Teratocarcinoma cell MHC antigen expression is regulated in vitro by a soluble noninterferon factor

The 402AX murine teratocarcinoma is a spontaneous testicular tumor of 129 (H-2b) origin which does not express MHC encoded antigens. Rejection of this tumor is immunologically mediated and the tumor cells are induced in vivo to synthesize H-2b antigens when passaged in genetically resistant host mice. The present studies demonstrate that serum from tumor primed genetically resistant host mice can induce tumor cell MHC antigen expression in vitro as measured by indirect immunofluorescence using monoclonal antibodies. The inducing factor is specific for 402AX tumor cells and is not interferon as shown by the lack of response of the 402AX tumor to gamma interferon, and the absence of significant interferon activity in inducer serum. These studies demonstrate another factor independent of interferon that can induce MHC class I antigen expression on tumor cells.     

402AX teratocarcinoma MHC class I antigen expression is regulated in vivo by Lyt 1, Lyt 2, and L3T4 expressing splenic T cells

The murine 402AX teratocarcinoma is a MHC class I antigen negative tumor of 129 strain origin. Host resistance to the 402AX tumor is genetically controlled. When passed intraperitoneally in genetically resistant mice, the tumor cells are induced to express MHC Class I antigens of the 129 genotype. When passed in genetically susceptible mice, the tumor cells remain MHC class I antigen negative. Earlier studies have demonstrated that resistance to the tumor and regulation of tumor cell MHC class I antigen expression are under the control of the host's immune system. The present studies indicate that splenic Lyt 1-, Lyt 2-, and L3T4-expressing cells regulate tumor cell MHC class I antigen expression, and that these cells require a genetically resistant host environment in which to differentiate. Splenic T cells primed to the 402AX tumor and transferred into genetically susceptible 129 mice give rise to GVHD, suggesting that immunity to the tumor involves reactivity to 129 minor histocompatibility antigens.     

Transfection and expression of syngeneic H-2 genes does not reduce malignancy of H-2 negative teratocarcinoma cells in the autologous host

Rejection of the MHC class I negative 402AX teratocarcinoma is accompanied by induction of tumor cell-encoded H-2K and H-2D antigens by the genetically resistant host. To determine whether MHC antigen expression is required for 402AX rejection, we have prepared H-2Db-transfected 402AX cells (402AX/Db). Transfectants express high levels of H-2Db, most of which is not associated with beta 2-microglobulin. MHC syngeneic and allogeneic mice susceptible to 402AX are resistant to 402AX/Db, suggesting that MHC class I antigen expression is required for tumor rejection. Autologous 129 hosts, however, are susceptible to 402AX/Db. 402AX cells transfected with the H-2Kb gene (402AX/Kb) are also lethal in the autologous 129/J host, but rejected by MHC syngeneic and allogeneic mice. Non-129 strain 402AX-susceptible mice pre-immunized with 402AX/Db or simultaneously challenged with 402AX/Db plus 402AX are immune to 402AX. Mice immunized with 402AX/Db produce MHC class I induction factor. 402AX/Db and 402AX cells are lysed equally by natural killer cells, indicating that in 402AX cells the expression of class I antigens is unrelated to NK susceptibility. These studies confirm the requirement for class I expression in 402AX immunity, but demonstrate that in the autologous host immunity requires additional factors beyond class I antigen expression.     

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.     

Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.     

The unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not due to a lack of H-2 antigen expression.

It is well documented that activated macrophages, but not nonactivated ones, kill tumor cells in vitro without damaging normal cells. We, however, have previously shown that embryo-derived teratocarcinoma cells (F9, P19, PCC4) are efficiently killed by nonactivated macrophages as well as by activated ones. Whereas other tumor cells are killed extracellularly by macrophages, we found that F9 teratocarcinoma cells are phagocytosed alive by macrophages and subsequently killed intracellularly by a process dependent on intact lysosomal function. Neither the H-2 antigens nor the mRNAs for the alpha-chain and beta 2-microglobulin are detectable in embryo-derived teratocarcinoma cells. An obvious explanation for this unique killing is that the nonactivated macrophages recognize and kill these cells due to their lack of class I MHC antigen expression, assuming that class I MHC gene products on the target cells switch off the cytolytic machinery of nonactivated macrophages. Our present findings demonstrate that there is no correlation between H-2 antigen expression on tumor cells and their susceptibility to killing by macrophages. Retinoic acid-differentiated F9 cells and P19 cells expressing H-2 antigen after exposure to MAF (IFN-gamma) were sensitive to the killing by nonactivated macrophages. Hybrids that arose from fusion of P19 teratocarcinoma cells with embryonal normal fibroblasts (C57BL/6), which displayed the morphology of embryonal carcinoma stem cells and expressed H-2 antigens, were also sensitive to the killing by nonactivated macrophages. On the other hand, the H-2-negative testicular 402AX teratocarcinoma cells and K1735P melanoma cells were both resistant to the killing by nonactivated macrophages. We concluded that the unique killing of embryo-derived teratocarcinoma cells by nonactivated murine macrophages is not related to a lack of H-2 antigen expression.     

