Qa alloantigen expression on functional T lymphocytes from spleen and thymus

The cell-surface expression of the class I alloantigen Qa-2 was analyzed on resting and activated spleen and thymus cells using cytotoxic elimination and immunofluorescence and flow cytometry. Spleen cells activated by mitogens or alloantigen were homogeneously positive for cell surface Qa-2, but activated splenic T cells expressed only about one-third as much Qa-2 per cell as did nonstimulated T cells. These data correlated with the ability to perform cytotoxic elimination with Qa-2-specific monoclonal antibodies (mAbs) in that cytotoxic T lymphocyte (CTL) activity was completely abrogated by pretreatment of spleen cells prior to in vitro culture but was only partially eliminated by treatment of CTL effectors. Qa-2-positive cells constituted only a small subpopulation of fresh normal thymocytes, but were enriched (>40% positive) among cortisone-resistant thymocytes (CRT). These Qa-2-positive CRT contained mature thymocytes as defined by Ly phenotype Ly-2^–, Ly-1^hi. When normal thymocytes were treated with Qa-2-specific mAb and complement prior to in vitro sensitization for generation of allogeneic CTL, CTL activity was completely abrogated despite the fact that the fraction of cells eliminated were undetectable as assessed by cell recovery. CTL effectors from alloantigen-stimulated thymocytes were also susceptible to cytotoxic elimination with Qa-2-specific mAb. These data suggest that the Qa-2 molecule may serve not only as a marker on resting and activated peripheral T cells, but also as a unique marker for functionally mature T cells in the thymus.     

H-2K/H-2D and Mls and I region-associated antigens stimulate helper factor(s) involved in the generation of cytotoxic T lymphocytes

This study examines the antigen that stimulate production or release of a soluble helper factor(s) involved in development of cytotoxic T lymphocytes (CTL). Antigens associated with the Mls locus, I and K/D regions of the MHC were all capable of stimulating responder cells in MLC to produce helper factor. These supernatant fluids were all capable of providing "help" for the generation of cytotoxic T lymphocytes in MLC in which spleen cells are stimulated by allogeneic heat-treated thymocytes or splenocytes. Previous reports from our laboratory as well as others have shown that heat-treated cells do not stimulate a cytotoxic response. Heat-treatment of Mls, I, and H-2K/H-2D region incompatible stimulatory cells in MLC eliminated their ability to induce responder cells to produce helper factor, suggesting this is the mechanism whereby heat-treatment reduces the ability of cells to stimulate cell-mediated lympholysis (CML). The inability of supernatant fluids, from MLCs in which heat-treated cells were the stimulators, to assist in the generation of cytotoxic T cells did not appear to be the result of any suppressive factor induced by such treatment. Further, the antigens that stimulate pre-killer cells appear functionally distinct from those heat labile antigens (Mls, I, H-2K/H-2D associated) that stimulate helper factor production since heat-treated allogeneic cells served as stimulators of cytotoxicity provided helper activity was added to the MLC.     

Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.     

Veto cell H-2 antigens: veto cell activity is restricted by determinants encoded by K, D, and I MHC regions

Responder cells from primary syngeneic and allogeneic one-way mixed-lymphocyte cultures (MLC) specifically inhibit the development of cytotoxic T lymphocytes (CTL) directed against the major histocompatibility complex (MHC) antigens of the MLC responder cells. This special kind of suppressor activity is known as veto suppression. Ia+ cells with veto activity obtained from H-2 recombinant mouse strains were shown to downregulate alloantigen (class II)-specific helper activity for class I-specific CTL development in a primary MLC provided that the veto cells expressed the same I-E alpha subregion as the MLC stimulator cells. The veto-induced suppression of allo-help was prevented by the addition of supernatant from concanavalin A-stimulated spleen cells (Con A-SN) and was inhibited considerably by very high amounts of recombinant interleukin-2 (IL-2). In the presence of Con A-SN, CTL precursors recognizing either the K end or the D end of the veto cell MHC were found to be inactivated. Thus, our results indicate that MLC responder cells include active veto cells expressing Ia region-encoded restriction elements for allospecific T helper cells, as well as K- or D-encoded restriction elements for allospecific T cytotoxic cells.     

