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.     

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.     

Sequential appearance of T-cell receptor gamma delta- and alpha beta-bearing intestinal intra-epithelial lymphocytes in mice after irradiation.

We have previously reported that T-cell receptor (TcR) gamma delta-bearing T cells precede TcR alpha beta-bearing T cells in appearance in the thymus after whole-body irradiation. In the present study, the kinetics of appearance of intestinal intra-epithelial lymphocytes (IEL) was examined in mice after whole-body irradiation with a lethal dose of 9.5 Gy or with a sublethal dose of 6 Gy. The number of CD3+ IEL decreased to the lowest value 4 days after irradiation with 9.5 Gy, and thereafter increased to half as many as the normal level by day 7. Thy-1+TcR alpha beta- IEL and Thy-TcR alpha beta- IEL recovered considerably by day 7 after the irradiation, whereas Thy-1+TcR alpha beta+ IEL and Thy-1+TcR alpha beta+ IEL hardly recovered at this stage. All mice died within 12 days after irradiation with a lethal dose of 9.5 Gy. On the other hand, when irradiation dose was decreased to 6 Gy, all mice survived beyond 40 days after irradiation. The number of CD3+ IEL recovered to the normal level by 10 days after irradiation with 6 Gy. Consistently with the results in mice irradiated with a lethal dose, the first cells to increase in IEL of mice irradiated with a sublethal dose were TcR gamma delta+ IEL expressing Thy-1 antigen. The number of Thy-1+TcR gamma delta+ IEL increased to approximately two-fold as many as that in normal mice by day 10, while TcR alpha beta+ IEL began to increase in number from day 20 after irradiation and recovered to the normal level by day 40 after irradiation. Thus, sequential appearance of TcR gamma delta+ and TcR alpha beta+ IEL was evident after irradiation, similar to that seen in the thymus after irradiation. The IEL on day 10 after a sublethal irradiation, which is composed mainly of Thy-1+TcR gamma delta+ IEL, exhibited a strong cytolytic activity against P815 in the presence of anti-CD3 mAb, suggesting that the early appearing Thy-1+TcR gamma delta+ IEL may play important roles in epithelial immunity at an early stage after irradiation.     

Phenotypical and Functional Characterization of Small Intestinal TcRγδ+ T Cells in Coeliac Disease

Increased numbers of TcR gamma delta + T cells are present in the small intestinal epithelium of patients with coeliac disease (CoD). Their function, however, is unknown. In order to facilitate detailed functional studies, intestinal gamma delta T cells have been isolated from small intestinal biopsies of patients with CoD (n = 18) and controls (n = 14). As expected, increased numbers of V delta 1+ TcR gamma delta + T cells were detected in freshly isolated intraepithelial cell suspensions (IEL) from CoD patients. Also, in the in vitro expanded IEL T-cell populations from CoD patients the numbers of V delta 1+ TcR gamma delta + T cells were increased compared with similar cell cultures from control patients. From IEL cultures derived from six CoD patients, 107 T-cell clones were generated by limiting dilution and analysed. Sixty of these clones were either CD4 or CD8 positive TcR alpha beta + clones. The remaining 47 clones expressed the TcR gamma delta. Further phenotypical analysis of the gamma delta T-cell clones indicated that the TcR gamma delta + T-cell population in the small intestinal epithelium of CoD patients is heterogeneous: four TcR gamma delta phenotypes could be detected and, although the majority of the TcR gamma delta + T cells were CD4 CD8, gamma delta T-cell clones expressing either a CD8 alpha alpha homodimer, a CD8 alpha beta heterodimer or CD4 were also identified. In contrast to the TCR alpha beta + IEL, most TcR gamma delta + IEL were CD5 negative. Furthermore, biochemical analysis indicated that the increase in V delta 1+ gamma delta T cells in the small intestinal epithelium of CoD patients was not the result of a monoclonal expansion. The small intestinal epithelium-derived gamma delta T-cell clones were functional in vitro since the majority of these clones were able to lyse target cell lines such as K562. Molt4 and Daudi. These novel findings therefore indicate that the gamma delta T cells in the small intestine of CoD patients represent a heterogeneous population and that such cells are functional in vitro. The isolation and the in vitro propagation and cloning of these cells may open new avenues for the study of the putative immune mechanisms leading to coeliac disease.     

