Ontogeny of cell-mediated cytotoxicity: induction of CTL in early postnatal thymocytes.

In this study we characterized the time when cytotoxic T lymphocytes (CTL) can be induced in the thymus and spleen from their immediate CTL precursors (CTL-P). In contrast to fetal or newborn thymus, the thymus of 1 to 2-day-old C57BL/6 mice contained cells that, after cultivation in vitro with allogeneic DBA/2 stimulating cells, exhibited high levels (as great or greater than that induced in adult thymocytes) of CTL activity as measured by the ability to lyse P815 (DBA/2) tumor target cells. However, CTL activity induced in spleen cells remained how during the first 5 days of life, increased sharply between 6 to 9 days, and reached adult levels at 11 to 20 days. Furthermore, early postnatal spleen cells did not suppress the adult splenic CTL response. These results suggest 1) that the full potential to generate CTL in response to an allogeneic stimulus commences in the thymus on the first day after birth and 2) a different temporal appearance of immediate CTL precursors in the thymus and spleen.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. III. Effect of priming on precursor frequencies.

The effect of specific priming with alloantigens on the frequency of cytolytic T lymphocyte precursors (CTL-P) has been investigated. Alloimmune lymphoid cells were obtained from the spleen of C57BL/6 (H-2b) mice primed with DBA/2 (H-2d) tumor cells or from 14-day unidirectional mixed leukocyte cultures (C57BL/6 anti-DBA/2). CTL-P frequencies directed against H-2d alloantigens were estimated by limiting dilution analysis in a sensitive micro MLC system. Under these conditions, an apparent increase of 3 to 4-fold in CTL-P frequency was observed in alloimmune (as compared with normal) C57BL/6 spleen cells. Evidence was obtained suggesting that this increase was specific for the priming alloantigens. A much greater increase in CTL-P frequency (25 to 100-fold) was observed after alloimmunization of C57BL/6 spleen cells in unidirectional MLC. Under the latter conditions, 5 to 20% of the surviving splenic MLC cells could be identified operationally as CTL-P. A similar enrichment in CTL-P frequency was obtained when lymph node, peripheral blood, or thymus cells were cultured for 14 days in MLC. These studies provide direct evidence that the pool of specific CTL-P can be expanded after alloimmunization. Furthermore, the very high frequencies observed after in vitro priming indicate that this system should be particularly useful for future studies of the progeny of individual CTL-P.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.     

Functional clonal deletion and suppression as complementary mechanisms operative in adult hapten-induced cytotoxic T cell tolerance

Immunologic tolerance to the hapten TNP was induced in adult mice through the i.v. injection of reactive TNBS. To probe the cellular basis of the tolerant state, splenic cytotoxic T lymphocyte precursors (CTL-P) were stimulated in vitro with haptenated, x-irradiated syngeneic spleen cells in the presence or absence of exogenously added growth factors derived from Concanavalin A-stimulated spleen cell conditioned medium (CAS). The cultures were either conventional bulk cultures or limit dilution cloning cultures. For the latter, cytotoxicity was assessed through a semi-automated, radioautographic 111In-release assay. Suppressive potential was assessed by mixing spleen cells from tolerant mice with normal spleen cells before culture. In the absence of CAS, bulk cultures showed profound tolerance, and suppressive capacity was clearly evident. Suppression was dependent on the presence of TNP-self during culture and affected the generation of CTL from CTL-P and not the effector function of CTL. Cyclophosphamide treatment did not prevent tolerance induction. In the presence of CAS, bulk cultures still showed marked tolerance, but mixing experiments now yielded no evidence of suppression. As documented previously, limit dilution cultures of tolerant spleen cells in the presence of CAS showed a functional clonal deletion of hapten-specific CTL-P. In the absence of CAS, limit dilution cultures became dependent on helper T cells as the limiting element. Tolerant populations showed a diminution of activatable helper T lymphocyte precursors (HTL-P), which may have been due to a functional clonal deletion of HTL-P and/or a concomitant activation of suppressor T cells. Adoptive transfer studies showed that cells from tolerant mice did not detectably influence the number of hapten-specific CTL-P in the spleens of host animals. Taken together, the results suggest that both functional clonal deletion of CTL-P and suppression of HTL-P contribute to the tolerant state induced.     