Functional heterogeneity in allospecific cytotoxic T lymphocyte clones. II. Development of syngeneic cytotoxicity in the absence of specific antigenic stimulation

Two out of four long-term murine allospecific cytotoxic T lymphocyte (CTL) clones tested could develop high levels of cytotoxicity against syngeneic target cells when cultured under appropriate conditions. All CTL clones maintained strict allospecificity so long as they were cultured with both appropriate allogeneic stimulator cells and growth factor (supernatant from secondary mixed lymphocyte cultures). In two of the clones, syngeneic reactivity rapidly developed when the allogeneic stimulator cells were replaced with syngeneic or third party stimulator cells, and when the supernatant from EL4 thymoma cells stimulated with phorbol ester was used as growth factor. In addition to killing the appropriate allogeneic target, clones with syngeneic reactivity could kill both syngeneic C57BL/6 targets and H-2-congenic BALB.B targets but not third party unrelated targets, suggesting that the self structure recognized was coded for within the major histocompatibility complex. Such clones did not kill the natural killer (NK) target YAC. The results obtained from cold target inhibition and from subcloning at limiting dilution of clones with syngeneic reactivity suggested that both allogeneic and syngeneic reactivity could be expressed by the same individual cell in the CTL clone. The specificity for syngeneic H-2 as opposed to third party H-2 and NK-sensitive target cells, and the observation that both allospecific and syngeneic killing could be partially blocked by anti-Lyt-2 antibody treatment of the CTL, strongly suggested that different recognition structures are involved in CTL-mediated syngeneic cytotoxicity and NK cytotoxicity.     

Induction and characterization of minor histocompatibility antigens. Specific primary cytotoxic T lymphocyte responses in vitro

A definite cytotoxic activity was developed in a BALB/c (H-2d) anti-DBA/2 primary mixed leukocyte culture (MLC), which received interleukin 2 (IL-2) on day 3 of culture. This cytotoxic activity was minor histocompatibility antigens (MIHA)-specific at the stimulator level, and was not developed in a syngeneic (BALB/c anti-BALB/c) MLC. The addition of IL-2 on day 3 of culture was crucial; no or very weak cytotoxic activity was developed in MLC receiving IL-2 on day 0 or on both day 0 and day 3. Only appropriate MIHA-allogeneic tumor cells were lysed as the target of the cytotoxic activity. The cytotoxic activity seemed MIHA-specific also at the target level; it lysed tumor cells of DBA/2 mouse origin but not those of BALB/c (syngeneic) origin. Phenotypes of the cytotoxic effector cell were Thy-1+ Lyt-2+. We concluded from these results that MIHA-specific cytotoxic T lymphocytes (CTL) were generated in the MIHA-allogeneic primary MLC. In this newly developed system, we studied genetic and antigenic requirements for primary anti-MIHA CTL responses in vitro. We demonstrated; among spleen cells (SC) of seven B10 H-2-congenic strains only SC of B10.D2 strain whose major histocompatibility complex (MHC) (H-2d) was compatible with the responder MHC effectively stimulated responder BALB/c (H-2d) SC for an anti-MIHA (DBA-C57BL-common) CTL response. Similarly, only SC of two out of seven C x B recombinant inbred strains (C x B.H and C x B.D), which were compatible at the MHC with responder SC, activated responder BALB/c SC for the response. The possibility that cells responding to H-2 alloantigens suppressed the anti-MIHA response was ruled out. Additional experiments showed that compatibility at the H-2K-end or the H-2D-end of the MHC was sufficient for a definite anti-MIHA response. These provided formal evidence that primary anti-MIHA CTL responses in vitro were MHC-restricted at the stimulator level. We then showed that sonication-disrupted SC or Sephadex G-10 column-passed nonadherent SC failed to stimulate responder SC for a primary anti-MIHA CTL response, whereas G-10-passed nonadherent SC responded well to adherent stimulator cells. Further study demonstrated that Ia+ adherent cells were the most active cell type as stimulator. Finally, we confirmed that the primary anti-MIHA CTL responses to adherent stimulator cells was MHC-restricted.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Cloned human cytotoxic T lymphocyte (CTL) lines reactive with autologous melanoma cells. I. In vitro generation, isolation, and analysis to phenotype and specificity