Listeria monocytogenes-induced gamma interferon secretion by intestinal intraepithelial gamma/delta T lymphocytes.

gamma/delta T cells represent a major proportion of intestinal intraepithelial lymphocytes (IEL), and it has been suggested that these IEL serve as a first immune barrier against microbial invasion and that they do so by destroying infected epithelial cells. In the present study, we confirm that both alpha/beta and gamma/delta IEL from naive mice express potent cytotoxicity and produce gamma interferon (IFN-gamma) after T-cell receptor (TCR) engagement by specific monoclonal antibodies (MAb). Intraperitoneal administration of the anti-gamma/delta TCR MAb GL3 caused downregulation of the gamma/delta TCR in IEL, and IEL from gamma/delta TCR-modulated mice failed to express cytotoxic activity and to secrete IFN-gamma after gamma/delta TCR engagement. In contrast, alpha/beta IEL from such mice were still cytolytic and secreted IFN-gamma. Mice were infected orally with virulent Listeria monocytogenes at doses which caused bacterial invasion through the intestinal epithelia. Although alpha/beta and gamma/delta IEL from these mice expressed high cytolytic activities in antibody-redirected killer assays, target cells pulsed with listerial antigens were not lysed. In contrast, IFN-gamma secretion by IEL from L. monocytogenes-infected mice was induced not only by anti-TCR MAb but also by target cells pulsed with listerial antigens, whereas irrelevant antigens, including heat shock protein 60, did not induce IFN-gamma secretion. Furthermore, the number of IFN-gamma-secreting IEL, as assessed by the enzyme-linked immunospot technique, was increased during listeriosis. gamma/delta TCR modulation by GL3 administration abrogated antigen-induced IFN-gamma secretion by IEL from infected mice. These findings suggest that L. monocytogenes induced IFN-gamma secretion by gamma/delta IEL from mice suffering from intestinal L. monocytogenes infection and invasion. Thus, the data provide evidence for a role of IFN-gamma-secreting IEL in local resistance against listeriosis and perhaps other food-borne diseases.     

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.     

Proliferative properties of murine intestinal intraepithelial lymphocytes (IEL): IEL expressing TCR alpha beta or TCR tau delta are largely unresponsive to proliferative signals mediated via conventional stimulation of the CD3-TCR complex.

Murine intestinal intraepithelial lymphocytes (IEL) were studied for their capacity to proliferate in vitro following stimulation of the T cell receptor (TCR)-associated CD3 epsilon molecule, or upon direct stimulation of the TCR complex itself. Although IEL consisted primarily of CD3+ T cells which included activated cytotoxic T lymphocytes as demonstrated in CD3- and TCR-mediated redirected cytotoxic assays, IEL displayed minimal proliferative responses following stimulation with anti-CD3, anti-TCR alpha beta, or anti-TCR tau delta monoclonal antibodies under soluble conditions, or under conditions which effect membrane cross-linking, including the addition of accessory cells to IEL cultures. The lack of proliferation induction could not be overcome by stimulation of IEL in the presence of T cell-dependent cytokines, phorbol ester, or interleukin-4. Moreover, unlike splenic T cells, stimulation of IEL failed to result in expression of interleukin-2 receptor, further demonstrating an inability of IEL to respond to exogenous proliferative signals. This study is the first to examine the proliferative potential of murine IEL following direct CD3 or TCR stimulation. The findings described here: (i) identify an important functional distinction between intestinal IEL and other peripheral alpha beta or tau delta T cells which generally respond well to proliferative signals mediated through the CD3-TCR complex, and (ii) demonstrate that on murine IEL the CD3-TCR complex can discriminate signals of lytic activity from those of cell proliferation.     

Intraepithelial TcR alpha/beta+ lymphocytes express CD45RO more often than the TcR gamma/delta+ counterparts in coeliac disease.

Expression of CD45RO on intraepithelial lymphocytes (IEL) bearing the T-cell receptor (TcR) alpha/beta or gamma/delta was studied in situ by three-colour immunofluorescence on jejunal tissue sections from 21 patients with coeliac disease and eight controls. CD45RA-TcR alpha/beta+ IEL expressed CD45RO significantly more often (75%) than the preferentially expanded TcR gamma/delta+ counterpart (59%). Triple staining for CD3, CD4/8 and CD45RA or CD45RB revealed that all CD3 + 4 - 8 - IEL (taken to be TcR gamma/delta+) expressed CD45RB and none were CD45RA. CD45RO positivity was of the same magnitude (66%) on the predominating monoclonal antibody delta TCS1-reactive fraction of TcR gamma/delta+ cells as on the remainder of the TcR gamma/delta+ subset. These results suggest that gluten exposition in patients with coeliac disease leads to accumulation of CD45RA-, putative antigen-primed memory cells of both TcR phenotypes. The less marked CD45RO expression within the preferentially expanded TcR gamma/delta+ subset of IEL may be of particular biological interest.     

