In vivo and in vitro death of mature T cells induced by separate signals to CD4 and alpha beta TCR.

To investigate whether a clonal deletion mechanism is responsible for the mature T cell tolerance that may be induced in vivo by TCR signal to anti-CD4 (H129.19 mAb) coated cells, we analyzed the T cell repertoire in anti-CD4 mAb treated BALB/c mice by flow cytometry following TCR signals through anti-alpha beta TCR mAb or SEB superantigen. Lymph nodes showed a strong reduction in the CD4+/CD8+ cell ratio, and a selective clonal loss of CD4+ V beta 8+ cells 4d following anti-alpha beta TCR or SEB injection, respectively. Following lymph node cell activation in a short-term in vitro assay with SEB or anti-V beta 8 mAb, a selective elimination of CD4+ V beta 8+ cells was again detected, and DNA fragmentation analysis disclosed a cell death by apoptosis. These findings suggest that TCR triggering transduces an apoptotic signal into CD4+ mAb saturated cells that in turn leads to specific holes in the mature T cell repertoire.     

The human T cell receptor V beta repertoire of normal peripheral blood lymphocytes before and after mitogen stimulation.

Mitogen stimulation of T cells in vitro has been employed in the analysis of the T cell antigen receptor (TCR) repertoire and as a method of generating T cell lines and clones. It has been suspected for some time that mitogen stimulation may bias the repertoire. We have addressed this problem employing a semi-quantitative technique utilizing the polymerase chain reaction (PCR) and flow cytometry. Using this PCR method and a panel of primers to 22 V beta subgroups, the V beta repertoire of both unstimulated and phytohaemagglutinin (PHA)-stimulated peripheral T cells from eight healthy individuals was investigated. The samples were also analysed by flow cytometry using anti-V beta 2, V beta 5 and V beta 8 MoAbs. A significant increase in the expression of V beta 6, V beta 7.2 and V beta 10.1 was found in all eight samples of PHA-stimulated T cells compared with unstimulated T cells using the PCR method. In contrast, no differences were found between unstimulated and PHA-stimulated T cells by flow cytometry. These results question the validity of using mitogen-stimulated T cells to investigate TCR gene usage.     

The human T cell antigen receptor repertoire: skewed use of V beta gene families by CD8+ T cells.

The TCR repertoire of human CD8+ peripheral blood lymphocytes has been determined using MoAbs to the V beta 2, 3, 5.1, 5.2/5.3, 6.7, 8, 12 and 19(17)V beta gene families. The CD8T cell repertoire for V beta 2 and V beta 3 is shown to be skewed, with an excess of individuals having higher values than are consistent with a normal distribution. A significant majority of these individuals are over the age of 40. High values of V beta CD8+ cells were found for each V beta family studied except for 6.7a. Individual high values are stable for at least 12 months. In addition, the total percentage of CD4 and CD8 cells reacting with this panel of reagents was determined. There is a significant excess of V beta + CD4+ cells (33%) over CD8+V beta + cells (21.9%). Thus the human CD8 V beta repertoire differs from the human CD4 repertoire in a number of important ways.     

Crosslinked staphylococcal enterotoxin B stimulates CD8+ T cells only in the presence of unlinked costimulator signals.

The superantigen staphylococcal enterotoxin B (SEB) binds to class II MHC expressing cells and subsequently causes selective activation of T cells carrying appropriate T cell receptor (TCR) V beta chains. Apparently SEB acts as a bifunctional molecule by bridging class II MHC structures with the appropriate TCR-V beta chains. This assumption predicts that immobilized SEB ought to stimulate purified, class II MHC negative murine T cells. We show here that immobilized SEB lacks the ability to trigger murine CD8 T cells. Responsiveness obtained at a high T cell concentration is due to contaminating class II MHC-positive lymphocytes. Complementation of the culture system with syngeneic irradiated B cells blasts effectively restores responsiveness. The proliferating cells exhibit SEB specific cytotoxicity and a bias for V beta 8 expression. Since no evidence for leakiness of SEB covalently bound to sephadex beads was obtained, the data imply that immobilized SEB in fact binds to the TCR of T cells expressing the appropriate V beta chains. However, for primary activation additional costimulatory signals are required which can be provided in an unlinked fashion by activated B cells. Resting B cells are activated by immobilized SEB to cells expressing high costimulator activity. As such, the data point out a third function of SEB.     

