Effect of in vivo administration of Lyt antibodies. Lyt phenotype of T cells in lymphoid tissues and blocking of tumor rejection.

After transplantation of B6RV2 leukemia, initial tumor growth was followed by tumor regression in B6 (CB6F1) female, but not male, mice. This indicated that H-Y antigen is involved in B6RV2 rejection by syngeneic female recipient mice. In the case of another leukemia, BALB.RL male 1, and Ir gene, probably identical to the Rgv-1 gene, is responsible for RL male 1 rejection. Thus, F1 hybrids of BALB/c with certain other strains of mice can reject RL male 1. Using these two different systems of tumor rejection, we investigated the effects of in vivo administration of Lyt and Thy-1 monoclonal antibodies (mAb). Results showed that Lyt-2 and -3 mAb blocked both B6RV2 rejection by B6 female mice and BALB.RL male 1 rejection by CB6F1 mice. The specificity of blocking was confirmed by use of Lyt-2 and -3 mAb to reciprocal alleles and mice from B6 Lyt-congeneic stocks. No blocking was observed with Lyt-1 and Thy-1 mAb. The Lyt phenotype of T cells in lymphoid tissues from mice treated with mAb was then studied. Blocking of the Lyt-2+3+ population was observed in the lymph node and spleen, but not in the thymus. These results indicate the involvement of Lyt-2+3+ cells (or Lyt-2,3 antigen) in tumor rejection. The precise mechanism of blocking is unknown, but it was observed after even a single injection of Lyt-2,3 mAb on day 9 after tumor transplantation, suggesting that effector cells were functionally blocked, rather than that the generation of these cells was inhibited.

Effector cells in allelic H-2 class I-incompatible skin graft rejection.

The cellular mechanisms of skin graft rejection with allelic H-2 class I differences were studied by examining the effect on graft survival of in vivo administration of anti-Lyt-2.2 mAb, anti-L3T4 mAb, or both to recipient mice. The injections of anti-Lyt-2.2 mAb and anti-L3T4 mAb caused selective depletions of Lyt-2+ cells and L3T4+ cells, respectively. Injection of anti-Lyt-2.2 mAb significantly prolonged graft survival in 7 of 12 combinations of H-2D-end difference, but did not prolong graft survival in 5 other combinations of H-2D-end difference, or in 2 combinations of H-2K-end difference. Injection of anti-L3T4 mAb did not prolong graft survival in any combinations with class I difference tested. Injection of anti-L3T4 mAb plus anti-Lyt-2.2 mAb markedly prolonged graft survival in the combinations with class I difference in which anti-Lyt-2.2 mAb had no effect and overcame the effect of anti-Lyt-2.2 mAb in those in which anti-Lyt-2.2 mAb had an effect in prolonging graft survival. These results indicated that in combinations in which anti-Lyt-2.2 mAb did not prolong graft survival, class I antigen stimulated L3T4+ effector cells when Lyt-2+ cells were blocked and Lyt-2+ effector cells when L3T4+ cells were blocked. On the other hand, in the combinations in which anti-Lyt-2.2 mAb prolong graft survival, these antigens initially caused preferential stimulation of Lyt-2+ but not L3T4+ effector cells, although delayed activation of L3T4+ effector cells occurred when Lyt-2+ cells were blocked. Furthermore, a significant correlation was found between the effect of anti-Lyt-2.2 mAb in prolonging graft survival and the failure of recipient mice to produce H-2 antibody. These results can be taken as evidence that L3T4+ effector cells are not involved in the initial phase of graft rejection in these combinations.

Identification of a unique antigen peptide pRL1 on BALB/c RL male 1 leukemia recognized by cytotoxic T lymphocytes and its relation to the Akt oncogene.

BALB/c radiation leukemia RL male 1 is an immunogenic tumor. We established bulk and cloned cytotoxic T lymphocyte (CTL) lines from regressor (BALB/c x C57BL/6)F1 (CB6F1) spleen cells that recognized RL male 1 specifically. We then obtained antigen peptide recognized by CTL from RL male 1 by acid extraction. Analysis of the acid extract by reversed-phase high performance liquid chromatography (HPLC) on a semipreparative C18 column revealed that fractions eluted in 23 min (peak a) and 26 min (peak b) showed sensitization activity on the P815 target for specific CTL. On further purification of these fractions by HPLC and direct sequencing by Edman degradation, we identified the CTL-recognizing RL male 1 peptide pRL1a (IPGLPLSL) in peak a and its possible precursor peptide pRL1b (SIIPGLPLSL) in peak b. Sequence homology indicated that these peptides were derived from the 5' untranslated region of c-akt oncogene.

