Specific triggering by concanavalin A of a secondary T killer cell-mediated anti-tumor immune response.

Spleen cells from C57 B1/6 mice which rejected a Moloney sarcoma virus (MSV)-induced tumor elicited a strong cytolytic reaction against MSV-associated antigens when incubated for 7 days with a mitogenic dose (5 μg/ml) of concanavalin A (Con A). The cytolytic activity evaluated in a chromium release test was shown to be mediated by T lymphocytes, by anti-Thy 1–2 serum treatment, and independent of remaining Con A, by treatment with d-[α-methyl]mannopyranoside. A similar reactivity was obtained with phytohemagglutinin (PHA) at mitogenic doses but not with the B-cell mitogen lipopolysaccharide (LPS). This “secondary-like” response was regenerated up to 50 days post-MSV inoculation but decreased regularly. The cytolytic activity had an antigenic pattern indistinguishable from that of the relevant tumor cell reactivation and was directed by the same H-2 restriction.     

Quantitation of the T cell deficiency in RFM mice with host versus graft disease.

Host versus graft (HVG) syndrome is the fatal complex of lesions which has been observed in six inbred strains of mice following the perinatal inoculation of related F₁ hybrid spleen cells. Morphological studies have indicated that the key lesion is the depletion of peripheral T lymphocytes due to inflammatory destruction and failure of the thymus to replace them. In the present studies, tests of T-cell function were done on RFM mice, which had developed HVG disease following perinatal inoculations of (T₆ × RFM)F₁ spleen cells. As compared to control values, HVG spleen cell suspensions showed loss of reactivity to phytohemagglutinin (PHA) = 90%, to concanavalin A (Con A) = 94%, to (T₆ × RFM)F, cells in the mixed lymphocyte reaction (MLR) = 82%, to DBA cells in MLR = 94%, and to DBA mastocytoma cells in cell-mediated lympholysis (CML) = 95%. Lymph node cell suspensions showed losses of reactivity to PHA = 83%, to Con A = 62%, to (T₆ × RFM)F₁ cells in the MLR = 91%, and to DBA cells in the MLR = 77%. The CML activity of nodal cells to DBA mastocytoma cells varied widely from 12 to 273% of control values, and averaged 121%. Filtration of HVG spleen cells through nylon fiber columns failed to restore low responses to PHA to normal values. This suggested that the macrophage-like, adherent accessory cells were not acting as suppressors of T-cell responses in HVG disease. The deficits in all T-cell-mediated functions tested so far, appeared to correlate very well with quantitative morphological studies which showed the loss of 98% of the small lymphocytes normally present in the thymic dependent portions of the splenic white pulp. It is suggested that experimental HVG disease may serve as a model for immunodeficiency syndromes of the Nezelof type which are also characterized by T-cell deficiency, poor primary antibody responses, and the presence of variable amounts of serum immunoglobulins.     

Possible mechanism of the preventive effect of BCG against diabetes mellitus in NOD mouse. I. Generation of suppressor macrophages in spleen cells of BCG-vaccinated mice.

With the aim of clarifying the mechanism of the suppressive action of BCG against insulitis and overt diabetes in NOD mice, we studied the effects of BCG on spleen cell populations and on the in vitro immune responses of spleen cells. The spleen cells of BCG-vaccinated mice showed much lower responsiveness to various mitogens such as Con A, PHA, PWM, and LPS than those of saline-treated mice. Low responsiveness to alloantigens was also observed. Flow cytometric analysis of the spleen cells revealed that Mac-1+ and Mac-2+ cells had increased while T and B cells had decreased in the BCG-vaccinated mice compared with the saline-treated mice at the time when the maximum level of inhibition of mitogen responses of BCG-vaccinated mice was observed. This suggests that the decreased in vitro immune response was due to the increase in macrophages which suppress lymphocyte functions. Support for this interpretation comes from the following two findings: (1) the restoration of mitogen responses of spleen cells when macrophages were eliminated by plastic adhesion or FACS sorting and (2) resuppression of PHA and Con A responses of plastic-nonadherent spleen cells by addition of adherent cells or flow cytometrically sorted Mac-1+ cells obtained from BCG-vaccinated mice. These results indicate the generation of suppressor macrophages after BCG vaccination and suggest that these macrophages prevent the autoimmune pathogenesis leading to diabetes in NOD mice.     