In vivo responses of CD4+ and CD8+ cells to bacterial superantigens.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex (MHC) class II molecules and specifically activates T cells bearing V beta 8 T cell receptor domains. We have compared several aspects of the response of CD4+ and CD8+ T cell subsets to SEB in vivo. V beta 8+ cells in both subsets proliferated to a similar extent upon SEB injection. Furthermore, mRNA for interferon-gamma was induced in both subsets with similar kinetics and SEB dose-response. Finally CD8+ (but not CD4+) T cells from SEB-injected mice exhibited SEB-specific lysis of MHC class II-bearing target cells. Collectively, these data indicate that the CD4: MHC class II interaction confers no detectable selective advantage to CD4+ cells in the in vivo response to SEB. The observed effector functions of both subsets may contribute to SEB-induced immunopathology.     

Comparison of activation marker and TCR V beta gene product expression by CD4+ and CD8+ T cells in peripheral blood and lymph nodes from HIV-infected patients.

Since lymphoid organs constitute the site of active and progressive HIV disease, analysis of their lymphocytes may provide more accurate information on T cell abnormalities than that obtained from studying peripheral blood lymphocytes. The objective of this study was to compare the expressions of activation markers and T cell receptor (TCR) V beta gene products by CD4+ and CD8+ T cells in lymph nodes (LN) and peripheral blood (PB) from healthy individuals and asymptomatic HIV-infected patients to determine whether anomalies that could be identified at the HIV replication site could support the hypothesis of T cell activation by HIV-encoded antigens or superantigens. CD4+ and CD8+ T cells in paired LN and PB obtained from six healthy controls and five asymptomatic HIV-infected individuals were analysed by flow cytometry, using anti-CD38, anti-HLA-DR and 13 anti-V beta MoAbs that cover, approximately, 45% of the T cell repertoire. Analysis of T cell activation marker expression indicated that the percentages of CD4+ and CD8+ T cells bearing CD38 or CD38 and HLA-DR molecules were higher in patients than in controls and, in patients, higher in LN than in PB. Comparison between the V beta repertoires of CD4+ and CD8+ T cells in LN and PB showed that, in each healthy individual, a limited number of V beta families expressed by CD4+ or CD8+ T cells had different repartition in LN and PB, whereas in each HIV+ patient, more V beta families exhibited different distributions and these differences recurred among certain V beta segments, such as V beta 5.3 and V beta 21 in the CD4+ T cell population and V beta 5.2/5.3, V beta 12 and V beta 21 in the CD8+ T cell population. Taken together, these data argue for a skewed TCR repertoire in HIV infection and sustained activation of T cells by HIV-encoded antigens at the site of HIV replication, and further demonstrate that a high proportion of CD4+ T cells are in an activation state that may, indirectly, participate in their functional abnormalities.     

Bacterial superantigens reactivate antigen-specific CD8+ memory T cells

Superantigens stimulate naive CD4+ and CD8+ T cells in a TCR V beta- specific manner. However, it has been reported that memory T cells are unresponsive to superantigen stimulation. In this study, we show that staphylococcal enterotoxins (SE) can activate influenza virus-specific CD8+ memory cytotoxic T cells. In vivo SEB challenge of mice that had recovered from influenza virus infection (memory mice) resulted in the generation of vigorous influenza-specific cytotoxic T lymphocyte (CTL) activity and in vitro SEA or SEB stimulation of splenic T cells from memory mice, but not naive mice, also induced influenza-specific CTL. Analysis of the mechanism of activation suggested that although there may be a component of cytokine-mediated bystander activation, the CTL activity is largely generated in response to direct TCR engagement by superantigen. Moreover, influenza-specific CTL could be generated from purified CD8+ CD62L loCD44hi (memory phenotype) T cells cultured in the presence of T cell-depleted splenic antigen-presenting cells and SE. Purified CD8+ memory T cells also secreted lymphokines and synthesized DNA in response to superantigen. These results definitively demonstrate that CD8+ memory T cells respond to SE stimulation by proliferating and developing appropriate effector function. Furthermore, the data raise the possibility that otherwise inconsequential exposure to bacterial superantigens may perturb the CD8+ T cell memory pool.      

Characterization of fresh (uncultured) tumour-infiltrating lymphocytes (TIL) and TIL-derived T cell lines from patients with renal cell carcinoma.

