Immune responses to weakly immunogenic virally induced tumors. V. Short in vitro cultivation of YAC changes its antigenic properties.

YAC tumor, a Moloney virus-induced lymphoma of A mice, considerably changes its immunogenic properties following cultivation for 12 to 14 hr in vitro. The cultivated tumor, designated YAC-1, generates anti-YAC and anti-YAC-1 reactive cells, while the original YAC tumor generates suppressor cells with abrogate anti-YAC and anti-YAC-1 cytotoxic responses. In this report, we demonstrate by cold target competition experiments that YAC-1 and YAC tumors have distinctive antigenic structures on their surfaces. We have also found that RBL5, a Rauscher virus-induced lymphoma of C57BL/6 mice, generates anti-YAC, anti-YAC-1, and anti-RBL5 reactive cells in A mice. YAC-1 also generates reactive cells against the allogeneic RBL5 tumor. The total population of antitumor reactive cells was found to contain separate subpopulations directed maximally toward each of the three target tumors. The subpopulation directed toward each tumor was shown to be partially, but not completely, cross-reactive with the other tumors. Further, RBL5 priming and YAC-1 priming were shown to stimulate separate populations of antitumor reactive cells.     

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.     

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.     

Isolated soluble fractions from the murine B16 melanoma induce primary in vitro syngeneic antitumor responses

Summary This paper extends our previous studies, which documented our ability to isolate immunogenic entities from nonimmunogenic or weakly immunogenic tumors.B16 melanoma cells failed, in our in vitro experimental system, to induce anti-B16 cytotoxic responses in spleen cells derived from normal syngeneic C57BL/6 mice. The B16 melanoma cellular homogenate was fractionated on an Ultrogel AcA 34 column, and the various fractions were tested for their ability to induce anti-B16 cytotoxic responses under the same conditions as those used for intact B16, the nonimmungenic tumor cells. Certain fractions, some of them with relatively low protein concentrations, induced anti-B16 cytotoxic responses in spleen cells of normal C57BL/6 mice, whereas others, some of them with relatively high protein concentrations, failed to induce such responses. One fraction (Fr.), designated Fr. 5/6, was examined in detail. It was found that in normal syngeneic spleen cells this fraction induced effector cells that efficiently killed (at various E : T ratios) the relevant B16 target cells and RBL5 syngeneic tumor cells, but not the YAC allogeneic tumor cells or C57BL/6 lymphoblasts. Furthermore, an excess of unlabeled B16 cells most efficiently blocked the ability of these anti-B16 effector cells to kill radiolabeled B16 target cells. RBL5 tumor cells, YAC tumor cells, or C57BL/6 lymphoblasts failed to block these effector cells efficiently. A significant fraction of the effector cells induced with Fr. 5/6 was characterized as thymus-derived cells (Thy-1⁺, Thy-2⁺3⁺ cells). It was suggested that another fraction of the cellular population was natural killer cells, which cytolyzed the RBL5 target cells. Various theoretical and practical aspects of these findings are discussed.     

The isolation of immunogenic molecular entities from immunogenic and nonimmunogenic tumor homogenates by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

Summary YAC, a Moloney-virus-induced tumor of A-strain mice, is a nonimmunogenic tumor. Mice injected with the inactivated neoplastic cells and challenged with viable tumor cells did not survive longer than mice that received the challenge dose alone. The homogenate of this nonimmunogenic tumor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the gel slices containing isolated molecular entities were injected into various groups of mice. The mice were challenged with low doses of viable tumor cells (10–30 cells) and their survival time was recorded. Small but significant numbers of mice injected with apparent 80–90 K SDS-PAGE-isolated molecular entity rejected the tumor or survived longer than the control groups of mice. Spleen cells from mice injected with 80–90 K molecular entity inhibited the YAC tumor cotransferred with them to naive recipients (Winn assay). Spleen cells from mice injected with monoclonal antibody against nonspecific T-cell helper factor and immunized with 80–90 K SDS-PAGE-isolated molecular entity failed to inhibit the tumor growth in naive recipients, indicating that helper T cells are involved in induction of the antitumor resistance. Nylon-wool-passed splenocytes from mice injected with 80–90 K inhibited tumor growth in some of the recipient mice. Spleen cells from these mice treated with anti-Thy-1 and complement also inhibited the tumor growth in some of the recipients, suggesting that the effector cells were both T and non-T cells. C57BL/6 mice immunized with apparent 20 K SDS-PAGE-isolated molecular entity of RBL5 tumor also induced in vivo resistance to the syngeneic viable RBL5 cells, but not to the syngeneic B16 melanoma cells, indicating the specificity of the protective effect. The practical and theoretical implications of these findings are discussed.     

