Ontogeny of Nk-1+ natural killer cells. I. Promotion of Nk-1+ cells in fetal, baby, and old mice

Using anti-Nk-1.1 serum, the alloantiserum specific for murine natural killer (NK) cells, we followed the ontogenetic development of Nk-1+ cells in fetal thymus, liver, and spleen. A transient population of Nk-1+ cells in fetal thymus was observed on day 14 but not on day 16 of gestation. On day 16 of gestation, Nk-1+ cells were detected only in liver and spleen. The proportion of Nk-1+ cells in spleen remained high (20 to 30%) at birth and persisted until 2 to 3 wk old. The Nk-1+ cells in "baby" (1 to 2 wk old) spleen bound to YAC cells but failed to lyse them in 51Cr-release assay. Upon induction with interferon (IF), the proportion of Nk-1+ cells increased, but the lytic activity remained low, suggesting that the "baby" NK-1+ cells are immature in lytic function. In old mice (12 to 14 mo), Nk-1+ cells were also detectable, even though NK activities were lower compared with those of the young adult (6 to 8 wk old) mice. The Nk-1+ cells of old mice were readily induced by IF to exhibit activities, and the induced NK cells were Nk-1+. We have thus established Nk-1.1 antigen as an early hemopoietic differentiation antigen. Splenic Nk-1- cells could be induce by IF to become NK-1+ cells, which could be inactive or active in NK assays, dependent on the age of the mice.     

Studies on the mechanism of low natural killer cell activity in infant and aged mice

NK activity in mice is high between about 6 and 10 weeks of age. In contrast, infant mice and mice older than 12-14 weeks of age usually have quite low or undetectable NK activity. Studies were performed to analyze the mechanisms underlying this characteristic age-related regulation of NK activity. Spleen cells from infant mice did not develop appreciable NK activity upon incubation for 12-18 h with either interferon (IFN) or interleukin-2 (IL-2). Analysis of the frequency of IL-2-dependent progenitors of NK cells, in a limiting dilution assay, also indicated that the spleens of infant mice are deficient in precursors of NK cells. In contrast, spleen cells from old mice (30 weeks old) developed substantial levels of NK activity upon incubation with either IFN or IL-2, and they showed a frequency of IL-2-dependent progenitors of effector cells that was similar to that of young mice. Both infant and old mice had plastic-adherent suppressor cells in their spleens, which could strongly inhibit NK activity. In addition, both infant and old mouse spleen cells contained nonadherent suppressor cells, which had a higher density on Percoll gradients than NK cells. Thus, several factors appear to contribute to the age-related regulation of NK activity in mice.     

Effector and regulatory cells in autoimmune oophoritis elicited by neonatal thymectomy.

(C57BL/6 x A/J)F1 (B6AF1) mice thymectomized between days 1 and 4 of age develop autoimmune oophoritis (D3TX oophoritis) 4 to 6 wk later. Oophoritis can be adoptively transferred to young recipients, and the disease in D3TX mice is prevented by reconstitution with normal adult spleen cells. The present study was further defined the nature of the effector and suppressor cells. Contrary to an earlier report, oophoritis is transferred to syngeneic and not allogeneic recipients. The spleen cells from D3TX mice when stimulated in vitro with Con A, also transfer oophoritis to adult recipients. The effector cells are CD4+: oophoritis transfer is abrogated by CD4 antibody and not by CD8 antibody and C. Spleen cells from D3TX male mice transfer disease less efficiently than female cells, thus endogenous ovarian Ag may be required for activation of effector T cells. T cells from normal adult spleen that suppress D3TX oophoritis also appear to be of CD4+ phenotype. These cells are likely to be derived from adult thymus because adult thymocytes also suppress D3TX disease. We were unable to substantiate the earlier claim that suppressor cells in normal mice are ovarian Ag specific. Thus male and female spleen cells suppress disease with comparable efficiency, and deprivation of endogenous ovarian Ag by neonatal ovariectomy of cell donors had no observable effect on disease suppression.     

