Preferentially expanding Vγ1+ γδ T cells are associated with protective immunity against Plasmodium infection in mice

γδ T cells play a crucial role in controlling malaria parasites. Dendritic cell (DC) activation via CD40 ligand (CD40L)‐CD40 signaling by γδ T cells induces protective immunity against the blood‐stage Plasmodium berghei XAT (PbXAT) parasites in mice. However, it is unknown which γδ T‐cell subset has an effector role and is required to control the Plasmodium infection. Here, using antibodies to deplete TCR Vγ1⁺ cells, we saw that Vγ1⁺ γδ T cells were important for the control of PbXAT infection. Splenic Vγ1⁺ γδ T cells preferentially expand and express CD40L, and both Vγ1⁺ and Vγ4⁺ γδ T cells produce IFN‐γ during infection. Although expression of CD40L on Vγ1⁺ γδ T cells is maintained during infection, the IFN‐γ positivity of Vγ1⁺ γδ T cells is reduced in late‐phase infection due to γδ T‐cell dysfunction. In Plasmodium‐infected IFN‐γ signaling‐deficient mice, DC activation is reduced, resulting in the suppression of γδ T‐cell dysfunction and the dampening of γδ T‐cell expansion in the late phase of infection. Our data suggest that Vγ1⁺ γδ T cells represent a major subset responding to PbXAT infection and that the Vγ1⁺ γδ T‐cell response is dependent on IFN‐γ‐activated DCs.     

Mechanisms determining cell membrane expression of different γδ TCR chain pairings

We investigated the ability of the most common TCR‐γ and δ chains to express on the cell surface. Vγ1Cγ4 and Vγ7Cγ1 chains paired with all TCR‐δ chains tested, whereas Vγ4Cγ1 chains were found with Vδ4 and Vδ5, but not with Vδ2 or Vδ6 chains, and Vγ2Cγ2 chains were expressed only with Vδ5. Mapping studies showed that up to four polymorphic residues influence the different co‐expressions of Vγ1 and Vγ2 chains with Vδ chains. Unexpectedly, these residues are not located in the canonical γ/δ interface, but in the outer part of the γδ TCR complex exposed to the solvent. Expression of functional Vδ4 or Vδ6 chains in Vγ2/Vδ5⁺ cells or of functional Vγ2Cγ2 in Vγ1⁺ cells reduced cell‐surface expression of the γδ TCR. Taken together, these data show that (i) the Vγ/Vδ repertoire of mouse γδ T cells is reduced by physical constraints in their associations. (ii) Lack of Vγ2/Vδ expression is due to the formation of aberrant TCR complexes, rather than to an intrinsic inability of the chains to pair and (iii) despite not being expressed at the cell surface, the presence of a functionally rearranged Vγ2 chain in γδ T cells results in reduced TCR levels.     

A novel subset of adult γδ thymocytes that secretes a distinct pattern of cytokines and expresses a very restricted T cell receptor repertoire

We have characterized the function, phenotype, ontogenic development, and T cell receptor (TCR) repertoire of a subpopulation of γδ thymocytes, initially defined by expressing low levels of Thy-1, that represents around 5 % and 30 % of total γδ thymocytes in adult C57BL/6 and DBA/2 mice, respectively. Activation of FACS-sorted Thy-1^dull γδ thymocytes from DBA/2 mice with anti-γδ monoclonal antibodies in the presence of interleukin-2 (IL-2) results in the secretion of high levels of several cytokines, including interferon-γ (IFN-γ), IL-4, IL-10, and IL-3. In contrast, only IFN-γ was detected in parallel cultures of Thy-1^bright γδ thymocytes. Virtually all Thy-1^dull γδ thymocytes express high levels of CD44 and low levels of the heat-stable antigen and CD62 ligand, while around half of them express the NK1.1 marker. Thy-1^dull γδ thymocytes are barely detectable in newborn animals, and their representation increases considerably during the first 2 weeks of postnatal life. The majority of Thy-1^dull γδ thymocytes from DBA/2 mice express TCR encoded by the Vγ1 gene and a novel Vδ6 gene named Vδ6.4. Sequence analysis of these functionally rearranged γ and δ genes revealed highly restricted Vδ-Dδ-Jδ junctions, and somewhat more diverse Vγ-Jγ junctions. We conclude that Thy-1^dull γδ thymocytes exhibit properties that are equivalent to those of natural killer TCRαβ T cells. Both cell populations produce the same distinct pattern of cytokines upon activation, share a number of phenotypic markers originally defined for activated or memory T cells, display similar postnatal kinetics of appearance in the thymus and express a very restricted TCR repertoire.     