Trypanosoma cruzi: regulation of mitogenic responses during infection in genetically resistant and susceptible inbred mouse strains

The outcome of Trypanosoma cruzi infection in inbred strains of mice is under genetic control. The lymphocyte responses to T-cell mitogens and their regulation were investigated in strains of mice resistant or susceptible to T. cruzi. Six to eight days after the inoculation of T. cruzi, resistant and susceptible mice had depressed responses to T-cell mitogens. In resistant B6 mice, suppression was maximal 18 days after infection and it persisted for at least 320 days. The duration of immunosuppression correlated with the persistence of a subpatent parasitemia. In cell mixing experiments, it was determined that the concanavalin A (Con A) responses in the resistant B6 and B6C3F1 mouse strains were suppressed by highly active T-suppressor cells. In the susceptible C3H mice, intense suppression of the Con A responses was detected 14 days after inoculation of T. cruzi. Nevertheless, only weak suppressor cell activity was detected in the infected C3H mice, and suppression was not abrogated by passage through a nylon wool column nor by treatment with antitheta antibodies and complement. Thus, it was suggested that, during the course of infection with T. cruzi, splenic T cells from C3H mice acquired a block in the metabolic pathway for cellular activation by Con A. The influences of T. cruzi epimastigotes on the Con A responses of spleen cells from uninfected mice were then studied. The Con A responses of spleen cells from C3H mice were depressed in the presence of epimastigotes, whereas they were either unaffected or enhanced in spleen cells from B6 mice. Hence, the immunoregulatory events provoked by T. cruzi infection differed in genetically resistant and susceptible mice, and lymphocytes from C3H mice were predisposed to a parasite-induced block in the responses to Con A. Thus, the gene(s) determining the outcome of infection with T. cruzi may be phenotypically expressed through an influence on immunoregulatory events.     

Splenic suppressor macrophages induced in mice by injection of Corynebacterium parvum.

Spleen cells from C57BL/6N mice injected with killed Corynebacterium parvum (CP) had a marked growth inhibitory effect on the in vitro proliferation of RBL-5 murine lymphoma cells. It was most marked 12 to 14 days after injection and was usually no longer detectable later than 21 days. It could be demonstrated at effector cell to target ratios between 20:1 and 5:1 at which normal spleen cells had a growth-promoting effect. Addition of CP to an in vitro mixture of spleen cells and tumor cells augmented the inhibitory effect of spleen cells from CP-injected mice although it conferred no inhibitory potential on normal spleen cells. Growth inhibiton by CP spleen cells was not mediated by T cells and various depletion experiments suggested that the effector cells of the phenomenon were macrophages. Spleen cells of CP-injected mice also showed strongly depressed responses to the T cell mitogens PHA and Con A and suppressed the mitogen responses of syngeneic normal spleen cells. The characteristics of the suppressor cells mediating this effect appeared to be very similar to those inhibiting lymphoma cell growth. The responses to LPS were also strongly suppressed in mice injected with 2.1 mg of CP. However, after injection of one-tenth of the dose a relative sparing of the LPS response was noted, whereas the PHA response was still suppressed.     

Altered lymphocyte functions in rats bearing syngeneic Moloney sarcoma tumors. I. Mitogen responses, mixed lymphocyte reactions (MLR) and mixed lymphocyte-tumor reactions (MLTR).

Impairment of mitogen responses to Con A and LPS and of MLR and MLTR was detected in the spleens of rats bearing syngeneic Moloney sarcoma tumors. Depressed responses of both T cell and Ig+ cell populations were observed. During the observation period of 6 to 10 days post-tumor inoculation when maximal T cell-mediated cytotoxicity was observed in spleen and draining lymph node cells, spleen cells showed marked impairment in response to stimuli mentioned above. By contrast, draining lymph node cell activity was either unaltered or somewhat elevated above the level of activity measured in normal control populations. Data presented in this and an accompanying paper strongly indicate that macrophages are activated as immunosuppressor cells in tumor-bearing rats.     

Immune suppression induced by protoscoleces of Echinococcus multilocularis in mice. Evidence for the presence of CD8dull suppressor cells in spleens of mice intraperitoneally infected with E. multilocularis.

