Helper cell analysis in cytotoxic T lymphocyte responses. I. Activation of specific and cross-reactive helper effects in two hapten-modified self responses

C3H/HeN mice were immunized by skin painting with trinitrochlorobenzene or by inoculation of fluorescein isothiocyanate (FITC)-conjugated syngeneic cells. Approximately four weeks later, spleen cells were removed, irradiated, and cocultured with spleen cells from normal mice. These cultures were stimulated with syngeneic cells conjugated with different concentrations of trinitrobenzene sulfonate (TNP-self) or FITC (FITC-self). The results demonstrate (a) appreciable helper cell activity from TNP-self and FITC-self-primed mice, as determined by enhanced hapten-specific cytotoxic T lymphocyte (CTL) responses, (b) that FITC helper activity enhanced the FITC-self CTL more efficiently than the TNP-self CTL response, and vice versa, (c) an alloantigenic stimulating population failed to activate TNP-self helper activity and (d) that the helper activity was not exclusively specific, since FITC helpers did enhance the anti TNP CTL response, and TNP helpers could enhance the anti-FITC-self CTL response. The results of this study indicate that these cell-mediated lympholysis models permit an analysis of hapten specificity and cross-reactivity at the different levels of cellular interaction involved in the generation of an H-2-restricted CTL response.     

Studies on In-vivo priming of the trinitrophenyl-reactive cytotoxic effector-cell system. III. are helper T Cells involved in the In-vitro generation of cross-reactive as well as restricted cytotoxic responses?

The present study investigates the role of helper cells from trinitrophenyl(TNP)-primed mice in the generation of cytotoxic T lymphocytes (CTL) which lyse TNP-modified syngeneic (TNP-self) targets and cross-reactively lyse TNP-allogeneic target cells. Anti-TNP CTL activities generated from primed (either by intraperitoneal injection of TNP-syngeneic cells or by skin painting with trinitrochlorobenzene) and unprimed spleen cells were compared. When effector populations with comparable lytic activities on TNP-self targets were tested against allogeneic TNP-modified targets, only the in vivo primed effector cells displayed significant cytotoxicity. Since radioresistant helper cells have been found to enhance the anti-TNP-self CTL response, the question was raised whether this helper population could be shown to be involved in the enhanced TNP-allogeneic cross-reactive lysis. Normal spleen cells co-cultured with radioresistant helper cells failed to induce any detectable lysis on TNP-allogeneic targets under conditions for which these helper cells did enhance the lysis detected on TNP-self targets. These results suggest that triggering of such TNP-cross-reactive effector cells during the in vivo priming stage is responsible for the in vitro generation of cross-reactivity.     

INCOMPATIBILITY FOR OR PRE-IMMUNIZATION AGAINST M1s DETERMINANTS DECREASES LETHAL GRAFT-VERSUS-HOST REACTION DEVELOPED ACROSS NON - H - 2 AND/OR H-2 BARRIERS