Cytomegalovirus drives Vδ2neg γδ T cell inflation in many healthy virus carriers with increasing age

Cytomegalovirus (CMV) usually causes lifelong asymptomatic infection, but over time can distort immune profiles. Recent reports describe selective expansion of Vδ2^neg γδ T cells in healthy and immunocompromised CMV carriers. Having shown previously that virus‐specific CD8⁺ and CD4⁺ T cell responses are increased significantly in elderly CMV carriers, probably driven by chronic stimulation, we hypothesized that Vδ2^neg γδ T cells may also be expanded with age. Our results show that Vδ2^neg γδ T cells are increased significantly in CMV‐seropositive healthy individuals compared to CMV‐seronegative controls in all age groups. The differences were most significant in older age groups (P < 0·0001). Furthermore, while Vδ2^neg γδ T‐ cells comprise both naive and memory cells in CMV‐seronegative donors, highly differentiated effector memory cells are the dominant phenotype in CMV carriers, with naive cells reduced significantly in numbers in CMV‐seropositive elderly. Although phenotypically resembling conventional CMV‐specific T cells, Vδ2^neg γδ T cells do not correlate with changes in magnitude of CMV‐specific CD4⁺ or CD8⁺ T cell frequencies within those individuals, and do not possess ex‐vivo immediate effector function as shown by CMV‐specific CD4⁺ and CD8⁺ T cells. However, after short‐term culture, Vδ2^neg γδ T cells demonstrate effector T cell functions, suggesting additional requirements for activation. In summary, Vδ2^neg γδ T cells are expanded in many older CMV carriers, demonstrating a further level of lymphocyte subset skewing by CMV in healthy individuals. As others have reported shared reactivity of Vδ2^neg γδ T cells towards tumour cells, the composition of γδ T cell subsets may also have implications for risk of developing cancer in elderly people.     

Functional and molecular characterization of B cell-responsive Vδ1+ γδ T cells

Cells expressing the Vδ1⁺ gene segment are a minor γδ T cell population in human peripheral blood but predominate in epithelia and (inflamed) tissues. The characteristic dendritic-like morphology of these γδ T cells is consistent with their putative immune surveillance role in epithelia. Their function, however, remains unknown. We and others previously reported that a subset of Vδ1⁺ γδ T cells proliferates after stimulation with Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines (LCL), but not with fresh peripheral blood-derived B cells. These responses were independent of the type of T cell receptor (TcR) γ chain co-expressed with the Vδ1 chain. The in vivo relevance of this LCL-mediated activation as well as the nature of the stimulatory ligand on the LCL is not well established. In this study, we tested the proliferative response of Vδ1⁺ LCL-responsive T cells against non-EBV-transformed B cells, activated through CD40 by murine EL4 B5 cells, and to a panel of B cell lines differing in the expression of EBV nuclear antigen proteins and adhesion/co-stimulatory molecules. The role of the Epstein-Barr virus-derived antigen in the induction of this response could be excluded as the activated (non-EBV-transformed) peripheral blood B cells were also able to induce a proliferative response in the LCL-responsive Vδ1⁺ T cells. Therefore, the stimulatory ligand on B cells is of cellular rather than of viral origin, and its expression is up-regulated upon activation of B cells. The expression of B7 and CD39 molecules on the surface of activated B cells appeared to be crucial since antibodies to these structures could block the induction of proliferation of the Vδ1⁺ T cells. Finally, we investigated the diversity of the responding Vδ1⁺ γδ T cell clones by sequence analysis of the TcRδ junctional regions. No restricted V-D-J sequences were found among the LCL-responsive Vδ1⁺ T cell clones, arguing strongly against a mono- or oligoclonal Vδ1⁺ γδ T cell response to LCL. These findings may explain the presence of polyclonally activated Vδ1⁺ T cells in inflamed tissues where activated B cells are often present.     