Immunoregulatory states induced by i.p. inoculation with the metazoan parasite Echinococcus multilocularis in the murine system were investigated. Proliferative responses and IL-2 production induced by Con A in spleen cells from BALB/c mice were significantly depressed at an early stage after infection with E. multilocularis protoscoleces (PSC). Addition of plastic-adherent cells from normal syngeneic mice to the nonadherent spleen cells from infected mice did not restore the depressed Con A responsiveness. On the other hand, exogenous IL-2 reconstituted completely the proliferative responses to Con A. Flow cytometry analysis revealed that CD4- CD8+ cells with a low density of CD8 Ag (CD8dull cells) increased in spleens from infected mice 2 weeks after inoculation. Addition of the spleen cell subpopulation containing the CD8dull cells, but not that depleted of the CD8dull cells, to normal spleen cells resulted in marked suppression of the Con A responses. These findings suggest that the CD8dull cells detected in spleens of mice inoculated with E. multilocularis PSC may play a key role in the suppressive regulation of immune responses. The relevance of the immune suppression seen in the early stages of experimental infection with E. multilocularis PSC to the eventual establishment of a host-parasite relationship is discussed.     

Effects of anti-interferon serum on growth and regression of Moloney sarcoma virus induced tumours in mice

Administration of sheep anti-mouse interferon serum or globulin to weanling BALB/c mice infected with Moloney sarcoma virus (MSV) accelerated the growth of tumours at the site of virus inoculation, increased the number and size of tumours, and inhibited their regression. The outcome of the MSV-induced disease after injection of anti-interferon globulin depended on the age of mice. The antigenic stimulation by normal sheep serum or globulin also enhanced the growth and mortality due to MSV but to a significantly lesser extent than anti-interferon globulin.     

Experimental cutaneous leishmaniasis. I. Nonspecific immunodepression in BALB/c mice infected with Leishmania tropica

Leishmania tropica in BALB/c mice causes a fatal infection accompanied by the development of multiple metastatic lesions. Spleen cells from these mice were shown to have depressed proliferative responses to concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharide (LPS). Coinciding with this immunodepression was the development of a cell population capable of suppressing normal spleen cell responses to Con A. This suppressor cell activity was first observed at 6 wk and was present throughout the remainder of the infection. At 12 wk the suppressor cells could be removed by Sephadex G-10 passage or carbonyl iron treatment; however, Sephadex G-10 passage could not reverse the suppression at 18 wk. Indomethacin, a prostaglandin synthetase inhibitor, was found to abrogate the activity of the adherent suppressor cell, suggesting that prostaglandin production may be involved in the immunosuppression seen in these mice. In addition, Sephadex G-10 passage and indomethacin were found to markedly augment spleen cell responses to leishmanial antigen, indicating that the adherent suppressor cell is capable of regulating specific immunologic responses.     

Cellular immunity in chronic Theiler's virus central nervous system infection.

After (IC) inoculation of the DA strain of TMEV, SJL/J mice develop chronic CNS infection with marked mononuclear cell infiltration of spinal cord leptomeninges and white matter and concomitant demyelination. In the present study the temporal course of cell-mediated and humoral immune responses to virus were measured in this infection. It was shown that chronic TMEV infection is associated with the development of immunologically specific spleen cell reactivity as judged by in vitro incorporation of 3H-TdR into DNA in response to inactivated TMEV antigen. Spleen cell reactivity is first detectable about 2 months after infection, persists for at least 1 year, and correlates with the temporal development of serum-neutralizing antibody. The late development of sensitized spleen cells is not the result of an immunosuppressive effect of this virus infection since infected mice exhibit normal spleen cell proliferative responses to T cell mitogens and produce normal antibody responses to a heterologous protein antigen, sheep red blood cells. In addition, anti-viral antibody inhibits virus-induced spleen cell reactivity. Finally, the antigen-reactive lymphocyte subpopulation within the spleen responsible for proliferation to TMEV antigen are T cells and not B cells.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Opposite modulation of lymph node cell and spleen cell responses to mitogens by muramyl dipeptide and its desmethyl analog

