functions of the targeted T cells. Specific mAbs targeting alphabeta T cells suppressed and those targeting gammadelta T cells enhanced AHR. Moreover, specific mAbs directed against subsets of gammadelta T cells varied in their effect on AHR. Using this approach of targeting either alphabeta or gammadelta T cells reduced airway eosinophila, although the effect of mAbs specific for alphabeta T cells was stronger. The use of aerosolized anti-TCR mAbs may offer an effective approach for the treatment of airway inflammation and AHR.

Detection of cell surface ligands for the gamma delta TCR using soluble TCRs.

The natural ligands recognized by gammadelta TCRs are still largely unknown, in part because immunization does not normally result in Ag-specific gammadelta T cell responses. Taking advantage of an established ligand for a particular gammadelta TCR, we demonstrated that a multimerized recombinant form of this gammadelta TCR can be used like a mAb to specifically detect its own ligand. Using the same approach for more common gammadelta TCRs whose ligands remain unknown, we detected on certain cell lines molecules that appear to be ligands for three additional gammadelta TCRs. One of these represents the mouse Vgamma6/Vdelta1 invariant gammadelta TCR, which predominates in the female reproductive tract, the tongue, and the lung, and other tissues during inflammation. The second represents the closely related Vgamma5/Vdelta1 invariant gammadelta TCR expressed by most epidermal T cells. The third is a Vgamma1/Vdelta6.3 TCR, representative of a variable type frequently found on lymphoid gammadelta T cells. We found evidence that ligands for multiple gammadelta TCRs may be simultaneously expressed on a single cell line, and that at least some of the putative ligands are protease sensitive. This study suggests that soluble versions of gammadelta TCRs can be as tools to identify and characterize the natural ligands of gammadelta T cells.

Different potentials of gamma delta T cell subsets in regulating airway responsiveness: V gamma 1+ cells, but not V gamma 4+ cells, promote airway hyperreactivity, Th2 cytokines, and airway inflammation.

Allergic airway inflammation and hyperreactivity are modulated by gammadelta T cells, but different experimental parameters can influence the effects observed. For example, in sensitized C57BL/6 and BALB/c mice, transient depletion of all TCR-delta(+) cells just before airway challenge resulted in airway hyperresponsiveness (AHR), but caused hyporesponsiveness when initiated before i.p. sensitization. Vgamma4(+) gammadelta T cells strongly suppressed AHR; their depletion relieved suppression when initiated before challenge, but not before sensitization, and they suppressed AHR when transferred before challenge into sensitized TCR-Vgamma4(-/-)/6(-/-) mice. In contrast, Vgamma1(+) gammadelta T cells enhanced AHR and airway inflammation. In normal mice (C57BL/6 and BALB/c), enhancement of AHR was abrogated only when these cells were depleted before sensitization, but not before challenge, and with regard to airway inflammation, this effect was limited to C57BL/6 mice. However, Vgamma1(+) gammadelta T cells enhanced AHR when transferred before challenge into sensitized B6.TCR-delta(-/-) mice. In this study Vgamma1(+) cells also increased levels of Th2 cytokines in the airways and, to a lesser extent, lung eosinophil numbers. Thus, Vgamma4(+) cells suppress AHR, and Vgamma1(+) cells enhance AHR and airway inflammation under defined experimental conditions. These findings show how gammadelta T cells can be both inhibitors and enhancers of AHR and airway inflammation, and they provide further support for the hypothesis that TCR expression and function cosegregate in gammadelta T cells.