Immunogenicity of subcellular fractions and molecular species of MuLV-induced tumors

Summary YBA, a Moloney virus-induced leukemia in CBA mice, and a relatively weak immunogenic tumor, was screened for the presence of immunogenic antigens. The tumor was subjected to homogenization and subcellular fractionation on sucrose gradients; the immunogenic subcellular fractions underwent further separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunogenicity of the subcellular fractions and the SDS-PAGE-isolated molecular species were tested by (their) subcutaneous injection into syngeneic mice and examination of their splenocytes examined against tumor cell and normal cell targets by the chromium release cell-mediated lympholysis assay. Tumor cell homogenates were also separated by SDS-PAGE and tested for immunogenicity without prior fractionation.Splenocytes from mice that had received injections of certain SDS-PAGE-isolated epitopes derived from YBA tumor homogenate or its light and heavy subcellular fractions generated effective cytotoxic responses against YBA target cells after 6 days in vitro cultivation. In contrast, intact YBA tumor cells or non-separated tumor homogenates failed to induce an efficient cytotoxic response. The effector cells induced with the immunogenic SDS-PAGE-isolated epitopes of YBA tumor were specific, since they cytolysed the homologous target cells more efficiently than unrelated target cells or syngeneic normal cells. The activity of these effector cells was affected by varying the effector : target ratio. Augmentation of the cytotoxic responses was obtained when the splenocytes of mice immunized with SDS-PAGE-isolated epitopes of YBA tumor were restimulated in vitro, with the homologous neoplastic cells.Immunogenic SDS-PAGE epitopes were isolated from YAC tumor also (YAC is a Moloney-induced tumor of A mice). The effector cells induced with these separated epitopes were characterized as thymus-derived cells and not as natural killer cells.The results suggest that (1) the molecular repertoire of YBA and YBA tumors contain immunogens that can induce a specific antitumor cell-mediated response; (2) the isolated molecular species injected are more efficient immunogens than the entire, unseparated homogenate sample or a dose of 10⁸ intact inactivated tumor cells; and (3) the gel matrix may be responsible for the enhanced cell-mediated response induced against the weakly immunogenic tumor.     

Immunosuppression induced by attenuated Salmonella. Reversal by IL-4

We previously demonstrated that an aroA- strain of Salmonella typhimurium, which provides excellent protection against virulent Salmonella challenge, also rendered immunized mice unable to mount in vivo and in vitro antibody responses to heterologous Ag. Coculture studies using transwell plates indicated that suppression was mediated by soluble factors. The suppressive cells were identified as belonging to the monocytic linkage. Macrophage precursors as well as mature adherent macrophages mediated the observed suppression. In the present study, the mechanism of immunosuppression was investigated. Suppression was found to be genetically nonrestricted as spleen cells from immunized C3HeB/FeJ mice (H-2k) suppressed the anti-SRBC plaque-forming cell (PFC) responses of normal spleen cells from two MHC noncompatible mouse strains, BALB/c (H-2d) and C57BL/6 (H-2b). Time course experiments demonstrated that the addition of spleen cells from immunized mice to normal splenocytes as late as day 4 of a 5-day assay was still markedly suppressive. Furthermore, suppression of the PFC responses was accompanied by a profound inhibition of the capacity of immune splenocytes to produce IL-2 in response to in vitro stimulation by Con A. Coculture studies showed that immune spleen cells were able to suppress IL-2 production by normal splenocytes in a dose-dependent fashion. However, the suppressed PFC responses of immune spleen cells could not be reversed by the exogenous addition of up to 200 U/ml of IL-2, suggesting that immune splenocytes are also defective in their ability to respond to IL-2. In marked contrast, suppression of PFC responses was reduced by more than 50% by the addition of as little as 1 U/ml of IL-4 and was completely abrogated when 5 U/ml of IL-4 were added to in vitro cultures of spleen cells from immunized mice. The antisuppressive action of IL-4 appeared to be via its inhibitory effect on activated macrophages. The implications of the above findings are discussed.     