Clonal expansions of Vδ1+ and Vδ2+ cells increase with age and limit the repertoire of human γδ T cells

We have investigated the complexity of the human γδ T cell repertoire by means of a VJ heteroduplex analysis method. cDNA obtained from peripheral blood mononuclear cells was amplified with Vδ1-Cδ or Vδ2-Cδ primers. The product was denatured and renatured to allow random reannealing of the strands and the heteroduplexes carrying mismatched junctional sequences were separated from the homoduplexes on polyacrylamide gels. Whenever one or more T cell clones were expanded to over 10% of the polyclonal background, discrete bands of homo- and heteroduplex appeared. This method was applied to the analysis of the peripheral γδ compartment from healthy donors and rheumatoid arthritis patients of different ages. While samples from young individuals showed a polyclonal pattern, a clear tendency towards oligoclonality appeared with increasing age, both in normal individuals and rheumatoid arthritis patients. We also show that the VJ junctional sequence derived from the heteroduplex fragments can be successfully used to isolate and characterize the corresponding T cell clones in vitro, even after a period of 1 year. In conclusion, our findings indicate that the complexity of the γδ T cell repertoire decreases with age as a consequence of the expansion of a few T cell clones.     

T cell receptor γδ repertoire is skewed in cerebrospinal fluid of multiple sclerosis patients: molecular and functional analyses of antigen-reactive γδ clones

To study the relevance of γδ T cells in multiple sclerosis (MS) we analyzed the T cell receptor (TCR) γδ repertoire and the antigen reactivity of γδ clones isolated from cerebrospinal fluid (CSF). In T cell cultures derived from CSF we found an increased percentage of Vδ1⁺ cells as compared to peripheral blood of the same donors. Phenotypic analysis of cells from MS CSF with Vγ- and Vγ-specific monoclonal antibodies (mAb) showed that the Vγ1 chain is most frequently associated with γ chains belonging to the VγI family. Sequence analysis of TCR genes revealed heterogeneity of junctional regions in both δ and γ genes indicating polyclonal expansion. γδ clones were established and some recognized glioblastoma, astrocytoma or monocytic cell lines. Stimulation with these targets induced serine esterase release and lymphokine expression characteristic of the T_H0-like phenotype. Remarkably, these tumor-reactive γδ cells were not detected in the peripheral blood using PCR oligotyping, but were found in other CSF lines independently established from the same MS patient. Altogether, these results demonstrate that in the CSF there is a skewed TCR γδ repertoire and suggest that γδ cells reacting against brain-derived antigens might have been locally expanded.     

Mycobacteria-reactive γδ T cells in HIV-infected individuals: lack of Vγ9 cell responsiveness is due to deficiency of antigen-specific CD4 T helper type 1 cells

Human γδ T lymphocytes expressing the variable T cell receptor elements Vγδ paired with Vδ2 are activated by antigen derived from Mycobacterium tuberculosis (M. tb.) and presented by antigen-presenting cells (APC). The subsequent proliferation is strictly dependent on the presence of CD4⁺TCRαβ⁺ T helper type 1 (Th1) cells producing interleukin-2 (IL-2). In this study, we report that the reactivity of Vγ9 cells to M. tb. stimulation in vitro was drastically decreased or absent in the majority of the analyzed HIV-1-infected individuals (CDC stages III and IV). We show that the failure of Vγ9 cells frim HIV^? individuals to proliferate following M. tb. stimulation was not due to an intrinsic qualitative or quantitative defect of γδ T cells but rather to a deficiency of M. tb.-reactive CD4 Th1 cells. Thus, Vγ9 responsiveness could be restored if cultures of M. tb.-stimulated T cells from HIV⁺ donors were reconstituted with one of the following: (i) exogenous IL-2; (ii) purified CD4T cells from allogeneic donors; or (iii) T cell-depleted APC from allogeneic donors. In the majority of HIV⁺ patients, the defective Th1 activity of M. tb.-stimulated CD4 T cells could be increased neither by cytokines known to favor Th1 development (IL-12, interferon-γ) nor by neutralization of the Th1-suppressing Th2 cytokine IL-10. We suggest that measurement of Vγ9 cell expansion within M. tb.-stimulated peripheral blood mononuclear cells provides a sensitive assay for the functional capacity of antigen (M. tb.)-specific CD4 Th1 cells in HIV-infected individuals.     

