Graft-vs-host reaction limited to a class II MHC difference results in a selective deficiency in L3T4+ but not in Lyt-2+ T helper cell function

Hybrid mice of the (B6 X bm12)F1 combination were inoculated i.v. with parental B6 spleen cells to induce a class II graft-vs-host disease (GVH). Such mice failed to generate in vitro cytotoxic T lymphocyte (CTL) responses that were dependent upon L3T4+ T helper cell (Th) function (e.g., anti-B6-TNP) but were capable of generating in vitro CTL responses that could be mediated by Lyt-2+ Th cells (anti-allo class I). When Th function was assayed directly by interleukin 2 (IL 2) secretion, class II GVH animals were found to be deficient in L3T4+ but not Lyt-2+ IL 2-secreting Th cells. This selective deficiency in L3T4+ Th function correlates with a selective decrease in class II GVH mice of host-derived derived L3T4+ T cells. In addition, it was found that the spleens of class II GVH mice contained cells capable of selectively suppressing L3T4+ Th function. In contrast, mice in which a class I + II GVH occurred were depleted of both L3T4+ and Lyt-2+ Th function as assessed by IL 2 production. The findings that class II GVH selectively depletes L3T4+ T cells and T cell functions are discussed with respect to the immune function of distinct T cell subsets in normal and diseased states.     

Role of cytotoxic T lymphocytes in the prevention of lupus-like disease occurring in a murine model of graft-vs-host disease

The inoculation of B6D2F1 mice with T lymphocytes from the C57BL/6 parental strain induces an "immunosuppressive" graft-vs-host reaction (B6 GVH), whereas inoculation of T cells from the other, DBA/2 parental strain induces an "immunostimulatory" GVH reaction and a lupus-like disease (DBA GVH). The present study compares cytotoxic T lymphocyte (CTL) function in the spleens of these GVH mice as well as differences in the donor inoculum that could account for these different types of GVH. We observed that the B6 GVH induces an immunodeficiency that encompasses CTL precursors (and possibly T helper cells) and results in suppressor cells that abrogate responses to both trinitrophenyl (TNP)-modified self and third party alloantigens. In contrast, the DBA GVH induces only a T helper cell immunodeficiency and results in suppressor cells selective for class II restricted L3T4+ T helper cells. Chimeric T cells were detected in both types of GVH. In the B6 GVH both L3T4+ and Lyt-2+ donor cells were observed, although Lyt-2+ cells predominated. In the DBA GVH, donor T cells were almost exclusively of the L3T4+ phenotype. The lack of appreciable donor Lyt-2+ cells in the DBA GVH can be explained by a defect in the DBA donor inoculum manifested by a naturally occurring two-fold reduction in Lyt-2+ cell numbers as well as a nine-fold reduction in CTL precursors with anti-F1 specificity. T cells in the DBA inoculum, therefore, are predominantly L3T4+. A similar defect induced in B6 donor cells by anti-Lyt2 antibody and complement not only converted the suppressive GVH to a stimulatory GVH, as measured by anti-DNA antibodies, but also resulted in a T cell immune deficiency characteristic of the DBA GVH, i.e., a selective loss of the TNP-self CTL response. Thus the presence or absence of adequate numbers of functioning Lyt-2+ cells in the donor inoculum is correlated with the development of either a suppressive or stimulatory GVH, respectively. That donor Lyt-2+ cells mediate a suppressive GVH through cytolytic mechanisms is evidenced by greater than 70% reduction in B6 GVH spleen cell numbers and readily demonstrable anti-F1 CTL activity by these spleen cells despite an inability to generate anti-allogeneic or anti-TNP self CTL activity even in the presence of added T helper factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of L3T4+ and Lyt-2+ donor cells in graft-versus-host immune deficiency induced across a class I, class II, or whole H-2 difference