Influence of interferon on the functional expression of natural killer target structures of murine lymphoma cells

Murine lymphoma cells (YAC-1), induced by Moloney leukemia virus, nontreated (YAC) or pretreated in vitro with interferon (YAC-IF), were tested for their susceptibility to natural killer (NK)-mediated cytolysis. In line with previous reports YAC-IF were less susceptible to NK lysis than YAC cells. In cold competition assay, YAC-IF inhibited cytotoxicity to a lesser extent than YAC lymphoma when labeled target YAC cells were used. However, when radioactive YAC-IF cells were used as targets, cold competition attained with both YAC and YAC-IF was essentially the same. Furthermore, effector splenocytes, depleted of NK effector cells through immunoabsorption on YAC monolayer, were inactive against both YAC and YAC-IF targets. On the other hand, effector lymphocytes, absorbed on YAC-IF monolayer, retained NK activity against YAC cells but not against YAC-IF targets. These results are compatible with the hypothesis that interferon (IF) modulates negatively a subset of "interferon-susceptible" (IFS) NK target structure(s) (TS) of YAC cells, which would then express membrane determinants not functionally present on YAC-IF cells. On the other hand YAC and YAC-IF cells share "interferon-resistant" (IFR) TS not affected by pretreatment with IF. In order to test whether IFS X TS and IFR X TS are present on the same cell or clonally distributed, YAC cells were cloned and tested for NK susceptibility following IF pretreatment. The results did not support the hypothesis of a clonal distribution of both IFS X TS and IFR X TS since IF pretreatment of all clones, obtained by limiting dilution, resulted in a net impairment of target susceptibility to NK effector cells.     

The genetic control of natural killer cell activity and its association with in vivo resistance against a moloney lymphoma isograft

Spleens of normal young mice of certain strains contain lymphocytes that can kill strain A-derived YAC-1 lymphoma cells in a⁵¹Cr release cytotoxic assay in vitro. We have previously classified mouse genotypes as high or low reactors, according to their responses in this test. In vivo resistance to small numbers of YAC ascites lymphoma cells is correlated with in vitro cytolytic activity. In vitro and in vivo tests were carried out on the same individual (A x C57BL)F₁ x A backcross mice. Natural in vitro killer cell activity appeared to be under polygenic control, including a strong H-2-linked factor. No linkage was found with five different isozyme loci, with theIg-l locus or with C5 serum activity. Also in vivo resistance showed strong linkage with theH-2 complex. In (A x CBA)F₁ x A backcross mice, a weak linkage was found with the coat color locusC. There was a correlation between in vitro killer activity and in vivo resistance in the same backcross mice. In vivo resistance was particularly strong in mice that combined theH-2 ^b -linked resistance factor with a high cytolytic activity in vitro.     