Phenotypic and functional properties of murine thymocytes. III. Kinetic analysis of the recovery of intrathymic cytolytic T lymphocyte precursors after in vivo administration of hydrocortisone acetate

The phenotypic and functional properties of cells in the C57BL/6 mouse thymus regenerating after a single dose of 100 mg/kg hydrocortisone acetate (H/C) are described. Functionally, the frequency of anti-H-2d cytolytic T lymphocyte precursors (CTL-P) in thymuses from individual mice was determined by limit dilution analysis of mixed leukocyte microcultures. The initial increase in CTL-P frequency, seen 48 hr post-H/C, was followed at 6 to 8 days by a phase of rapid decrease. The CTL-P frequency returned to a normal level by 28 days post-H/C. Analysis of the results from individual mice suggested that changes in total thymic CTL-P content were independent of the kinetics of thymus regeneration. Phenotypically, whereas the thymus 48 hr after H/C was considerably depleted of Lyt-2+ cells, there followed a rapid increase in the proportion of such cells to normal levels by 14 days post-H/C. In addition, as measured by FLS, a subpopulation of larger, predominantly Lyt-2+ cells was found during the phase of rapid thymic regeneration. With the use of a monoclonal anti-Thy-1.2 antibody, the weakly Thy-1-staining subpopulation of cells was absent from the thymus at 14 days post-H/C. These changes in the phenotypic properties of the post-H/C regenerating thymus were correlated with changes in their functional properties.     

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)     

Naturally occurring cytotoxic T lymphocyte precursors with specificity for an Ig idiotype

The humoral response to the p-azobenzenearsonate hapten in the A/J mouse includes the major cross-reactive idiotype associated with anti-p-azobenzenearsonate (CRIA) found in all immunized mice. Limiting dilution cultures of non-immunized spleen cells of A/J mice with irradiated B hybridoma cells bearing the Ig idiotype, CRIA, in the presence of T cell growth factors developed cytotoxic activity against the CRIA-bearing hybridoma; in some wells this activity was completely abrogated by an anti-idiotype mAb specific for CRIA or by a univalent hapten antigen, tyrosine-p-azobenzenearsonate, indicating the existence of cytotoxic T cell precursors (CTL-P) specific for one or more idiotopes of CRIA in normal spleen cells. The CTL clones lysed targets in a H-2D-restricted manner and were cytotoxic for CRIA-bearing hybridoma lines, but not for CRIA-non-bearing, IgG1k-bearing hybridoma lines. These CTL-P were detected at a high frequency (1/4,500 to 1/10,000) in a spleen cell population of non-immunized, relatively aged A/J mice (16 to 30 wk of age), and at a lower frequency in spleen cells of younger A/J mice (8 wk of age). However, they were not detected in normal spleen cells of B10.A (CRIA-non-producer) mice at any age (less than 1/6 x 10(5)). Normal Ighd-congenic C.AL-20 mice (16 wk of age), that are CRIA producers had as a high frequency of the CTL-P as did A/J mice, whereas normal Ighb-congenic C.B-20 mice (CRIA-non-producers) had none. In the spleen cells of the CRIA-producers, cytotoxicity of the CTL-P developed only in cultures with small numbers of seeding cells. They were completely absent in cultures with greater numbers of cells; this may be due to the presence of suppressor cells of lower frequency but greater potency. In lymph node cells or PBL of relatively aged A/J mice, the CTL-P were also detected, but only in cultures containing higher cell numbers, and at low frequency (between 1/5 x 10(5) to 1/2 x 10(6)). In thymocytes of 8-wk-old A/J mice, they were occasionally detected at very low frequency (less than or equal to 1/1 x 10(6)), but were not present in the bone marrow cells at any age. These results demonstrate the high incidence of the generation of CTL-P specific for an autologous Ag, and indicate that CRIA on B cells may induce CTL specific for CRIA. However, the development of CTL-P may be inhibited by co-existent suppressor cells under normal conditions.     