The i.v. injection of parental T cells into F1 hybrid mice can result in a graft-vs-host (GVH)-induced immune deficiency that is Ag nonspecific and of long duration. The effect of the GVH reaction (GVHR) on the host's immune system depends on the class of F1 MHC Ag recognized by the donor cells. To determine the role of different subsets of donor-derived T cells in the induction of GVHR, donor spleen cells were negatively selected by anti-T cell mAb and C, and the cells were injected into F1 mice that differed from the donor by both class I and II MHC Ag or by class I or class II MHC only. The induction of GVHR across class I + II differences was found to require both L3T4+ and Lyt-2+ parental cells. Induction of GVHR across a class II difference required only L3T4+ parental T cells in the combination tested [B6-into-(B6 x bm12)F1]. In contrast, B6 Lyt-2+ cells were sufficient to induce GVHR across a class I difference in (B6 x bm1)F1 recipients. In addition, a direct correlation was observed between the cell types required for GVH induction and the parental T cell phenotypes detected in the spleens of the GVH mice. The number of parental cells detected in the unirradiated F1 hosts was dependent upon the H-2 differences involved in the GVHR. Induction of a class I + class II GVHR resulted in abrogation of both TNP-self and allogeneic CTL responses. In contrast, induction of a class II GVHR resulted in only a selective loss of TNP-self but not of allogeneic CTL function. Unexpectedly, the induction of a class I GVHR also resulted in the selective loss of the TNP-self CTL response. Thus, these class I and class II examples of GVH both result in the selective abrogation of L3T4+ Th cell function. The data are discussed in terms of respective roles of killer cells and/or suppressor cells in the induction of host immune deficiency by a GVHR, and of the selective deficiency in host Th cell function induced by different classes of GVHR.     

Differential helper and effector responses of Lyt-2+ T cells to H-2Kb mutant (Kbm) determinants and the appearance of thymic influence on anti-Kbm CTL responsiveness

The goal of this study was to assess and compare the allorecognition requirements for eliciting Lyt-2+ helper and effector functions from primary T cell populations. By using interleukin 2 (IL 2) secretion as a measure of T helper (Th) function, and cytolytic T lymphocyte (CTL) generation as a measure of effector function, this study compared the responses of Lyt-2+ T cells from wild-type B6 mice against a series of H-2Kb mutant determinants. Although all Kbm determinants stimulated B6 Lyt-2+ T cells to become cytolytic effector cells, the various Kbm determinants differed dramatically in their ability to stimulate Lyt-2+ T cells to function as IL 2-secreting helper cells. For example, in contrast to Kbm1 determinants that stimulated both helper and effector functions, Kbm6 determinants only stimulated B6 Lyt-2+ T cells to become cytolytic and failed to stimulate them to secrete IL 2. The distinct functional responses of Lyt-2+ T cells to Kbm6 determinants was documented by precursor frequency determinations, and was not due to an inability of the Kbm6 molecule to stimulate Lyt-2+ Th cells to secrete IL 2. Rather, it was the specific recognition and response of Lyt-2+ T cells to novel mutant epitopes on the Kbm6 molecule that was defective, such that anti-Kbm6 Lyt-2+ T cells only functioned as CTL effectors and did not function as IL 2-secreting Th cells. The failure of Lyt-2+ anti-Kbm6 T cells to function as IL 2-secreting Th cells was a characteristic of all Lyt-2+ T cell populations examined in which the response to novel mutant epitopes could be distinguished from the response to other epitopes expressed on the Kbm6 molecule. The absence of significant numbers of anti-Kbm6 Th cells in Lyt-2+ T cell populations was examined for its functional consequences on anti-Kbm6 CTL responsiveness. It was found that primary anti-Kbm6 CTL responses could be readily generated in vitro, but unlike responses to most class I alloantigens that can be mediated by Lyt-2+ Th cells, anti-Kbm6 CTL responses were strictly dependent upon self-Ia-restricted L3T4+ Th cells. Because the restriction specificity of L3T4+ Th cells is determined by the thymus, in which their precursors had differentiated, anti-Kbm6 CTL responsiveness, unlike responsiveness to most class I alloantigens, was significantly influenced by the Ia phenotype of the thymus in which the responder cells had differentiated.(ABSTRACT TRUNCATED AT 400 WORDS)     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Developmental sequence of T200 antigen modifications in murine T cells