Activation of peripheral blood lymphocytes (PBL) from a melanoma patient either in secondary MLC in which EBV-transformed B cells from the cell line JY were used as stimulator cells, or by co-cultivation with the autologous melanoma cells in a mixed leukocyte tumor cell culture (MLTC) resulted in the generation of cytotoxic activity against the autologous melanoma (O-mel) cells. From these activated bulk cultures four cloned cytotoxic T lymphocyte (CTL) lines were isolated. The CTL clone O-1 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+), and O-36 (T3+, T4-, T8+, OKM-, HNK-, and HLA-DR+) were obtained from MLC, whereas the CTLC clones O-C7 (T3+, T4+, T8-, OKM-1-, HNK-, and HLA-DR+) and O-D5 (T3+, T4-, T8+, OKM-1-, HNK, and HLA-DR+) were isolated from autologous MLTC. All four CTL clones were strongly cytotoxic for O-mel cells but failed to lyse autologous fibroblasts and autologous T lymphoblasts. Moreover, the CTL clones lacked NK activity as measured against K562 and Daudi cells. Panel studies indicated that the CTL clones also killed approximately 50% of the allogeneic melanoma cells preferentially, whereas the corresponding T lymphoblasts were not lysed. Monoclonal antibodies against class I (W6/32) and class II (279) MHC antigens failed to block the reactivity of the CTL clones against O-mel and allogeneic melanoma cells, indicating that a proportion of human melanoma cells share determinants that are different from HLA antigens and that are recognized by CTL clones. In contrast to the CTL clones isolated from MLTC, the clones obtained from MLC also lysed JY cells, which initially were used as stimulator cells. The reactivity of O-36 against JY could be inhibited with W6/32, demonstrating that this reactivity was directed against class I MHC antigens. These results suggest that the lysis of O-mel and JY cells by O-36 has to be attributed to two independent specificities of this CTL clone. The specificity of the other cross-reactive CTL clone (O-1) could not be determined. The notion that individual CTL clones can have two specificities was supported by the following observations. The cytotoxic reactivity of both O-1 (T4+) and O-36 (T8+) against JY was blocked by monoclonal antibodies directed against T3 and human LFA-1, and against T3, T8, and human LFA-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell types required for H-2-restricted cytotoxic responses generated by trinitrobenzene sulfonate-modified syngeneic cells or trinitrophenyl-conjugated proteins.

Murine spleen cells were fractionated over nylon wool or Sephadex G-10 columns, and the cell types involved in the generation of trinitrophenyl (TNP)-specific, H-2 restricted (TNP-self) cytotoxic effector cells were studied from cultures stimulated with trinitrobenzene sulfonate (TNBS)-modified syngeneic cells, TNP-conjugated soluble proteins such as bovine gamma-globulin (TNP-BGG), or bovine serum albumin (TNP-BSA). Unfractionated or nylon nonadherent responding cells generated such effectors, irrespective of whether the cultures were stimulated with TNBS-modified cells or TNP-conjugated proteins. TNP-modified T lymphocytes, B lymphocytes, and phagocyte-enriched spleen cells were all capable of stimulating TNP-self effectors. TNP-self effectors. TNP-self as well as allogeneic cytotoxic responses were dependent on the presence of a radioresistant non-T cell that was removed by Sephadex G-10 fractionation and was replaced by irradiated, Thy 1.2-negative, glass adherent spleen cells, enriched in phagocytic cells. Results obtained by using glass adherent cells that were allogeneic or semi-syngeneic to the responding cells indicated that H-2 homology was not required for efficient glass adherent cell function, and that the H-2 restriction of TNP-self effectors is not determined by these glass adherent cells.     

Intrathymic differentiation of cytotoxic T lymphocyte (CTL) precursors. I. The CTL immunocompetence of peanut agglutinin-positive (cortical) and negative (medullary) Lyt 123 thymocytes