Enhanced H-2 expression and T-cell-dependent rejection after intracerebral transplantation of the murine lymphoma YAC-1

The relationship between MHC class I (H-2) expression and tumorigenicity was investigated after intracerebral inoculation of the murine lymphoma YAC-1 and its H-2 negative variant, A.H-2-. YAC-1 was less tumorigenic than A.H-2- in normal as well as NK-depleted syngeneic A/Sn mice. However, in T-cell-depleted syngeneic mice YAC-1 was as tumorigenic as A.H-2-. Following intracerebral growth, the H-2 expression of YAC-1 was markedly enhanced in a similar fashion as after intraperitoneal passage. The A.H-2- variant remained H-2 negative after intracranial passage. The H-2 negative variant cells were not rejected from the brain even when intermixed with wild-type YAC-1 cells prior to intracerebral inoculation, excluding an "innocent bystander" effect. In vitro, the intracerebrally passaged YAC-1 line showed enhanced sensitivity to lysis by H-2 Kk Dd (H-2a) specific CTLs but decreased sensitivity to NK cells. The A.H-2- line was unchanged. Our data suggest that the lack of H-2 molecules may facilitate the growth of antigenic tumor cells in the brain due to escape from T-cell-mediated immunosurveillance. Our data also suggest, in line with other recent findings, that intracerebrally growing tumor cells are sheltered from NK cell-mediated rejection.     

Immunological response in the mouse melanoma model after local hyperthermia

Our study was aimed to characterize the phenotype and functional endpoints of local microwave hyperthermia (LHT, 42 degrees C) on tumor infiltrating and spleen leukocytes. The effectiveness of LHT applied into the tumor of B16F10 melanoma-bearing C57/BL6 mice was compared with anesthetized and non-treated animals. Subpopulations of leukocytes were analyzed using the flow cytometry, and the cytotoxic activity of splenocytes against syngeneic B16F10 melanoma and NK-sensitive YAC-1 tumor cell lines was evaluated in (51)Cr-release assay. Similarly, the in vitro modification of the heat treatment was performed using healthy and melanoma-bearing splenocytes. We found a 40 % increase of activated monocytes (CD11b+CD69+) infiltration into the tumor microenvironment. In the spleen of experimental animals, the numbers of cytotoxic T lymphocytes (CTLs-CD3+CD8+) and NK cell (CD49b+NK1.1+) raised by 22 % and 14 %, respectively, while the NK1.1+ monocytes decreases by 37 %. This was accompanied by an enhancement of cytotoxic effector function against B16F10 and YAC-1 targets in both in vivo and in vitro conditions. These results demonstrate that LHT induces better killing of syngeneic melanoma targets. Furthermore, LHT evokes the homing of activated monocytes into the tumor microenvironment and increases the counts of NK cells and CTL in the spleen.     

Characterization of T lymphocyte clones isolated from BCNU-cured LSA mice.

1,3-Bis(chloroethyl)-1-nitrosourea (BCNU) has been shown to "cure" over 90% of the mice bearing the syngeneic tumor LSA, and the cured mice acquire elevated levels of tumor-specific immunity. In the present study, we report for the first time the establishment and characterization of several tumor-specific CD8+ cytotoxic T cell (CTL) clones from splenic T cells of BCNU-cured LSA mice. Many of these clones were found to be strongly cytotoxic to LSA but not to a different H-2b tumor target such as EL-4, or the natural killer (NK)-susceptible target YAC-1, NK-resistant target P815, or con A or LPS blasts from H-2b mice. Some of the clones showed a moderate level of cytotoxicity to the NK-susceptible target YAC-1. The relative roles of interleukins such as IL-2, IL-4 or IL-6 in supporting the proliferative response of some LSA-activated CTL clones were analyzed. As expected, recombinant human (rh) IL-2 alone supported the proliferative response of activated CTL clones. Addition of recombinant murine (rm) IL-4 or rhIL-6 alone to the culture failed to influence the response. Also, in combination with rhIL-2, neither rmIL-4 nor rhIL-6 appreciably augmented rhIL-2-supported proliferative response of CTL clones. These studies may provide insights for the development of effective approaches to modulate function and activity of effector T cells.     

Further characterization of thymic suppressor cells and a factor that suppress the generation of cells cytotoxic for a syngeneic tumor in DBA/2 mice.