The T200 glycoproteins of T cells were analyzed at different stages of T cell development. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that Lyt-2-L3T4-, and Lyt-2+L3T4+ thymocytes had similar T200 proteins, whereas Lyt-2+L3T4- and Lyt-2-L3T4+ thymocytes expressed a distinct set of T200 molecules. This result indicated a molecular switch in regulation of T200 protein expression upon differentiation of thymocytes to mature phenotype T cells. Further modifications were evident when the T200 proteins of peripheral T cell subsets were examined. In particular L3T4+ T cells expressed T200 proteins of m.w. 220,000, 200,000, and 175,000, whereas Lyt-2+ lymph node T cells expressed an additional T200 protein of m.w. 235,000. Antigenic differences in the T200 glyco-proteins of peripheral L3T4+ and Lyt-2+ T cells were also detected. The anti-B220 monoclonal antibody, 14.8, reacted with lymph node Lyt-2+ T cells but did not react with lymph node L3T4+T cells or with Lyt-2+L3T4- thymocytes. This finding demonstrated a lineage-specific modification of the T200 protein of Lyt-2+ T cells that occurred after exit of these cells from the thymus into peripheral lymphoid organs. This modification apparently occurred on the m.w. 235,000 and 220,000 proteins since these species were precipitated by 14.8, whereas the others were not. In vitro growth and activation also resulted in further T200 antigen alterations. The monoclonal antibody, RA3, which reacts with the B220 antigen of B cells but, unlike 14.8, does not react with any peripheral T cells, showed significant reactivity with Lyt-2+ cytotoxic T cell (CTL) clones but not with L3T4+ T helper cell clones. CTL clones were also 14.8+ but T helper cell clones were not. Immunoprecipitation by 14.8 and RA3 of T200 proteins from CTL clones yielded a single protein of m.w. 240,000 that co-migrated with the B cell form of T200. Overall, the results indicate the presence of developmentally regulated mechanisms that control T200 glycoprotein expression during T cell differentiation in the thymus and in peripheral lymphoid organs.     

Down-regulation of cytotoxic T lymphocyte development by a minor stimulating locus-induced suppressor cascade that involves Lyt-1+ suppressor T cells, IA- macrophages, and their factors

Down-regulation of the development of CTL has been studied in mice both in vivo and in vitro. To generate CTL to hapten-altered self Ag in vivo, an immunization protocol has been used in which the host's Th cells are stimulated by a minor locus histocompatibility Ag (Mlsd) and its precursor CTL are activated by trinitrophenylated syngeneic spleen cells. Injecting the H-2 compatible Mls-disparate spleen cells along with the TNP-coupled self cells into the hind paws causes TNP-self specific CTL to appear in popliteal lymph nodes within 5 days. We have previously reported that inducing Ts cells by i.v. injecting Mlsd-bearing cells prevents in vivo generation of TNP-self specific CTL after immunization in this way. Here the induced Ts cell as well as the mechanism by which it functions have been further examined. The suppression was seen to extend to allogeneic as well as TNP-self Ag, provided the Mlsd-tolerized animal was reexposed to Mlsd-bearing cells at the time of immunization for CTL. By transferring the Mlsd-induced suppression adoptively we have learned that the splenic suppressor cell bears Thy-1.2 as well as Lyt-1.1 Ag and inhibits the generation of CTL at the afferent limb. In addition, Mlsd-induced PEC of Mlsd-tolerized mice, but not of normal mice, mediated suppression of development of CTL in vivo. The active cells within the tolerized PEC have been identified as T cells and macrophages (M phi). Furthermore, PEC from mice tolerized to Mlsd suppressed generation of CTL directed toward TNP-self targets in vitro. T cells and M phi separated from PEC of Mlsd-tolerized mice achieved suppression best in culture when present together. In addition, Lyt-1+ splenic cells from tolerized but not normal mice cooperated to down-regulate CTL generation in vitro with peritoneal M phi from either tolerized or normal mice. Supernatants of 24- to 72-h cultures of PEC from tolerized mice were suppressive of CTL generation when incorporated at 40 to 50% of culture volume. Supernatants of T cells from tolerized PEC or spleen were suppressive in culture only when M phi from normal mice were also present. To achieve suppression dialyzed supernatants of M phi from tolerized mice could replace the M phi.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intrathymic differentiation and tolerance induction of lymphokine-secreting Lyt-2+ T helper cells