Fresh (uncultured) TIL from 12 untreated patients with primary renal cell carcinoma were prepared from tumour specimens by enzymatic digestion, and were characterized by immunofluorescence using MoAbs recognizing leucocyte differentiation antigens or particular V alpha or V beta segments of the T cell receptor (TCR). These fresh TIL comprised CD3+ (20-84%); CD4+ (3-15%); CD8+ (13-35%); alpha beta TCR+ (20-50%); gamma delta TCR+ (3-17%); CD16+ (1-18%) and CD56+ (3-10%) cells. Significant proportions of V alpha 2+, V beta 5.1+ and V beta 6+ cells were found in TIL of certain patients with renal cell carcinoma, suggesting that they comprised oligoclonal T cells. T cell lines were developed in low concentrations of rIL-2 (200 U/ml) from TIL from 11 patients with renal cell carcinoma, and were characterized by immunofluorescence and cell-mediated cytotoxicity. These T cell lines consisted primarily of CD3+ (51-94%); CD4+ (1-80%); CD8+ (0-84%); alpha beta TCR+ (65-87%); gamma delta TCR+ (0-25%); CD16+ (0-16%) and CD56+ (2-57%) cells. These T cell lines exhibited non-specific cytotoxicity against autologous and allogeneic renal tumour cells, with the exception of one T cell line that exhibited preferential cytotoxicity against autologous renal tumour cells. These results suggest that fresh TIL from patients with renal cell carcinoma contain significant proportions of oligoclonal T cells that may have accumulated at the tumour site as a result of a clonal expansion.     

Microbial colonization influences composition and T-cell receptor V beta repertoire of intraepithelial lymphocytes in rat intestine.

Studies in mice have shown that the composition of intestinal intraepithelial lymphocytes (IEL) may be markedly altered by gut microbial colonization. Such modulation was studied in a rat model by the use of germ-free and conventionalized animals from which IEL from the small intestine were isolated and analysed by flow cytometry. Conventionalization caused expansion as well as phenotypic alterations of T-cell receptor (TCR) alpha/beta + IEL in that the proportions of CD4+ and CD8 alpha beta + TCR alpha/beta + cells were increased, while the double negative (CD4- CD8-) fraction was reduced. microbial colonization also influenced the TCR V beta repertoire of CD8+ IEL in that the proportions of V beta 8.2+ and V beta 10+ cells were increased, whereas V beta 8.5+ and V beta 16+ cells were relatively decreased. Moreover, conventionalization influenced the levels of TCR cell surface expression in the same V beta subsets. Three-colour flow-cytometric analysis demonstrated that skewing of the V beta repertoire was most pronounced in the CD8 alpha alpha + subset, although the numerical increase of IEL mainly included the CD8 alpha beta + subset. In contrast to IEL, the TCR V beta repertoire in mesenteric lymph nodes was unchanged after intestinal colonization. These results confirm that TCR alpha/beta + IEL subpopulations respond dynamically to the microbial gut flora and suggest that their V beta repertoire can be shaped by luminal microbial antigens.     

Activation of primary T lymphocytes results in lysosome development and polarized granule exocytosis in CD4+ and CD8+ subsets, whereas expression of lytic molecules confers cytotoxicity to CD8+ T cells

Lytic granule exocytosis is the major cytotoxic mechanism used by CD8(+) cytotoxic lymphocytes. CD8(+) T cells acquire this effector function in the process characterized by lysosomal biogenesis, induction of expression of cytolytic molecules, and their selective sorting into the lysosomal vesicles. However, temporal relation of these differentiation stages during T cell activation has not been defined precisely. Also, although CD4(+) T cells typically do not express lytic molecules as a consequence of activation, and therefore, do not acquire granule exocytosis-mediated lytic function, it is not clear whether CD4(+) T cells are able to degranulate. By using in vitro TCR stimulation of primary mouse lymphocytes, we found that polyclonally activated CD4(+) T cells degranulate upon TCR ligation and polarize enlarged lysosomal granules in response to target cell recognition, despite the lack of granule exocytosis-mediated cytotoxicity. Upon TCR stimulation, resting CD8(+) T cells rapidly express lytic molecules and acquire potent lytic function early in activation. Maximal cytolytic potential, however, depends on enlargement of lysosomal granules during the subsequent activation stages. Thus, polyclonal TCR stimulation of resting T cells results in development of lysosomal granules and their release upon TCR engagement in CD4(+) and CD8(+) T cells, but only CD8(+) T cells acquire lytic function as a result of induction of expression of lytic molecules.     

