Distinct structure and signaling potential of the gamma delta TCR complex

Alpha beta and gamma delta T cells are distinguished by the clonotypic subunits contained within their TCRs. Although the alpha beta TCR has been well characterized, much less is known about the gamma delta TCR. Here, we report that, unlike alpha beta T CRs, most gamma delta TCRs expressed on ex vivo gamma delta T cells lack CD3 delta. Despite this structural difference, signal transduction by the gamma delta TCR is superior to that of the alpha beta TCR, as measured by its ability to induce calcium mobilization, ERK activation, and cellular proliferation. Additionally, the TCR complexes expressed on primary gamma delta T cells contain only zeta zeta homodimers; however, following activation and expansion, Fc epsilon R1 gamma is expressed and is included in the gamma delta TCR complex. These results reveal fundamental differences in the primary structure and signaling potential of the alpha beta- and gamma delta TCR complexes.     

Induction of human TCR gamma delta + and TCR gamma delta-CD2+CD3- double negative lymphocytes by bacterial stimulation

When human blood mononuclear cells (MNC) were incubated with heat-killed bacteria, proliferation of MNC was observed 5 days after stimulation, showing a peak on day 7. Interestingly, the bioassay of the culture supernatant and Northern blot analysis of mRNA demonstrated that no IL-2 production was associated with these proliferative responses. The induced lymphoblasts consisted predominantly of TCR gamma delta + (22.4 +/- 9.3%) and TCR gamma delta-CD2+CD3-(33.2 +/- 11.8%) double negative lymphocytes (n = 10), which were initially minor populations (less than 10%) in freshly isolated MNC. The prominent induction of TCR gamma delta + cells was confirmed by Northern blot analysis. TCR gamma delta + cells induced by bacterial stimulation seemed to generate from lymphocytes lacking the apparent expression of gamma delta TCR. The inducing capability for double negative cells is present in a large number of species of bacteria, especially Gram-positive bacteria. Gel filtration analysis of ultrasonicated filtrates of Staphylococcus aureus and Streptococcus pyogenes revealed that a substance with an Mr of 25-26 kd could be substituted for whole bacterial particles in the cell proliferative responses. In contrast to the purified protein derivative (PPD)-induced response, the response described here was inducible in the cord blood of neonates who had not yet been exposed to the corresponding bacterial infection. The physicochemical properties of the sonicated filtrates were different from those of PPD. These results suggested that the present phenomenon may be nonspecific, polyclonal (or oligoclonal) activation of TCR gamma delta + and TCR gamma delta -CD2+CD3- cells by bacterial stimulation.     

An appropriate in vitro culture condition for the induction of human TCR gamma delta + cells by heat-killed bacteria.

TCR gamma delta + cells proliferated when MNC were stimulated with various heat-killed bacteria. We investigated here the culture conditions for their maximum proliferation. MNC were cultured for 6 days with Streptococcus pyogenes, and for 3 days with T cell mitogens, PHA and anti-CD3 mAb, in medium supplemented with various concentrations (0.05-50%) of human sera. TCR gamma delta + and TCR gamma delta- CD2+3- double negative cells induced by Str. pyogenes required high concentrations of sera (greater than 6%) for their proliferation. Moreover, increased sera (up to 50%) greatly augmented their proliferation. On the other hand, TCR alpha beta + cell proliferation induced by T cell mitogens was supported by a small concentration (even 0.1%) of the sera, and the addition of high concentrations of sera (greater than 6%) somewhat suppressed responses. Similar serum requirement patterns were evident for the induction of cytotoxic cells. These results clearly demonstrated the existence of an appropriate culture condition for the proliferation of TCR gamma delta + cells induced in vitro.     

Increased division of alpha beta TCR+ and gamma delta TCR+ intestinal intraepithelial lymphocytes after oral administration of cholera toxin.