The present study has assessed thymic influence on the differentiation and recognition specificity of developing Lyt-2+ lymphokine-secreting T cells, and compared it with those of developing Lyt-2+ CTL. It was demonstrated that development of Lyt-2+ lymphokine-secreting Th cells requires an intrathymic differentiation step, and that peripheral Lyt-2+ lymphokine-secreting Th cells, unlike peripheral Lyt-2+ CTL, are profoundly tolerant to intrathymically expressed alloantigens. These data are interpreted as demonstrating that functionally distinct Lyt-2+ T cell populations are heterogeneous in their requirements for differentiation and/or activation.     

Il-4 is an endogenous T cell growth factor during the immune response to a syngeneic retrovirus-induced tumor

The relative contributions of IL-2 and IL-4 during the immune response to the retrovirus-induced tumor, FBL, were examined. Both proliferative and cytolytic responses to FBL were measured and compared to similar responses to minor histocompatibility Ag. The addition of alpha IL-2 partially inhibited FBL-stimulated proliferation of purified L3T4+ T cells and nearly completely inhibited the response of Lyt-2+ T cells, whereas alpha IL-4 partially inhibited the proliferative response of the L3T4+ subset but had no effect on the response of the Lyt-2+ subset. The addition of exogenous IL-4 augmented the proliferative response of both subsets. Therefore, IL-4 is an endogenous growth factor for FBL-induced specific proliferation of the L3T4+ and not the Lyt-2+ population, but both subpopulations can respond to IL-4. Similar examination of anti-FBL CTL responses revealed that alpha IL-2, but not alpha IL-4, inhibited FBL-specific Lyt-2+ CTL generation. However, exogenous IL-4 partially replaced the L3T4+ Th cell activity necessary for optimal Lyt-2+ FBL-specific CTL generation. Therefore, IL-4 is not required but can participate in the CTL response. The role of IL-4 during the immune response of B6 mice to minor histocompatibility Ag disparate BALB.B cells was analyzed. alpha IL-4 had no detectable effect on the proliferative or cytolytic response to BALB.B cells, suggesting that endogenous IL-4 does not have a significant role in these responses. The extent of involvement of endogenous IL-4 in the T cell responses to retrovirus-induced tumor Ag and minor histocompatibility Ag presumably reflects the nature of the stimulating Ag, and detection of an IL-4 response may correlate with induction of an antibody response. Thus, the immunizing Ag and/or host B cell repetoire may influence which subsets of L3T4+ Th cells are activated during priming in vivo.     

Role of L3T4 antigen in the activation of various functions of Lyt-1+2- T cells against vaccinia virus

The present study defines assay systems for vaccinia virus-reactive Lyt-1+2- T cells mediating various functions and investigates the positivity of L3T4 antigen on these Lyt-1+2- T cells as well as the role of L3T4 antigen in the activation of these T cells with respect to their functions. C3H/He mice were immunized against vaccinia virus by inoculating viable virus intraperitoneally (i.p.). Anti-vaccinia virus reactivity in lymphoid cells from these immunized mice was assessed by proliferative response, helper T cell activities involved in cytotoxic T lymphocyte (CTL) and B cell (antibody) responses, delayed type-hypersensitivity (DTH) response, and production of lymphokines such as interleukin 2 (IL2) and macrophage-activating factor (MAF). The results demonstrate that all of the above anti-vaccinia virus responses were mediated by Lyt-1+2- T cells and that these Lyt-1+2- T cells expressed L3T4 antigens on their cell surfaces. Moreover, such anti-vaccinia Lyt-1+2- T cell responses were inhibited in the presence of anti-L3T4 antigen antibody. These results indicate that there is a reciprocal relationship between Lyt-2 and L3T4 markers, and that L3T4 antigen is closely related to the activation of various functions of anti-vaccinia virus Lyt-1+2- T cells.     