To analyze the developmental and functional interrelationship between cortical and medullary thymocytes, the peanut agglutinin-(PNA) binding capacity was used to separate thymocytes into PNA+ (cortical) and PNA- (medullary) thymocytes. Virtually, all positively selected PNA+ thymocytes (90% of the overall thymocyte population) expressed the Lyt 123 phenotype, whereas 90% of negatively selected PNA- thymocytes expressed Lyt 1 alloantigens, about 10% being Lyt 123 thymocytes. Provided, the requirement of Lyt 1 T helper cells was bypassed by Interleukin 2, a nonspecific mediator of T help, PNA+ Lyt 123 thymocytes mounted cytotoxic T cell responses comparable in magnitude to that of peripheral T cells. Their repertoire included antigenic disparities coded for by the complete MHC complex, H-2K, I-A, H-2D, mutational events at H-2K, as well as antigenic disparities expressed on TNP conjugated- and Sendai virus-infected syngeneic cells. PNA- Lyt 123 thymocytes represent a highly reactive pool of primary cytotoxic T lymphocyte (CTL) precursors for both alloreactive and H-2-restricted CTL responses. Since PNA- thymocytes include also Lyt 1 T helper cells, PNA- responder thymocytes are able to mount autonomously (CTL responses. Our data are first to provide direct evidence that Lyt 123 cells represent a common source of alloreactive and H-2-restricted CTL precursors in unprimed lymphocyte populations. Moreover, the apparent immunocompetence of cortical PNA+ thymocytes is now explained by their lack of T helper cells.     

Regulation of the cytotoxic activity of alloreactive cytotoxic T lymphocytes by helper cells and lymphokines

The cytotoxic activity of alloreactive cytotoxic T lymphocytes (CTL) was maintained and augmented by transferring cells from a 5-day mixed lymphocyte culture MLC into a host culture (HC) containing indomethacin, freshly explanted normal spleen cells, and peritoneal cells which were syngeneic to the MLC cells. The MLC cells used in the transfer experiments were generated by culturing untreated H-2b splenic responders with irradiated H-2d stimulators, or were generated by culturing Lyt-2-depleted H-2b splenic responders with irradiated H-2d stimulators. The allo-CTL were found to be derived from the donor MLC (first culture) when unfractionated MLC cells were transferred into a host (second) culture and incubated for 5 days. In contrast, the allo-CTL were derived from host culture cells when Lyt-2-depleted MLC cells were transferred and the combined cultures incubated for 5 days. In the former case, the augmentation of MLC-derived cytotoxicity did not result from nonspecific expansion of all donor T cells; instead it was mediated by lymphokine(s), distinct from IL-2, produced by helper T cells generated in host culture, which appeared to selectively expand the antigen-specific CTL or to increase the cytotoxic activity of these CTL. The helper T cells were Thy-1+, L3T4+, and Lyt-2-. These findings indicate that antigen-nonspecific help was provided by helper cells or helper factors (lymphokines) generated in the host culture, which maintained and augmented the cytotoxic activity of the fully generated allo-CTL. This helper effect was also seen in the induction of primary allo-CTL responses which could be generated with fewer stimulating cells and with a stronger cytotoxic response at different R/S ratios tested. The generation of allo-CTL in second culture following transfer of Lyt-2-depleted MLC cells to host cultures appears to have involved antigen carryover from the MLC; however, antigen carryover alone was not sufficient. It appears that in the absence of Lyt-2+ suppressor T cells, antigen-specific help might be generated in donor cultures (Lyt-2-depleted MLC) which promoted or recruited the generation of antigen-specific CTL in host culture.     

Auto-tumor lysis by blood lymphocytes in vitro

Summary Selectivity of the lysis of the tumor cells by autologous blood lymphocytes and its various subsets was investigated by means of the cold target competition assay. The effectors were autologous lymphocytes passed through a nylon-wool column (unfractionated: U) and their low-and high-density subsets, either without or after activation. The lymphocytes were activated (a) in autologous mixed lymphocyte tumor cell culture in autologous (MLTC), (b) in mixed lymphocyte culture (MLC), without and with interleukin-2, for 6 days, or (c) by phytohaemagglutinin for 3 days. Autologous-lymphocyte-mediated cytotoxicity (auto-tumor lysis: ALC) by the unfractionated, unmanipulated blood lymphocyte (U) population, its high-density fraction and those induced for auto-tumor lysis in the MLTC is regularly weak and affects only the autologous tumor cells. Their ALC function was inhibited only by the target identical unlabelled cells while the effect of separated low-density lymphocytes was inhibited also by allogeneic tumor cells.The cold-target competition assay indicated that several subsets with different specificities exist simultaneously in the effector populations activated in MLC, because the various targets did not cross-compete or did so only partially. Whenever interleukin-2 was added, at the start of the mixed cultures (MLTC or MLC), the lytic effects were no longer selective. Phytohaemagglutinin-activated effectors lysed several targets. These targets were inhibitory in a criss-cross fashion. Generally, populations showing auto-tumor selectivity had weak lytic effects, while the strongly activated effectors, with strong cytotoxic function, were not selective.     

