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)     

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)     

Cloned auto-Ia-reactive T cells elicit lichen planus-like lesion in the skin of syngeneic mice

To explore the physiologic or pathologic roles of autoreactive T cells, we examined immunological functions of several autoreactive mouse T cell clones in vitro and in vivo. All of the T cell clones were Lyt-2-, L3T4+ and showed self-I region-restricted proliferative responses (one clone was self-I-E restricted, the other clones were self-I-A restricted). One clone derived from C57BL/6 mouse and reactive to the self-I-Ab product (clone bb1-2) showed cross-reactivity to the I-Ak product. Among four such auto-Ia-reactive T cell clones examined, one clone produced fairly large amounts of interleukin 2 (IL 2) in response to syngeneic stimulator cells, and mediated help for the in vitro cytotoxic T cell (CTL) responses of syngeneic thymocytes, whereas this clone did not mediate in vitro antibody responses of syngeneic B cells. The other three clones were producers of small amounts of IL 2 and did not mediate the in vitro CTL responses. Among the three clones, clone bb1-2 showed strong regulatory function, and clone kk-1 (B10.BR origin and self-I-Ak reactive) showed weak regulatory function in vitro antibody responses of syngeneic B cells. The physiologic or pathologic roles of autoreactive T cells in vivo were explored by injecting subcutaneously clone kk-1 T cells or clone bb1-2 T cells into the footpads of the respective syngeneic mice. Clone kk-1 T cells injected into syngeneic mice elicited swelling of the footpad and marked accumulation of mononuclear cells in the dermis, leaving the epidermis intact, as in the delayed-type hypersensitivity reaction. As a notable finding, clone bb1-2 T cells injected into syngeneic mice elicited marked swelling of the footpad and lichen planus-like skin lesions, i.e., infiltration of lymphocytes in the epidermis and epidermal cell damage. The lymphocytes infiltrating in the epidermis were evaluated, as were the injected clone bb1-2 T cells expressing the Lyt-1.2 phenotype, by examination of the skin lesions elicited in C3H/He mice (H-2k, Lyt-1.1, 2.1) by the clone T cells. Clone bb1-2 T cells exerted in vitro cytotoxicity against H-2b and H-2k target cells, whereas clone kk-1 T cells did not show any cytotoxic activity, indicating a correlation between the cytotoxic activity of clone bb1-2 T cells and their ability to elicit lichen planus-like lesions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin 2 enhancement of lymphokine secretion by T lymphocytes: analysis of established clones and primary limiting dilution microcultures

The effect of exogenous interleukin 2 (IL 2) on lymphokine production by T lymphocytes was examined in two systems: the secretion of macrophage-activating factor (MAF) and interferon (IFN) by cloned long-term T cell lines, and a limiting dilution system for estimating the frequency of precursors of MAF-secreting cells in normal spleen. An IL 2-containing, MAF- and IFN-free supernatant from the EL-4 thymoma (EL-4 SN) significantly enhanced release of MAF and IFN by mitogen- or antigen-stimulated, cytolytic or noncytolytic T lymphocyte clones directed against alloantigens or Moloney leukemia virus-associated antigens. Highly purified IL 2 produced equivalent enhancement as EL-4 SN in cultures of alloreactive clones stimulated with concanavalin A. Kinetics experiments showed that EL-4 SN increased both the rate and duration of MAF release by T cell clones. EL-4 SN also increased MAF production when added during restimulation of limiting dilution cultures of positively selected Lyt-2+ and Lyt-2- C57BL/6 splenic T lymphocytes activated against DBA/2 alloantigens. This enhancement resulted in a threefold increase in the apparent precursor frequency of MAF-secreting cells among Lyt-2+ lymphocytes, but did not affect the frequency among Lyt-2- cells. Additional analysis indicated that average MAF production in cultures of Lyt-2-+ cells was sixfold lower than in cultures of Lyt-2- cells, and hence that EL-4 SN allowed detection of a significant proportion of Lyt-2+ cell cultures secreting low levels of MAF. Under these improved conditions, the MAF assay detected the majority of responding Lyt-2+ and Lyt-2- lymphocytes.     