Age-related changes in the capacity of bone marrow cells to differentiate in thymic organ cultures

The capacity of the bone marrow to give rise to T cells in advanced age was studied in vitro by reconstituting fetal thymic lobes from 14-day C57BL/Ka (Thy-1.1) mice with bone marrow cells from old (24-month) or young (3-month) C57BL/6 (Thy-1.2) mice. The use of these congenic strains enabled distinguishing between donor and host contribution to the developing T cells. We found that bone marrow cells from aged mice maintained their capacity to reconstitute fetal thymic explants and to differentiate into various T-cell subsets as assessed by distinct T-cell-specific surface markers (Thy-1, Lyt-1, Lyt-2, and L3T4) and functions (concanavalin A-induced proliferative and cytotoxic responses). However, when mixtures of old and young bone marrow cells reconstituted fetal thymic explants, the cells of old mice were less efficient than those of young in their capacity to give rise to T cells. These results indicate that bone marrow cells from aged mice can reconstitute the thymus and differentiate into T cells; however, their reconstituting capacity is inferior to that of bone marrow cells from young mice.     

Generation of helper cell-independent cytotoxic T lymphocytes is dependent upon L3T4+ helper T cells

The role of L3T4+ (CD4+) Th cells in generation of CTL specific for discrete minor histocompatibility Ag was investigated. Suppression of the function of Th cells in vivo by chronic treatment with anti-L3T4 mAb prevented congenic strains of mice from being primed and from generating CTL specific for Ag encoded by the minor histocompatibility loci--H-3, H-1, and B2m. Analysis of proliferative responses and lymphokine secretion of cells from animals primed with one of these minor H Ag, beta 2-microglobulin, but not treated with anti-L3T4 antibodies, indicated that L3T4- class I MHC-restricted T cells were themselves responsible for the very great majority of the observed minor H Ag-specific proliferation and secretion of lymphokines associated with both T cell proliferation and activation of CTL. All together, the data indicate that in responses against discrete minor H Ag, L3T4+Th-independent CTL are generated through an L3T4+Th-dependent pathway.     

The origin of cytotoxic T lymphocyte precursors (CTL-P): MHC-restricted and alloreactive CTL-P in the spleen during regeneration after a sublethal dose of cyclophosphamide (Cy)

The regenerating spleen 8 days after an injection of a sublethal dose (300 mg/kg) of cyclophosphamide (Cy) had a defective capacity to give rise to cytotoxic T lymphocytes (CTL) in response against allogeneic cells, whereas the cytotoxicity against 2,4,6-trinitrophenyl-(TNP) modified syngeneic cells was at the normal level. Alloresponse was first obtained 2 wk after the Cy treatment. The limiting dilution analysis showed this at the clonal level: the frequency of anti-TNP-specific CTL precursors (CTL-P) in the spleen treated with Cy 8 days previously was the same as the frequency in the normal spleen. The defective alloresponse was due to a decreased number of allospecific CTL-P that was later increasing. The regenerative capacity was not abolished by adult thymectomy or treatment of the mice with a bone marrow-seeking isotope, 89Sr, suggesting that these CTL-P are derived from Cy-resistant splenic precursors rather than from the thymus or bone marrow. These precursors have probably been under thymic education: the dominance of H-2k-restricted CTL over H-2d-restricted CTL in the response of (H-2k X H-2d)F1 mice to TNP-self is known to be influenced by the H-2 genotype of the thymus, and this dominance was also demonstrated with anti-TNP CTL derived from these F1 mice pretreated with Cy. The CTL-P in the regenerating spleen (day 8) were not hydrocortisone sensitive, and nylon wool-purified T cells from this stage had a lactate dehydrogenase (LDH) isoenzyme pattern of the mature T cell type (rather than of the thymocyte type). Thus, in these aspects the T cells of the regenerating spleen resembled normal splenic cells. These data suggest that the Cy-resistant spleen population contains cells that can give rise to CTL-P that have a defective specificity repertoire at the beginning of the regeneration, but later mature to a normally alloreactive population.     