Human B cell differentiation induced by microbial superantigens: unselected peripheral blood lymphocytes secrete polyclonal immunoglobulin in response to Mycoplasma arthritidis mitogen.

Microbial superantigens (SA) activate a significant portion of the T cell repertoire based on their dual avidity for MHC class II antigens and T cell receptor (TCR) epitopes common to products of one or several TCR beta chain variable gene families. While SA that induce massive T cell proliferation and cytokine secretion have been implicated in clinical syndromes characterized by shock and generalized immunosuppression, SA activation of a more restricted T cell response may also have significant, perhaps immunostimulatory, effects on the immune system. To investigate this issue, we measured 3H-thymidine incorporation and polyclonal IgM and IgG secretion by normal human peripheral blood mononuclear cells (PBMC) cultured with a panel of microbial SA, including the Staphylococcus aureus-derived SA, SEA, SEB, SEC-1, SEC-2, SEC-3, SEE, TSST-1, and the Mycoplasma arthritidis-derived SA, MAM. The S. aureus-derived SA induce vigorous proliferation by PBMC, while optimal MAM-induced proliferation is significantly lower in magnitude. In all 12 subjects tested, mitogenic concentrations of MAM reproducibly stimulate unselected PBMC to secrete polyclonal IgM and IgG. In contrast, the S. aureus-derived SA induce Ig production only in cultures containing isolated B cell populations and either very low numbers of untreated autologous T cells, larger numbers of X-irradiated autologous T cells, or very low concentrations of the SA. No difference in the activation of helper (CD4) versus suppressor/cytotoxic (CD8) T cells by MAM and the S. aureus-derived SA was noted. Taken together, these data suggest that MAM's capacity to induce B cell differentiation correlates with its induction of a relatively weak proliferative response by unselected human T cells. MAM-like SA, when encountered in vivo, may result in a significant perturbation of the human immune system and potentially contribute to clinical syndromes characterized by immunostimulation and hypergammaglobulinemia.     

Reactivity of Human γδ T Cells to Staphylococcal Enterotoxins: a Restricted Reaction Pattern mediated by Two Distinct Recognition Pathways

Staphylococcal enterotoxins (SEs) are known superantigens for T cells expressing the αβ T-cell receptor (TCR). They bind to MHC class II molecules on antigen-presenting cells and can subsequently trigger T-cell responses by binding to Vβ-gene products. The reactivity of γδ T cells with enterotoxins is less well defined although both proliferative and cytotoxic responses have been described. In the present study we have tested the cytotoxic reactivity of a panel of 41 γδ T-cell clones against target cells coated with the enterotoxins SEA, SEB, SEC1, SEC2, SEC3, SED, SEE or TSST. Three reaction patterns were observed with the γδ T-cell clones: (1) clones that specifically lysed SEA-coated target cells only; (2) clones that specifically lysed SEE-coated target cells only, and (3) clones that specifically lysed SEA-coated target cells only in the presence of certain human sera. The presence of SEA-specific antibodies in such human sera could be demonstrated. Moreover, γδ T- cell clones of this third category expressed the IgG FcRIII (CD16) which indicates that these clones are capable of mediating antibody-dependent cellular cytotoxicity towards SEA-coated target cells. Thus, the cytotoxic response of γδ T cells to SEs is mediated by two distinct pathways: an antibody-independent and an antibody-dependent pathway. The antibody-independent reactivity of γδ T cells was directed to either SEA or SEE, whereas antibody-dependent reactivity was found only towards SEA. The capacity of γδ T-cell clones to respond to stimulation with SEs, combined with their high cytolytic capacity in vitro, suggests that these cells can be involved in SE-directed immune responses and efficiently kill SE-coated target cells in vivo.     

Clonal expansion precedes anergy and death of V beta 8+ peripheral T cells responding to staphylococcal enterotoxin B in vivo

Staphylococcal enterotoxin B (SEB) selectively stimulates T cells bearing T cell receptor V beta 8 domains and hence provides a useful model to study immunity and tolerance in vivo. We show here that V beta 8+ T cells in both CD4+ and CD8+ subsets expand dramatically (fivefold) in lymphoid tissues of mice 2-4 days following injection with SEB. This initial clonal expansion, which is accompanied by a transient hyper-reactivity to SEB, is followed by a rapid decrease in V beta 8+ cells and a concomitant induction of specific non-responsiveness which persists for at least 30 days. Selective death of V beta 8+ cells occurs during this latter phase. Taken together, our data indicate that clonal expansion, anergy and death can occur as sequential stages of an immune response in vivo.     