Property of class I H-2 alloantigen-reactive Lyt-2+ helper T cell subset. Abrogation of its proliferative and IL-2-producing capacities by intravenous injection of class I H-2-disparate allogeneic cells

The present study investigates the distinctiveness of Class I H-2 alloantigen-reactive Lyt-2+ helper/proliferative T cell subset in the aspect of tolerance induction. Primary mixed lymphocyte reactions (MLR) revealed that Lyt-2+ and L3T4+ T cell subsets from C57BL/6 (B6) mice were exclusively capable of responding to class I H-2 [B6-C-H-2bm1 (bm1)]- and class II H-2 [B6-C-H-2bm12 (bm12)]-alloantigens, respectively. Anti-bm12 MLR was not affected by i.v. injection of bm12 spleen cells into recipient B6 mice. In contrast, a single i.v. administration of bm1 spleen cells into B6 mice resulted in the abrogation of the capacity of recipient B6 spleen and lymph node cells to give anti-bm1 MLR. This suppression was bm1 alloantigen-specific, since lymphoid cells from B6 mice i.v. presensitized with bm1 cells exhibited comparable anti-bm12 primary MLR to that obtained by normal B6 lymphoid cells. Such tolerance was rapidly (24 h after the i.v. injection of bm1 cells) inducible and lasting for at shortest 3 wk. Addition of lymphoid cells from anti-bm1-tolerant B6 mice to cultures of normal B6 lymphoid cells did not suppress the proliferative responses of the latter cells, indicating that the tolerance is not due to the induction of suppressor cells but attributed to the elimination or functional impairment of anti-bm1 proliferative clones. The tolerance was also demonstrated by the failure of tolerant lymphoid cells to produce IL-2. It was, however, found that anti-bm1 CTL responses were generated by tolerant lymphoid cells which were unable to induce the anti-bm1 MLR nor to produce detectable level of IL-2. These results demonstrate that class I H-2 alloantigen-reactive Lyt-2+ Th cell subset exhibits a distinct property which is expressed by neither Lyt-2+ CTL directed to class I H-2 nor L3T4+ Th cells to class II H-2 alloantigens.     

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.     

Selective binding of Dolichos biflorus agglutinin to L3T4-, Lyt-2- thymocytes. Expression of terminal alpha-linked N-acetyl-D-galactosamine residues defines a subpopulation of fetal and adult murine thymocytes

Using histochemical and immunocytochemical techniques, a lectin with nominal specificity for alpha-linked N-acetyl-D-galactosamine, Dolichos biflorus agglutinin (DBA), was found to preferentially label thymocytes with an L3T4-, Lyt-2- phenotype from fetal/newborn and adult mice. Through days 14 to 16 of gestation, virtually all thymocytes bound DBA, followed by a dramatic reduction of DBA labeling during the last 4 days of gestation, reaching adult levels of about 2 to 4% of total thymocytes. At later stages of fetal development, the DBA+ cells were confined to the subcapsular area of the thymus. This apparent loss of DBA+ cells was caused by an expansion of the thymocyte population not labeled with this lectin. Affinity purification of thymocyte cell surface components with insolubilized DBA indicated that virtually all of the lectin binding to fetal thymocytes was mediated by a 120-kDa glycoprotein. In addition to thymocytes, DBA also labeled about 5% of bone marrow cells from both normal or nude mice and a small population of spleen cells as well. These results suggest that this lectin may be useful to positively select for LT34-, Lyt-2- thymocytes, and, possibly, other immature populations within the T cell lineage.     

Cell-mediated immune responses in vitro. II. Simultaneous generation of cytotoxic lymphocyte responses to two sets of alloantigens of limited cross-reactivity.