Relationship among function, phenotype, and specificity in primary allospecific T cell populations: identification of phenotypically identical but functionally distinct primary T cell subsets that differ in their recognition of MHC class I and class II allodeterminants

The goal of the present study was to evaluate the relationship among function, Lyt phenotype, and MHC recognition specificity in primary allospecific T cell populations. By using Lyt-2+ and L3T4+ T cells obtained from the same responder populations, we assessed the ability of T cells of each phenotype to generate cytotoxic effector cells (CTL) and IL 2-secreting helper T cells in response to either class I or class II MHC allodeterminants. It was found that a discordance between Lyt phenotype and MHC recognition specificity does exist in primary allospecific T cells, but only in one T cell subpopulation with limited functional potential: namely, Lyt-2+ T cells with cytotoxic, but not helper, function that recognize class II MHC alloantigens. Target cell lysis by these Lyt-2+ class II-allospecific CTL was inhibited by anti-Ia monoclonal antibodies (mAb), but not anti-Lyt-2 mAb, indicating that they recognized class II MHC determinants as their "restriction" specificity and not as their "nominal" specificity even though they were Lyt-2+. A second allospecific T cell subset with limited functional potential was also identified but whose Lyt phenotype and MHC restriction specificity were not discordant: namely, an L3T4+ T cell subset with helper, but not cytotoxic, function specific for class I MHC allodeterminants presented in the context of self-Ia. Thus, the present study demonstrates that primary allospecific T cell populations contain phenotypically identical subpopulations of helper and effector cells that express fundamentally different MHC recognition specificities. Because the recognition specificities expressed by mature T cells reflect the selection pressures they encountered during their differentiation into functional competence, these findings suggest that functionally distinct but phenotypically identical T cell subsets may be selected independently of one another during ontogeny. Thus, the existence of Lyt-2+ CTL specific for class II allodeterminants can be explained by the hypothesis that the association of Lyt phenotype with MHC recognition specificity results from the process of thymic selection that these Lyt-2+ effector cells avoid.     

Lethal graft-vs-host disease across major histocompatibility barriers: requirement for leucyl-leucine methyl ester sensitive cytotoxic T cells

L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) is selectively toxic for human natural killer (NK) cells and cytotoxic T lymphocytes (CTL) at both the precursor and effector stage of differentiation. The present studies explored the effects of Leu-Leu-OMe on murine spleen cell function. Leu-Leu-OMe exposure removed NK function from murine spleen cells but spared their capacity to proliferate in response to lipopolysaccharide and Con A. The capacity to generate CTL from both L3T4 (+) and Lyt-2 (+) precursors was lost after Leu-Leu-OMe treatment, whereas alloantigen-induced proliferation and interleukin 2 (IL 2) production by L3T4 (+) T helper cells remained intact. Lethal graft vs host disease (GVHD), which developed in irradiated (C57BL/6 X DBA/2)F1 recipients of C57BL/6 bone marrow and spleen cells was completely prevented by Leu-Leu-OMe treatment of donor cells. In contrast depletion of Lyt-2 positive cells from the donor inoculum did not prevent acute GVHD in this fully major histo-compatibility complex (MHC) incompatible strain combination. However, Leu-Leu-OMe treatment of the Lyt-2 depleted inoculum completely prevented lethal GVHD, although the treated cells retained the capacity to proliferate and secrete IL 2 normally after in vitro stimulation with (C57BL/6 X DFA/2)F1 spleen cells. These findings indicate that L3T4 (+) T helper cells alone are unable to initiate lethal GVHD in this H-2 incompatible strain combination. Rather, lethal GVHD requires the transfer of a Leu-Leu-OMe sensitive T cell subset, likely to be thymus educated pre-CTL. Leu-Leu-OMe treatment should provide a useful way to delineate subpopulations of cells involved in the production of lethal GVHD and an approach to preventing this complication of bone marrow transplantation.     