Antigen-specific suppressor cells and suppressive extracts obtained from the thymuses of DBA/2 mice bearing small syngeneic P815 mastocytomas were compared for their immunogenetic properties and requirements. The assay for specific suppression involved the ability of either cells or extracts to inhibit the primary in vitro cytotoxic response of normal DBA/2 splenocytes to mitomycin-treated P815 cells. It was shown that pretreatment of suooressor cell populations with anti-Iad antiserum plus rabbit complement removed the suppressive activity. Similarly, absorption of the suppressor factor with anti-Iad antiserum removed the suppressive properties of the material. It was found that the suppressor cells, generated in DBA/2 tumor bearers, were capable of specifically suppressing the anti-P815 response of B6D2 F1 radiation chimeras possessing lymphoid cells of the H-2b or H-2t2 haplotype equally as well as they could suppress the response of H-2d-bearing effector cells. This indicates that the suppressor cells are not H-2 restricted with respect to K or D markers on the responder cells in this system.     

Two types of allograft-induced cytotoxic macrophage, one against allografts and the other against syngeneic or allogeneic tumor cells

In the 1990s, based on the results of studies using beta(2)M, CD4 or CD8 knockout mice, several groups reported that the main effector cells responsible for skin or organ allograft rejection were non-T, non-NK cells. Similarly, we demonstrated that in an animal model of transplantation of BALB/c (H-2(d)) skin onto or Meth A (H-2(d)) tumor cells into C57BL/6 (H-2(b)) mice, AIM, which expressed iNOS, IL-12, and IL-18, were the main effector cells and also that they were cytotoxic against syngeneic tumor cells. Here, we examined whether the same population of macrophages could react with two distinct types of target cell. When BALB/c skin or Meth A tumor cells were transplanted into C57BL/6 mice, cytotoxic activity against the allograft was induced in the transplantation site on days 5-14 and was recovered in non-adherent cells after a 20-min incubation in a serum-coated dish, suggesting the induction of a type of AIM (AIM-1) in the transplantation site. The AIM-1-expressing receptors for H-2D(d)K(d) antigens had no cytotoxic activity against syngeneic tumor cells. In contrast, AIM-2, which were recovered in the fraction adherent to the serum-coated dish, exhibited cytotoxic activities against various types of tumor cells, whereas they were inactive toward BALB/c skin. AIM expressed iNOS (AIM-1 < AIM-2), IL-12 (AIM-1 > AIM-2), and IL-18 (AIM-2 alone) mRNAs. These results indicate that after allografting, two distinct types of cytotoxic AIM were induced in the transplantation site, one against the allografted skin or tumor (AIM-1) and the other against allogeneic or syngeneic tumor cells (AIM-2).     

Mechanisms of leukemogenesis. I. Generation of autoreactive lymphocytes in response to a murine leukemia virus.

Examined in this paper is the capacity of 334C murine leukemia virus (MuLV) to stimulate the generation of virus-specific cytotoxic effector cells in mice of the C57BL/6 strain that are relatively resistant to Friend, Moloney, and Rauscher (FMR) MuLV-induced leukemia, and in BALB/c mice that are relatively susceptible to leukemia induced by FMR MuLV. Generation of cytotoxicity requires in vivo administration of the virus followed by in vitro culture of lymphoid cells from virus-injected animals. Lymphoid cells from MuLV-resistant C57BL/6 donors develop high levels of specific cytotoxicity after secondary in vitro stimulation with syngeneic MuLV-induced tumor cells. Cells derived from these same donors, cultured in the absence of MuLV-induced tumor cells, fail to exhibit cytotoxicity. Secondary in vitro stimulation of lymphocytes from MuLV-susceptible BALB/c animals results not only in generation of cytotoxic reactivity against syngeneic MuLV-induced tumor cells but also induces apparently autoreactive effector cells capable of lysing other H-2d tumor cells as well as normal peritoneal cells bearing H-2d antigens. Moreover, generation of cytotoxicity by BALB/c lymphocytes occurs whether or not MuLV-induced tumor cells are included in the secondary culture system.     