The effect of incompatibility for Mls determinants was studied in lethal graft-versus-host reaction (GVHR) in the mouse. GVHR was induced in adult recipients of the following H-2^k strains: (AKR x B10.BR)F1 (Mls^B/Mls^b); (C3H x B10.BR)F1 (Mls^c/Mls^b); (CBA/J x B10.BR)F1 (Mls^d/Mls^b) and (CBA/H x B10.BR)F1 (Mls^b). Recipient mice were heavily irradiated and grafted with bone marrow and spleen cells from H-2 compatible B10.BR (H-2^k, Mls^b) or H-2 incompatible B10.D2 and B10 donors were normal, while those from B10.BR donors were either normal or pre-immunized against the recipient strains. In all experiments the survival of recipients with Mls^a/Mls^b and Mls^d/Mls^b phenotypes, and only in one experiment of those with Mls^c/Mls^b phenotype was greater and/or the survival time longer than that of recipients expressing only Mls^b. However, late deaths (> 120 days post grafting) observed after grafting of normal B10.BR cells were more frequent in Mls^d/Mls^b than in Mls^b strains. On the other hand, when B10.BR donor cells were pre-immunized against H-2^k compatible (AKR x B10.BR)F1 (Mls^a/Mls^b) or (CBA/J x B10.BR)F1 (Mls^d/Mls^b) strains, the survival time of H-2 incompatible (B10 x B10.BR)F1 (H-2^b/k, Mls^b) recipients was longer than when donor cells were pre-immunized against (CBA/H x B10.BR)F1 (Mls^b) strain. We conclude that donor incompatibility for Mls^a or Mls^d or donor-pre-immunization against Mls^a or Mls^d exerts a protective effect on lethal GVHR developed across non-H-2 or H-2 barriers; the protective effect of Mls^c is less efficient or absent. The Mls-induced protective effect shows the following properties: (a) efficiency in vivo correlates with the capacity of the corresponding alleles to stimulate an in vitro MLR; (b) is efficient in either primary or secondary response to other minor antigens; (c) is not H-2 restricted; (d) is nonspecific; (e) disappears late after grafting; (f) with respect to the genetic background, the early protective effect is followed, late after grafting, by an opposite effect which increases the mortality, suggesting that M/s locus determinants are capable of activating several cell populations with different biological functions.     

The augmentation of tumor-specific immunity by virus help. I. Demonstration of vaccinia virus-reactive helper T cell activity involved in enhanced induction of cytotoxic T lymphocyte and antibody responses

C3H/He mice were immunized to vaccinia virus by inoculating i.p. viable virus. Their spleen cells (SC) were tested for vaccinia virus-reactive helper T cell activity capable of augmenting (a) anti-trinitrophenyl (TNP) cytotoxic T lymphocyte (CTL) response generated from unprimed C3H/He SC (responding cells) or (b) anti-TNP antibody response generated from TNP-primed C3H/He SC (responding cells) by the stimulation with syngeneic SC infected with vaccinia virus and subsequently modified with TNP (virus-self-TNP). The results demonstrate that cultures of responding cells plus 850 rds X-irradiated vaccinia virus-primed SC failed to enhance anti-TNP CTL or plaque-forming cell (PFC) responses when in vitro stimulation was provided by either virus-self or TNP-self alone. In contrast, these cultures resulted in appreciable augmentation of CTL and PFC responses when stimulated by virus-self-TNP. Such a helper activity provided by vaccinia virus-primed SC was revealed to be T cell mediated and antigen specific. These results are discussed in the context of (a) nature of virus helper antigens, (b) mechanism of help and (c) potential of virus help in augmenting CTL and antibody responses to tumor antigens.     

Experimental Autoimmune Thyroiditis (EAT) Induced by the Thyroglobulin Peptide (2596–2608): Influence of H-2 and Non H-2 Genes

We have previously identified five thyroglobulin (Tg) peptides with A^k-binding motifs that induce experimental autoimmune thyroiditis (EAT) in CBA/J (H-2^k) mice. In this study, we have examined whether H-2 or non H-2 genes can influence the immunopathogenicity of peptide p2596 (a.a. 2596–2608), which earlier elicited considerable pathology in CBA/J hosts. The p2596 peptide induced mild EAT—(infiltration index range=1–2)— in H-2-compatible AKR/J, B10.BR, and C3H/HeJ mice. Moreover, p2596-primed LNC from these mice exhibited peptide-specific proliferative responses and secreted significant amounts of IL-2 and IFN-γ in recall in vitro assays. Priming and boosting of these strains with p2596 resulted in the generation of specific IgG responses five weeks after the initial challenge. In contrast, s.c. challenge of H-2-incompatible strains such as DBA/1J (H-2^q), SJL (H-2^s), DBA/2J (H-2^d) and C57BL/6 (H-2^b) with the same peptide dose did not elicit EAT pathology and peptide-specific B- or T-cell responses. These data demonstrate the thyroiditogenic potential of p2596 in H-2^k strains of diverse non-H-2 backgrounds but not in mice carrying H-2^{b, d, q?or?s} haplotypes.     