Deletion of self-reactive T cells in nude mice grafted with neonatal allogeneic thymus

The cellular basis of tolerance induction has been investigated in BALB/c(H-2d, thy 1.2, M1s1b2a) nude mice grafted with thymus of neonatal AKR/J mice(H-2k,Thy1.1,M1s1a2b). The spleen cells from nude mice grafted with AKR/J thymus showed a significantly decreased level of primary cytotoxic T cell response when stimulated with AKR/J cells, although these cells lysed well target cells of a third party C57BL/6 when stimulated with C57BL/6 cells. Consistent with CTL responses, T cells bearing V beta 6, that is important for recognizing M1s1a-encoded products of the thymic phenotype, were virtually abolished in the spleen and lymph node cells of nude mice 8 wk after grafting with AKR/J thymus. However, a substantial number of V beta 6-bearing T cells were detected in the peripheral organs of nude mice 23 wk after grafting with AKR/J thymus and in those of nude mice grafted with AKR/J fetal thymus depleted of macrophages/dendritic cells by incubating with 2'-deoxyguanosine in vitro before grafting. On the other hand, T cells bearing V beta 3, which are selectively related to M1s2a-encoded products of the host phenotype, were expressed neither on the peripheral T cells of nude mice grafted with AKR/J thymus at any stage after grafting nor on those of nude mice grafted with 2'-deoxyguanosine-treated AKR/J thymus. These data suggested that both V beta 6 and V beta 3 T cells were eliminated in the thymus of nude mice grafted with AKR/J thymus, presumably on the basis of interaction with both of graft-derived persisting and host-derived hemopoietic cells in the thymus and that thymic epithelium appears to have little capacity to eliminate T cells reactive to minor lymphocyte stimulating-encoded products.     

In vitro propagation and cloning of murine natural suppressor (NS) cells

During a short period of time after birth or after radiotherapy, the spleens of neonatal and adult TLI-treated mice contain suppressor cells of the mixed leukocyte reaction (MLR) and of graft-vs-host disease. The present report shows that the MLR suppressive activity of spleen cells from TLI-treated adult BALB/c mice can be maintained in long-term tissue culture by using conditioned medium. The suppressor cells can be cloned by limiting dilution, and reproducibly inhibit the [3H]TdR incorporation in the MLR at responder-to-suppressor cell ratios of 50:1. There is no antigen specificity or H-2 haplo-type restriction of the MLR suppression. The suppressor cells do not inhibit [3H]TdR per se, because no inhibition was observed in co-culture experiments with the EL4 tumor line or the IL 2-dependent HT-2 cell line. By using immunofluorescent staining techniques, the surface phenotype of the suppressor cells was found to be similar to that reported previously for cloned NK cells (Thy-1+, Lyt-1-, Lyt-2-, Ig-, Ia-, MAC-1-, asialo-GM1+). However, the suppressor lines showed no natural killer activity when YAC-1 target cells were used. Thus, the suppressor lines have been termed "natural suppressor" cells to indicate surface marker similarities to NK cells, both in vivo and in vitro, but different effector functions.     

Mechanism of nonresponsiveness to AKR/Gross leukemia virus in AKR.H-2b:Fv-1b mice. An analysis of precursor cytotoxic T lymphocyte frequencies in young versus moderately aged mice