Influence of IgH V-region genes on the growth kinetics of a murine B cell leukemia (BCL1)

The growth kinetics of an IgM-bearing B cell leukemia of BALB/c (Ig-1a) origin, designated BCL1, has been investigated in 2 allotype immunoglobulin (Ig) heavy (H) chain congenic strains, C.B-20 (Ig-1b) and C.AL-20 (Ig-1d), and an (H) chain recombinant strain, BAB-14 (Ig-1a/1b), that carries Ig-1a genes in the variable (V)-region and Ig-1b genes in the constant (C)-region. When large numbers (10(6) to 10(7)) of BCL1 cells were injected into these mice, leukemia, as measured by the appearance of leukemic cells in peripheral blood with subsequent mortality, did not occur in C.B-20, was delayed in C.AL-20, and progressed at the same rate in BAB-14 relative to BALB/c control mice. These results indicate that an immune response directed against an antigen encoded for by an H chain V region gene (idiotype or variable-region allotype) or linked gene (minor histocompatibility antigen) prevents the growth of the BCL1 leukemia in the C.B-20 mice. Tumor resistance appears to be due to T cell activity since adoptive transfer of such cells from C.B-20 tumor rejectors protected sublethally irradiation recipients from subsequent tumor challenge. Although H-2 restricted, anti-BCL1 cytotoxic cells were detected in C.B-20 mice challenged in vivo and restimulated in vitro with BCL1 cells, evidence is discussed that suggests that the resistance observed is not due to these effector cells. The resistance of allotype congenic mice to BCL1 was not absolute; a small inoculum (10(2)) was as lethal in C.B-20 and C.AL-20 as BALB/c mice. Thus, Ig-encoded cell surface antigens, although immunogenic, in no way ensure ultimate host survival.     

Teratocarcinoma cells cultured on embryonic substrates show accelerated migrating behavior in vitro and increased metastatic activity in vivo

Teratocarcinoma cells (402AX) were grown on feeder layers of whole mouse embryos (Day 4) or mouse embryonic fibroblasts and then were either examined in vitro or transplanted in vivo. After twenty-four hours of coculture, teratocarcinoma cells demonstrate accelerated cell migration in vitro. Furthermore, transplantation of teratocarcinoma cells with embryonic substrates into syngeneic hosts produces grossly detectable lymph node metastases. These effects appear to be due to soluble factor(s) produced by embryonic substrates which enhance tumor cell proliferative/migratory activity. This suggests that tumor cell invasion and metastasis may be stimulated by soluble factors produced by host tissues.     

Inhibition of tumor growth mediated by lymphocytes sensitized in vitro to a syngeneic murine teratocarcinoma 402AX

TerC, a cell line derived from a strain 129 teratocarcinoma 402AX, was used to sensitize syngeneic 129 (H-2bc) splenic lymphocytes in vitro. The effector cells generated inhibited in vitro growth of TerC as measured by an 125I-IUDR ost-labeling technique. It was also shown, with a modified Winn assay, that the sensitized cells were effective in preventing TerC growth in vivo. The effector lymphocyte was nonadherent to nylon wool was sensitive to anti-Thy-1.2 + C, and was phenotypically Ly 1-2+. The anti-TerC effector T lymphocytes were not functional in a 51Cr-release assay. However, this failure to lyse appears not to be due to some intrinsic membrane resistance since both BCG and ConA-activated killers were able to lyse TerC. The TerC-sensitized lymphocytes displayed no H-2 restriction and were able to growth inhibit in vitro a wide range of tumorigenic cell lines, e.g., P815 (H-2d), EL-4 (H-2b),Sal (H-2a), and BALB/c (H-2d) 3T12. Mouse blastocyst cell lines were also inhibited. BALB/c 3T3 and mouse fibroblast cell strains were not growth inhibited. Thus, it appears that oncofetal antigens expressed on TerC are capable of initiating a cell-mediated response and that these antigenic specificities are shared by many transformed cell lines.     

