Purine metabolic enzymes in lymphocytes. I. Adenosine deaminase and purine nucleoside phosphorylase activities in mouse lymphocyte subpopulations

The distribution of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities in lymphoid organs and lymphocyte subpopulations in mice, and the effect of phytohemagglutinin P (PHA-P) and concanavalin A (Con A) on the enzyme activities were studied. ADA activity was distributed equally in cells from all organs used and no mouse strain differences were observed. In contrast, PNP activity varied with the mouse strain, being highest in C57BL/6 mice and lowest in BALB/c mice, and with the organ in ICR mice, being high in peripheral blood lymphocytes and spleen lymphocytes, low in mesenteric lymph node cells and absent or very weak in thymus cells. T and B lymphocytes were prepared from spleen of ICR mice. High ADA activity was found in both T and B lymphocytes, whereas PNP activity in the T lymphocytes was about one-third of that in the B lymphocytes. PNP activity in thymus cells was increased to the normal level of T lymphocytes in the spleens by cultivation without stimulant. The development of PNP activity in thymus cells was partially inhibited by Con A but was not affected by PHA-P. ADA activity in thymus cells was enhanced by in vitro stimulation with PHA-P but not with Con A. In contrast, in spleen lymphocytes the development of ADA activity was enhanced by stimulation with PHA-P and Con A, and that of PNP activity was enhanced by PHA-P but not by Con A.     

The effect of cyclophosphamide and gamma irradiation on adenosine deaminase and purine nucleoside phosphorylase in mice

Changes in ADA and PNP activities in the spleens and thymuses of mice were studied after a single administration of cyclophosphamide (CY, 200 mg/kg) and after whole-body gamma irradiation (5.5 Gy), applied alone or three days after CY application. In the first days after the treatment the enzyme activities were significantly depressed (p less than 0.01) with the exception of ADA in the spleen, where a high elevation (220-380%) in relation to controls was observed. During the regeneration period a pronounced rise of PNP activity in the spleen occurred mainly after a combined application of CY and irradiation (270%). In the thymus the regeneration was manifested by a mild increase of both ADA and PNP activities towards control values. The findings suggest that the expressive changes of ADA and PNP activities, participating in the purine salvage pathway, may, after a cytotoxic treatment, influence the nucleotide pool and DNA synthesis in lymphoid organs.     

Purine nucleoside metabolizing enzyme activities in mouse thymocytes at different stages of differentiation and maturation

The activities of the enzymes involved in purine nucleoside metabolism, adenosine deaminase (ADA), adenosine kinase (AK), purine nucleoside phosphorylase (PNP) and deoxycytidine kinase (deoxyCRK), were determined in mouse thymocytes at various stages of differentiation and maturation, and compared with those in other tissues. The thymocytes were characterized by high ADA and deoxyCRK activities with high ADA/AK and ADA/PNP ratios and low PNP/deoxyCRK ratio. In fetal thymocytes of 16 gestational days, ADA activity was lower, and PNP, AK and deoxyCRK activities were higher than those in the adult thymocytes. During differentiation of fetal thymocytes, ADA activity increased while PNP and AK activities decreased. DeoxyCRK activity decreased after birth. In spleen T lymphocytes, ADA and deoxyCRK activities were lower and PNP activity was about 2.5-fold higher than in the thymocytes. Thus the differentiation stages of T lymphocytes may be characterized by the absolute levels and the ratios of these enzymes.     

Purine salvage pathway enzyme activities in human T-, B-, and null lymphocyte populations

Deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) result in distinctly different immunodeficient states. In order to better understand the roles of these two purine salvage pathway enzymes in normal immunity we have characterized their activities in peripheral blood T-, B-, and null (non-T, non-B) cell populations. We have found that T lymphocytes have significantly higher ADA activity than B or null cells (P < 0.001) only when expressed as a function of cell protein. Contrary to other reports B lymphocytes cannot be distinguished from T lymphocytes on the basis of PNP activity. When the enzyme activities are expressed per cell number null cells have a much higher PNP activity (mean ± SD = 461 ± 174 ng/hr/10⁶ cells) than either T (57 ± 10) or B (93 ± 51) lymphocytes (P < 0.001 and 0.002, respectively). Null cells also have a significantly greater protein content per cell (55.0 ± 9.6 μg/10⁶ cells) than T (13.3 ± 4.6) or B (18.7 ± 13.8) lymphocytes. When expressed as a function of cell number null cells have a higher mean, though not statistically significant, ADA activity than T and B lymphocytes. These results indicate that normal human peripheral blood T, B, and null cells can be distinguished on the basis of their ADA and PNP activities and that the method of expression of the activities is critical. The significantly higher PNP activity and protein content per cell in null lymphocytes is compatible with a less differentiated cell population which may include B- as well as T-cell precursors.     