UNEXPECTED LYMPHO-CYTOTOXIC REACTIONS OF ANTI-H-2 SERA ON NORMAL LYMPH-NODE CELLS: ARE THEY DUE TO ALTERED H-2 STRUCTURES OR ANTI-VIRAL ANTIBODIES?

When testing the serum of an individual anti-H-2 immunized mouse (B10 x A.SW)F₁ anti-B10.M by the routine micro-lymphocytotoxicity test on lymph-node cells, unexpected antibodies were found. The most striking finding was that after absorption of anti-H-2.8 antibodies with B10.A(2R) (K^k) cells, antibodies remained which reacted with AKR, B10.AKM and B10.A V + mice while B10.A V-, B10.BR and C3H mice were negative. While all these strains share the K^k allele, only the positively reacting strains express high titres of infectious RNA turnover viruses. Unexpected reactions were observed also with H-2^d, H-2^j and H-2^r cells and absorption experiments indicated two or three antibody populations. These reactions could be interpreted by two different possibilities: (1) anti-H-2 antibodies react with virus-altered H-2 structures; and (2) antiviral antibodies react with H-2 structures complexed with viruses. These possibilities should be taken into account when H-2 sera are tested on tumour or virus-infected cells.     

POLYGENIC INFLUENCES ON THE LENGTH OF OESTROUS CYCLES IN INBRED MICE INVOLVE MHC ALLELES*

Genetic influences on female reproductive cycles were analysed in histocompatibilitycongenic strains of mice. Oestrous cycles of young, virgin mice of inbred-congenic strains, hybrid crosses (F₁, and parental-hybrid backcrosses (F₂) were monitored for 3 months. Oestrous cycles were categorized by length (inter-oestrous interval): 4,5,6, or 7–14 days. Mice with the following H-2 haplotypes had a greater proportion of 5-day oestrous cycles: H-2^b, H-2^r, H-2^h2, H-2^h4, and H-2ⁱ⁵. In contrast, the H-2^k and H-2^d haplotypes had mostly 4-day oestrous cycles. Influences of H-2 haplotype were seen on two genetic backgrounds, C57BL/10Sn and C3H. Non-H-2 alleles were also implied by different patterns of cycles between strains with the same H-2^b haplotype: C57BL/10Sn with predominantly 5-day cycles vs. C57BL/6J with a mix of 4- and 5-day cycles. The genetic basis for strain differences was investigation in F₁ hybrids and their backcrosses. F₁ hybrids of an H-2^b (C57BL/10Sn; 5-day cycles) and an H-2^k (B10.BR; 4-day cycles) strain had mostly 5-day cycles, indicating dominance of an H-2^b allele(s). However, F₁ hybrids from the reciprocal B6 × B10 cross (both H-2^b) also display a preponderance of 5-day cycles, indicating dominance of a non-H-2 autosomal allele from the C57BL/10Sn strain. Among F₂ mice, a ‘4-day’ phenotype segregated with homozygosity for the k haplotype (P < 0.05, χ²). These findings demonstrate the influence of genetic differences at the major histocompatibility complex on oestrous cycles.     

GENETIC CONTROL OF THE IMMUNE RESPONSE TO HAEMOGLOBIN: IV. Ly-1+ T-CELLS AND APPROPRIATE NON-H-2 GENES ARE REQUIRED FOR IN VITRO RESPONSES TO α - AND β-SUBUNITS OF HUMAN ADULT HAEMOGLOBIN

Experiments were conducted to determine if non-H-2 gene effects could be demonstrated in mice which had been primed to either the α-subunit or β-subunit of human haemoglobin. It was found that C3H.SW (H-2^b) and Balb/c (H-2^d) mice are low responder mice to α-chain of a haemoglobin when compared to H-2 identical B10 (H-2^b) and B10.D2(H-2^d) mice. B120.S and A.SW (both H-2^s) are responsive to β-chain challenge while Balb/c mice are low responders in contrast to high responder B10.D2 mice. Ly-1⁺ cells were demonstrated to be required (by cell depletion experiments) for an in vitro T-cell proliferative response to either subunit. In these experiments, Ly-2⁺ cells were not of crucial importance.     