Cholera toxin (CT) or its subunits were given orally to mice and division of intestinal intraepithelial lymphocytes (IEL) in vivo measured by double immunofluorescence using 5-bromo-2'-deoxyuridine (BRdU) and membrane alpha beta T-cell receptors (TCR) or gamma delta TCR staining in frozen sections. Cholera toxin (10 micrograms) produced a two- to eightfold-increase in the uptake of BRdU in alpha beta TCR+ IEL in the duodenum and a two-to fivefold increase in gamma delta TCR IEL in the ileum. Increased uptake of BRdU was also seen after a dose of 100 micrograms of CT but this dose was also associated with the loss of alpha beta TCR+ IEL and gamma delta TCR+ IEL in the duodenum. CT-A and CT-B subunit produced increased BRdU incorporation by alpha beta TCR in the duodenum and by gamma delta TCR IEL in the ileum. Cholera toxin therefore appears to be mitogenic for IEL probably due to an indirect mechanism.     

Strain-specific TCR repertoire selection of IL-4-producing Thy-1 dull gamma delta thymocytes

Thy-1 dull gamma delta thymocytes constitute an unusual subset of mature TCR gamma delta cells which share with NK T cells the expression of cell surface markers usually associated with activated or memory cells and the simultaneous production of high levels of IL-4 and IFN-gamma upon activation. In DBA / 2 mice, Thy-1 dull gamma delta thymocytes express a restricted repertoire of TCR that are composed of the V1 gene product mainly associated with V6.4 chains exhibiting very limited junctional sequence diversity. In this study we have characterized this gamma delta T cell population in different strains of mice and show that Thy-1 dull gamma delta thymocytes are present in every strain tested, albeit at different frequencies. Moreover IL-4 production by gamma delta thymocytes is mainly confined to the Thy-1 dull population in every strain tested. Finally, the repertoire of TCR expressed by Thy-1 dull gamma delta thymocytes varies in different strain of mice, although a biased expression of Vgamma1 and Vdelta6 chains was observed in all strains studied. However, the extent of junctional diversity of the V1 and V6 chains expressed by Thy-1 dull gamma delta thymocytes varied from oligoclonal in DBA/2 mice to polyclonal in FVB/N mice. Thy-1 dull gamma delta thymocytes from mouse strains such as C3H/HeJ and BALB/c contain cells with diverse Vdelta6(D)Jdelta junctions together with cells with relatively homogeneous Vdelta6(D)Jdelta junctions, similar to those found in DBA/2. Thus, the Thy-1 dull gamma delta population appears to contain two subsets of cells which differ in the diversity of their TCR.     

Regional variation in the proliferative rate and lifespan of alpha beta TCR+ and gamma delta TCR+ intraepithelial lymphocytes in the murine small intestine.

Using double staining for T-cell receptor (TCR) and 5-bromo-2'-deoxyuridine (BRdU) we have examined the proliferation rates and lifespan of murine intraepithelial lymphocytes (IEL's) in vivo. After a 24-hr pulse of BRdU the number of labelled alpha beta TCR+ IEL was significantly higher in the ileum than the duodenum. In contrast, incorporation of BRdU into gamma delta TCR+ IEL was significantly higher in the duodenum than the ileum. This regional variation was also seen after a 4-hr pulse of BRdU indicating that the differences probably reflect local rates of proliferation in the epithelium. Over a 6-day labelling period, the accumulation of labelled alpha beta TCR+ and gamma delta TCR+ IEL was linear, which allowed IEL lifespan to be calculated. There was considerable variation between groups of mice but the 50% population renewal time for alpha beta TCR+ IEL was 12-36 days in the duodenum and 9-11 days in the ileum, and for gamma delta TCR+ IEL was 12-21 days in the duodenum and 26-100 days in the ileum. The incorporation of BRdU into V beta 8+ IEL showed the same regional variation as alpha beta TCR+ IEL and the V delta 4 population behaved like the total gamma delta TCR+ IEL population. In contrast V beta 11+, potentially self-reactive IEL, showed a regional pattern of labelling like gamma delta TCR+ IEL. Incorporation of BRdU into both alpha beta TCR+ and gamma delta TCR+ IEL in germ-free mice was very low and did not show marked regional variation. alpha beta TCR+ and gamma delta TCR+ IEL from both proximal and distal bowel were cytotoxic. Therefore alpha beta TCR+ and gamma delta TCR+ IEL show different rates of division in different sections of the gut, perhaps reflecting responses to different antigens. Both alpha beta TCR+ and gamma delta TCR+ IEL reside in the epithelium for weeks during which time the gut epithelial population will have been renewed many times.     