Augmentation by serum-induced helper cells of T cell-mediated cytotoxic response against tumor associated antigens and alloantigens

Summary In vitro T cell-mediated cytotoxic responses to tumor associated antigens or alloantigens can be augmented by the addition of small amounts (0.1 to 1%) of syngeneic (mouse) or xenogeneic (rabbit) serum in the standard lymphocyte culture medium. Further studies showed that the augmentation is mediated by helper cells, which are induced by culturing the spleen cells or lymph node cells in the presence of these sera. In the syngeneic system performed with mixed lymphocyte tumor cell cultures (MLTC), the serum-induced helper cells are found to be resistant to the lysis of anti-Thy 1.2 antibody and are radioresistant; thus they have the characteristics of macrophages. In the allogeneic system performed with mixed lymphocyte culture (MLC), the serum-induced helper cells are also found to be resistant to the lysis of anti-Thy 1.2 antibody but are radiosensitive. In the latter case, however, removal of T cells abolishes the helper cell generation and only the T cell-enriched fraction provides for the generation of helper cells, indicating that the helper cells for MLC are probably derived from T cells but lose their susceptibility to anti-Thy 1.2 antibody lysis upon culturing in vitro. A study of the mode of action of the helper cells for MLC showed that they are probably needed at a later stage of cytotoxic response for the amplification of the killing efficiency of the T effector cells whereas the helper cells for MLTC are needed in the early induction phase of the immune response. These results indicate that although serum can augment the cytotoxic responses both in the syngeneic and in the allogeneic systems, the mechanism for the augmentation differs: macrophagelike helper cells are responsible for the augmentation of cytotoxic response to tumor associated antigens, whereas augmentation of cytotoxic response to alloantigens appears to be mediated by a subpopulation of T helper cells. Supported by a grant from the Japan Society for the Promotion of Science (T. I.).     

Mechanism of target cell lysis by cytotoxic T lymphocytes (CTL) employing RSV-induced H-2 congenic and recombinant mouse tumor cells: demonstration of soluble mediators released from H-2-restricted CTL clones

The secretion and the specificity of cytotoxic mediators from H-2-restricted cytotoxic T lymphocytes (CTL) were examined using non-virus-producing target tumor cells induced by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) in B10 congenic and recombinant mice. By using rat concanavalin A supernatant, two H-2-restricted CTL clones were established from cytotoxic effector cells of B10.A(5R) mice primed with SR-RSV-induced syngeneic tumor Cell-free supernatants from the H-2-restricted CTL clones cocultured with syngeneic tumor cells had selectively high cytotoxic activity for syngeneic and H-2-compatible tumor cells, but not for H-2-incompatible tumor cells. YAC-1 cells, and B10.A(5R) blasts as defined in the 5-hr 51Cr-release assay. The cytotoxic activity was detected in the cell-free supernatants from the CTL clones cocultured with the CTL-sensitive syngeneic and H-2-compatible tumor cells, but not with the CTL-insensitive tumor cells and YAC-1 cells. The cytotoxic activity of the cell-free supernatant could be adsorbed by the syngeneic tumor cells, but not by YAC-1 and L(s) cells. Thus, the H-2-restricted CTL clones against SR-RSV-induced tumor cells were capable of releasing cytotoxic mediators by coculturing with syngeneic or H-2-compatible tumor cells, and the cytotoxic mediators showed a certain H-2-restricted manner in killing the target cells. These results suggest that the lysis of RSV-induced tumor cells by H-2-restricted CTL can at least in part be mediated by cytotoxic factors.     