Peripheral (somatic) expansion of the murine cytotoxic T lymphocyte repertoire. I. Analysis of diversity in recognition repertoire of alloreactive T cells derived from the thymus and spleen of adult or aged DBA/2J mice

Cytotoxic T lymphocyte precursors (CTLp) in the spleen or thymus of individual adult (8 to 10 wk) or aged (greater than 20 mo) DBA/2J mice have been activated by irradiated H-2Kb antigens under limiting dilution conditions such that cytotoxic cells in responder wells result from stimulation of a single CTLp. After division into several equal samples and expansion in the presence of IL 2 and more irradiated H-2Kb stimulators, the contents of replicate individual wells were tested for their ability to lyse a panel of selected H-2Kb mutant targets. The heterogeneity within a given age group, and the similarity of CTLp repertoires between different age groups were then compared for splenic and thymic CTLp repertoires. Our data indicate a far greater mouse-to-mouse variation for the splenic CTLp repertoire of aged mice compared with young mice, despite the greater heterogeneity of the repertoire in the latter case. Less difference was seen for the thymic CTLp repertoire. When we studied the correlation between the repertoires present in the thymus and spleen within a given age group, it seemed that the most striking difference in aged mice was a loss of systematic expansion of the early appearing thymic CTLp repertoire. These findings are discussed in terms of a two-stage model of T cell differentiation.     

Defective thymic education of L3T4+ T helper cell function in graft-vs-host mice

Our study investigates the effect of a prior graft-vs-host (GVH) reaction on the subsequent ability of irradiated, bone marrow-re-populated mice to develop T cell function. The results indicate that such GVH-bone marrow transplanted (BMT) mice do not generate CTL responses to trinitrophenyl-modified syngeneic cells (TNP-self), but do generate strong CTL activity to H-2 alloantigens. This selective deficiency in TNP-self CTL response potential appeared as early as 10 days after GVH, and required both L3T4+ and Lyt-2+ donor T cells. The in vitro addition of either soluble Th factors or L3T4-enriched spleen cells from normal mice circumvented the defect in the TNP-self response in GVH-BMT mice. These results indicate that T effector function was not defective, and instead suggest a Th defect. Cell depletion and antibody-blocking, as well as IL-2 production experiments, indicate that the Th defect was selective for L3T4+ Th population and not for Lyt-2+ Th population. This defect in L3T4 Th function is not accounted for by the approximate twofold reduction in L3T4 cell numbers in GVH-BMT mice, because IL-2 production and CTL generation to L3T4-dependent Ag were at least eightfold below control levels. Rather, defective L3T4 Th function appears to be the consequence of a GVH-induced defect in thymic maturation because the defect was corrected in vivo by a neonatal parental thymus graft before irradiation and bone marrow transplantation. This system may be useful for elucidating the role of the thymus in the maturation of Th cells. Our findings raise the possibility that impaired development of T cell function occurring in marrow grafted patients who have undergone a GVH reaction could be partly due to a GVH-induced thymic defect.     

Heterogeneity of CD4+ T cells involved in anti-allo-class I H-2 immune responses. Functional discrimination between the major proliferating cells and helper cells assisting cytotoxic T cell responses.