Changing jejunal gamma delta T cell receptor (TCR)-bearing intraepithelial lymphocyte density in coeliac disease.

The function of jejunal intraepithelial gamma delta+ T cells is obscure, but they are commonly implicated as playing a role in inflammatory and autoimmune conditions. In coeliac disease (CoD), there are controversial reports as to gluten dependency of these cells. We have now studied the small bowel mucosal intraepithelial T cell densities, and the ratios of gamma delta+ to CD3+ T cells and gamma delta+ to alpha beta+ T cells during early disease development and on a gluten-free diet. Nine children initially excluded for CoD were followed up and rebiopsy after 0.8-4.5 years showed mucosal deterioration. Further, 21 biopsy specimens from newly diagnosed CoD patients were studied, together with 20 specimens taken from children on a gluten-free diet. During CoD development the density of gamma delta+ and alpha beta+ T cells as well as the ratios of gamma delta+ to CD3+ T cells and gamma delta+ to alpha beta+ T cells increased. In the latent stage of CoD when the small bowel mucosal architecture was still normal, two children had clearly normal densities of gamma delta+ (< 2.5 cells/100 epithelial cells) and alpha beta+ (< 25.0 cells/100 epithelial cells) T cells, and low ratios as well. In patients with newly diagnosed CoD the densities decreased significantly on a long-term gluten-free diet. We conclude that the density of intraepithelial gamma delta+ T cells as well as alphabeta+ T cells in CoD is gluten-dependent. CoD can develop in a child ingesting normal amounts of gluten and having normal jejunal mucosal morphology on biopsy and a normal density of gamma delta+ T cells.     

Exacerbation of Plasmodium chabaudi malaria in mice by depletion of TCR alpha beta+ T cells, but not TCR gamma delta+ T cells.

Although gamma delta T cells are found in increased numbers in the spleens of humans and mice infected with malaria, it is not known if these cells are necessary components of an effective immune response. The surface phenotype of spleen cells obtained from mice infected with avirulent Plasmodium chabaudi adami or virulent Plasmodium chabaudi chabaudi were examined using anti-delta or anti-alpha beta T-cell-specific reagents and flow cytometry. Levels of parasitaemia, red blood cell (RBC) counts, and survival times were followed in mice depleted of tumour necrosis factor (TCR)gamma delta+ or TCR alpha beta+ T cells. Numbers of gamma delta T cells increased in the spleens of control antibody-treated infected mice, but not in mice depleted of TCR gamma delta+ or TCR alpha beta+ T cells. Mice depleted of gamma delta T cells had levels of parasitaemia, RBCs, and survival rates similar to control antibody-treated mice. However, mice depleted of TCR alpha beta+ T cells had higher levels of parasitaemia, lower RBC counts, and decreased survival rates. These results indicate that TCR alpha beta+ but not TCR gamma delta+ T cells play an essential role in host defense against P. chabaudi infection in mice.     

delta beta-Thalassaemia in Sicily: report of a case of double heterozygosity for A gamma delta beta-thalassaemia and A gamma G gamma delta beta-thalassaemia.

A case of double heterozygosity for A gamma delta beta-thalassaemia and A gamma G gamma delta beta-thalassaemia was found during a screening programme in Sicily. The proband, a 4-year-old girl, showed a clinical picture of thalassaemia intermedia. Hb F (85.12% by the Singer method) was G gamma A gamma type. The parents and the brother were delta beta-thalassaemia carriers. Structural analysis of Hb F showed both G gamma and A gamma chains in the father, but only A gamma chains in the mother.     