Tissue-specific segregation of TCRgamma delta+ NKT cells according to phenotype TCR repertoire and activation status: parallels with TCR alphabeta+NKT cells

Whereas the majority of NKT cells in the mouse express an alpha beta TCR (NKTalpha beta cells), a small subset of NKT cells express a gamma delta TCR (NKTgamma delta). Here we have systematically analyzed the phenotype, TCR repertoire and activation status of NKTgamma delta cells in the thymus, liver, spleen and bone marrow of normal C57BL/6 mice. Our data indicate that NKTgamma delta cells segregate in a tissue-specific manner according to these parameters. While most NKTgamma delta cells in the thymus and liver have a recently activated CD62L(lo) phenotype and a TCR repertoire that is heavily biased to Vgamma1.1 and Vdelta6.3, the majority of NKTgamma delta cells in the spleen and bone marrow are CD62L(hi) and have a much less biased TCR repertoire. Moreover, expression of NK markers is high on NKTgamma delta cells in spleen and bone marrow but low in thymus and liver. Collectively our results reveal a tissue-specific segregation of NKTgamma delta cells that is strikingly similar to that recently described for CD1d-dependent and Cd1d-independent NKTalpha beta cells. We therefore propose that chronic TCR activation by tissue-specific endogenous ligands is a generic property of NKT cells of both the alpha beta and gamma delta lineages.     

Down syndrome (DS) peripheral blood contains phenotypically mature CD3+TCR alpha, beta+ cells but abnormal proportions of TCR alpha, beta+, TCR gamma, delta+, and CD4+ CD45RA+ cells: evidence for an inefficient release of mature T cells by the DS thymus.

Down syndrome (DS) thymocytes have a markedly diminished proportion of cells expressing high levels of the alpha, beta T cell receptor (TCR alpha, beta) and the associated CD3 molecule. Thus, we examined the surface expression of TCR alpha, beta and CD3 as well as TCR gamma, delta, CD4, CD8, CD16, and CD45RA on peripheral blood lymphocytes (PBL) from 13 noninstitutionalized subjects with DS and 13 closely age-matched sibling controls using immunofluorescence and flow cytometry. DS PBL expressed high surface levels of TCR alpha, beta and CD3, but, as compared to controls, they had a lower proportion of cells expressing TCR alpha, beta (61% vs. 68%, respectively; P less than or equal to 0.05). Moreover, the absolute number of TCR alpha, beta+ cells was considerably lower for DS subjects than for controls (1634 +/- 229 vs. 2763 +/- 530, respectively; P less than or equal to 0.05). DS subjects had a markedly higher proportion of cells expressing TCR gamma, delta than did the controls (12% vs. 7%, respectively; P less than or equal to 0.02). In addition, DS subjects had a lower proportion of CD4+CD45RA+ cells than controls (22% vs. 35%, respectively; P less than or equal to 0.02), representing naive T cells which have recently emigrated from the thymus. The imbalance in the proportions of T cell subpopulations we have observed in DS PBL may contribute to the increased susceptibility to infection associated with DS and may represent a diminished efficiency in the production of newly differentiated T cells by the DS thymus.     

Expression of T cell receptors alpha beta and gamma delta in the ileal mucosa of patients with Crohn''s disease and with spondylarthropathy.

The expression of the alpha beta and gamma delta heterodimer of the T cell receptor (TCR) was studied in normal human ileal mucosa or in ileal biopsies featuring Crohn's disease or acute and chronic spondylarthropathy-related gut inflammation. With an immunohistochemical technique we demonstrated that the increase of mucosal lymphocytes per mm mucosa in Crohn's disease and spondylarthropathy-related ileitis is exclusively due to expansion of the alpha beta + T cell compartment. In Crohn's disease and chronic ileitis observed in some spondylarthropathy patients the alpha beta + T cells were increased amongst intraepithelial lymphocytes (IEL). The lamina propria lymphocytes (LPL) were augmented in all studied inflammatory conditions. The gamma delta + T cells showed no changes in IEL or LPL and their proportions were not altered. They were evenly dispersed throughout the ileal mucosa and did not seem to participate in the inflammatory process. This study confirms that gamma delta T cells are a distinct subset in the intestinal mucosa. The increase in alpha beta + T cells suggests augmented mucosal antigen handling and involvement of the major histocompatibility complex in the pathogenesis of spondylarthropathy-related gut inflammation and Crohn's disease.     