Suppression of Ia-unrestricted primary anti-Qa-1 cytotoxic T lymphocyte responses by class II major histocompatibility complex-restricted cellular interactions

A model has been established for investigating the cellular interactions for the generation and regulation of primary cytotoxic T lymphocyte (CTL) responses to Qa-1 alloantigens. Although NZB anti-BALB/c one-way mixed leukocyte cultures (MLC) generate anti-Qa-1b CTL, anti-Qa-1 CTL responses are not generated during BALB/c anti-NZB one-way MLC or during two-way MLC with NZB and BALB/c spleen cells. However, depletion of L3T4+ cells from the spleens of BALB/c mice before two-way MLC with NZB spleen cells resulted in anti-Qa-1b CTL responses. Likewise, the addition of anti-L3T4 monoclonal antibody (mAb) or anti-I-Ad mAb to two-way MLC with NZB and BALB/c spleen cells resulted in the generation of anti-Qa-1b CTL. Conversely, anti-Lyt-2 mAb inhibited the generation of anti-Qa-1 CTL. These data indicate that class II major histocompatibility complex-restricted cellular interactions are capable of suppressing the generation of Ia-unrestricted anti-Qa-1 CTL responses by Lyt-2+ responder cells. This model provides a novel opportunity to both characterize the cellular interactions responsible for regulating primary CTL responses to the Qa/Tla-encoded class I molecule Qa-1, and determine the contribution of this L3T4+ Ts-dependent defect in NZB mice to the pathogenesis of autoimmunity.     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). I. Analysis of bulk populations and establishment of Lyt-2+L3T4- and Lyt-2-L3T4+ bulk CTL lines

Murine allogeneic cytolytic T lymphocytes (CTLs), including long-term bulk CTL lines, were induced in I-region-incompatible combinations of strains in vitro in order to study the phenotypes of class II major histocompatibility complex (MHC) antigen-specific CTLs, as well as the possible functional involvement of accessory cell interaction molecules such as Lyt-2 and L3T4. This report shows that class II-specific allogeneic CTL populations consist of two types of T cells. Lyt-2+L3T4- (2+4-) and Lyt-2-L3T4+ (2-4+), in variable proportions depending on the strain combination, that in vitro bulk CTL lines with each of these phenotypes can be established, that the killing function of 2-4+ CTL is sensitive to the blocking effect of anti-L3T4 antibodies, suggesting functional involvement of this molecule in the CTL-target interaction, that anti-Lyt-2 antibodies fail to block killing by 2+4- cells, suggesting that such CTLs do not utilize this molecule in their killing function, and that while I-A-specific CTLs of both phenotypes are detectable, 2-4+ cells could not be detected among I-E-specific CTL populations.     

Intrathymic T cell differentiation in radiation bone marrow chimeras and its role in T cell emigration to the spleen. An immunohistochemical study

Immunohistochemical studies were made on the regeneration of T cells of host- and donor-type in the thymus and spleen of radiation bone marrow chimeras by using B10- and B10.BR-Thy-1 congenic mice. Both the thymic cortex and the medulla were first repopulated with thymocytes of irradiated host origin, restoring the normal histologic appearance by days 11 to 14, regardless of the H-2 compatibility between the donor and the host. In Thy-1 congenic chimeras, thymocytes of donor bone marrow origin, less than 100 cells in one thymic lobe, were first recognized at day 7, when the thymus involuted to the smallest size after the irradiation. The thymocytes of donor-type then proliferated exponentially, showing a slightly faster rate when higher doses of bone marrow cells were used for reconstitution, reaching a level of 100 million by day 17 and completely replacing the cortical thymocytes of host origin by day 21. The replacement of cortical thymocytes started from the subcapsular layer in a sporadic manner. The replacement of medullary thymocytes from host- to donor-type occurred gradually between days 21 and 35, after the replacement in the cortex was completed. In the spleen, about 1 million survived cells were recovered at day 3 after the irradiation, and approximately 60% of them were shown to be host-type T cells that were observed in the white pulp areas. The host-type T cells in the spleen increased gradually after day 10, due to the influx of host-type T cells from the regenerating thymus. Thus a pronounced increase of T cells of host-type was immunohistochemically observed in the splenic white pulp between days 21 and 28, when thymocytes of host-type were present mainly in the thymic medulla. These host-type T cells were shown to persist in the spleen for a long time, as long as 420 days after the treatment. Phenotypically, they were predominantly Lyt-1+2+ when examined at day 28, but 5 mo later, they were about 50% Lyt-1+2+ and 50% Lyt-1+2-. Donor-type T cells in the spleen began to appear at about day 14 in chimeras that were transplanted with a larger dose of bone marrow cells, whereas this was slightly delayed in those grafted with a smaller dose of bone marrow cells, starting at about day 28.(ABSTRACT TRUNCATED AT 400 WORDS)     