An unusual cardiomelic syndrome

We report on a patient with pre- and postnatal growth retardation, bilateral symmetrical ulnar agenesis with monodactyly, atrial septal defect, two ventricular septal defects, Wolff-Parkinson-White conduction abnormality, and abnormal configuration of the pancreas. Although she had some facial features reminiscent of the Brachmann-de Lange syndrome, relatively normal head size and motor development indicate a distinct syndrome.     

T gamma lymphocytosis is clinically non-progressive but immunologically heterogeneous.

Immunological studies were performed with the expanded T gamma cells of five patients with T gamma lymphocytosis. All patients showed a stable clinical picture with persistent T gamma lymphocytosis and neutropenia, with or without recurrent infections. The expanded T gamma cells in the blood had the T1-3+4-8+11+M1- phenotype, with the exception of one patient whose cells lacked the T8 marker. The expanded T gamma cell population did not proliferate in response to T cell mitogens and did not show immunoregulatory activity on pokeweed mitogen driven immunoglobulin synthesis. In four of the five patients the T gamma cells had killer cell activity against IgG sensitized mouse mastocytoma cells. Taken together, these results and the data from the literature, it is concluded that T gamma lymphocytosis represents a spectrum of T cell expansions clearly distinct from clinically progressive mature T cell neoplasias.     

Dysregulation of the host mevalonate pathway during early bacterial infection activates human TCR gamma delta cells

Primates, but not rodents, have T cell receptor Vgamma9-Vdelta2 T cells bridging innate and adaptive antimicrobial immunity. This T cell population is activated by prenyl pyrophosphates isolated from microbial or eukaryotic cells. Although the microbial metabolites are more active than the cellular ones, their involvement in TCR gammadelta activation during infection has not been studied. Here, we show that, during the initial phases of infections with Escherichia coli and Staphylococcus aureus, TCR gammadelta cells are activated by endogenous mevalonate metabolites. Infections with low bacteria inocula up-regulate the production and accumulation of host-derived TCR gammadelta stimulatory antigens within 1 h, which is followed by a peak of TCR gammadelta cell activation at 5 h. Infections induce the accumulation and dephosphorylation of the hydroxymethylglutaryl-coenzyme A reductase, the rate-limiting enzyme of the mevalonate pathway, resulting in increased activity of this enzyme and in increased synthesis of intermediate metabolites. Thus, primates have evolved the ability to readily respond to bacterial infection by sensing the dysregulation of the mevalonate pathway within infected cells, as a mechanism of immediate antimicrobial immunity.     

Functional analysis of TCR gamma delta+ T cells in tumour-infiltrating lymphocytes (TIL) of human pancreatic cancer.

The effect of HIV infection on intestinal lamina propria macrophage subsets was investigated in 41 patients at various stages of HIV infection (asymptomatic HIV infection, n = 17; AIDS, n = 24). Duodenal biopsies taken from HIV patients at endoscopy were snap frozen and cryostat sections cut for immunohistochemical staining. MoAbs CD68 (EBM11, pan-macrophage marker), RFD1 (antigen-presenting cells) and RFD7 (mature phagocytic macrophages) were used to identify cell subsets using indirect immunoperoxidase or alkaline phosphatase. Double immunofluorescence using MoAbs to HIV proteins (p24, p17 and gp120) and RFD1 were used to identify HIV-infected antigen-presenting cells. Double immunofluorescence was also used to identify macrophages that expressed both RFD1 and RFD7 ('suppressor' macrophages). Intensity of HLA-DR expression in lamina propria cells was investigated using a MoAb to HLA-DR directly conjugated to glucose oxidase. The results show that there was no difference in overall density of macrophages, but there was a significant decrease in dendritic cells (RFD1+) in all clinical stages of HIV. There was no difference in the density of RFD7+ macrophages, nor was there a difference intensity of HLA-DR expression in lamina propria cells. Only four HIV-infected cells were positively identified in the 41 patients. This result suggests that the antigen-presenting arm of mucosal immune defences may be seriously compromised in HIV infection, and represents a further insult to mucosal immunity already impaired as a result of loss of CD4+ T lymphocytes. This may contribute to development of opportunist infection in the gut.