Differential involvement of CD4+ cells in mediating skin graft rejection against different amounts of transgenic H-2K(b) antigen.

Differential involvement of CD4+ cells in mediating class I-disparate skin graft rejection was investigated using quantitatively different Kb transgenic mice as donors under conditions in which CD8+ cells were blocked in vivo by administration of anti-CD8 monoclonal antibody (mAb). Tg.H-2Kb-1 and -2 are C3H transgenic mice with 14 and 4 copies, respectively, of the H-2Kb gene. Cell surface expression of Kb antigen and the Kb antigenicity of skin for eliciting graft rejection with homozygous and heterozygous transgenic mice were correlated with the copy number. In vivo administration of anti-Lyt-2.1 (CD8) mAb markedly prolonged survival of heterozygous and homozygous C3H Tg.H-2Kb-2 skin grafted onto C3H mice, but prolonged survival of heterozygous Tg.H-2Kb-1 skin grafts much less and did not prolong survival of homozygous Tg.H-2Kb-1 grafts. Administration of anti-L3T4 (CD4) mAb alone did not have any effect on skin graft rejection. Administration of anti-L3T4 (CD4) mAb with anti-Lyt-2.1 (CD8) mAb blocked rejection in all combinations. These findings indicate that a quantitative difference of class I antigen caused differential activation of CD4+ cells under conditions in which CD8+ cells were blocked.

Detection of a unique antigen on radiation leukemia virus-induced leukemia B6RV2.

Radiation leukemia virus-induced leukemia of a male C57BL/6 mouse, B6RV2, is immunogenic to female BALB/c X C57BL/6 F1 mice. In these mice, B6RV2 tumors regressed after initial growth, and after tumor regression the mice were resistant to repeated inocula of up to 10(8) B6RV2 cells. Serum from these mice reacted with B6RV2 in mixed hemadsorption or protein A assays, and absorption analysis indicated that the antigen was restricted to B6RV2; it could not be detected in normal thymocytes or spleen concanavalin A blasts from different inbred strains, nor in 16 C57BL/6 or BALB/c leukemias. Spleen cells from mice in which the tumor had regressed were cytotoxic to B6RV2 after in vitro stimulation with B6RV2, as shown by 51-chromium release assay. This cytotoxicity was eliminated by pretreatment of the cells with anti-Thy-1.2, anti-Lyt-2.2, anti-Lyt-3.2, and complement, indicating that the effector cells were T-cells. The specificity of T-cell killing of B6RV2 was examined by competitive inhibition assays with unlabeled cells; only B6RV2 inhibited killing, while eight other C57BL/6 leukemias did not inhibit. Thus, the antigen on B6RV2 defined serologically and by cytotoxic T-cells is a unique antigen. However, it was not revealed by antibody-blocking test whether the unique determinant defined serologically was related to that recognized by T-cells; B6RV2 antiserum did not block lytic activity in the absence of added complement, irrespective of whether the target cells were untreated or anti-H-2b-treated B6RV2. H-2Kb antisera, but not H-2Db antisera, blocked lysis. This indicated that the H-2Kb molecule was exclusively involved in recognition of B6RV2 by cytotoxic T-cell.

Rejection antigen peptides on BALB/c RL male 1 leukemia recognized by cytotoxic T lymphocytes: derivation from the normally untranslated 5' region of the c-akt proto-oncogene activated by long terminal repeat.

Tumor antigen peptides on BALB/c leukemia RL male 1 that were recognized by cytotoxic T lymphocytes were shown to be derived from a normally untranslated region of the akt proto-oncogene (Uenaka, A. et al., J. Exp. Med., 180: 1599, 1994). We show here that the murine leukemia virus (MuLV) long terminal repeat (LTR) was inserted directly into the exon of c-akt in RL male 1 leukemia and that transcription started from the cap site of the LTR. Translation appeared to start from the ATG codon created in the six nucleotides of unknown origin, which were inserted into the LTR/akt junction. The deduced molecular size is approximately M(r) 59,000 due to the addition of 33 amino acid residues to the normally expressed c-AKT protein. Western blot analysis demonstrated the presence of M(r) 59,000 molecules in an RL male 1 lysate, and their expression at about ten times the level of normal AKT molecules of M(r) 56,000, which is consistent with the increased expression of akt mRNA demonstrated by Northern blot analysis. The findings show that the molecular alteration of AKT protein by insertion of MuLV LTR is the mechanism for creating rejection antigen peptides derived from the untranslated region of akt.