Inverse relationship between adenosine deaminase and purine nucleoside phosphorylase in rat lymphocyte populations

The levels of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in rat lymphoid cell populations. The specific activities of the two enzymes in thymus, lymph node, spleen, and bone marrow were found in inverse proportion to each other. Thy mocytes had the highest ADA activity and the lowest PNP activity, whereas spleen and bone marrow lymphocytes had the lowest ADA activity (six- to sevenfold lower than thymocytes) and the highest PNP activity (fourfold higher than thymocytes). This reciprocal relationship was also found in cells of the T lymphocyte lineage at various stages of differentiation. These results suggest that specific stages of T-cell development may be characterized by the levels of the two enzymes, and that deficiencies of each of these enzymes might affect T cells at separate stages of differentiation.     

Purine metabolizing enzyme activities in radiosensitive tissues of mice after sublethal whole-body irradiation

The activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were determined between days 1-14 in the spleen, thymus and femoral bone marrow of mice subjected to whole-body gama irradiation with a dose of 5.5 Gy. In control animals, the highest activity of ADA (as related to 10(6) cells) was recorded in the thymus (58.9 pmol.s-1), the lowest one in the femur (34.8 pmol.s-1), the PNP activity was the lowest in the thymus (14.5 pmol.s-1) and the highest in the femur (96.0 pmol.s-1). In the spleen, an elevation of ADA activity (up to 379%) was observed during the first postirradiation days; PNP activity was reduced (to 58%) on postirradiation day 3, followed by the return and even elevation on day 14 (265%). In the thymus, a parallel reduction of the activities of both enzymes appeared during the first postirradiation days, with a subsequent increase during the regeneration phase. In the femoral bone marrow, ADA and PNP activities were increased on postirradiation day 1 (275% and 201%, respectively). Reference is made to the possible relationship between the observed characteristic changes in activities and the degree of damage and/or renewal of cell population in the hemopoietic tissues after irradiation.     

Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

The role of suppressor macrophages (S-M phi) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2b/k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of M phi from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the M phi isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic M phi obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in M phi isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-M phi expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.     

Distribution of enzymes of purine metabolism in lymphocytes of horse, Equus caballus

A microassay requiring as few as 2 X 10(5) cells per assay was developed for systematic analysis of 9 purine enzymes in lymphocytes from equine peripheral blood, spleen, lymph node, thymus and bone marrow. The activities of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by this microassay in lymphocytes from peripheral blood from four different breeds of horses (Arabian, Quarter Horse, Thoroughbred and Shetland Pony). There were no significant differences in the enzyme activities among the various breeds. Peripheral blood lymphocytes (PBL) from foals exhibited enzyme activities similar to those observed for adult animals. All lymphoid tissue contained similar levels of activity for each kinase (AK, dAK and dCK). Spleen had the highest activity for ADA, PNP, 5'-N, and HGPRT. The lowest activity for ADA, APRT, PNP and AMP deaminase was found in thymus. Enzymatic activities that varied the most among the tissue were 5'-N, ADA, APRT, HGPRT and AMP deaminase.     

SV40 T antigen acts as a minor histocompatibility antigen of SV40 T antigen tolerant transgenic mice

The ability of normal mice to mount an SV40 T antigen-specific cytolytic T lymphocytes response when immunized in vivo with splenocytes from the SV40 T antigen transgenic 427-line mice and restimulated in vitro with SV40-transformed fibroblasts, or when immunized with SV40 and restimulated with 427-line splenocytes, was analyzed. Both immunization schemes resulted in an SV40 T antigen-specific immune response, indicating the presence of SV40 T antigen-positive cells in the spleens of these transgenic mice. Normal mice engrafted with skin from 427 donors showed no rejection of the graft. Thus, SV40 T antigen in transgenic 427-line mice is expressed on an undefined cell type in the spleen and acts as a tissue-specific minor histocompatibility antigen.     