The conditions for generation of simultaneous and independent cytotoxic lymphocyte (CL) responses to each of two sets of alloantigens of limited cross-reactivity by mouse spleen cells in vitro have been investigated. Responder spleen cells were incubated with mitomycin C-treated C57BL/6 (H-2b) or DBA/2 (H-2d) stimulator spleen cells and day 5 CL responses were assayed with 51Cr-labeled EL-4 leukemia (H-2b) and P815 mastocytoma (H-2d) as target cells. Spleen cells from mice of the various H-2 haplotypes tested differed greatly in their ability to develop specific CL responses against alloantigens on the stimulator spleen cells and in the degree of cross-reactive cytotoxic activity against target cells bearing alloantigens not present on the stimulator spleen cells. In contrast to the other strains examined, DBA/1 (H-2q) spleen cells developed specific CL responses to either H-2b or H-2d alloantigens without exhibiting significant cross-reactive activity on the inappropriate target cell. The CL responses to H-2b and H-2d alloantigens by DBA/1 spleen cells were comparable in magnitude and had similar stimulator cell-dose requirements. Further, DBA/1 spleen cells developed CL responses of normal magnitude simultaneously against both target cells when incubated with both mitomycin C-treated C57BL/6 and DBA/2 stimulator cells.     

Interleukin 2-mediated induction of lytic activity in a cloned murine CTL line

We have analyzed the ability of interleukin 2 (IL 2) to induce lytic activity within a cloned murine H-2Dd-specific CTL line. Weakly lytic CTL harvested 6 to 7 days after previous stimulation with irradiated DBA/2J spleen cells and conditioned medium from secondary MLC (MLC SN) could be reactivated to high antigen-specific lytic activity with highly purified gibbon IL 2 or E. coli-produced human recombinant DNA IL 2. Dose-response curves with IL 2 and MLC SN suggest that IL 2 may be the principal detectable activity in MLC SN that is active on these CTL. Doses of IL 2 or MLC SN that were saturating for the induction of lytic activity were suboptimal for the expression of DNA synthesis measured by 3HTdR incorporation. This is consistent with a mechanism in which different threshold IL 2 concentrations are required to induce these two biologic responses. Finally, we show that IFN-gamma has little effect on the expression of lytic activity either alone or in combination with IL 2 in this bioassay.     

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.     

Thymocyte differentiation activity from the cloned monocyte/macrophage cell line RAW 264.7. Alterations in the expression of immature thymocyte surface antigens

The cloned monocyte/macrophage cell line RAW 264.7 was previously shown to produce thymocyte mitogenic and co-mitogenic activity that eluted from a Sephadex G-75 column not only at approximately 16,000 daltons, the m.w. described for interleukin 1 (IL 1), but also at 30,000 to 40,000 daltons. The studies reported here indicate that the 30,000 to 40,000 dalton molecule has thymic differentiating activity. Thymocytes from A/J mice were fractionated on discontinuous BSA gradients, which yielded populations of cells enriched for immature and mature cells. The cells found at the interface between 35 and 29% BSA (band 1 cells), which are the most immature, were cultured for 48 hr with highly purified IL 1, with the 30,000 to 40,000 dalton form of thymocyte co-mitogenic activity obtained after Sephadex G-75 chromatography and chromatofocusing chromatography, or with media alone. The surface antigens TL-3, H-2Kk, Thy-1.2, Lyt-1, and Lyt-2 were examined by immunofluorescence. It was found that the highly purified 30,000 to 40,000 dalton species of co-mitogenic activity induced a significant increase in the content of surface H-2Kk, a decrease in TL-3, and a very small decrease in Thy-1.2 on the cell surface, whereas IL 1 was not capable of inducing a change in these surface antigens. There was no change in Lyt-1 on the surface of band 1 thymocytes after incubation with either IL 1 or the 30,000 to 40,000 dalton species. The 30,000 to 40,000 dalton species caused a significant decrease in the percentage of cells staining positive for Lyt-2, whereas IL 1 caused a smaller but significant decrease in Lyt-2. These changes in the surface markers TL-3, H-2Kk, and Thy-1.2 are consistent with changes that occur during thymocyte differentiation. It was also observed that the proliferative response to the 30,000 to 40,000 dalton form and IL 1 increased with increasing functional maturity of each band of thymocytes when used in the thymocyte mitogenic assay. However, only the 30,000 to 40,000 dalton form was capable of inducing a proliferative response in the immature band 1 thymocytes in the thymocyte co-mitogenic assay. These results indicate that the RAW 264.7 cells produce a factor that has, in addition to thymocyte co-mitogenic activity, thymocyte differentiation activity, and this factor is distinct from IL 1.     