Oligoclonal T cell expansions in patients with Behçet's disease

Behçet's disease (BD) is a multisystem disorder with oral and genital ulcers, mucocutaneous, ocular, joint, vascular and central nervous system involvement. In this study, the peripheral T cell repertoire was analysed in patients with BD with MoAbs against T cell receptor (TCR) Vβ gene products in CD4⁺ and CD8⁺ T cell compartments, and these were compared with rheumatoid arthritis (RA) patients and healthy controls (HC). In the CD4⁺ T cell compartment, oligoclonal TCR Vβ expression was observed in 56% of BD (10/18), 71% of RA (5/7) patients and 21% (3/14) of HC. In the CD8⁺ T cell group 50% of BD (9/18), 57% of RA patients and 28% of HC (4/14) had an oligoclonal TCR repertoire. An increase of TCR Vβ5.1 subset was observed in five BD patients among CD8⁺ T cells. Other elevations of TCR Vβ subsets were heterogeneously distributed with one to three different Vβ subsets. Our results suggest an antigen-driven oligoclonal increase of T cells in BD. There was no overall increase in any Vβ group to suggest a superantigen effect. Analysis of the responsible antigens causing the increase in T cell subsets may give insights into the aetiopathogenesis of BD and immunomodulation of these T cells may lead to new treatments.     

TCR repertoire of suppressor CD8+CD28- T cell populations.

The cellular basis of graft rejection and the development of strategies for specific suppression of T cell responses against allogeneic and xenogeneic transplants represents an area of active investigation. Recently, a population of MHC-class I restricted CD8+CD28- T suppressor cells (Ts) which are able to inhibit specifically the proliferative response of allospecific, xenospecific and nominal-antigen specific CD4+ T helper cells (Th) has been identified. We have studied the TCR V beta gene repertoire expressed by CD8+CD28- Ts isolated from allospecific, xenospecific, and nominal antigen-specific T cell lines (TCL). A limited V beta repertoire has been found in all TCLs studied. The most restricted TCR V beta usage was observed within the population of Ts from xenospecific TCLs. The TCR V beta usage within the Ts subset of TCL differs from the TCR repertoire expressed by the CD4+ Th subset of the same TCL. This is consistent with the fact that Ts and Th cells recognize distinct MHC/ antigen complexes. The finding that the TCR repertoire used by Ts is limited opens new avenues for studying the mechanisms of transplant rejection.     

Contributions of CD4+, CD8+, and CD4+CD8+ T cells to skewing within the peripheral T cell receptor beta chain repertoire of healthy macaques.

Diversity in the peripheral T cell receptor repertoire of rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) has been studied by examining the profile of CDR3 lengths in TCR beta chains. Expressed CDR3 length distribution profiles for individual TCRBV families were obtained from total peripheral blood mononuclear cells (PBMC) and T cell subsets isolated from PBMC. These studies reveal that the T cell receptor repertoire of PBMC from healthy macaques often exhibits skewing in TCRBV family CDR3 profiles. The skewing of TCRBV family CDR3 profiles was evident as discrete expanded length(s) and was detected in up to 50% of the PBMC profiles. Analyses of separated T cell populations demonstrated that the CD8+ T cell subset was responsible for the majority of observed skewing in CDR3 length profiles. However, CD4+ T cells were also shown to contribute to the skewed peripheral PBMC repertoire in these animals. While certain TCRBV families frequently displayed skewed profiles, there was no concordance in the particular CDR3 lengths expanded among the different animals. Furthermore, an additional feature of the peripheral blood of the animals studied was the presence of an unusual population of extrathymic CD4 and CD8+ (double-positive) T cells (up to 9.6% in the PBMC of rhesus macaques). The double-positive T cells could be differentiated from CD4 single-positive and CD8 single-positive T cells by their increased surface expression of LFA-1 and decreased CD62L expression. The percentage of the double-positive T cells was higher in rhesus than pig-tailed macaques and contributed substantially to the peripheral T cell repertoire.     

T cell receptor (TCR) V gene usage in patients with systemic necrotizing vasculitis.