MLR in various combinations with class I H-2 disparity revealed that there are three patterns of MLR in the aspect of responding T subset (CD4 vs CD8) dominance. Irrespective of the CD8 vs CD4 dominance, a single i.v. administration of class I-disparate allogeneic spleen cells resulted in almost complete abrogation of anti-class I proliferative capacity of both CD4+ and CD8+ T cells in six combinations. The suppression of proliferative responses was correlated with the striking reduction in the ability to produce IL-2 upon stimulation with the relevant class I alloantigens. In contrast, i.v. presensitized recipient mice exhibiting only marginal MLR/Il-2 production could generate comparable magnitudes of anti-allo class I CTL as well as graft rejection responses to those induced by normal unpresensitized mice. The administration in vivo of anti-CD4 antibody along with the i.v. presensitization not only suppressed the generation of CTL responses by spleen cells but also induced appreciable prolongation of allo-class I-disparate skin grafts under conditions in which neither alone did it. These results demonstrate that 1) the suppression of graft rejection responses is not necessarily reflected on the reduction of MLR; 2) CD8+ CTL precursors responsible for graft rejection can be activated by either allo-class I-reactive CD8+ or CD4+ Th cells; 3) i.v. presensitization induces functional elimination of CD8+ and CD4+ proliferative/IL-2-producing T cells but not of CD8+ CTL precursors and CD4+ Th whose capacity is expressed by assistance of CTL induction but not by their own proliferation. Thus, this study illustrates the heterogeneity of class I alloantigen-reactive CD4+ T cells in the aspect of their capacity to proliferate themselves vs contribute to CTL induction as well as graft rejection.     

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.     

Regulation of primary cytotoxic T lymphocyte responses generated during mixed leukocyte culture with H-2d identical Qa-1-disparate cells

Cytotoxic lymphocyte (CTL) responses are not usually generated during primary mixed leukocyte culture (MLC) with H-2 identical cells. Thus NZB mice are unusual in that their spleen cells do mount CTL responses during primary MLC with H-2d identical stimulator cells; the predominant target antigen for these NZB responses is Qa-1b. Considering the numerous immunoregulatory defects in NZB mice, we postulated that these NZB anti-Qa-1 primary CTL responses were due to an abnormality in T suppressor cell activity. Cellular interactions capable of suppressing NZB anti-Qa-1 primary CTL responses were investigated by using one-way and two-way MLC with spleen cells from NZB mice and other H-2d strains. Although H-2d identical one-way MLC with the use of NZB responders resulted in substantial CTL responses, only minimal CTL responses were detected from two-way MLC with the use of NZB spleen cells plus nonirradiated spleen cells from other H-2d mice. Thus the presence of non-NZB spleen cells in the two-way H-2d identical MLC prevented the generation of NZB CTL. Noncytotoxic mechanisms were implicated in the suppression of the NZB CTL responses during two-way MLC, because only minimal CTL activity was generated when NZB spleen cells were cultured with semiallogeneic, H-2d identical (e.g., NZB X BALB) F1 spleen cells. The observed suppression could be abrogated with as little as 100 rad gamma-irradiation to the non-NZB spleen cells. The phenotype of these highly radiosensitive spleen cells was Thy-1+, Lyt-1+, Lyt-2-, L3T4+. The functional presence of these cells in the spleens of semiallogeneic, H-2d identical F1 mice indicated that their deficiency in NZB mice was a recessive trait. These data suggest that NZB mice lack an L3T4+ cell present in the spleens of normal mice that is capable of suppressing primary anti-Qa-1 CTL responses. This model system should facilitate additional investigations of the cellular interactions and immunoregulatory mechanisms responsible for controlling primary CTL responses against non-H-2K/D class I alloantigens. The model may also provide insight into the immunoregulatory defects of autoimmune NZB mice.     