Ir gene expression on T cells: Effect of a monoclonal antibody directed against I region-controlled determinants on T cells

A monoclonal antibody directed at an I region-controlled epitope uniquely expressed on T cells (Iat) was studied for its in vivo effect on the antibody response under the control of an Ir gene. The antibody was produced by a hybridoma made from A.TH spleen cells immune to A.TL (anti-I^k), that was selected for its reactivity with T but not B cells and macrophages, and thus was designated as anti-Iat^k. The injection of this anti-Iat^k into H-2^k, H-2^b and H-2^k×bF₁ mice resulted in the suppression of antibody response to poly-L-(His,Glu)-poly-D,L -Ala–poly-L -Lys [(H,G)-A–L] in H-2^k and F₁ mice but not that to poly-L -(Tyr,Glu)-poly-D,L -Ala–poly-L -Lys [(T,G)-A–L] both in H-2^b and F₁ mice. The adoptive cell transfer of the combinations of anti-Iat^k-or normal mouse serum-treated T and B cells into irradiated hosts demonstrated that anti-Iat^k primarily affected (H,G)-A–L-specific helper T cells but not B cells and macrophages, resulting in the specific elimination of the antibody response. Suppressor T cells were not induced by the treatment with anti-Iat^k. The antibody specifically eliminated the (H,G)-A–L-specific but not (T,G)-A–L-specific helper T cells in F₁ spleen cells that had been primed with both (H,G)-A–L and (T,G)-A–L. The results indicated that anti-Iat^k affected the H-2^k-associated Ir gene function born by T cells but not by antigen-presenting cells, which was expressed on F₁ helper T cells with apparent exclusion of the other allele, and implied that the Iat antigen on helper T cells is one of the sites of expression of Ir genes.     

Association Between H-2 Antigens and Thyroglobulin Influences the Immunogenicity of Thyroglobulin in Mice

To ascertain the role of H-2 in the immunogenicity of thyroglobulin, congenic strains of mice, B10.BR (H-2^khigh-responder) and B10.02 (H-2^d, low-responder) were immunized with purified thyroglobulin (from B10.BR and B10.D2) which was or was not passed through affinity columns of Sepharose coupled to anti-H-2^k or anti-H-2^d sera. Thyroglobulin absorbed with the same anti-H-2 as its source did not induce thyroglobulin antibodies or thyroid infiltrates in mice of the same strain and induced thyroid lesions but no antibodies in mice of the opposite strain. These experiments suggest that thyroglobulin, an autoantigen, is associated with syngeneic histocompatibility antigens in vivo and this association is important for the (auto) antigenicity of thyroglobulin.     

T-T cell interaction in the in vitro induction of delayed-type hypersensitivity (DTH) responses: demonstration of vaccinia virus-reactive helper T cell activity involved in enhanced induction of DTH responses

C3H/HeN mice were inoculated i.p. with viable vaccinia virus to generate virus-reactive helper T cell activity. 850R X-irradiated spleen cells from vaccinia virus-primed or unprimed mice as helper cells were stimulated in vitro with either trinitrophenyl (TNP)-modified syngeneic spleen cells (TNP-self), vaccinia virus-infected spleen cells (virus-self), or cells modified with TNP subsequent to virus infection (virus-self-TNP) in the presence of normal C3H/HeN spleen cells (responding cells). After 5 days of culture, effector cells were tested for anti-TNP delayed-type hypersensitivity (DTH) responses by adoptive transfer into footpads of syngeneic C3H/HeN recipient mice together with TNP-self. The results demonstrate that spleen cells from virus-primed mice failed to enhance anti-TNP DTH responses when in vitro stimulation was provided by either virus-self or TNP-self alone. In contrast, spleen cells from vaccinia virus-primed mice, but not from unprimed mice, could augment anti-TNP DTH responses when stimulated by virus-self-TNP. Such a helper activity provided by vaccinia virus-primed mice was shown to be antigen-specific, and mediated by Lyt-1+2-T cells. DTH effector cells enhanced by helper cells were also antigen-specific and Lyt-1+2-T cells. Furthermore, vaccinia virus-reactive helper T cell activity could be applied to augmented induction of anti-tumor DTH responses by stimulation with virus-infected syngeneic fibrosarcoma tumor cells. Thus, these results provide evidence for the role of antigen-specific helper T cells in augmenting the development of DTH responses to cell surface antigens including tumor antigens.     