Cytolytic T lymphocyte clones that proliferate autonomously to specific alloantigenic stimulation. II. Relationship of the Lyt-2 molecular complex to cytolytic activity, proliferation, and lymphokine secretion

Previous analyses of the inhibitory effects of anti-Lyt-2 monoclonal antibodies (mAb) on cytolytic activity suggested that Lyt-2/3 antigens expressed on the surface of murine cytolytic T lymphocytes (CTL) are involved in antigen recognition. In the present study, we investigated the effects of anti-Lyt-2 mAb (in the absence of complement) on the functional activities of H-2K/D-specific Lyt-2+ CTL clones that proliferate to antigenic stimulation in the absence of helper T cells or added interleukin 2 (IL 2) and secrete lymphokines. For those clones that were inhibited in cytolysis by anti-Lyt-2 mAb, a parallel inhibition of antigen-dependent proliferation and lymphokine secretion (interferon, macrophage-activating factor) was observed. Inhibition of proliferation or lymphokine secretion could be overcome by the addition of IL 2 or lectin, respectively. Collectively, these results would strongly suggest that anti-Lyt-2 mAb were inhibiting CTL antigen recognition. Not all CTL clones, however, were inhibited in cytolysis by anti-Lyt-2 mAb, in which case proliferation and lymphokine secretion were similarly unaffected. This heterogeneity of Lyt-2+ CTL clones in their susceptibility to inhibition of cytolytic activity, proliferation, and lymphokine secretion by anti-Lyt-2 mAb is discussed in the context of a model proposing that Lyt-2/3 molecules function to stabilize the interaction between CTL receptors and the corresponding target/stimulating cell antigens. Such a stabilization may be required by CTL possessing few and/or low affinity receptors.     

Recognition of Rous sarcoma virus-induced tumor antigens by cytotoxic T lymphocytes (CTL): studies on specificity of killing by CTL employing H-2 congenic and recombinant mouse tumor cells

The specificities of cytotoxic T lymphocytes (CTL) were studied for the analysis of CTL against tumor-specific cell surface antigen(s) (TSSA) of non-virus-producing tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. Eight CTL clones were established from immune spleen cells of B10.A(5R) mice. These clones demonstrated six patterns of cytotoxic reactivity in vitro: Two clones showed H-2 restriction in tumor cell lysis. Two other clones had the capacity to lyse syngeneic, H-2K-compatible B10 and H-2-incompatible B10.A(4R) tumor cells, but not YAC-1 cells. One clone had cytotoxic activity against syngeneic, H-2D-compatible B10.D2 tumor cells and YAC-1 cells, but not against H-2-incompatible tumor cells. One clone had cytotoxic activity against syngeneic and YAC-1 tumor cells, but not against either H-2-compatible or H-2-incompatible tumor cells. One clone had lytic activity to syngeneic, H-2-compatible, H-2-incompatible, and YAC-1 tumor cells. Another clone killed H-2-incompatible B10.A(4R) tumor and YAC-1 cells, but not syngeneic or H-2-compatible tumor cells. All these clones strongly expressed surface Thy-1.2 antigens, whereas the expression of Lyt-1.2 and Lyt-2.2 antigens was different from clone to clone. These results demonstrate heterogeneity of both lytic specificity and phenotype of CTL against RSV-induced mouse tumor cells, suggesting the existence of multiple antigenic sites on the RSV TSSA recognized by CTL populations.     

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.     