Mouse T lymphocytes that express a gamma delta T-cell antigen receptor contribute to resistance to Salmonella infection in vivo.

Mice depleted of lymphocytes expressing the alpha beta or the gamma delta T-cell receptor for antigen (TCR) by antibody treatment were infected orally with Salmonella enteritidis. In both groups of treated mice, the 50% lethal dose decreased, suggesting that both the alpha beta TCR+ and the gamma delta TCR+ subsets contribute to resistance to oral infection. These data provide further evidence for the contribution of gamma delta T cells in the response to bacterial infections.     

T cell receptor-bearing cells among rat intestinal intraepithelial lymphocytes are mainly alpha/beta+ and are thymus dependent

Rearrangement of both the beta and gamma chain T cell receptor (TcR) genes was detected in intestinal intraepithelial lymphocytes (IEL) from normal euthymic rats. Flow cytometric analyses showed that about 73% of the IEL were CD3+ (1F4) and that 67% were TcR alpha/beta+ (R73). About 5% of the IEL were found to be CD3+, TcR alpha/beta- in double-labeling experiments suggesting that a small fraction of IEL in the rat express the alternative TcR gamma/delta. More than 70% of the IEL were granular implying that many CD3+ IEL are granular. In IEL from athymic nude rats no rearrangement of either the TcR beta or gamma chain genes or surface expression of CD3 or TcR alpha/beta was detected despite the fact that about 95% of the cells were granular and morphologically similar to those in normal rats. Taken together our data suggest that the majority of IEL in the rat express the conventional TcR alpha/beta and that TcR-bearing cells in the gut epithelium are thymus dependent.     

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.     

Phenotypic and functional assessment of intraepithelial lymphocytes bearing a 'forbidden' {alpha}{beta} TCR

Differences in the surface antigen phenotype, such as the expressionCD8 as an homodimer or the lack of Thy-1, on Intestinal Intraepitheliallymphocytes (IEL) are related, In part, to alternative differentiationpathways. The relationship of IEL lacking the pan-T cell markerCD5 to these IEL, their TCR repertoire and function has notbeen examined directly. We explored the TCR repertoire and functionof the CD5^– IEL subset In relation to the expression ofthe ‘autospecific’ V_ß6 TCR in Mls-1^a miceand to TCR. The results indicate that CD5 expression was absenton the majority of TCR IEL (96.9%) and on a significant proportionof TCR ß IEL (25.0%). Virtually all IEL In DBA/2 (Mls-1^a)mice that expressed the ‘autospecific’ V_ß6TCR were CD5^–, and this correlated with the expressionof CD8 . To assess the functional capacity of this subset ofIEL, we examined proliferation and IL-2 production in responseto TCR activation. Although CD5^– IEL proliferated in responseto anti-CD3, IEL bearing TCR V_ß6, In Mls-1^a mice,were not responsive to TCR-mediated activation. Similarly, TCR IEL were not responsive to stimulation by anti-TCR antibodies.The addition of exogenous IL-2, however, reconstituted the prollferativeresponse of both TCR IEL and the TCR V_ß6 expressingIEL. We conclude that the lack of CD5 defines a unique subsetof intraepithelial T cells expressing either TCR or ßthat Include potentially autoreactive cells that remain anergicin the absence of IL-2.     

Analysis of clones derived from human CD7+CD4-CD8-CD3- thymocytes.