Activation of Helper T Cells for B Lymphocytes in Primary Mixed Lymphocyte Cultures

Normal B10.BR (H-2k, C57B1/6 background) spleen cells, enriched in primary mixed lymphocyte culture (MLC) for antigens of C3H/Tif mice (H-2k, C3H background), induced normal C3H/Tif but not B10.BR B lymphocytes to proliferate and produce Ig. In contrast, normal B10.BR spleen cells enriched in parallel B10.BR anti-C57B1/6 (H-2b) MLC were not able to activate either B10.BR or C57B1/6 B lymphocytes. However, normal B10.BR spleen cells depleted of Lyt2+ cells before initiation of the MLC, and subsequently enriched either for C3H/Tif or C57B1/6 antigens, activated B lymphocytes of the respective mouse strains specifically and equally well. These experiments show that primary MLC gives rise to effector T helper cells that, on recognition of specific alloantigens, activate normal B lymphocytes of the 'stimulator' strain. In response to major histocompatibility complex (MHC) alloantigens, this help is not revealed because of interference by Lyt 2+ lymphocytes. MHC-reactive T helper cells for B lymphocytes, however, participate in these reactions and constitute the predominant population in long-term cultures that are maintained by consecutive in vitro restimulations.     

Original H-2d and alien H-2k-like antigens of a BALB/c fibrosarcoma as defined by in vitro and in vivo studies of cell-mediated immunity

The present study examines the immunosensitivity and the immunogenicity of both original H-2^d and alien H-2^k-like antigens of the BALB/c (H-2^d) fibrosarcoma C-1 as detected by in vitro and in vivo cell-mediated cytotoxicity (CMC) assays. It was found that ⁵¹Cr-labeled C-1 cells were lysed in vitro by C 57 BL/6 anti-H-2^d lymphocytes. The specificity of this reaction was shown by cold inhibition experiments in which the anti-H-2^d cytotoxic activity on YC8 (H-2^d) targets was inhibited by unlabeled YC8 or C-1 but not by C3UR11 (H-2^k) tumor cells. Both D^d- and K^d-encoded antigens were recognized by appropriate cytotoxic effectors. The immunogenicity of H-2^d antigens of C-1 was revealed by the ability of C 57 BL/6 anti-C-1 lymphocytes to lyse YC8 targets. The expression of H-2^k-like alien alloantigens on C-1 was indicated by the finding that anti-H-2^k cytotoxic T lymphocytes (CTL), generated by culturing BALB/c spleen cells immune to BALB.K (H-2^k), C3Hf (H-2^k) or A (H-2^a = H-2^k/d) tissues with the cells of the same strain used for immunization, lysed C-1 targets. The cytotoxicity of these anti-H-2^k CTL against C 3 UR11 (H-2^k) targets could be specifically inhibited by cold C 3 UR 11 or C-1 cells but not by two other BALB/c tumors. Using recombinant H-2-congenic mice, it was shown that both D^k and K^k antigens were recognized by CTL on C-1 cells. The immunogenicity of the H-2^k-like antigens, however, could not be detected in vitro. In fact, effector spleen cells from BALB/c mice immune to C-1 did not develop any detectable cytotoxicity against C 3 UR 11 targets as assayed either by a direct in vitro test or after in vitro restimulation with C-1 sarcoma cells. A similar experimental design was adopted in Winn assays carried out by mixing spleen cells of BALB/c immune mice with either C-1 or C3 UR 11 targets and injecting the mixtures in BALB/c or hybrid recipients. These in vivo tests revealed the presence of both H-2^d (K^d and D^d) and H-2^k-like (K^k and D^k) antigens on C-1. At variance with the in vitro CMC assays, however, the Winn assay also detected the immunogenicity of the H-2^k alien antigens, since BALB/c anti-C-1 spleen cells were able to significantly reduce the growth of C 3 UR 11 lymphoma cells in (BALB/c × C 3 Hf)F₁ hosts.     