Production and characterization of T-cell clones specific for trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self)

Using serial antigenic challenge as the method of selection and stimulation, continuous lines of cytotoxic T-lymphocytes (CTL) directed against TNBS-modified syngeneic spleen cells (TNP-self) have been generated. Spleen cells from C3H/HeJ (H-2k) mice were primed in vitro with autologous spleen cells modified with TNBS, and subsequently cloned by limiting dilution and in soft agar in the presence of IL2. These CTL clones grew continuously in medium supplemented with IL2 and in the presence of antigen. They are antigen specific and H-2 restricted in their target cell recognition. They all express Thyl and Lyt2 surface markers. None of the clones exhibit natural killer (NK) cell activity. All CTL clones tested so far are restricted in their target cell recognition to H-2Kk-TNP and none were found to be restricted to H-2Dk-TNP. These findings demonstrate at the clonal level the H-2K/D restriction of TNP-self specific CTL. These clones provide tools that may facilitate an understanding of the development and regulation of antigen specific CTL. They may also serve as models useful towards an understanding of the mechanism of lysis by CTL.     

The generation of cytolytic activity in mixed leukocyte cultures: cortisone-resistant thymocytes are deficient in helper T lymphocytes

Cortisone-resistant (CR) thymocytes did not generate cytolytic activity toward H-2 K or D alloantigen unless they were also stimulated by H-2 I or non-H-2 alloantigens, even though spleen cells generated brisk cytolytic activity toward H-2 K or D alone. Backstimulation by stimulating strain T lymphocytes accounted for neither the response of spleen cells toward H-2 K or D alloantigen nor the response of CR thymocytes to a full set of alloantigens. In addition, lack of non-T accessory cells did not account for the CR thymocyte pattern of reactivity. Rather, CR thymocytes appeared to be relatively deficient in helper T lymphocytes (HTL). CR thymocytes generated specific cytolytic activity toward H-2 D alloantigen when T cell growth factors (TCGF) or cloned alloreactive helper T lymphocytes were added to culture. CR thymocytes contained fewer HTL precursors detected at limit dilution than spleen cells did. Thus spleen cells generated cytolytic activity toward class I alloantigens alone, but under the same culture conditions CR thymocytes had to be stimulated by both class I and class II alloantigens. Class II alloantigens may be required to stimulate cytolytic activity only under culture conditions in which class I-reactive HTL are not sufficient to provide a minimal threshold of help.     

Immunoregulation by mouse T-cell clones. I. Suppression and amplification of cytotoxic responses by cloned H-Y-specific cytolytic T lymphocytes

H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.     

Tolerance induction of allo-class I H-2 antigen-reactive Lyt-2+ helper T cells and prolonged survival of the corresponding class I H-2-disparate skin graft

C57BL/6 (B6) mice were i.v. presensitized with class I H-2-disparate B6-C-H-2bm1 (bm1) spleen cells. Such presensitization resulted in almost complete abrogation of bm1-specific Lyt-2+ T cell-mediated proliferative and IL-2-producing capacities as measured by MLC of lymphoid cells from presensitized B6 mice with stimulating bm1 cells. In contrast, comparable magnitude of CTL responses was generated in bulk cultures from presensitized B6 lymphoid cells to that obtained in unpresensitized B6 responding cultures. These differential influences of Lyt-2+ T cell functions were also demonstrated by limiting dilution assays; frequencies of proliferative and IL-2-producing T cell precursors were as low as undetectable in presensitized B6 lymphoid cells, whereas an appreciable frequency of CTL precursors in a portion of the same lymphoid cells was observed. When bm1 skin grafting was performed in B6 mice i.v. presensitized with bm1 cells, the strikingly prolonged survival of bm1 skin grafts was observed. It was also demonstrated that the bm1 skin graft-bearing B6 mice which had been presensitized with bm1 cells not only exhibited a continuing suppressive state of bm1-specific helper (proliferative and IL-2-producing) function but also failed to generate anti-bm1 CTL responses. These results indicate that 1) i.v. presensitization with class I H-2 alloantigens results in selective tolerance of Lyt-2+ Th cells which is adequate for inducing prolonged graft survival, 2) the induction of complete abrogation of CTL potential is not absolute requirement for the prolongation of graft survival, and 3) residual CTL potential is attenuated after grafting so far as Th cells are rendered tolerant.     

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)