4–1BB signal stimulates the activation,expansion, and effector functions of γδ T cells in mice and humans

We show here that the expression of 4–1BB is rapidly induced in γδ T cells following antigenic stimulation in both mice and humans, and ligation of the newly acquired 4–1BB with an agonistic anti‐4–1BB augments cell division and cytokine production. We further demonstrate that γδ rather than αβ T cells protect mice from Listeria monocytogenes (LM) infection and 4–1BB stimulation enhances the γδ T‐cell activities in the acute phase of LM infection. IFN‐γ produced from γδ T cells was the major soluble factor regulating LM infection. Vγ1⁺ T cells were expanded in LM‐infected mice and 4–1BB signal triggered an exclusive expansion of Vγ1⁺ T cells and induced IFN‐γ in these Vγ1⁺ T cells. Similarly, 4–1BB was induced on human γδ T cells and shown to be fully functional. Combination treatment with human γδ T cells and anti‐hu4–1BB effectively protected against LM infection in human γδ T cell‐transferred NOD‐SCID mice. Taken together, these data provide evidence that the 4–1BB signal is an important regulator of γδ T cells and induces robust host defense against LM infection.     

The surface epithelium of recurrent infected palatine tonsils is rich in γδ T cells

Using a large panel of MoAbs in quantitative morphometric analysis of immunohistochemically stained tissue sections, we compared the frequency and distribution of immune cells in palatine tonsils from patients with recurrent tonsillitis (RT) and patients with idiopathic tonsillar hypertrophy (ITH). We found that differences between the two patient groups in leucocyte populations were limited to the surface epithelium, whereas the cellular composition of interfollicular and follicular areas was similar. Most intraepithelial lymphocytes were CD8⁺ T cells in both groups. However, the number of intraepithelial T cells was significantly higher in RT compared with ITH. This was due to a selective increase in the number of intraepithelial CD8⁺γδ T cells utilizing Vδ1 and Vγ9. In both patient groups the majority of the intraepithelial γδ T cells expressed Vδ1 and Vγ9. Subepithelially, γδ T cells utilizing Vγ9 dominated over cells utilizing Vγ8, while equal proportions expressed Vδ1 and Vδ2. These results suggest that cells utilizing the otherwise rare combination Vδ1/Vγ9 in their T cell receptors (TCR) may constitute a major γδ T cell population in palatine tonsils and are probably reactive to antigens specific to the tonsillar milieu. Furthermore, they indicate that preferentially this γδ T cell subpopulation is involved in immune reactions within the surface epithelium in RT. We speculate that γδ T cells are involved in clearing infectious bacteria at the tonsillar surface and in limiting inflammatory responses in the tonsils. Both local expansion and infiltration of blood cells probably contribute to the high numbers of γδ T cells in RT patients.     

T cell receptor heterogeneity in γδ T cell clones from intestinal biopsies of patients with celiac disease

In celiac disease large numbers of γδ T lymphocytes infiltrate the intestinal epithelia. We have isolated intestinal γδ T cell clones from patients with celiac disease and have analyzed their T cell receptor repertoire. T cell lines and clones were obtained from jejunal biopsies of 14 celiac patients and 12 individuals without celiac disease. These were analyzed by staining with monoclonal antibodies against CD3, αβ and γδ T cell receptor, by Southern blot with γ and δ specific probes and by polymerase chain reaction using Vδ-specific oligonucleotides. Intestinal γδ cells from patients with celiac disease differed from those of controls with normal jejunal histology in that Vδ1⁺ cells and Vδ Vδ2 cells were significantly increased. There was no evidence of the expansion of one or more clones expressing particular types of γδ T cell receptor.     