As young adult AKR.H-2b:Fv-1b mice reach about 9 wk of age, they begin to develop a nonresponsiveness to AKR/Gross leukemia virus. Unlike young mice that are responders, moderately aged AKR.H-2b:Fv-1b mice, after immunization and secondary in vitro restimulation in bulk culture with AKR/Gross virus induced tumors, can not generate anti-AKR/Gross virus-specific CTL. The mechanism of conversion to nonresponsiveness in moderately aged AKR.H-2b:Fv-1b mice is not understood, but it is correlated with increased expression of endogenous ecotropic viral antigens. Our present investigation focuses on determining the frequency of anti-AKR/Gross virus precursor CTL in AKR.H-2b:Fv-1b mice as a function of age. This was achieved by performing limiting dilution cultures of immune spleen cells obtained from young and moderately aged AKR.H-2b:Fv-1b mice. Although spleen cells obtained from immune moderately aged mice can not differentiate in bulk cultures into anti-AKR/Gross virus-specific CTL, there was no evidence of substantially decreased frequencies of virus-specific precursor CTL, relative to precursor CTL frequencies observed in young responder AKR.H-2b:Fv-1b mice.     

Induction of anti-AKR/gross virus cytolytic T lymphocytes in AKR.H-2b:Fv-1b congenic mice: age-dependent conversion to a nonresponder phenotype

Previously, we reported that the generation of cytolytic T lymphocytes (CTL) specific for syngeneic tumors induced by AKR/Gross leukemia viruses was under multi-gene control. Thus, although carrying the required immune response gene(s) encoded by the H-2b haplotype and characteristic of responder strains such as C57BL/6, AKR.H-2b congenic mice failed to mount antiviral CTL responses. Young adult AKR.H-2b:Fv-1b "doubly congenic" mice, however, were able to generate specific anti-AKR/Gross virus CTL activity. These results demonstrated that the positive effect of MHC-encoded immune response gene control could be overcome by the action of the Fv-1n allele. The responder status of the B6.Fv-1n congenic, however, indicated that this Fv-1n-mediated inhibition was dependent on the interaction of Fv-1n with another gene(s) encoded by the AKR background. The results of experiments performed with AKXL recombinant inbred mice further suggested that a single additional genetic locus, encoding the Akv-1 provirus, was necessary along with Fv-1n to cause inhibition of antiviral CTL generation. Here we show that the responsiveness of AKR.H-2b:Fv-1b mice is dependent on their age. Thus, with moderate aging these doubly congenic mice converted to a nonresponder status with respect to anti-AKR/Gross virus CTL production: 85% of mice less than or equal to 9 wk of age responded compared with 0% of mice greater than 9 wk old. As with nonresponder AKR.H-2b mice, an inverse correlation was observed between CTL responsiveness and the expression of CTL-defined viral antigens by normal cells. Namely, spleen cells from young AKR.H-2b:Fv-1b mice showed little or no expression of such viral antigens, whereas with moderate aging there was a steady increase in their display. These results are discussed with reference to possible mechanisms of unresponsiveness of AKR.H-2b vs moderately aged AKR.H-2b:Fv-1b mice, and with respect to the utility of this system as a model for naturally occurring retrovirus infections and the interactions of retroviruses with the immune system.     

Natural suppressor (NS) cells found in the spleen of neonatal mice and adult mice given total lymphoid irradiation (TLI) express the null surface phenotype

We studied the surface markers of suppressor cells of the mixed leukocyte reaction (MLR) that are transiently present in the spleens of neonatal mice after birth and of adult mice after total lymphoid irradiation (TLI). Approximately 80% of the mononuclear cells in the spleen, within the first few days after birth or after TLI, express neither the Thy-1 antigen nor surface immunoglobulin (Ig). After 30 days, less than 20% of mononuclear cells bear this null phenotype. With the use of the panning technique, we showed that the suppressors of the MLR are confined to the null cell population. The null suppressor cells are not macrophages because they did not carry macrophage markers identified by the monoclonal antibodies anti-MAC-1 and F4/80. In addition, the suppressor cells did not stain for nonspecific esterase and did not adhere firmly to plastic or glass. Spleen cells from TLI-treated mice maintained their suppressive capacity after culture in vitro for 6 to 8 wk. The cultured suppressor cells did not develop mature T cell, B cell, or macrophage markers during this time interval. Thus, the suppressor cells did not appear to be precursors of the latter cells. There was no clear relationship between the suppressor activity of the spleens and natural killer (NK) activity; the kinetics of these activities in newborn spleen appear to be inversely related. The suppressor cells, however, are similar to NK cells in that both are found in the absence of antigenic challenge, lack antigen specificity, and bear the null surface phenotype. Thus, we have termed the former cells natural suppressor (NS) cells.     