Class II antigen-specific murine cytolytic T lymphocytes (CTL). II. Genuine class II specificity of Lyt-2+ CTL clones

Class II-specific allogeneic cytolytic T lymphocytes (CTL) consist of two types of cells, i.e., Lyt-2+L3T4- and Lyt-2-L3T4 T cells. The Lyt-2+L3T4- class II-specific CTL population constitutes a conspicuous exception to the general correlation observed between the class of major histocompatibility complex antigen recognized and the type of accessory molecules expressed by T cells. In order to examine the specificity of such an exceptional T cell population, CTL clones were established by limiting dilution of a bulk CTL line developed in an I region incompatible combination of mouse strains, B10.QBR anti-B10.MBR. These CTL lines showed single genetic specificity indicating their clonal nature with respect to CTL activities. Lyt-2+L3T4- (2+4-), Lyt-2-L3T4+ (2-4+) and Lyt-2-L3T4- (2-4-) clones were obtained. Among many CTL clones showing a spectrum of genetic specificities, 2+4- and 2-4+ clones with apparent I-Ak-specificity, were studied further and four lines of evidence confirmed their class II specificity: 1) genes encoding the target antigen for these CTL clones were mapped within the I-A subregion by simple genetics; 2) an I-Ak-specific monoclonal antibody readily blocked specific cytolysis by these clones; 3) the clones failed to react with cells expressing mutated I-Ak antigens; and 4) a B cell tumor transfected with alpha- and beta-chain genes of I-Ak was specifically lysed by these CTL clones. These data therefore establish the existence of Lyt-2+ CTL with genuine class II specificity. All 2-4+ CTL were sensitive to the blocking effect of an antibody to L3T4, whereas none of the 2+4- class II-specific CTL were sensitive to blocking by an anti-Lyt-2 antibody, indicating that class II-specific CTL with "wrong phenotype" is not dependent on the function of the accessory molecule. Besides true class II-specific CTL clones, 2+4- clones with a spectrum of genetic specificities were obtained, including clones recognizing a combination of an I-Ak product and the Kb molecule. Two 2-4- clones were also specific for the combination of Kb + I-Ak. These clones most likely recognize an allogeneic class II antigen in the context of a class I antigen and therefore would more appropriately be included in the class I-restricted T cell population.     

Effect of ionizing radiation on thymic epithelial cell function. I. Radiation-spared thymic epithelial grafts expedite the recovery of T cell function in lethally irradiated and fetal liver reconstituted mice

A murine model system was developed to determine whether ionizing radiation has a detrimental influence on thymic epithelium, cell function. Normal mice were lethally irradiated, grafted intracamerally with normal fetal thymic epithelium, and then reconstituted with fetal liver cells. These animals were compared with a group of animals who received their thymic grafts before the irradiation protocol. Analysis of the reconstitution of T cell function in peripheral lymph nodes and spleens at various times post transplantation demonstrated that animals with radiation-spared thymic grafts had superior proliferative responses to T cell mitogens and alloantigens. It was also determined that the capacity of these animals to elicit contact hypersensitivity responses was significantly greater when compared with animals whose thymic grafts had been radiated. The observed difference in T cell function could not be ascribed to a difference in the rate of export of mature T cells from the thymic grafts since the absolute number of Thy-1+, L3T4+, or Lyt-2+ lymphocytes present in the peripheral lymphoid compartment of our two groups of animals was equivalent. Immunohistologic analysis of the thymic grafts demonstrated a marked reduction in the medullary compartment of the repopulated grafts that had been exposed to ionizing radiation. The results of this study suggest: 1) that irradiation of the thymic microenvironment during marrow ablative preparative regimens may be in part responsible for some of the immune alterations observed in marrow transplant recipients, and 2) that our model system may provide a valuable tool for delineating the roles played by medullary and cortical epithelial cells of the thymus on the T cell maturation and education processes.