Impaired germinal center maturation in adenosine deaminase deficiency

Mice deficient in the enzyme adenosine deaminase (ADA) have small lymphoid organs that contain reduced numbers of peripheral lymphocytes, and they are immunodeficient. We investigated B cell deficiency in ADA-deficient mice and found that B cell development in the bone marrow was normal. However, spleens were markedly smaller, their architecture was dramatically altered, and splenic B lymphocytes showed defects in proliferation and activation. ADA-deficient B cells exhibited a higher propensity to undergo B cell receptor-mediated apoptosis than their wild-type counterparts, suggesting that ADA plays a role in the survival of cells during Ag-dependent responses. In keeping with this finding, IgM production by extrafollicular plasmablast cells was higher in ADA-deficient than in wild-type mice, thus indicating that activated B cells accumulate extrafollicularly as a result of a poor or nonexistent germinal center formation. This hypothesis was subsequently confirmed by the profound loss of germinal center architecture. A comparison of levels of the ADA substrates, adenosine and 2'-deoxyadenosine, as well resulting dATP levels and S-adenosylhomocysteine hydrolase inhibition in bone marrow and spleen suggested that dATP accumulation in ADA-deficient spleens may be responsible for impaired B cell development. The altered splenic environment and signaling abnormalities may concurrently contribute to a block in B cell Ag-dependent maturation in ADA-deficient mouse spleens.     

Recovery from chronic rotavirus infection in mice with severe combined immunodeficiency: virus clearance mediated by adoptive transfer of immune CD8+ T lymphocytes.

Severe combined immunodeficient (SCID) mice lack both functional T and B cells. These mice develop chronic rotavirus infection following an oral inoculation with the epizootic diarrhea of infant mice (EDIM) rotavirus. Reconstitution of rotavirus-infected SCID mice with T lymphocytes from immunocompetent mice allows an evaluation of a role of T-cell-mediated immunity in clearing chronic rotavirus infection. Complete rotavirus clearance was demonstrated in C.B-17/scid mice 7 to 9 days after the transfer of immune CD8+ splenic T lymphocytes from histocompatible BALB/c mice previously immunized intraperitoneally with the EDIM-w strain of murine rotavirus. The virus clearance mediated by T-cell transfer was restricted to H-2d-bearing T cells and occurred in the absence of rotavirus-specific antibody as determined by enzyme-linked immunosorbent assay, neutralization, immunohistochemistry, and radioimmunoprecipitation. Temporary clearance of rotavirus was observed after the transfer of immune CD8+ T cells isolated from the intestinal mucosa (intraepithelial lymphocytes [IELs]) or the spleens of BALB/c mice previously infected with EDIM by the oral route. Chronic virus shedding was transiently eliminated 7 to 11 days after spleen cell transfer and 11 to 12 days after IEL transfer. However, recurrence of rotavirus infection was detected 1 to 8 days later in all but one SCID recipient receiving cells from orally immunized donors. The viral clearance was mediated by IELs that were both Thy1+ and CD8+. These data demonstrated that the clearance of chronic rotavirus infection in SCID mice can be mediated by immune CD8+ T lymphocytes and that this clearance can occur in the absence of virus-specific antibodies.     

Distribution of terminal deoxynucleotidyl transferase and purine degradative and synthetic enzymes in subpopulations of human thymocytes

Terminal deoxynucleotidyl transferase (TdT) and purine metabolic enzymes were examined in subsets of human infant thymocytes (defined by surface cell antigens) and normal peripheral T lymphocytes. Putative prothymocytes (RFB-1+, HTA-1+/- large blast-like cells), medium and high density cortical thymocytes (RFB-1+, HTA-1+), and medullary thymocytes (RFB-1-, HTA-1-, OKT3+) were isolated by density gradient centrifugation, monoclonal antibody and complement-mediated cytolysis, and cell-antibody affinity chromatography. Peripheral T lymphocytes were isolated from normal adult mononuclear cells using nylon fiber technique. Adenosine deaminase (ADA) and TdT were highest in prothymocytes 48.8 +/- 14.7 mumol/hr/10(8) cells (mean +/- SE) and 22.9 +/- 1.4 U/10(8) cells, respectively. Both enzymes decreased progressively down the maturation pathway. In peripheral T lymphocytes, ADA was 3.9 +/- 1.5 mumol/hr/10(8) cells, and TdT was undetectable. Purine nucleoside phosphorylase (PNP) and ecto-5'nucleotidase (5'NT) were lowest in cortical thymocytes (27.5 +/- 11.0 nmol/hr/10(6) cells and 2.8 +/- 1.3 nmol/hr/10(6) cells, respectively) and increased with T cell maturation. The PNP level was 124.9 +/- 17.2 nmol/hr/10(6) cells and 5'NT was 30.1 +/- 3.9 nmol/hr/10(6) cells in peripheral T lymphocytes. The deoxynucleoside kinases (deoxyguanosine, deoxyadenosine, and deoxycytidine kinases) paralleled the changes in ADA and TdT activity among the different T subsets. The proliferative activity (labeling index) was highest in the prothymocyte fraction and lowest in peripheral T cells. Variation in the distribution of these enzymes in T cell subsets may explain their different sensitivities to deoxyadenosine and deoxyguanosine toxicity and the different effects on T cell development of ADA or PNP deficiency.     