Host H-2 genotype regulates the metastatic ability of H-2-associated variants of B16 melanoma: defense systems screening for absence of self H-2 components by natural killer cells and host-associated homing barrier

The mechanisms of host H-2-associated resistance against metastasis of tumor cells were evaluated in relation to the H-2 phenotype of tumor cells. We used H-2 heterozygous H-2a/b and H-2d/b, and H-2 homozygous H-2b/b hosts, and H-2-associated variant lines of B16 cells (H-2b+, H-2b-). In H-2b/b hosts, H-2+ cells were highly metastatic in vivo, and were resistant to host NK effectors in vitro. Therefore, H-2a/b and H-2d/b hosts showed resistance to metastasis of H-2+ cells and their effectors showed killing activity to these cells in vitro. Though the host resistance was reduced by anti-asialo GM1 serum treatment, these hosts continued to demonstrate a considerable resistance against early survival and metastasis of the B16 cells. To evaluate this natural resistance, aside from the NK system, radiation bone marrow chimeras of F1-parental combinations were used. The data suggest that host MHC-associated resistance involves not only the NK defense system but also the host environmental resistance. Both exert resistance by recognizing the H-2 mismatch in relation to the host.     

Stimulation of genetic resistance to marrow grafts in mice by interferon-alpha/beta

Lethally irradiated mice were infused with syngeneic, H-2 allogeneic, parental strain, or H-2 heterozygous bone marrow cells. They were injected daily with rabbit anti-mouse interferons (IFN)-alpha/beta or gamma or with IFN-alpha/beta. The growth of donor-derived cells was judged 5 days later by measuring splenic incorporation of 5-iodo-2'-deoxyuridine-125I into DNA. Antibodies to IFN-alpha/beta, but not to IFN-gamma, weakened genetic (both hybrid and allogeneic) resistance to marrow cell grafts. IFN-alpha/beta stimulated hybrid and allogeneic resistance, the latter even in genetically "poor responder" mice. Mice pretreated with silica, which weakens genetic resistance, were stimulated by IFN-alpha/beta to resist incompatible marrow cell grafts; however, IFN-alpha/beta failed to reverse the effects of antiasialo GM1 serum on marrow graft rejection. IFN-alpha/beta did not inhibit the growth of syngeneic marrow cells and did not stimulate resistance to H-2 heterozygous bone marrow cells. We propose that genetic resistance occurs in two discrete steps. In the first step, hemopoietic histocompatibility (Hh) antigens are recognized by one host cell type, and this recognition leads to IFN-alpha/beta secretion by a silica-sensitive cell. In the second step, asialo GM1-positive natural killer cells stimulated by IFN-alpha/beta recognize Hh antigens on marrow stem cells and cause rejection. The defects in resistance observed in genetically poor responder mice and in mice treated with silica appear to involve the first step in recognition. The lack of rejection of H-2 heterozygous (Hh-) marrow cells by parental strain mice injected with IFN-alpha/beta indicated that specific Hh recognition is critical in the second step of genetic resistance.     

Spontaneous fusion in vivo between normal host and tumor cells: possible contribution to tumor progression and metastasis studied with a lectin-resistant mutant tumor.

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGAr) mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAs) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4 (WGAr) tumor cells and normal host cells, at least some of which are of bone marrow origin. Thus, growth of the H-2d MDW4 tumor cells in (C3H X DBA/2)F1 (H-2k X H-2d) or (C57BL/6 X DBA/2)F1 (H-2b X H-2d) mice led to the appearance of WGAs revertants bearing the H-2k or H-2b major histocompatibility complex antigens associated with the C3H or C57BL/6 parental strains, respectively. Similarly, WGAs revertants of MDW4 were found to express H-2k antigens after growth in CBA/HT6T6 (H-2k) leads to DBA/2 bone marrow radiation chimeras. Attempts to mimic the in vivo hybridization process were successful in that in vitro somatic cell fusion between an ouabain-resistant (OuaR), 6-thioguanine-resistant (Thgr) derivative of the MDW4 mutant and either normal bone marrow or spleen cells resulted in loss of the WGAr phenotype in the hybrids (thus showing its recessive character) and increased malignant properties in vivo. An analysis of spontaneous frequencies of re-expression of various drug resistance genetic markers in several hybrid metastatic cells was also consistent with chromosome segregation of the sensitive alleles. The results show that tumor progression and the emergence of metastatic cell variants could arise as a consequence of tumor X host cell fusion followed by chromosome segregation. We also discuss the possibility that this type of event may normally be a very rare one during the growth of tumors, the frequency of which can be artificially amplified by the use of certain classes of lectin-resistant mutants carrying particular cell surface alterations.     