Suppression of in vitro maintenance and interferon-mediated augmentation of natural killer cell activity by adherent peritoneal cells from normal mice

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.     

Development of self-tolerance in normal mice. Appearance of suppressor cells that maintain adult self-tolerance follows the neonatal autoantibody response

T cell populations from BALB/c mice at different ages were analyzed to determine when in development Ts cells specific for the anti-mouse RBC (MRBC) autoantibody response become activated. Previous studies have shown that adult CD8+ T cells actively suppress this autoimmune response and adult spleen cells depleted of CD8+ cells can generate an anti-MRBC response in culture with MRBC. The present results demonstrate that T cells from mice less than 1 wk of age do not suppress the in vitro anti-MRBC response of adult spleen cell populations depleted of CD8+ Ts cells. By 2 wk of age Ts cells are detectable in this anti-self response and reach adult levels by 3 wk of age. Non-specific "natural suppressor" cells normally present in neonatal spleen cell populations are unable to suppress this autoantibody response, although they are active in suppressing anti-SRBC responses in the same cultures. Before the appearance of Ts cells active in the anti-MRBC response, neonatal spleen cell populations can generate anti-MRBC antibody-forming cells, both spontaneously in vivo and in vitro. The in vitro anti-MRBC response of neonatal spleen cells was shown to be Ag driven and Ag specific. The ability of unfractionated spleen cells to generate this response in vitro declines with age and is relatively low by 3 wk. This decline in responsiveness occurs simultaneously with the appearance of suppression specific for the anti-MRBC response, suggesting that the two events may be causally related.     

Changes in the host natural killer cell population in mice during tumor development. 2. The mechanism of suppression of NK activity

Our earlier studies revealed that a rapid and progressive loss of splenic NK activity in mice during the development of a number of transplanted tumors as well as of spontaneous tumors was due to an inactivation of natural killer (NK) lineage cells rather than to their disappearance. The mechanism of this inactivation have now been explored in CBA/J mice receiving transplanted Ehrlich ascites tumors and in C3H/HeJ mice bearing spontaneous mammary tumors or receiving transplants of syngeneic mammary tumor lines of recent origin. A poor activation state or maturation arrest of NK lineage cells due to a low interferon level in vivo was ruled out, since the host NK activity could not be restored after administration of either an interferon inducer poly(I:C) or interferon-alpha, although such treatments enhanced the activity in tumor-free mice by four- to eightfold. Possible presence of host suppressor cells acting on the effector or preeffector stage of NK cells was explored by mixing spleen cells from tumor bearers with normal spleen cells either during the NK assay, or for a 20-hr period of in vitro short-term culture prior to the NK assay. Mixing during the NK assay led to a reduction of NK activity explicable by a simple dilution of active NK cell concentration rather a suppression of active NK cells. On the other hand, a 20-hr coculture of the mixed population at various ratios led to a complete abrogation of the NK activity, indicating that the suppressor cells acted on the preeffector stage of the NK Lineage. A further characterization of suppressor cells revealed that they were (1) contained in the adherent fraction of the spleen of tumor bearers, (2) of monocyte/macrophage morphology, (3) capable of phagocytosing latex particles, and (4) positive for surface Mac-1 antigen, as noted from a radioautographic binding of 125I-labeled monoclonal anti-Mac-1 antibody. The mechanism of the suppression was identified, at least in part, as being mediated by prostaglandin-like molecules, since the presence of indomethacin, a prostaglandin-inhibitor, during the 20-hr coculture period completely abrogated the suppression. Indomethacin exerted no direct effect on the recruitment or killer activity of NK lineage cells in vitro. NK cell suppression may be another normal immunoregulatory mechanism which alters the host-tumor balance in favor of the tumor rather than the host.     