The immunocompetence of murine stromal cell-associated thymocytes

Thymocyte subpopulations that associate in vivo with distinct nonlymphoid cells of the thymus have been isolated, and their immunocompetence was analyzed. Previous studies have indicated that greater than 95% of such cells bear a surface antigen phenotype representative of immature thymocytes, and are among the earliest thymic compartments repopulated by bone marrow-derived cells after lethal and sub-lethal irradiation. Stromal cell-associated thymocytes may be activated in vivo because they proliferate well in vitro with no additional stimulus, and show little increase in proliferation with the addition of T cell mitogens or allogeneic spleen cells. Stromal cell-associated T cells contain cytotoxic T lymphocyte (CTL) precursors that are indistinguishable from mature peripheral T cells by the parameters of self tolerance, alloreactivity, H-2 restriction, and stringency of self H-2 preference. CTL precursor frequencies and the cytotoxic activity of cells further separated on the basis of high levels of Thy-1 expression argue against the possibility that stromal cell-associated CTL activity is due solely to contaminating mature lymphocytes. Our data suggest that stromal cell-associated thymocytes represent an intermediate subpopulation of thymocytes that is functionally mature and that expresses an immature surface phenotype. Furthermore, the imposition of self tolerance and MHC restriction specificity appears to be tightly associated with the acquisition of immunocompetence in these thymocyte subpopulations.     

Immune responses against allogeneic and syngeneic tumors in aged C57BL/6 mice

Aged C57BL/6 (B6) mice could reject allogeneic BALB/c RL male 1 tumor as efficiently as young B6 mice. However, in vitro analysis showed impaired generation of cytotoxic T cell response in aged B6 mice against allogeneic tumor. The reaction could be augmented by the addition of recombinant interleukin-2 (rIL-2). Enzyme-linked immunospots (ELISPOT) produced by CD8+ T cells purified from spleen cells showed no reduction in aged mice. The findings suggested that the number of CD8+ T cells capable of reacting against allogeneic H-2 antigens was similar in young and aged B6 mice. Low cytotoxic T lymphocyte (CTL) responsiveness in aged B6 mice appeared to have resulted from low responsiveness of CD4+ T cells producing IL-2. Although CTL generation was apparently impaired, strong multiple antigenicity of allogeneic tumor evoked a rejection response in aged B6 mice. On the other hand, no rejection response was observed against syngeneic EL4 tumor in aged B6 mice even after depletion of CD4+ CD25+ immunoregulatory cells. Depletion of CD4+ CD25+ cells caused rejection of EL4 tumor in young B6 mice. The findings suggested that aged B6 mice were incapable of inducing effector cells against weak tumor antigens. Only marginal CTL response and small number of ELISPOTs were generated in young but not aged B6 mice against EL4. Addition of rIL-2 to the culture augmented EL4 killing and ELISPOTs in spleen cells from young and aged B6 mice.     

Induction of preferential cytotoxicity against allogeneic mouse lymphoma cells: in vitro and in vivo studies

The aim of this study was to activate, in mixed leukocyte/tumor cell cultures (MLTC), cytotoxic lymphocytes exhibiting preferential activity in vitro and in vivo towards allogeneic mouse lymphoma cells. Whereas the lymphoma target cells were readily lysed by the MLTC-derived lymphocytes, the cytotoxicity against the corresponding allogeneic concanavalin-A(ConA)-induced lymphoblasts was more than tenfold lower. Both activities were mediated by CD3⁺, TCR⁺, CD8⁺, CD4⁻ cytotoxic T cells (CTL). ConA-induced lymphoblasts were readily lysed by anti-Thy1.2 antibodies and complement, by CTL derived from mixed leukocyte cultures (MLC) and by the MLTC-derived CTL in the presence of ConA, indicating that the lymphoblasts are not merely less lysable than the lymphoma cells but that the latter are specifically recognized by the CTL. Lymphoblasts poorly competed with ⁵¹Cr-labeled lymphoma cells in a “cold”-target competition assay, suggesting that the MLTC-derived CTL largely recognize epitopes expressed only by the lymphoma cells. Furthermore, analysis of the cytotoxic activity of more than 500 MLTC-derived CTL oligoclones and over 30 clones revealed that one-third of them were cytotoxic only against the allogeneic lymphoma cells, one-third were reactive against both the lymphoma and the allogeneic lymphoblast target cells and the remainder were not cytotoxic at all. Upon injection into sublethally irradiated, lymphoma-bearing allogeneic mice, the MLTC-derived CTL cured 56% of the recipients and caused graft versus host disease (GVHD) is only 22%, whereas CTL activated in MLC against allogeneic splenocytes were therapeutically ineffective and caused lethal GVHD in 89% of the recipients. Although the therapeutic efficacy of the in vitro-generated antitumor CTL was demonstrated against experimental lymphoma lines, this strategy might prove effective in tumor immunotherapy in conjunction with other modalities. Received: 24 December 1998 / Accepted: 5 April 1999     