The Development of Haematopoietic Cells Is Biased in Embryonic Stem Cell Chimaeras

The haematopoietic development of embryonic stem (ES) cell injection chimaeras was analysed using β-galactosidase expression from an X-linked transgene as a marker to distinguish the ES-derived cell population from the host cells. The number of cells in the different haematopoietic cell subpopulations was determined by flow cytometry. When the proportions of ES-derived cells in the antigen-positive lineages were compared to the ES cell contribution to all cells in the organs, we found an unexpected bias in the haematopoietic differentiation of ES-derived cells. ES descendants were overrepresented in the bone marrow B lymphoid cell population and the splenic myeloid cells but were underrepresented in the CD4-positive T lymphoid cells in the spleen. These results were obtained by comparison with control female animals that were X chromosome mosaic for β-galactosidase expression. These findings of uneven contribution to haematopoietic development by ES cells indicate that the commitment of ES cell descendants may be different from that of the host cells.     

Immune responses to weakly immunogenic virally induced tumors. III. Genetically unrestricted cytolysis of allogeneic tumor target cells.

Splenocytes from A mice injected with YAC-1 or RBL5 could generate, after in vitro culture with or without stimulation, a genetically nonrestricted cytotoxic response against the allogenic tumor RBL5. YAC-1 tumor is an in vitro carried tumor induced in A mice (H-2a) by Moloney virus. RBL5 tumor is a Rauscher virus-induced tumor of C57BL/6 mice (H-2b). These tumors cross-react serologically. The effector cells that were generated after the in vitro cultivation recognized tumor-associated antigens on the target cells. H-2 alloantigens were not recognized by the effector cells. The effector cells that killed RBL5 tumor in a genetically nonrestricted manner were identified as T cells. The in vivo carried tumor YAC, in contrast to the in vitro carried tumor YAC-1, could not induce anti-RBL5 reactive cells in A mice. Instead, YAC tumor induced suppressor cells in A mice, which could abrogate the anti-RBL5 cytotoxic response of RBL5-primed splenocytes, but not that of YAC-1 primed splenocytes.     

Genetically determined resistance to foreign bone marrow transplantation in mice: characterization of the effector cells

Mice of most strains show a genetically determined ability to reject a variety of foreign marrow grafts even after lethal irradiation. The phenomenon is both host strain and donor marrow graft-dependent. To characterize the effector cell responsible for graft rejection, attempts were made to 1) determine to what morphologic subclass it belongs; 2) determine its life span; and 3) establish whether genetically different host environments influence the functioning of the effector cell. Mice of the 129/J strain (normally nonresistant), C57BL/6 strain (made non-resistant), and the homozygous mutants of C57BL/6, i.e., C57BL/6 (bg/bg), were recipients of C57BL/6 marrow or spleen cells. After lethal irradiation, hosts were given marrow or spleen cells from normal, strongly resistant C57BL/6 donors pretreated with a) 950 R whole body irradiation or b) twice daily injections for 4 days of the cell cycle toxic drug hydroxyurea followed by 950 R. In other cases, hosts were recipients of the lymphoid cell-rich fraction of marrow from irradiated C57BL/6 donors or adherent cells taken from cultures of marrow cells of unirradiated C57BL/6 donors. Three hours after receiving C57BL/6 marrow or spleen cells, irradiated hosts were given allogeneic DBA/2 marrow (always strongly rejected by C57BL/6 mice and always accepted by 129/J strain mice). Seven days later, host spleens were removed and the numbers of microscopic colonies were counted from subserial sections. The results demonstrate that 1) mice either normally or rendered nonresistant to a marrow allograft can be made to develop resistance by the administration of either whole spleen cells or marrow lymphoid cells from lethally irradiated strongly resistant donors; 2) adherent cells from cultures of marrow from strongly resistant mice are ineffective in conferring resistance; 3) the cell effective in conferring resistance has a life span greater than 4 but less than 7 days; and 4) the effector cell can function in genetically different environments of nonresistant strains.     

A requirement for helper T cells in the induction of delayed-type hypersensitivity

This paper describes a model system for studying the role of helper T cells in the induction of delayed-type hypersensitivity (DTH). Cyclophosphamide- (CP) treated mice sensitized with antigen 3 days later develop high levels of delayed-type immunity; however, DTH cannot be demonstrated in mice that are sensitized with antigen 1 day after drug treatment. The inability to respond to antigen 1 day after CP treatment can be restored if either normal or low-dose primed spleen cells are transferred at the time of sensitization. Although irradiated (1500 rad) normal spleen cells are unable to restore DTH, such treatment has no effect on the primed spleen cell population. The lymphocytes responsible for restoring the DTH response were identified as T cells, in that treatment with anti-Thy-1.2 serum and C abrogated their effect. Furthermore, restoration of the DTH response was dependent on the presence of antigen at the time of lymphocyte transfer; irradiated primed cells could not transfer DTH alone. The DTH effector cells in reconstituted mice were identified as originating from the host and not from the transferred cell population. This was accomplished by using anti-H-2 serum to identify the source of the DTH effector cells after transferring parental (H-2b) irradiated primed spleen cells into CP-treated F1 mice (H-2b,k). Thus, the irradiated transferred cells are behaving as helper T cells and promoting the development of DTH effector cells in the host.     