Metabolism of purine nucleosides and phosphoribosylpyrophosphate in thymocytes and splenocytes of various mammalian species

1. Activities of ADA, PNP and AK were measured in splenocytes and thymocytes of newborn children, young horses, pigs, sheep, rats and mice and compared with the activities previously found in peripheral lymphocytes. 2. With all species, except horse, the activity of ADA (per 10(6) cells) was higher in thymocytes than in lymphocytes. Activity of ADA was highest in splenocytes of pig and sheep. Activity of ADA was lowest in all lymphoid cells of the horse and only about 10% of the activity in human splenocytes and lymphocytes. 3. With all species, except horse, the activity of PNP was lower in thymocytes than in lymphocytes. Activity of PNP was highest in human lymphocytes and lowest in ovine thymocytes. 4. Activity of AK is comparable in thymocytes of all species and always lower than the ADA activity. In splenocytes of man, horse and pig the activity of AK is comparable to that in thymocytes. 5. Activity of deoxyguanosine kinase was lowest in rat thymocytes and highest in those of man. 6. When enzyme activities are expressed per milligram of protein, the differences between thymocytes and lymphocytes are less pronounced. 7. Activity of PRPP synthetase per 10(6) cells was comparable in thymocytes, splenocytes and lymphocytes of the same species and between the various species. 8. The concentration of PRPP was lowest in ovine thymocytes and higher in splenocytes than in thymocytes of the same species, except man.     

Contact sensitivity to picryl chloride: the occurrence of B suppressor cells in the lymph nodes and spleen of immunized mice.

Mice were immunized for contact sensitivity and antibody production by painting the skin with picryl chloride. Lymph node and spleen cells taken 4 days later transferred contact sensitivity. However, cells taken at 7–8 days failed to transfer but were able to block the transfer by 4 day immune cells. These suppressor cells occurred in the regional lymph nodes, spleen and thymus. The suppressor activity of lymph node and spleen cells was due to B cells as shown by the effect of anti-θ serum and complement, nylon wool filtration and separation of EAC positive and negative cells by centrifugation on a discontinuous gradient. The transfer of fractions rich or poor in macrophages showed that the suppressor cell in the transferred population was not a macrophage. Separation using EAC rosettes suggested that B cells were responsible for the suppressor activity in the thymus.T cells isolated from the lymph nodes and spleen 7–8 days after immunization transferred contact sensitivity although the initial population was inactive. This indicates that passive transfer cells are present in the regional lymph nodes and spleen at later times after immunization but cannot be demonstrated because of the presence of suppressor B cells. However, no passive transfer cells were found in the thymus. The production of B suppressor cells required little or no T cell help and following immunization the spleens of reconstituted (B) mice were at least as active as control cells in causing suppression. There are several different suppressor cells which act in the picryl system and the B suppressor cells in immunized mice described here are distinct from the T suppressor cells in mice injected with picryl sulphonic acid.     

Activity of adenosine deaminase and purine nucleoside phosphorylase in erythrocytes and lymphocytes of man, horse and cattle.

1. Activities of ADA and PNP were measured in erythrocytes and lymphocytes of man, horse and cattle. 2. In bovine hemolysates both enzyme activities are low when compared with activities in human hemolysates. In horse hemolysates both enzyme activities are virtually absent. 3. Enzyme activities are consistently lower (about 50%) in intact lymphocytes than in sonicated lymphocytes. This finding suggests that the uptake of nucleosides is rate-limiting for both enzymes in intact lymphocytes. 4. The activity of ADA in horse lymphocytes is comparable to that in lymphocytes of patients with severe combined immunodeficiency associated with ADA deficiency.     