The differentiation of human thymocyte precursors was studied by analysis of clonal progeny of CD4-CD8-CD3- (triple negative or TN) thymocytes. Using a culture system of phytohemagglutinin, IL-2, and irradiated allogeneic lymphoid feeder cells, we found that 48% of clones (104 total) derived from TN thymocyte suspensions were TCR gamma delta cells, 12% of clones were TCR alpha beta cells, and 34% were CD16+CD3- cells. Importantly, 6% of clones were novel subsets of CD4+CD8-CD3- or CD4-CD8+CD3- thymocytes. The majority of TCR alpha beta, TCR gamma delta, and CD16+CD3- clones expressed low levels of CD4. Molecular analysis of freshly isolated TN- thymocytes prior to in vitro culture demonstrated that up to 40% of cells had TCR gamma, delta, and beta gene rearrangements, but were negative in indirect immunofluorescence assays for cytoplasmic TCR delta and beta. These data provide evidence at the clonal level for the presence of precursors of the TCR alpha beta and TCR gamma delta lineages in the human TN thymocyte pool. Moreover, a substantial proportion of freshly isolated human TN thymocytes had already undergone TCR gene rearrangement prior to in vitro culture. Whether these precursors of the TCR alpha beta and TCR gamma delta lineages mature from cells already containing TCR gene rearrangements into sTCR+ cells or differentiate in vitro from cells with TCR genes in germline configuration remains to be determined. Nonetheless, these data demonstrate that the predominant clone types that grow out of human TN thymocytes in vitro are TCR gamma delta and NK cells.     

Gamma delta T cell receptor-positive cells of the human gastrointestinal mucosa: occurrence and V region gene expression in Heliobacter pylori-associated gastritis, coeliac disease and inflammatory bowel disease.

T cells expressing the gamma delta heterodimer of the T cell receptor (TCR) were studied with respect to their occurrence and expression of gamma delta TCR variable region (V) genes in the normal gastrointestinal mucosa and in a variety of inflammatory conditions. In controls, gamma delta TCR+ cells were a minority population confined to the epithelial compartment of stomach, small bowel and colonic mucosae. Unlike in the periphery, gastro-intestinal gamma delta TCR+ intraepithelial lymphocytes (IEL) were mainly V delta 1+ (89.98 +/- 17.70%); few were V delta 2+ (6.04 +/- 13.8%) or V gamma 9+ (11.38 +/- 10.73%). All gamma delta TCR+ IEL were CD5low; nearly half were CD8+ and the remainder were CD4-CD8- 'double negatives'. There was no significant change from normal in percentages of gamma delta TCR+ IEL in H. pylori-associated gastritis, Crohn's disease and ulcerative colitis. However, in coeliac disease, gamma delta TCR+ IEL were elevated from 2.54% (+/- 1.71) in controls to 29.6% (+/- 16.1) in untreated patients (P less than 0.001) and 18.5% (+/- 7.2) in treated patients (P less than 0.001) and more were CD4-CD8-. Otherwise, gamma delta TCR+ IEL phenotypes were little changed: the majority remained V delta 1+V delta 2-V gamma 9- and all were CD5low. These data suggest that increased gamma delta TCR+ IEL are not a generalized response to intestinal inflammation or to stress proteins, although the typical V delta 1+V delta 2-V gamma 9- CD5low phenotype is retained.     

Evidence for the early recruitment of T-cell receptor gamma delta+ T cells during rat listeriosis.

We have previously reported that heat-shock protein (hsp) 60-reactive T-cell receptor (TCR)gamma delta+ T cells appear in the peritoneal cavity during the early stage of infection with Listeria monocytogenes in mice. In this study, we examined the kinetics of TCR gamma delta+ T cells during listeriosis in F344 rats by flow cytometry using a V65 monoclonal antibody (mAb) directed to a constant determinant of rat TCR gamma delta chains. TCR gamma delta+ T cells significantly increased in the peritoneal cavity on day 6 and then decreased by day 10 after infection, in parallel with the kinetics of hsp60 expression in the peritoneal macrophages during listeriosis in F344 rats. Most of the early appearing TCR gamma delta+ T cells were of the CD4- CD8 alpha beta+ CD5+ lymphocyte function-associated antigen (LFA)-1 alpha high CD45RC- interleukin-2 receptor (IL-2R) alpha- phenotype, although a significant fraction of the TCR gamma delta+ T cells expressed CD8 alpha only. The increase in TCR gamma delta+ T cells during listeriosis was prominent in F1 (F344 x Lewis) rats but only marginal in Lewis rats, which was correlated with the expression level of hsp 60 in the peritoneal macrophages. The peritoneal TCR gamma delta+ T cells in naive F344 rats appeared to proliferate significantly in response to recombinant hsp 60 (rhsp 60) derived from Mycobacterium bovis bacillus Calmette-Guérin (BCG). These results imply that the early appearance of hsp 60-reactive TCR gamma delta+ T cells during listerial infection can be generalized across species.