γδ T cells come to stay: Innate skin memory in the Aldara model

The term immunological memory has long been a trademark restricted to adaptive lymphocytes such as memory B cells and plasma cells as well as memory CD8⁺ αβ T cells. In recent years, innate lymphocytes such as NK cells have also been shown to adapt to their environment by antigen‐specific expansion and selective survival. However, whether γδ T cells mount comparable memory responses to pathogenic stimuli is less well understood. In this issue of European Journal of Immunology, Hartwig et al. [Eur. J. Immunol. 2015. 45: 3022–3033] identify a subset of IL‐17‐producing γδ T cells that are capable of establishing long‐lived memory in the skin of mice exposed to imiquimod in the Aldara psoriasis model. These γδ T cells uniformly express a Vγ4⁺Vδ4⁺ TCR. They produce IL‐17A/F and persist in the dermis for long periods of time, also at untreated distal sites. Upon secondary challenge, experienced Vγ4⁺Vδ4⁺ cells show enhanced effector functions and mediate exacerbated secondary inflammation. These findings showcase innate γδ T‐cell memory that uses a single conserved public TCR combination. Furthermore, they provide mechanistic insight to the observed psoriatic relapses in patients in response to topical treatment with imiquimod.     

Fine antigen specificity of human γδ T cell lines (Vγ9+) established by repetitive stimulation with a serotype (KTH-1) of a gram-positive bacterium,Streptococcus sanguis

We have established human γδ T cell lines specific for Streptococcus sanguis (S. sanguis) KTH-1 present in normal oral cavity flora. The CD4^?CD8^? CD3⁺Vγ9⁺Vδ1^?CD45RO⁺ CD25⁺ T cell lines showed a proliferative response to the streptococcal antigen (Ag) in the presence of autologous antigen-presenting cells without apparent evidence of HLA restriction. The proliferative response of the γδ T cell lines was completely blocked by anti-TcRγδ monoclonal antibody (mAb) and anti-HLA class I mAb (W6/32), whereas anti-HLA classical class Ia mAb (B-H9; anti-HLA-A,B,C), anti-HLA class II mAb (anti-DR, anti-DQ, and anti-DP) and anti-CD4 mAb did not have any inhibitory effects. Surprisingly, the γδ T cell lines showed the proliferative response against the original bacterial Ag KTH-1 exclusively, and exhibited no cross-reactivity with nominal Ag such as purified protein derivative of tuberculin, tetanus toxoid and Mycobacterium tuberculosis, or the same species but different strain of S. sanguis, American Type Culture Collection (ATCC) standard strain (10556), or even with the same strain but different serotype of S. sanguis, KTH-3. Moreover, cytokine production of the γδ T cell lines was similar to the Th1 pattern [interferon-γ, tumor necrosis factor (TNF)-α and TNF-β]. They also produced interleukin-8 that functions as one of chemoattractants for polymorphonuclear cells. Using direct sequencing technique of the polymerase chain reaction products, we found that junctional diversity of the T cell receptor (TcR) used by the parental KTH-1 specific γδ T cell line and its subclones is rather limited. It is suggested that γδ T cells with canonical TcR could preferentially respond to KTH-1 Ag. Thus, in addition to a broad or cross-reactivity of γδ T cells against phylogenetically conserved stress/heat-shock protein, which is well characterized by others, some peripheral blood γδ T cells could recognize and kill exogenous agents with fine antigenic specificity to protect the body against them.     