Cross reactive antigens of Acholeplasma laidlawii and L-form of Staphylococcus aureus]

We demonstrated that the membrane of Acholeplasma laidlawii PG8 and L-form of Staphylococcus aureus, both of which induce cellular immunity in BALB/c mice, were antigenically related each other. Foodpad responses of the mice immunized with a mixture of either antigen and Freund's complete adjuvant showed clearly a cross reaction when challenged with the other antigen. Cross responses to incorporate 3H-thymidine to the spleen lymphocytes of the mice immunized with either antigen occurred in the presence of the other antigen. Furthermore, the purified T cells, but not B cells, of the spleen were activated in the presence of antigen-presenting cells. These antigens existing in the membrane fractions of both microorganisms were purified by Razin's method. Finally, these membrane components of A. laidlawii and L-form of S. aureus were subjected to gel electrophoresis and transferring to nitrocellulose membrane and used to stimulate the spleen lymphocytes of the mice immunized with A. laidlawii or of non-immunized mice. The fractions representing molecular weights of approximately 45 kD, 25 kD, and 13 kD of both microorganisms consistently stimulated the lymphocytes of the immunized mice but not those of non-immunized mice.     

Ribonucleic acid-dependent ribonucleic acid polymerase in the immune response.

Ribonucleic acid (RNA)-dependent RNA polymerase activity was demonstrated in the microsomal and ribosomal fraction from the spleen cells of immunized mice. The enzyme activity was solubilized by Triton X-100 from the fraction and partially purified by Biogel A 1.5 m column chromatography. The RNA-dependent RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RNA polymerase activity was eluted in a single peak from the column. High activity was demonstrated with an RAN preparation (iotaRNA) as template made from the spleens of immunized mice but very low activity was found with an RNA preparation made from the spleens of normal mice. Incorporation of 3H-UTP markedly decreased in the presence of RNase but not in the presence of DNase. DNA preparations made from the spleens of immunized mice were inactive as template for this enzyme. The iotaRNA preparation was fractionated by sucrose density gradient centrifugation. A fraction corresponding to 12-13 S was most active as a template. It was followed by a fraction corresponding to 6-7 S. Sucrose gradient analysis of the 3H-UTP-labeled product was attempted. Some properties of this enzyme are described.     

Cellular basis of tolerance to serum albumin in adult mice. I. characterization of T suppressor and T helper cells.

The surface markers and size of suppressor cells were determined in adult (BALB/c x C57BL/Ka)F1 mice which were tolerized with a single injection of deaggregated bovine serum albumin (BSA). Suppressor cells from the spleens of tolerazided donors were assayed in a cell transfer system in which graded numbers of cells were injected into irradiated syngeneic mice along with limiting numbers of T cells primed to BSA and an excess of B cells primed to DNP-BSA. Adoptive hosts were challenged with DNP-BSA in saline, and the anti-DNP response was measured. Suppressor cells were antigen specific as shown by the inhibitory activity of BSA-tolerant spleen cells on the response to DNP-BSA, but not to DNP-BGG. Suppressor cells were eliminated by in vitro treatment with anti-Thy 1.2, anti-Ly-2.2, anti-I-J subregion antisera and C, but not with anti-Ly-1 or anti-I-A subregion antisera. Neither unprimed nor primed helper T cells were detected in the spleen of tolerized donors after in vitro treatment with anti-Ly-2.2 antisera. Both helper and suppressor T cells from the spleens of primed or tolerized donors, respectively, showed a rapid sedimentation velocity (S greater than 3.7 mm/hr).     

Influence of aging on intracellular free calcium and proliferation of mouse T-cell subsets from various lymphoid organs.

The influence of aging on T-cell activation and proliferation was examined in lymphocytes derived from peripheral blood, spleen, and lymph nodes of WBB6F1 C57B1/6J x WB/Re) mice. Following activation with anti-CD3 monoclonal antibodies, the greatest age-related changes were seen in CD4+ cells derived from spleens of 27- to 30-month-old mice. These CD4+ lymphocytes showed reduced [Ca2+]i signaling and decreased proliferation in the presence of exogenous interleukin 2. CD8+ cells from spleens of old animals showed reduced [Ca2+]i but not altered proliferation. Both CD4+ and CD8+ cells derived from peripheral blood of old mice showed decreased peak [Ca2+]i, but no defect in cell proliferation. In contrast, age-related deficits in either [Ca2+]i or proliferation were not observed in CD4+ and CD8+ cells from lymph nodes. Additionally, the percentage of CD4+ cells was decreased in all lymphoid organs from old mice, while the percentage of CD8+ cells was similar in lymphoid organs of old and young mice. Old mice had a significant increase in expression of Pgp-1 in CD4+ cells from spleen and peripheral blood and CD8+ cells derived from lymph node. Our studies indicate that there are differential effects of aging in T lymphocytes derived from different lymphoid organs in mice. Among the cell sources and subsets examined, the age-related changes noted in CD4+ cells from mouse peripheral blood were the most similar to those previously observed in the corresponding peripheral blood lymphocyte subset in humans.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.