Suppression of natural killer cell cytotoxicity by splenocytes from Corynebacterium parvum-injected, bone marrow-tolerant, and infant mice.

Natural killer (NK) cell cytotoxicity to YAC-1 lymphoma was investigated in mice tolerant to bone marrow grafts (BM-tolerant), Corynebacterium parvum- (C. parvum) treated mice, and infant mice. Also the comparison was made between the NK cell and the hemopoietic-resistance effector (HR-E) cells. It was found that the BM-tolerant mice and C. parvum-treated mice showed either no or markedly decreased NK cell cytotoxicity. These mice were also nonresponders to bone marrow grafts in vivo. The lack of or decreased reactivity was apparently caused by the regulatory cell activities of the suppressor cell since the splenocytes from C. parvum-treated and BM-tolerant mice suppressed significantly the cytotoxic activities of otherwise fully functional NK cells. Similar suppressive effect on NK cells was mounted by splenocytes from infant mice, indicating again the suppressor cell regulation of NK cell cytotoxicity.     

Cyclosporine-induced transplantation unresponsiveness in rat cardiac allograft recipients: in vitro determination of helper and suppressor activity

Lymphocytes from spleen, peripheral blood, thymus, and lymph node of naive rats, nonimmunosuppressed recipients of MHC-incompatible heart grafts, and cyclosporine-treated recipients of MHC-incompatible heart grafts were tested for their ability to augment or suppress proliferation of naive cells in an in vitro MLR co-culture assay. Rats treated with cyclosporine for only 7 days maintained their grafts indefinitely. Potent suppressor activity was found in the peripheral blood and spleen of adult naive rats. In untreated engrafted rats, increased suppressor activity was found 1 wk after transplantation and increased helper activity 2 wk after transplantation. In contrast, subnormal helper and suppressor activity was found in cyclosporine-treated rats 1 wk after transplantation. Subsequently, suppressor activity peaked at 2 to 3 wk and helper activity at 4 wk after transplantation. Beyond 5 wk, the cyclosporine-treated rat was indistinguishable from naive ungrafted rats. Two types of suppressor activity were identified that differed in buoyant density and cyclophosphamide sensitivity. Neither suppressor activity demonstrated antigen specificity. These data suggest that one role of cyclosporine in this rat model is to delay the initial helper mechanisms until generalized suppressor activity is operable. The increased antigen-nonspecific activity is only transient, presumably until the final antigen-specific mechanisms become operative.     

Effects of adriamycin on the activity of mouse natural killer cells

Adriamycin, a widely employed anti-neoplastic agent, was found to have either inhibitory or stimulatory effects on NK activity, depending on the site examined. A single i.p. administration of ADM resulted in a rapid increase of cytolytic activity by PEC of various mouse strains. The effector cells appeared to be NK cells, being nonadherent and nonphagocytic; they expressed low amounts of Thy 1.2 antigen and had the same pattern of specificity as splenic NK cells. In contrast to the stimulatory effects of NK activity of PEC, ADM caused a transient dose-dependent depression of NK activity in the spleen, with a peak reduction at day 3 and recovery within a few days thereafter. The depressed NK activity could be reversed by removal of adherent cells by passage through a nylon column. Moreover, ADM induced cytostatic activity against tumor cells by macrophages, suggesting that activated macrophages may be responsible for suppression of splenic NK activity. The possible modulation of the levels of NK activity by ADM-induced macrophages was supported by mixture experiments, in which plastic adherent spleen cells from ADM-treated mice, but not from normal mice, inhibited the NK activity of normal spleen cells.     

Evidence for extrathymic development of TNK cells. NK1+ CD3+ cells responsible for acute marrow graft rejection are present in thymus-deficient mice.