The target minor H antigen for F1 cytotoxic T lymphocytes induced by Igh-congenic parental spleen cells is coded for by gene linked to H-2

Graft-vs-host reaction (GvHR) induced in (B10.BR X CWB)F1 (BWF1; H-2k/b, Ighb/b) by i.v. injection with CWB (H-2b, Ighb) spleen cells resulted in complete suppression of cytotoxic T lymphocyte (CTL) responsiveness of the F1 host spleen cells (GvHR-associated immunosuppression). In contrast, GvHR induced in BWF1 mice with CSW (H-2b, Ighj; Igh-congenic to CWB) spleen cells did not affect CTL responsiveness of the F1 host spleen cells at all. The BWF1 hosts undergoing the CSW-induced GvHR generated anti-CSW CTL in their spleens, and the subsequent culture of such BWF1 spleen cells with CSW stimulator cells, augmented the CTL activity. The BWF1 anti-CSW CTL lysed both Con A- and LPS-induced splenic blasts from mouse strains carrying the Ighj allele in the context of self H-2Kb. However, determination of the Igh haplotype in the serum IgG and of the susceptibility of the splenic lymphocytes to the BWF1 anti-CSW CTL on backcross mice, which carry either Ighb/j or Ighb/b in the context of H-2b/b or H-2b/k, showed clearly that Ighj and the gene coding for the target antigen for the BWF1 anti-CSW CTL segregated at ratios close to 1:1. The study in which linkage between the gene(s) coding for the target antigen for the BWF1 anti-CSW CTL and H-2 was examined on CWB X (C3H X CWB)F1 backcross mice and (B10.BR X CSW)F1 X B10 mice demonstrated that the gene, most likely a single gene, coding for the target antigen for the BWF1 anti-CSW CTL is located at 8.5 +/- 4.3 cross-over units to the right or left of the H-2 complex. We designated the minor H antigen to be recognized by the BWF1 anti-CSW CTL as H-X+, and we discuss the distinction between the H-X+ locus and the other minor H loci on chromosome 17.     

Limiting dilution analysis of alloantigen-reactive T lymphocytes. IV. High frequency of cytolytic T lymphocyte precursor cells in MLC blasts separated by velocity sedimentation

A sensitive limiting dilution microculture system was used to obtain minimal estimates of the frequency of cytolytic T lymphocyte precursor cells (CTL-P) directed against DBA/2 alloantigens, after priming of spleen cells in unidirectional mixed leukocyte cultures (MLC, C57BL/6 anti-DBA/2). The mean CTL-P frequency in day 4 to 5 MLC populations was found to be approximately 50- to 100-fold greater than the frequency in normal spleen, and up to 25% of the cells present in such MLC could be identified operationally as CTL-P. Even higher frequencies (up to 50%) of CTL-P were obtained in a population of large-sized cells separated from day 4 MLC by velocity sedimentation. Furthermore, since a strikingly quantitative correlation was observed between CTL activity and CTL-P frequency in such separated MLC populations, it is likely that mature CTL in MLC are not end cells, but can further proliferate and thus behave operationally as CTL-P.     

Cellular events during radiation-induced thymic leukemogenesis in mice: abnormal T cell differentiation in the thymus and defect of thymocyte precursors in the bone marrow after split-dose irradiation

Cellular events during the development of thymic lymphomas in young B10.BR mice given leukemogenic split-dose irradiation were studied by examining the differentiation of functional T lymphocyte precursors in the regenerating thymus. It was found that leukemogenic radiation treatment resulted in a sustained depression of the level of thymic cytotoxic T lymphocyte precursors (CTLp) and of mixed lymphocyte reactivity of thymus cells when assessed between 1 and 4 mo after irradiation, in spite of the fact that the total number of thymocytes was restored to the normal level within 2 mo and continued to increase thereafter. In vitro mixing studies of normal thymocytes with thymus cells from split-dose irradiated mice provided no evidence for active suppression as a mechanism for this depressed activity. The ability of bone marrow cells from split-dose irradiated mice to regenerate the thymus and to differentiate into functional CTLp was examined by use of supralethally irradiated Thy-1 congenic recipients. Reconstitution of supralethally irradiated B10.BR Thy-1.2 mice with normal bone marrow from B10.BR Thy-1.1 mice resulted in the complete repopulation of host-thymus with donor-derived cells when assessed at 4 wk after reconstitution. Lymphocytes from the regenerating thymus of these animals were shown to contain high levels of CTLp which were donor-derived. On the other hand, when the recipient mice were reconstituted with bone marrow cells from donor mice which had been split-dose irradiated 1 mo earlier, regeneration of the recipient thymus was severely depressed when assessed at 4 wk to 3 mo after reconstitution. Although variable but small numbers of donor-derived Thy-1+ cells were detected, CTL activity for alloantigen could not be induced in these donor-derived cells. The results suggest that T cell precursors derived from split-dose irradiated donor mice were unable to undergo active proliferation and differentiation into functional CTLp. The significance of these findings on radiation-induced thymic leukemogenesis is discussed.     