N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), the minimal structure necessary for adjuvant activity of mycobacterial cell wall preparations, was evaluated as an immunostimulant in mice. MDP treatment, which increased carbon clearance and nonspecific resistance to lethal Klebsiella challenge, induced lymph node cellular hyperplasia (4-fold). In contrast, spleen and resident peritoneal cell recovery was comparable to controls. Lymph node cells (LNC) from MDP-treated mice had enhanced [³H]thymidine uptake in unstimulated (4-fold) and lipopolysaccharide (LPS) (5-fold)-, concanavalin A (Con A) (2-fold)-, and phytohemagglutinin (PHA) (1.5-fold)-stimulated cultures. In contrast, spleen cells exhibited depressed responses when stimulated with LPS (2-fold), Con A (2- to 5-fold), and PHA (3-fold). Depressed responses of spleen cells to mitogens were demonstrated over a range of mitogen concentrations. The desmethyl analog produced similar effects, although spleen cells were not as hyporeactive. Opposing modulations of the immune system by MDP resembles that reported after BCG infection, and correlates with increased nonspecific host resistance to microbial challenge.     

Studies of the T-lymphocytes resident in the spleens of thymectomized, irradiated, bone marrow-reconstituted (TXB) mice.

The T-lymphocytes resident in the spleens of thymectomized, lethally irradiated mice that had been reconstituted with syngeneic bone marrow (TXB) were characterized. Both recently reconstituted N-TXB, (approximately 3 weeks after bone marrow injection) and aged (>6 months after reconstitution) A-TXB animals were studied. The T-lymphocytes from spleens of recently reconstituted N-TXB donors did not respond to PHA but did react significantly to Concanavalin A (Con A). The lack of PHA sensitivity was not due to dilution of reactive cells by other cell types. Removal of adherent cells, likewise, did not restore N-TXB spleen cell PHA responsiveness. N-TXB splenic T-cells were cortisone resistant. N-TXB spleen cells by themselves did not cause a graft vs host response. However, N-TXB spleen cells amplified the graft vs host response of normal lymph node cells but not N-TXB lymph node cells. Addition of cyclic GMP enhanced [³H]thymidine uptake of N-TXB spleen cells caused by Con A. N-TXB spleen cells were exclusively spleen seeking. The Con A reactive cell within N-TXB spleens was demonstrated to be of donor origin. Fetal liver as well as syngeneic bone marrow contained cells capable of reconstituting the Con A response. Spleen cells from aged. (>6 months) A-TXB were found to be PHA sensitive. Competitive inhibition assays measuring θ expression in A-TXB spleen cells indicate a significant increase in the θ positive lymphocyte population occurred with time. The data indicate that considerable reconstitution of θ positive cells had occurred in A-TXB donors. The results also suggest that the T-lymphocyte population of the TXB spleen may be a unique subpopulation of T-lymphocytes that resides exclusively in spleen and bone marrow.     

Immunoregulation in experimental filariasis. I. In vitro suppression of mitogen-induced blastogenesis by adherent cells from Jirds chronically infected with Brugia pahangi

Nonspecific immunoregulatory events were examined in inbred jirds chronically infected with Brugia pahangi. The responsiveness of spleen cells from infected animals to the T cell mitogens PHA and Con A and to the B cell mitogens, LPS and PWM, was found to be suppressed by as much as 90% when compared with the reactivity of lymphocytes from normal animals. Furthermore, spleen cells from infected jirds were capable of suppressing the mitogen reactivity of normal spleen cells. Depletion of cells adherent to nylon wool, glass wool, or plastic alleviated the regulatory activity exerted by spleen cells from infected jirds. Addition of indomethacin, an inhibitor of prostaglandin synthetase, to cultures of spleen cells from infected animals did not alter the suppression observed. In contrast, lymphocytes from the peripheral lymph nodes of infected jirds did not exhibit depressed T cell mitogen reactivity and were incapable of suppressing the PHA or Con A responsiveness of normal lymph node cells. However, the reactivity of lymph node cells from infected jirds to B cell mitogens, LPS and PWM, was suppressed. These results imply the existence of multiple regulatory mechanisms, at least one of which is restricted to the spleen. The relevance of nonspecific regulation to development of parasite-specific immunologic reactivity and to the infection is discussed.     