The predominant mechanism responsible for acute specific rejection of allogeneic and parental bone marrow by irradiated mice is due to a cell (TNK) that expresses the NK cell surface markers NK1 and ASGM1 as well as TCR. Here we analyze the question as to whether TNK cells require a functional thymus for their development. Using adoptive cell transfer assays, evidence is presented that, as is the case in normal mice, NK1+ CD3+ effector cells are responsible for rejection in thymus-deficient nude mice and that the specificity of rejection is indistinguishable from that of normal mice. To reveal the presence of TNK cells in the spleen of nude mice, double staining for NK1 and CD3 followed by FACS analysis was done. It is shown that NK1+ CD3+ cells are present in the spleens of nude but not euthymic mice, suggesting that the lack of a functional thymus stimulates either Ag expression or the number of TNK cells. In support of this finding, the treatment of irradiated marrow reconstituted mice with cyclosporin A leads to the appearance of TNK cells in the spleen. The relative efficiency of spleen cells from nude and cyclosporin A-treated mice to transfer resistance in adoptive cell transfers was assessed and found to be higher than that of normal spleen, consistent with the higher frequency of these cells in thymus-defective mice. The fate of NK1+ CD3+ cells subsequent to stimulation with an allogeneic marrow graft indicates that these cells proliferate in nude mice without gaining cytolytic activity. In euthymic mice, however, NK1+ CD3+ cells appear transiently but disappear in favor of CD4+ and CD8+ cells that proliferate in response to an allogeneic marrow graft. The CD8+ cells express cytolytic activity with specificity similar to that of the acute rejection mechanism, consistent with the suggestion that TNK cells differentiate into CD8+ killer cells. The reason why TNK cells in nude mice fail to differentiate into CD8+ CTL is explained by the lack of Th cells.     

Difference in thymus dependency between effector and suppressor T cells for delayed footpad reaction to sheep erythrocytes in mice

Effects of thymectomy at various times after birth on effector and suppressor T cells for a delayed footpad reaction were determined in 6-week-old mice immunized intraperitoneally (ip) with sheep erythrocytes (SRBC). Mice thymectomized 1 day after birth (Tx-1 mice) gave delayed footpad reactions weaker than those of mice thymectomized 7 days after birth (Tx-7 mice) or sham operated (SH mice) after immunization with a low dose of SRBC. After immunization with a high dose of SRBC, on the other hand, Tx-1 mice showed reactions stronger than those of Tx-7 or SH mice. Pretreatment with cyclophosphamide (CY) augmented the delayed footpad reaction in Tx-7 or SH mice, but not in Tx-1 mice, immunized with a high dose of SRBC. The presence of T cells suppressive for the delayed footpad reaction in the spleen of Tx-7 or SH mice was confirmed by cell transfer experiments. These results suggest that effector T cells responsible for a delayed footpad reaction to SRBC are less thymus dependent and require the presence of the thymus for a shorter period in their development compared to suppressor T cells.     

Cytotoxic and morphologic profile of endogenous and pyrimidinone-activated murine NK cells

We investigated the effect of therapeutically relevant pyrimidinone molecules on murine natural killer (NK) cell cytotoxicity. Our studies demonstrated that pyrimidinones augmented or induced substantial levels of NK cell anti-YAC-1-directed cytotoxic potential in the spleen, bone marrow, peripheral blood, peritoneal exudate, lungs, and liver of adult and infant mice. The NK cell stimulating effect of pyrimidinones was not restricted to a single mouse strain, but was displayed by six different inbred strains of mice. Percoll density gradient separation studies demonstrated that activated effector cells were of low density, displayed morphology of large granular lymphocytes (LGL), and expressed asialo GM-1 cell surface antigen. The analysis of the mechanism of NK cell potentiation showed that the increase in the cytotoxic activity was manifested on several levels, including an increased kinetics of lysis and an increase in the number of LGL and in their tumor-binding and killing capacity. Furthermore, the pyrimidinone-mediated NK cell-augmenting effect was abolished by anti-interferon serum, indicating the role of interferon in NK cell potentiation. In the light of possible role of NK cells in cancer defense, pyrimidinones may have therapeutic value in defense against primary and metastatic tumors.     