Interleukin-15 preferentially promotes the growth of intestinal intraepithelial lymphocytes bearing γδ T cell receptor in mice

Several cytokines including stem cell factor (SCF) and interleukin (IL)-7 are known to be required for development of γδ T cell receptor (TCR) intestinal intraepithelial lymphocytes (i-IEL) in mice. We show here the effects of IL-15 on the proliferation and maintenance of murine γδ i-IEL in vitro. γδ i-IEL constitutively expressed a high level of IL-15 receptor α mRNA and proliferated in response to IL-15 more vigorously than αβ i-IEL. Vγ/δ repertoire analysis revealed that IL-15, like IL-2, induced polyclonal expansion of γδ i-IEL, whereas γδ i-IEL responding to IL-7 showed a Vγ/δ repertoire skewed towards Vγ1/Vδ4, Vδ5. IL-15 efficiently prevented γδ i-IEL from apoptosis induced by growth factor deprivation. This rescue was accompanied by up-regulation of Bcl-2 expression. These results suggest that IL-15 plays important roles in proliferation and maintenance of γδ i-IEL.     

The homogeneity of the TCRδ repertoire expressed by the Thy-1dull γ δ T cell population is due to cellular selection

Thy-1^dull γ δ T cells are an unusual subset of mature TCRγ δ T cells characterized by their highly restricted TCR repertoire. In DBA/2 mice, they predominantly express the product of the Vγ1 gene together with that of a member of the Vδ6 subfamily (the Vδ6.4 gene) and their junctional sequences show very little diversity. To address the mechanisms underlying the expression of the restricted TCRγ δ repertoire, we have cloned all Vδ6 subfamily members present in DBA/2 mice and studied their frequency of expression in Thy-1^dull and Thy-1^bright γ δ thymocyte populations. Furthermore, we have also cloned non-functional Vδ6DδJδ1 rearrangements present in the Thy-1^dull γ δ T cell population and compared their Vδ6 gene utilization and their junctional sequences with those expressed by this population. Our results indicate that the restricted TCRδ repertoire expressed by the Thy-1^dull γ δ thymocytes results from cellular selection, rather than molecular constraints suggesting the existence of a limited set of self-ligands. Finally, phenotypic, functional and TCRγ δ repertoire analysis of Thy-1^dull γ δ T cells in β2 -microglobulin (β₂m)-deficient mice indicated that these putative ligands are not β₂m-dependent major histocompatibility complex class I or class I-like molecules.     

A regulatory cross‐talk between Vγ9Vδ2 T lymphocytes and mesenchymal stem cells

The physiological functions of human TCRVγ9Vδ2⁺ γδ lymphocytes reactive to non‐peptide phosphoantigens contribute to cancer immunosurveillance and immunotherapy. However, their regulation by mesenchymal stem cells (MSC), multipotent and immunomodulatory progenitor cells able to infiltrate tumors, has not been investigated so far. By analyzing freshly isolated TCRVγ9Vδ2⁺ lymphocytes and primary cell lines stimulated with synthetic phosphoantigen or B‐cell lymphoma cell lines in the presence of MSC, we demonstrated that MSC were potent suppressors of γδ‐cell proliferation, cytokine production and cytolytic responses in vitro. This inhibition was mediated by the COX‐2‐dependent production of prostaglandin E2 (PGE₂) and by MSC through EP2 and EP4 inhibitory receptors expressed by Vγ9Vδ2 T lymphocytes. COX‐2 expression and PGE₂ production by MSC were not constitutive, but were induced by IFN‐γ and TNF‐α secreted by activated Vγ9Vδ2 T cells. This regulatory cross‐talk between MSC and Vγ9Vδ2 T lymphocytes involving PGE₂ could be of importance for the antitumor and antimicrobial activities of γδ T cells.     

Human peripheral blood γδ T cells are uniformly sensitive to destruction by the lysosomotropic agents leucine methyl ester and leucyl leucine methyl ester