MULTIPLE FOREIGN NON-H-2 DETERMINANTS ON THE SURFACE OF A CHEMICALLY-INDUCED MURINE SARCOMA*

By immunizing BALB/c (H-2^d mice against normal tissues from C57BL/6J (H-2^b), C3Hf (H-2^k) and DBA/2 (H-2^d), but not from AKR (H-2^k) strains, resistance was induced to the subsequent challenge of the ‘syngeneic’ methyl-cholanthrene-induced BALB/c sarcoma ST5; lymph node cells from allo-immune BALB/c mice were also able to exert a parallel cytotoxic effect against in vitro cultured ST5 cells. The involvement of foreign H-2 specificities in the observed cross-reactions was ruled out by absorptions of H-2 monospecific sera and by the interallelic combinations used, thus suggesting that non-H-2 histocompatibility antigens were responsible for the above findings. By using the indirect isotopic antiglobulin assay, BALB/c anti-C57BL/6J and anti-C3Hf polyspecific sera were found to bind specifically to cultured ST5 cells. C57BL/6J and C3Hf, but not DBA/2, lymph node cells were able to absorb the anti-ST5 activity of the anti-C57BL/6J serum. These results indicated that ST5 cells expressed on their surface at least two different sets of foreign non-H-2 antigens: one shared by C57BL/6J and C3Hf tissues, and detected by both cell-mediated and serological techniques; the other one belonging to DBA/2 tissues, and revealed mainly at the cell-mediated level.     

Generation of hen egg lysozyme-specific and major histocompatibility complex class I-restricted cytolytic T lymphocytes: recognition of cytosolic and secreted antigen expressed by transfected cells

Syngeneic cells exogenously supplied with hen egg lysozyme (HEL) or endogenously synthesizing HEL were used as antigen-presenting cells to induce major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL). Immunization of C57BL/6 mice followed by repeated stimulation of their splenocytes in vitro with trypsinized HEL peptides led to the generation of CTL lines specific for trypsinized HEL peptides and restricted by H-2K^b. Immunization of C3H mice with a mixture of soluble native HEL and irradiated syngeneic spleen cells followed by in vitro stimulation of immune spleen cells with soluble HEL could in a few cases result in HEL-specific CTL able to kill syngeneic transfectant L cells secreting HEL (HELs) or expressing cytosol-targeted HEL (HELc). The use of HELs or HELc transfectant L cells as in vivo and in vitro immunogens was a potent way for eliciting HEL-specific polyclonal CTL. These CTL and two CD8⁺ clones were found to be H-2K^k restricted and specific for the 1-17 N-terminal HEL peptide. In addition, the anti-HEL CTL could also exhibit a significant cross-reactivity against unsensitized and HEL-untransfected targets expressing the K restriction element. This cross-reactivity was likely due to recognition of unidentified HEL mimicking peptides (self-derived ?) presented by the MHC class I (H-2K^b or H-2K^k) molecule used as the restriction element for the specific recognition of HEL. The CTL raised after immunization with HELs or HELc transfectant cells were found to recognize both the HELs and HELc transfectant cells even though HEL was not detected in the latter after a 2- or 5-min radiolabeling pulse. Recognition of both HELs and HELc transfectant cells by a given CTL clone suggests that HEL subjected to two separate processing pathways, each depending on the initial subcellular localization, can ensure the generation of similar MHC class I peptide complexes.     