Cell surface expression of cytotoxic T lymphocyte-defined, AKR/Gross leukemia virus-associated tumor antigens by normal AKR.H-2b splenic B cells

We previously described a system in which H-2Kb-restricted C57BL/6 (B6) cytotoxic T lymphocytes (CTL) could be raised that were specific for tumors, such as the thymic lymphoma AKR.H-2b SL1, that were induced by endogenous AKR/Gross murine leukemia virus and that expressed the Gross cell surface antigen. In this study, certain normal lymphoid cells from AKR.H-2b mice were also found to express target antigens defined by such anti-AKR/Gross virus CTL. AKR.H-2b spleen, but surprisingly not thymus, cells stimulated the production of anti-AKR/Gross virus CTL when employed at either the in vivo priming phase or the in vitro restimulation phase of anti-viral CTL induction. This selective stimulation by spleen vs thymus cells was not dependent on the age of the mice over the range (3 to 28 wk) tested. Both AKR.H-2b spleen and thymus cells, however, were able to stimulate the generation of H-2-restricted B6 anti-AKR minor histocompatibility (H) antigen-specific CTL. Thus, AKR.H-2b spleen cells appeared to display the same sets (minor H and virus-associated) of cell surface antigens recognized by CTL as the AKR.H-2b SL1 tumor, whereas AKR.H-2b thymocytes were selectively missing the virus-associated target antigens, a situation analogous to that of cl. 18-5, a variant subclone of AKR.H-2b SL1 insusceptible to anti-AKR/Gross virus CTL. Like AKR.H-2b thymocytes, neither AKR spleen cells or thymocytes nor B6.GIX + thymocytes were able to stimulate the generation of anti-AKR/Gross virus CTL from primed B6 responder cell populations. In contrast, both T cell-enriched and B cell-enriched preparations derived from AKR.H-2b spleen cells were able to stimulate at the in vitro phase of induction, although B cell-enriched preparations were considerably more efficient. The discordant results obtained with AKR.H-2b spleen cells vs thymocytes were confirmed and extended in experiments in which these cells were employed as target cells to directly assess the cell surface expression of virus-associated, CTL-defined antigens. Thus, AKR.H-2b spleen cells, but not thymocytes, were recognized by anti-AKR/Gross virus CTL when fresh normal cells were tested as unlabeled competitive inhibitors, or when mitogen blasts were tested as labeled targets. Fresh or lipopolysaccharide-stimulated B cell-enriched spleen cells were as efficiently recognized as unseparated spleen cell preparations. Unexpectedly, fresh or Lens culinaris hemagglutinin-stimulated T cell-enriched spleen cell preparations, although susceptible to anti-minor H CTL, were almost as poor as targets for anti-viral CTL as were thymocytes. Together, these results demonstrate the H-2-restricted expression of CTL-defined, endogenous, AKR/Gross virus-associated target antigens by normal AKR.H-2b splenic B cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytological identification of the lymphocytes in highly leukemic AKR/J mice in ontogenesis. Effect of somatotropin on the cellular composition of the lymphoid organs]

In AKR/J mice lymphocytes of thymus, spleen and lymph nodes can be classified according to their morphology as revealed by the Stockinger and Kellner staining: I--basophilic cytoplasm, 1--3 large basophilic nucleoli (blast cells), II--basophilic cytoplasm, 1--4 small clearly visible nucleoli (T-lymphocytes), III-weakly basophilic cytoplasm, 1-4 small fairly visible nucleoli (B-lymphocytes). The number of cells of types I and II increases with the progressing of leucaemia. After the administration of somatotrophic hormone the number of cells of types I and II increases only in the lymphoid organs of 5 months old AKR/J mice.