Allotype-linked production of an idiotype-specific factor that promotes idiotype expression in nonresponder strains

We had previously demonstrated that expression of the cross-reactive idiotypes (CRI) in the phenyltrimethylammonium (TMA) system depends on the presence of a second-order T helper (Th2) cell. Furthermore, we showed that this cell type can be replaced by an idiotype-specific helper factor derived from either a 24-hr concanavalin A supernatant (Con A) or the T cell hybridoma LOP 1.4. This factor, regardless of its source, is idiotype-specific, I-J+, and promotes in vitro expression of the cross-reactive phenyltrimethylammonium idiotype (CRI+-TMA) found on anti-trinitrophenyl antibodies. Because the expression of this idiotype in antigen-primed immune sera is linked to the Ig-1e heavy chain locus, experiments were conducted to test whether the production of this factor was also linked to the same locus. Of the strains tested, only splenocytes derived from the Ig-1e mice, irrespective of their background genetics, produced the factor upon Con A stimulation. Furthermore, the function of the factor is not major histocompatibility complex (MHC)-restricted because Con A supernatants derived from the C57.Ige (H-2b, Ig-1e), NZB (H-2d, Ig-1e), and A.SW (H-2s, Ig-1e) strains promoted CRI+ trinitrophenyl plaque-forming cells in A/J (H-2a, Ig-1e) cultures. Further experiments were carried out to determine if the idiotype-specific factor could promote CRI+ TNP plaque-forming cells in non-Ig-1e strains. To this end, A/J Con A and LOP 1.4-derived supernatants were added to primed C57Bl/6 (H-2b, Ig-1b) and DBA/2 (H-2d, Ig-1c) splenic cultures, both of which do not express serum CRI-TMA or produce the idiotype-enhancing factor. The cultures from either strain in the presence of the factor produced CRI+-TMA trinitrophenyl plaque-forming cells of comparable numbers to the A/J prototype strain. The results suggest an important regulatory role for this factor in allotype-linked expression of dominant idiotypes.     

In vivo treatment with monoclonal anti-I-A antibodies: disappearance of splenic antigen-presenting cell function concomitant with modulation of splenic cell surface I-A and I-E antigens

A single injection of anti-I-Ak antibody (AB) into H-2k mice resulted in abrogation of splenic antigen-presenting cell (APC) function for protein antigen-primed T cells or alloantigen-specific T cells. Spleen cells from anti-I-A-treated mice are not inhibitory in cell mixing experiments when using cloned antigen-specific T cells as indicator cells, thus excluding a role for suppressor cells in the observed defect. Also, nonspecific toxic effects and carry-over of blocking Ab were excluded as causes for the defect. Experiments with anti-I-Ak Ab in (H-2b X H-2k)F1 mice showed abrogation of APC function for T cells specific for both parental I-A haplotypes. In homozygous H-2k mice, anti-I-Ak treatment not only abrogated APC function for I-Ak-restricted cloned T cells but also for I-AekE alpha k-restricted cloned T cells. FACS analysis of spleen cells from anti-I-Ak-treated (H-2b X H-2k)F1 mice revealed the disappearance of all Ia antigens (both I-A and I-E determined), whereas the number of IgM-bearing cells was unaffected. The reappearance of APC function with time after injection was correlated with the reappearance of I-A and I-E antigen expression. In vitro incubation of spleen cells from anti-I-A-treated mice led to the reappearance of Ia antigen expression and APC function within 8 hr. Thus, it appears that B cells (as determined by FACS analysis) and APC (as determined by functional analysis) behave similarly in response to in vivo anti-I-A Ab treatment. We interpret these findings as suggesting that in vivo anti-I-A treatment temporarily reduces the expression of Ia molecules through co-modulation on all Ia-bearing spleen cells, thereby rendering them incompetent as APC. Such modulation of Ia molecules does not occur when spleen cells are incubated in vitro with anti-I-A antibodies. These results imply that a primary defect purely at the level of APC in anti-I-A-treated mice may be responsible for the observed T cell nonresponsiveness when such mice are subsequently primed with antigen.     

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)