Abnormal thymocyte development and production of autoreactive T cells in T cell receptor transgenic autoimmune mice.

Development of a C57BL/6-+/+ TCR transgenic mouse containing the rearranged TCR alpha- and beta-chain specific for the Db + HY male Ag results in production of a nearly monoclonal population of early thymocytes expressing the Db + HY reactive TCR. These thymocytes are autoreactive in H-2Db male mice and undergo clonal deletion and down-regulation of CD8. To study the effect of the lpr gene on development of autoreactive T cells, these transgenic mice were backcrossed with C57BL/6-lpr/lpr mice. T cell populations in the thymus and spleen were analyzed by three-color flow cytometry for expression of CD4, CD8, and TCR. The thymus of TCR transgenic H-2b/b lpr/lpr male mice had an increase in percent and absolute number of CD8dull thymocytes compared to TCR transgenic H-2b/b +/+ male mice. However, there was not a complete defect in clonal deletion, because clonal deletion and down-regulation of CD8 was apparent in both +/+ and lpr/lpr H-2Db HY+ male mice compared to H-2Db HY- female mice. The phenotype of splenic T cells was almost identical in TCR transgenic +/+ and lpr/lpr males with about 50% CD4-CD8- T cells and 50% CD8+ T cells. However, there was a dramatic increase in the SMLR proliferative response of splenic T cells from TCR transgenic lpr/lpr males compared to TCR transgenic +/+ males. To determine the specificity of this response, spleen cells from TCR transgenic lpr/lpr and +/+ mice were cultured with irradiated H-2b/b and H-2k/k male and female spleen cells. T cells from TCR transgenic C57BL/6-lpr/lpr male mice had an increased proliferative response to H-2b/b male spleen cells compared to T cells from TCR transgenic C57BL/6(-)+/+ male mice, but both lpr/lpr and +/+ mice had a minimal response to irradiated H-2b/b female or H-2k/k male or female stimulator cells. The splenic T cells from TCR transgenic lpr/lpr mice also had an increased specific cytotoxic activity against H-2b/b male target cells compared to TCR transgenic +/+ mice. These results demonstrate that there is a defect in negative selection of self-reactive T cells in the thymus of lpr/lpr mice and a defect in induction or maintenance of clonal anergy of self-reactive T cells in the periphery of lpr/lpr mice.     

Accessory cells in the in vitro generation of type C virus-specific T-killer lymphocytes. III. Two methods of antigen presentation of cytolytic T-lymphocyte precursors

F₁ hybrid mice primed in vivo with tumor cells bearing the virus-induced FMR antigen and the H-2 specificities of each parent are able to produce in vitro in secondary response cytolytic T lymphocytes (CTL) reacting with FMR in the context of the H-2 antigens of both parents. This suggests that the processing in vivo of the immunizing cells by f₁ macrophages results in the presentation of FMR antigens in the context of both H-2 specificities. It has also been suggested that FMR antigens are recognized by cytolytic T-lymphocyte precursors (CTL-P) at the surface of tumor cells and not of macrophages (4). The results reported here show that there are two methods of CTL-P priming: (a) in most cases, FMR antigens are presented directly by the tumor cells; (b) however, in the absence of antigen-presenting tumor cells in vivo or in vitro, macrophages present FMR to CTL-P. The presentation by macrophages appears less efficient but is probably sufficient to explain the priming of memory cells corresponding to both parental H-2.     