Wegener's granulomatosis (WG) and polyarteritis nodosa (PAN) are systemic necrotizing vasculitides of unknown etiology. These disorders run a fatal course if untreated. T lymphocytes are implicated in the pathogenesis of WG, since they have been found to infiltrate affected organs, and sIL-2R correlates with disease activity. To elucidate further the role of T cells in necrotizing vasculitis, we have used a panel of 12 TCR V-specific MoAbs to investigate the number of cells expressing certain V alpha and V beta gene segments in the CD4+ and CD8+ subsets of altogether 11 patients with WG or PAN. In the group of patients, we found abnormal expansions of T cells using particular TCR V alpha or V beta gene products. These T cell expansions were more numerous, of a dramatically higher magnitude, and frequently more often found in the CD4 subset, compared with T cell expansions identified in healthy individuals. In long-term studies of the T cell expansions for up to 18 months, a heterogeneous pattern was revealed, with no obvious correlation to clinical features such as disease activity or treatment. Studies of TCR V gene usage in this group of patients may help in understanding the pathogenesis of necrotizing vasculitis, and in the identification of unknown antigens, and may open the possibility to a highly selective immunotherapy by targeting disease-mediating T cells.     

Superantigen-induced anergy of V beta 8+ CD4+ T cells induces functional but non-proliferative T cells in vivo.

The response profile of staphylococcal enterotoxin B (SEB)-primed murine V beta 8+ CD4+ and V beta 8+ CD8+ T cells was analysed upon rechallenge in vitro. While in vitro responses to secondary stimulation with SEB were reduced to background levels, the in vivo reactivity after rechallenge with SEB was retained, in that SEB-primed mice succumbed to lethal T-cell shock, lymphokines [interleukin-1 (IL-1), IL-2, Il-4, IL-6, IL-10, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha)], and lymphokine-specific mRNA accumulation could be detected in V beta 8+ CD4+ and V beta 8+ CD8+ T cells. However, V beta 8+ CD4+ T cells failed to enter the cell cycle. While the phenotype of V beta 8+ CD8+ T cells was indistinguishable from that of their counterparts from naive mice, V beta 8+ CD4+ T cells exhibited in vivo an unusual phenotype as non-proliferative but functional T cells. We conclude that in vitro-defined anergy does not disclose the functional abilities of ligand-reactive V beta 8+ T cells in vivo, and that priming with superantigen (SAg) induces in vivo a differentiation of SEB-reactive V beta 8+ CD4+ T cells into a non-proliferative but functional phenotype.     

Human CD4+ and CD8+ T lymphocytes are both cytotoxic to Toxoplasma gondii-infected cells.

Studies to determine if Toxoplasma gondii-specific human T cells lyse parasite-infected cells have yielded conflicting results. Furthermore, attempts to obtain human cytotoxic CD8+ T lymphocytes have been difficult because of the lack of a reproducible system for their generation. By using paraformaldehyde-fixed, T. gondii-infected peripheral blood mononuclear cells as antigen-presenting cells, we developed a method whereby T. gondii-specific T-cell lines can be reproducibly generated. Six T. gondii-specific T-cell lines were generated from an individual chronically infected with T. gondii. Cytofluorometric analysis of these lines revealed > 99% CD3+, 85 to 95% CD3+ alpha beta T-cell-receptor-positive (TCR+), 5 to 9% CD3+ gamma delta TCR+, 50 to 70% CD4+, and 20 to 40% CD8+ cells when cells were examined during the first 3 weeks of stimulation and >99% CD3+, >99% CD3+ alpha beta TCR+, < 1% CD3+ gamma delta TCR+, 20 to 40% CD4+, and 60 to 80% CD8+ cells when cells were examined between 5 and 11 weeks. Both CD4+ and CD8+ T cells had remarkable cytotoxic activity against T. gondii-infected target cells (30 to 50% specific Cr release at an effector-to-target ratio of 30:1) but not against uninfected target cells ( < 10% at an effector-to-target ratio of 30:1). Cytotoxic activity by the whole T-cell lines was not T. gondii strain specific. Whole T-cell lines were cytotoxic for target cells infected with the C56 and ME49 strains and the RH strain (which was used to infect peripheral blood mononuclear cells). T. gondii-specific T-cell lines displayed the predominant expression of V beta 7 TCR. The CDR3 regions of the V beta 7 TCRs of these T-cell lines showed a striking degree of sequence identity (oligoclonality). T-cell lines obtained by the method reporter here can be used to characterize functional activity of T-lymphocyte subsets in humans infected with T. gondii.