Treatment of peripheral blood mononuclear cells (PBMC) with the lysosomotropic agent leucine methyl ester (Leu-OMe) eliminates monocytes/macrophages and cytotoxic lymphocytes including CD3^? CD16⁺ natural killer (NK) cells and a fraction of T cell receptor (TcR)αβ⁺ CD8⁺ T cells. We report that freshly isolated peripheral blood γδ T cells are highly sensitive to Leu-OMe treatment as well. After incubation of PBMC with 5 mM Leu-OMe or incubation of purified T cells with 50 μ.M leucyl leucine methyl ester (Leu-Leu-OMe) and subsequent overnight culture, CD3^?CD16⁺ NK cells and γ^δ T cells were no longer detectable by immunofluorescence analysis. The two major γ^δ T cell subsets Vγ9⁺Vδ2⁺ and Vγ9-Vδ1⁺ were equally susceptible to Leu-OMe and Leu-Leu-OMe treatment. The elimination of Vγ9⁺ T cells by Leu-OMe treatment was confirmed in functional assays. Stimulation of peripheral blood T cells with killed mycobacteria resulted in selective expansion of Vγ9⁺ T cells. In contrast, no activation of γ^δ T cells was elicited in Leu-OMe-treated responder T cells stimulated with killed mycobacteria.     

Helper activity of CD4+ αβ T cells is required for the avian γδ T cell response

We have studied the in vitro activation of chicken γδ T cells. Both splenic αβ and γδ T cells obtained from complete Freund's adjuvant-primed chickens proliferated in vitro when stimulated with mycobacterial sonicate or purified protein derivative of Mycobacterium tuberculosis. When CD4⁺ cells or αβ T cell receptor (TcR)-positive cells were removed, both the proliferation and the blast formation of γδ T cells in response to mycobacterial antigens were abrogated. The response was restored if supernatant from concanavalin A (Con A)-activated lymphocyte cultures (CAS) as a source of helper factors was added together with the specific antigen purified protein derivative. The CD4- or αβ TcR-depleted cells still proliferated in response to Con A, although a decrease of the response was observed. To analyze the γδ T cell response more specifically we stimulated peripheral blood cells with immobilized monoclonal antibodies against T cell receptor. Anti-γδ TcR antibody alone did not induce significant proliferation. When CAS was added together with the anti-γδ TcR monoclonal antibody, a strong proliferation of γδ T cells was observed. In contrast, both Vβ1- and Vβ2-expressing αβ T cells proliferated in vitro in response to stimulation with the relevant anti-TcR monoclonal antibody alone. Depletion of either Vβ1⁺ or Vβ2⁺ T cell subset alone had no negative effect on the proliferation or blast formation of γδ T cells stimulated with mycobacterial antigens. Taken together our results suggest that CD4⁺ αβ T cells (both Vβl- and Vβ2-expressing) play a role in the activation and response of chicken γδ T cells.     

Control of self-reactive cytotoxic T lymphocytes expressing γδ T cell receptors by natural killer inhibitory receptors

The majority of peripheral blood γδ T cells in human adults expresses T cell receptors (TCR) with identical V regions (Vγ9 and Vδ2). These Vγ9Vδ2 T cells recognize the major histocompatibility complex (MHC) class I-deficient B cell line Daudi and broadly distributed nonpeptidic antigens present in bacteria and parasites. Here we show that unlike αβ or Vγ9^? γδ T cells, the majority of Vγ9Vδ2T cells harbor natural killer inhibitory receptors (KIR) (mainly CD94/NKG2A heterodimers), which are known to deliver inhibitory signals upon interaction with MHC class I molecules. Within Vγ9δ2 T cells, KIR were mainly expressed by clones exhibiting a strong lytic activity against Daudi cells. In stark contrast, almost all Vγ9Vδ2 T cell clones devoid of killing activity were KIR^?, thus suggesting a coordinate acquisition of KIR and cytotoxic activity within Vγ9Vδ2 T cells. In functional terms, KIR inhibited lysis of MHC class I-positive tumor B cell lines by Vγ9Vδ2 cytotoxic T lymphocytes (CTL) and raised their threshold of activation by microbial antigens presented by MHC class I-positive cells. Furthermore, masking KIR or MHC class I molecules revealed a TCR-dependent recognition by Vγ9Vδ2 CTL of ligands expressed by activated T lymphocytes, including the effector cells themselves. Taken together, these results suggest a general implication of Vγ9Vδ2 T cells in immune response regulation and a central role of KIR in the control of self-reactive γδ CTL.