MEMBRANE ANTIGENS RECOGNIZED BY HETEROANTISERA AGAINST MOUSE TUMOURS,TESTED IN CELL LINES OF DIFFERENT H - 2 HAPLOTYPES

The pattern of reactivity of two heteroantisera against mouse tumour cells (rabbit anti-Meth A sarcoma: RAMA; and rabbit anti-RBL-5 leukaemia: RAR-5) has been studied in various cell lines of different H-2 haplotypes in complement-dependent cytotoxicity tests using a microadioassay. RAMA was cytotoxic not only for Meth A (H-2^d) but also for MCG4, P815Y, LSTRA, YC8 (H-2^d), L929, TLC5, Gardner (H-2^k), RBL-5 and TLX/9 (H-2^b). RAR-5 similarly killed RBL-5, as expected, as well as Meth A, P815Y, MCG4, SL2, YC8, LSTRA (H-2^d), L929 (H-2^k), TLX/9, MBL2, Gil IV, EL4 (H-2^b) and YAC (H-2^a). The cross-cytotoxicity RAMA-RBL-5 and RAR-5-Meth A was abolished when RAMA or RAR-5 were absorbed with allogeneic tissues from C57B1/6, BALB/c, CBA/H, C57B1/10 (B10), B10.BR, B10.D2, but not with rat lymphoid cells. A species-specific antigen present in these strains of mice thus appeared to be responsible for this cytotoxicity. The direct killing obtained with RAMA on Meth A and RAR-5 on RBL-5 was not absorbed by: (a) allogeneic cells of C57B1/6, CBA/H, AKR, B10, B10.BR, B10.D2 for RAMA, and CBA/H, BALB/c, B10, B10.BR, B10.D2 for RAR-5; or (b) syngeneic normal lymphoid cells (BALB/c and C57B1/6 respectively). This suggests the presence of a heteroantibody in the sera recognizing structures at the tumour cell surface that are absent in normal lymphoid cells—a tumour-specific antigen (TSA). Finally, the TSA of Meth A (recognized by BALB/c-absorbed RAMA) did not cross-react with eight mouse cell lines of different aetiology and origin: MCG4, P815Y, Gardner, TLC5, L929, TLX/9, LSTRA and RBL-5. However, the TSA of RBL-5 (recognized by C57B1/6 absorbed RAR-5) reacted not only with RBL-5 but also with tumour cells induced by viruses antigenically related to Rauscher, belonging to the group FMR Gi. It reacted also with EL4, a tumour cell line not virus induced, and weakly with SL2, a spontaneous tumour cell line. It did not react with TLX/9 (X-ray induced) or Meth A (chemically induced). From these results it is concluded that heteroantisera against mouse tumour cells recognize at least species-specific antigens and tumour-specific antigens.     

IMPORTANCE OF NON-H-2 GENES IN THE CONTROL OF THE IN VIVO GROWTH OF THE VIRUS INDUCED LYMPHOMA YC8

The genetic control of the in vivo growth of the Moloney virus-induced BALB/c lymphoma YC8 was studied in F1 hybrids between BALB/c and several strains differing at the MHC and/or at the level of non-H-2 genes. Parental strains of the B10 and C3Hf but not of A, BALB/c or DBA/2 backgrounds introduced a significant resistance to the growth of 10² YC8 cells (a dose able to kill 100% of BALB/c mice) in semisyngeneic hosts. This resistance appeared to be due to non-H-2 genes although a modulation of the tumour growth by genes encoded by the MHC was also evident. The study of backcrosses between susceptible BALB/c and resistant (BALB/c x B10.BR)F1 crosses revealed that 83% of animals developed lethal tumors after injection of 10² YC8 cells. This high frequency of tumour takes was not linked to genes of the MHC. Adult thymectomy plus sublethal irradiation was able to abrogate the resistance of (BALB/c x B10.BR) or (BALB/c x B10.RIII)F1 mice to YC8 growth. Since the injection of silica also impaired the resistance to YC8, we tentatively concluded that the genetic control of resistance to YC8 is mediated both by T cells and macrophage-like cells.     