Three different signals are required for the induction of cytolytic T lymphocytes from resting precursors

The requirement for the signals in induction of cytolytic T lymphocytes (CTL) has been investigated. C57BL/6 X CBA/T6 F1 spleen cells stimulated with the lectin leukoagglutinin (L-A) failed to show CTL activity in a PHA-facilitated assay, although L-A-activated splenic T cells were able to respond to T cell growth factor (TCGF). Concanavalin A (Con A) on the other hand was able to induce cytolytic activity from CTL-P, as well as to render splenic T cells responsive to TCGF. Furthermore, L-A-activated splenic T cells could generate cytolytic activity upon subsequent culture in secondary mixed leukocyte culture supernatant (2 degrees MLC SN). In contrast, EL-4-derived SN (EL-4 SN) was unable to induce cytolytic activity from L-A-activated spleen cells. In addition, proliferation of L-A-activated spleen cells cultured in EL-4 SN was similar to those cultured in 2 degrees MLC SN. Nonactivated spleen cells were totally unresponsive to both SN in proliferation and generation of CTL. Analysis of T cell clones for the production of a factor necessary for induction of cytolytic activity revealed that both cytolytic and noncytolytic T cell clones were able to produce a factor(s) for the generation of cytolytic activity from L-A-activated T cells. On the other hand, SN from certain antigen-stimulated T cell clones produced factors capable of inducing cytocytic activity by L-A-activated T cells only in the presence of EL-4 SN. Neither EL-4 SN nor cloned T cell SN alone had such a capacity. The nature of the necessary lymphokines in the SN from the clone cells or from the EL-4 is unknown. In the case of the EL-4 SN, it is not known whether the presence of TCGF plays a role or whether that role is perhaps more differentiative than proliferative. This study provides evidence that the induction of CTL from CTL-P can be dissociated into activation, which is required to render T cells responsive to second signals, and differentiation, which is mediated by two different factors.     

Antigen-presenting T cells. I. Class I alloantigen (bm1)-bearing T lymphoblasts efficiently stimulate a primary clonal response of Lyt-2+ cytotoxic lymphocyte precursors of B6 origin

The cytotoxic response of splenic Lyt-2+ T cells to class I H-2 alloantigen-bearing stimulator cells was analyzed under limiting dilution conditions. One of 50 to one of 200 nylon wool-nonadherent (FACS-purified), small Lyt-2+ spleen cells of B6 origin gave rise in vitro to a cytotoxic T lymphocyte clone that specifically lysed targets bearing bm1 alloantigen. This population of alloantigen-specific cytotoxic lymphocyte precursors (CLP) was activated by different types of bm1 stimulator cells with different efficiency: 2 X 10(5) nonfractionated spleen cells, 5000 normal peritoneal cells, 400 to 10(4) L3T4+ helper T blasts, or 2000 to 10(4) Lyt-2+ T blasts induced clonal growth of this CLP pool. Irradiated or mitomycin-treated small (L3T4+ or Lyt-2+) bm1-derived T cells were inefficient stimulator cells for this response. Supplementation of culture medium with (recombinant) interleukin 2 was necessary and sufficient to support clonal development of alloantigen-triggered CLP in the presence of allogeneic T blasts. Under these limiting dilution conditions, we observed comparable cloning efficiencies for (wild-type) Kb-allospecific splenic Lyt-2+ CLP from bm1 mice generated in response to either irradiated B6 spleen cells or inactivated B6-derived T cell lines (EL4 and RBL-5 lymphoma cells). The data indicate that normal T lymphoblasts as well as tumor T cell lines stimulate clonal development in vitro of class I H-2-allospecific cytotoxic T lymphocytes in the presence of interleukin 2.     

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.