Studies on the mechanisms of action of BCG

Summary Bacillus Calmette-Guérin was given intravenously (i.v.) or subcutaneously (s.c.), 14 days before antigenic or mitogenic stimulation.When given i.v., BCG increased plaque-forming cell production against thymus-dependent antigens (sheep red blood cells and a hapten coupled to hemocyanin) and against a thymus-independent antigen (the same hapten coupled to polymerized flagellin). BCG given s.c. was generally ineffective in potentiating humoral responses.In contrast, a marked depression of spleen cell reactivity in vitro to a T-cell mitogen, concanavalin A (Con A) but an increased response to a B-cell mitogen, dextran sulfate, were observed after BCG i.v., and to a lesser extent after BCG s.c. This inhibitory effect was reversed after removal of cells adherent to nylon or plastic. Normal spleen cell reactivity to Con A was strongly depressed when the cells were cultivated in presence of unfractionated or plastic-adherent BCG-treated spleen cells, suggesting that BCG induces suppressor cells which inhibit T-cell responses in vitro. Similar impairment of T-cell functions in vivo was not observed since BCG given i.v. significantly increased the intensity of delayed-type hypersensitivity to picryl chloride.BCG also induced macrophage activation, measured by macrophage tumoricidal activity in vitro, more strongly and more rapidly when given i.v. than s.c.Modifications of the distribution of lymphocytes from the circulation to the organs were observed. An early increase of lymphocyte migration to the spleen and a long-lasting depression of cell migration to mesenteric lymph nodes and bone marrow were seen after BCG given i.v. When given s.c., BCG induced a progressive increase of cell migration to the draining lymph nodes concomitant with a depression of migration to mesenteric nodes.These results demonstrate the complexity of the mode of action of BCG, but many of its effects on immune responses may result from a primary action on macrophages.     

Suppression of Con A mitogen-induced proliferation of normal spleen cells by macrophages from chickens with hereditary muscular dystrophy

Spleen cells from chickens with hereditary muscular dystrophy (MD) give low blastogenic responses to the T cell mitogen concanavalin A (Con A) while exhibiting normal mitogen stimulated blastogenic responses to the T cell mitogen phytohemagglutinin (PHA). The addition of MD spleen cells to normal spleen cells caused a marked suppression of the Con A response of the normal cells while not affecting the PHA response of the normal cells. The suppressive activity by the MD spleen cells requires viable cells and is contact mediated. The suppressive activity is attributed to the presence in MD spleens of a population of suppressor cells with characteristics typical of macrophages. The suppressor cell activity was not removable by complement-mediated lysis using anti-T or anti-B sera, but it was reversible by treatment with carrageenan or carbonyl iron magnet, by passage through a Sephadex G-10 column, and by adherence to plastic petri dishes or glass beads. MD spleen cells depleted of the suppressor cell population remained unable to respond to Con A.     

Asynchronous depression of responses to T- and B-cell mitogens during acute infection with cytomegalovirus in the guinea pig

The nonspecific functional capacity of spleen cells, taken from female guinea pigs with primary acute cytomegalovirus (CMV) infection, was assessed using lipopolysaccharide (LPS), a B-cell mitogen, and concanavalin A (Con A), a T-cell mitogen. Proliferative responses to the two mitogens were found to be significantly depressed in animals inoculated with CMV as compared to control animals. The defect in Con A responsiveness occurred earlier during the course of the infection than the defect in LPS responses. Although responses to the mitogens were depressed at the time of peak virus activity in the spleen, the possibility of lytic destruction of the spleen cells by the virus during in vitro culture was excluded. In addition, the depression in Con A responsiveness was noted with a wide range of Con A concentrations, and preculture studies failed to result in enhanced reactivity of the cells from infected animals. We conclude that reductions of both B- and T-cell functions, which differ in their timing during the course of acute CMV infection, occur concurrently with an enhanced viral specific immune response in guinea pigs acutely infected with CMV.     