Polymorphism of a Qa-1-associated antigen defined by cytotoxic T cells. I. Qed-1a and Qed-1d

Tla^d mice have a distinct Qed-1 allele, Qed-1^d. Its product is detected by cytotoxic T cells raised in C57BW6 (H-2^b, Tla^b) mice against cells from a new recombinant, B6-TL.123⁺ (H-2^b, Tla^d/b). Qed-1^d is also found on cells from B10.M, A.CA and B10.STC90 mice. It cross-reacts weakly with Qed-1^b. (C57BL/6 X BALB/c) F₁ anti-B10. A (SR) effectors discriminate Qed-1^a and Qed-1^d, while C3H/HeJ anti-B10. BR effectors cross-react extensively. CB6F₁ anti-5R effector cells also discriminate between the Qed-1 antigens of B6-Tla^a (H-2^b, Tla^a) and those of B10. BR and other H-2k, Tla^a strains.     

Evaluation of functional heterogeneity in the CD8 subset with T cells from T cell receptor-transgenic mice

The question of functional differentiation within the CD8 subset has been addressed in a model of TcR-transgenic (TcR-tg) mice expressing a TcR specific for H-2K^b (Ti). CD8⁺ Ti⁺ T cells present in the periphery of these mice have no cytotoxic T lymphocyte (CTL) activity unless they are stimulated with H-2K^b-expressing cells. In contrast to T cells from normal H-2^k littermates, alloantigen induction of CTL from TcR-tg mice is independent of CD4⁺ T helper (T_h) cells and is accompanied by high level secretion of interleukin-(IL)-2 by Ti⁺ CD8⁺ T cells. Precursor frequency analysis performed on CD8⁺ cells from TcR-tg mice revealed a high frequency of T_h as compared to CTL precursors. This raised the possibility of the existence of distinct subpopulations within CD8⁺ precursors with different requirements for differentiation to functional CTL. FACS analyses (performed on resting and on in vitro stimulated T cells from normal and TcR-tg mice) demonstrated a heterogeneous expression of Ly-6C on CD8⁺ cells with a large enrichment of Ly-6C^? cells among the Ti⁺ cells which persisted after stimulation with H-2^b cells in conditions that led to a homogeneous expression of the activation markers pgp-1 and CD69. The possibility that Ly-6C expression could mark functionally different subpopulations in CD8⁺ T cells was investigated. Stimulation of sorted populations of Ly-6C^? and Ly-6C⁺ cells allowed detection of CTL precursors in both these subsets and the majority of limiting dilution wells containing one pCTL also scored positive for IL-2 secretion. Thus, for CD8⁺ T cells expressing the same TcR, differentiation led to acquisition of both IL-2 secretion and CTL function and there was no evidence for the existence of a distinct population of helper-dependent CTL precursors.     

Adoptive transfer of unresponsiveness to allogeneic skin grafts with hepatic gamma delta + T cells.

C3H/HEJ mice injected with irradiated multiple minor incompatible B10.BR lymphoid cells via the portal vein showed delayed rejection of subsequent B10.BR skin grafts. Similar delayed rejection was produced by lateral tail vein injection of B10.BR hepatic mononuclear cells or H-2k cells pulsed in vivo with B10 minor histocompatibility antigens. Inhibition of C3H anti-B10.BR immunity in vivo (assessed by delayed graft rejection) and in vitro (assessed by B10.BR-induced lymphokine production) can be transferred by radioresistant, plastic-adherent F4/80+33D1-CD4-CD8-alpha beta TcR-gamma delta TcR- mononuclear hepatic cells from (C3H/HEJ x C3H.SW)F1 mice injected 36 hr earlier with 100 x 10(6) irradiated spleen cells. By 10 days post-injection, cells transferring delayed rejection are radiosensitive, plastic non-adherent, F4/80-33D1-CD4-CD8- alpha beta Tc+- gamma delta TcR+ cells. Injection of interleukin-2 (IL-2) in vivo into mice receiving pretreatment with B10.BR cells via the portal vein, or adoptive transfer into such mice of immune anti-B10.BR lymphoid cells, abolished delayed rejection on subsequent skin grafting. Delayed rejection or modulation of lymphokine production was associated in all cases with suppression of IL-2 production and preferential retention of IL-4 production from cells stimulated in vitro.