Lymphokine-activated killer cells are generated before classical cytotoxic T lymphocytes after bone marrow transplantation in mice

Lymphokine-activated killer (LAK) cells are demonstrable within 2 wk after syngeneic or allogeneic (H-2-compatible) bone marrow transplantation in mice. Classical cytotoxic T lymphocytes (CTL) are not active until at least 4 wk after transplant. Both LAK cells and CTL bear the Thy-1 marker and do not possess the murine natural killer cell marker asialo GM.     

Cyclosporine A and peripheral tolerance. Inhibition of veto cell-mediated clonal deletion of postthymic precursor cytotoxic T lymphocytes.

Veto cell-mediated suppression of CTL responses has been proposed as one mechanism by which self tolerance is maintained in mature T cell populations. We have reported that murine bone marrow cells cultured in the presence of high-dose IL-2 (activated bone marrow cells) mediate strong veto suppressor function in vitro and in vivo, and that such veto activity is effected through clonal deletion of cytotoxic T cell precursors. In our studies, we have determined that bone marrow cell populations from athymic NCr-nu mice (H-2d) mediate strong veto cell activity without exposure to exogenous IL-2 in vitro. To examine mechanisms by which these naturally occurring veto cell populations in BM suppress precursor CTL (pCTL) responses, we used as a responding cell population in MLC, spleen cells of transgenic mice expressing at high frequency TCR specific for H-2 Ld encoded Ag with stimulation by H-2d-expressing cells in culture. Flow cytometric analysis was performed by staining the responding MLC cell population with the mAb 1B2 specific for the transgene-encoded TCR and determined changes of 1B2+ T cells. Such experiments demonstrated that the anti-H-2d cytotoxic response by these cell populations was specifically suppressed by NCr-nu (H-2d) bone marrow, and that 1B2+ pCTL were in fact specifically deleted from the responding cell population by incubation with such naturally occurring veto cell populations expressing the appropriate target Ag. In addition, to further understand the interactions of pCTL and veto cells and possible contributions by the latter to peripheral tolerance, we evaluated the effect of cyclosporine A (CsA) on veto cell-mediated suppression of pCTL of the transgenic mice. CsA inhibited veto cell-mediated suppression of cytotoxic T cell responses, and this inhibition correlated with a lack of clonal deletion of pCTL by veto cells in the presence of CsA. Furthermore, CsA exerted its effect through pCTL and not through veto cells, indicating that pCTL may play an active role in their own deletion by veto cells.     

Mutations in H-2K b influence the specificity of alloreactive effector cells included in the repertoire of H-2D b -restricted cytotoxic T lymphocytes against Moloney leukemia virus

Moloney leukemia virus-specific cytotoxic T lymphocytes (CTL), generated by secondary in vitro stimulation of spleen cells with syngeneic virus-infected cells, frequently lysed not only syngeneic virus-infected cells, but also noninfected allogeneic target cells. This phenomenon was studied with B6(H-2 ^b ) responder cells and a series of H-2K ^b -mutant responder cells. Thus, B6 Moloney-specific CTL lysed noninfected K ^b -mutant cells, but not B6 cells, whereas K ^b -mutant Moloney-specific CTL lysed noninfected B6 cells and not noninfected cells of the same mutant. Cold-target-inhibition studies showed that the CTL reactions against different allogeneic cells were mediated by different subpopulations of virus-specific CTL: lysis of allogeneic target cells was fully inhibited only by the same allogeneic and by syngeneic virus-infected cells, but not by another allogeneic cell, also lysed by the same effector-cell population. Lysis of syngeneic virus-infected cells could not be inhibited by allogeneic target cells. These data imply that a minority of virus-specific CTL shows cross-reactivity with a given allogeneic target cell. It is concluded that limited amino acid substitutions in the K^b molecule alter the repertoire of Moloney virus-specific CTL, as reflected in alloreactive CTL populations, even though the virus-specific CTL response. of B6 and all K ^b mutants is mainly D^b-restricted. Thus, the development of tolerance to self class-I major histocompatibility complex (MHC) molecules affects the repertoire of self-restricted cytotoxic T cells.