Failure or success in the restoration of virus-specific cytotoxic T lymphocyte response defects by dendritic cells

C57BL/6 (B6, H-2b) mice are CTL responders to both Sendai virus and Moloney leukemia virus. In the former response the H-2Kb class I MHC molecule is used as CTL restriction element, in the latter response the H-2Db molecule. B6 dendritic cells (DC) are superior in the presentation of Sendai virus Ag to CTL in comparison with B6 normal spleen cells. Con A blasts have even less capacity to present viral Ag than NSC, and LPS blasts show an intermediate capacity to present viral Ag. H-2Kb mutant bm1 mice do not generate a CTL response to Sendai virus, but respond to Moloney leukemia virus, as demonstrated by undetectable CTL precursors to Sendai virus and a normal CTL precursor frequency to Moloney virus. Compared to B6 mice, other H-2Kb mutant mice show decreased Sendai virus-specific CTL precursor frequencies in a hierarchy reflecting the response in bulk culture. The Sendai virus-specific CTL response defect of bm1 mice was not restored by highly potent Sendai virus-infected DC as APC for in vivo priming and/or in vitro restimulation. In mirror image to H-2Kb mutant bm1 mice, H-2Db mutant bm14 mice do not generate a CTL response to Moloney virus, but respond normally to Sendai virus. This specific CTL response defect was restored by syngeneic Moloney virus-infected DC for in vitro restimulation. This response was Kb restricted indicating that the Dbm14 molecule remained largely defective and that a dormant Kb repertoire was aroused after optimal Ag presentation by DC. In conclusion, DC very effectively present viral Ag to CTL. However, their capacity to restore MHC class I determined specific CTL response defects probably requires at least some ability of a particular MHC class I/virus combination to associate and thus form an immunogenic complex.     

The effect of a high external temperature on cellular immunity]

The study was made of spleen cells proliferative response to mitogens PHA, Con A or alloantigens in relation to hyperthermia effects. Acute hyperthermia (rectal temperature 42 degrees) enhanced lymphocyte function, proliferative responses to allo-antigens, PHA and Con A increased. Thermal shock was associated with suppression of the spleen cell response. Mice suffering from hyperthermia for 20 min (43-44 degrees) daily during 10, 20 and 30 days showed suppressed T-cell immune response. Normal splenocyte proliferation recovered 40 days after hyperthermia induction.     

Ontogeny of con A and PHA responses of chicken blood cells in MHC-compatible lines 6(3) and 7(2)

The development of T cell responsiveness to Con A and PHA was examined in two MHC-compatible inbred chicken lines, RPRL 6(3) and 7(2), at ages 2 to 118 days posthatching. These lines are respectively resistant or susceptible to Marek's disease, a naturally occurring, virally induced T cell lymphoma. Between-line comparisons were made of optimal in vitro responses of diluted serum-free blood cells to each mitogen in two groups of chicks tested over ages 2 to 63 and 41 to 118 days. Over 2 to 63 days, Con A responses increased with age at the same rate in each line, but 7(2) responses averaged 2.3 times higher than 6(3). The increase with age was dependent on blood lymphocyte counts, which also increased with age in parallel in both lines. In contrast, the between-line difference in responsiveness was dependent on intrinsic reactivity of cells as well as lymphocyte counts. Covariance analysis was used to estimate that line 7(2) was 1.4 times higher than 6(3) in intrinsic cell reactivity, after accounting for the effect of the twofold higher blood lymphocyte counts in 7(2), and that this intrinsic difference contributed almost one-half the total difference. Over 41 to 118 days Con A responses no longer increased with age, although lymphocyte counts were still increasing, and the line difference (2.6 times) was now almost entirely contributed by a 2.3-fold superiority of 7(2) blood cells in intrinsic reactivity. The line difference in PHA responses was the reverse of the above in young chicks, with 6(3) responses greater than 7(2) in spite of lower lymphocyte counts. In additional chicks tested over 5 to 26 days, intrinsic reactivity of 6(3) cells to PHA averaged 4.5 times higher than 7(2). There was an abrupt decline in intrinsic reactivity of line 6(3) blood cells between 26 and 41 days to a level equal with 7(2). After this age, line 7(2) responses were 1.8 times greater than those of 6(3), and this difference was dependent solely on lymphocyte count differences. The results suggest that different gene systems mediate blood cell responses to PHA as compared with Con A. The pattern of developmental differences between inbred lines indicates the existence of distinct or partly overlapping T cell subsets with different reactivities to PHA or Con A, and of higher suppressor activity of adherent cells in line 6(3) blood. Both these differences may be related to line 6(3) inherited resistance to Marek's disease.