In vivo generation of suppressor T cells by thymosin in congenitally athymic nude mice

Treatment of nude mice with thymic factors such as thymosin has been mostly ineffective in generating effector T cells. This study examined the effects of treating nude mice with thymosin fraction 5 on the induction of cells that could participate in and/or regulate cytotoxic T lymphocyte (CTL) generation by normal spleen cells in vitro. Splenic lymphocytes from BALB/c nude mice injected with thymosin fraction 5 every other day for 2 wk were tested for their ability to generate CTL in vitro. Two days after the last subcutaneous injection of thymosin, nude spleens were removed, mixed with normal BALB/c spleen cells, and placed into a mixed lymphocyte tumor culture (MLTC) against allogeneic RBL 5 tumor cells. After a 5-day incubation, cultures were tested for the presence of CTL in a 4-hr 51Cr-release assay. Spleen cells from thymosin-treated nude mice did not generate CTL but suppressed the ability of normal spleen cells to generate CTL in vitro. Characterization of the thymosin-induced nude mouse suppressor cells showed them to be Thy 1 positive, nonadherent, cyclophosphamide-sensitive T cells. These data demonstrate that some T cell maturation occurs in vivo under thymosin influence. However, the activity of these cells is initially limited to a regulatory function. These studies suggest that maturation of functional suppressor T cells occurs before CTL. Further immunologic manipulation appears to be necessary in order to induce CTL effector cells in nude mice.     

Herpes-Simplex-virus-specific, H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2k) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected taraget cells expressing the Dd alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2Dk determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for Dd alloantigen could be positively selected on H-2d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing Dd alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

Characterization of cytotoxic cells generated from bone marrow culture

Bone marrow (BM) harbors precursors (Pre-NK) to NK cells. Recently, we devised an in vitro culture system that induces differentiation of the presumptive BM Pre-NK cells into cytotoxic cells to YAC in the presence of rat concanavalin A (Con A) conditioned medium. We have now analyzed the antigenic phenotype of the effector cells, precursor cells, and the target specificity of these cytotoxic cells. The cytotoxic cells had antigenic profiles similar to endogenous NK cells with the exception of Lyt-2 antigen. They are strongly positive for Qa-5, Thy-1, and partially positive for NK-1, Ly-5, Ly-6, Ly-10, and AsGm-1 and Lyt-2 antigens. The Pre-NK or accessory cells are positive for Qa-5, Ly-10, and Ly-20 and partially positive for NK-1, Thy-1, and AsGm-1 antigens. These Qa-5+ NK cells do not exhibit cytotoxic activity to WEHI or P815. They could also be generated from BM of nude mice as well as beige mice. We concluded from these studies that rat Con A-conditioned medium contained factors that could differentiate Pre-NK cells to mature NK cells and that these cells are heterogeneous. This in vitro culture system is useful in delineating the ontogeny of NK cells.     

Resistance to 402AX teratocarcinoma involves immunity to minor histocompatibility antigens

The 402AX teratocarcinoma is a 12/J-derived mouse major histocompatibility complex (MHC) antigen negative tumor that is induced to express H-2^b class I antigens during rejection. Resistance to 402AX by MHC allogeneic and syngeneic mice is immunologically mediated and involves the recognition of tumor-associated antigens (TAA) in the context of induced MHC class I antigens. The current studies were undertaken to define the 402AX TAAs. Reconstitution of irradiated susceptible hosts (129/J) with 402AX-primed resistant spleen cells (C57BL/6) results in acute graft-versus-host disease, suggesting that tumor-primed C57BL/6 splenocytes are reactive to tumor genotype (129/J) minor histocompatibility (Hm) antigens. C57BL/6 anti-129/J effector cells, although not directly cytotoxic for 402AX cells, are specifically cold target inhibited by 402AX cells. Genetically susceptible hosts (C3H.SW) immunized to 129/J Hm antigens by skin grafting become resistant to an i.p. challenge of 402AX cells. These results suggest that 129/J Hm antigens may be the TAAs recognized during genetically controlled rejection of the 402AX teratocarcinoma.