Vaccination with multiple antigen peptide as rejection antigen peptide in murine leukemia.

pRL1a (IPGLPLSL) is the Ld-binding tumor rejection antigen peptide recognized by CTLs on BALB/c radiation leukemia RL(male)1. We demonstrated that in vivo and in vitro sensitization with pRL1a multiple antigen peptide (MAP), but not with the pRL1a peptide itself, generated pRL1a-specific CTLs in the spleen cells of BALB/c mice. No enhancement of cytotoxicity was observed by emulsifying pRLla MAP in incomplete Freund's adjuvant or in complete Freund's adjuvant for in vivo sensitization. Selective depletion of CD4+ T cells in mice by treatment with anti-L3T4 (CD4) monoclonal antibody and that of macrophages by treatment with carrageenan on in vivo sensitization with pRL1a MAP abrogated CTL generation. The findings suggest that CD4+ T cells and antigen-presenting cells were necessary for the in vivo priming of CD8+ T cells with pRL1a MAP. Furthermore, we demonstrated that in vivo sensitization of BALB/c mice with pRL1a MAP, but not with pRL1a peptide, showed an inhibitory effect on RL(male)1 tumor growth. No growth-inhibitory effect was observed on control RVA, RVD, or Meth A tumors.

Correlation of c-myc expression with nuclear pleomorphism in human renal cell carcinoma.

The expression of the c-myc gene product in renal cell carcinomas was examined by immunostaining with monoclonal antibody (mAb) MYC-1. The effects of preservation and fixation of tissues on staining were first examined. In cryostat sections fixed with 4% buffered formalin for 15 min, staining was observed in the nucleus. On the other hand, in paraffin sections after fixation with 10% formalin, staining was observed in the cytoplasm, but not in the nucleus. Because c-myc protein has been shown to be a nuclear protein, the finding that c-myc protein was not detectable in the nucleus appeared to be due to the preservation or fixation procedures used. Therefore, cryostat sections fixed with 4% formalin were used to investigate the correlation between the reaction of MYC-1 mAb and nuclear pleomorphism in primary and metastatic renal cell carcinomas. Among 41 primary tumors, positive staining was observed in 2 of 17 tumors (12%) of grade 1, 17 of 21 (81%) of grade 2, and all 3 (100%) of grade 3. Among 17 metastatic tumors, positive staining was not observed in any of the 5 (0%) of grade 1 but was observed in 2 of 4 (50%) of grade 2 and all 8 (100%) of grade 3. Thus, the frequency of the positive reaction with MYC-1 mAb was correlated with nuclear pleomorphism in primary and metastatic renal cell carcinomas. The reaction of Ki-67 mAb, which recognized a nuclear antigen present in proliferating cells, was also correlated with nuclear pleomorphism. These findings suggest that the c-myc gene product plays a role in cell proliferation in renal cell carcinomas.

Molecular cloning and sequence analysis of the cDNA for the mouse counterpart to adult hamster liver purified growth inhibitory factor.

A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.

ELISPOT cloning of tumor antigens recognized by cytotoxic T-lymphocytes from a cDNA expression library.

The methodology of cloning genes coding for antigens recognized by T-cells from cDNA expression libraries was improved technically by using enzyme-linked immunospot (ELISPOT) assays instead of enzyme-linked immunosorbent assays (ELISA) or bioassays to detect cytokines produced by T-cells in response to antigens. Combining large and small scale ELISPOT assays for expression cloning has the following advantages compared to conventional cDNA expression cloning: i) the number of recombinant plasmids which can be screened is greater than 10,000 per well in a 24-well plate in a large scale ELISPOT assay compared to fewer than 100 per well in a 96-well plate in an IFN-gamma ELISA or a TNF-alpha bioassay; ii) the total number of recombinant plasmids which can be screened in a routine assay is 2 x 10 (5) in only one 24-well plate in a large scale ELISPOT assay compared to 1 x 10 (5) in ten 96-well plates in an IFN-gamma ELISA or a TNF-alpha bioassay. Thus the screening efficiency of large scale ELISPOT cloning is approximately 200 times that of conventional expression cloning approaches. The efficiency of the method was confirmed by detecting the model gene RLakt from a cDNA library of a murine leukemia RL male 1.

The mechanism of discordant xenograft rejection.

The mechanism of discordant xenograft rejection using the guinea pig-to-rat heart graft model was studied. In this model, we found that (A) Rejection occurred rapidly, in 17.5 +/- 8.3 min (mean +/- SD) (n = 8). (B) The graft survived longer when the recipient rat was pretreated with cobra venom facter (CVF). (C) Complement hemolytic titers in serum showed significant reduction of C3 in rejection without consumption of C4 and C2, suggesting complement activation through the alternative pathway. (D) No natural antibodies were detected in this combination. Complement-dependent cytotoxicity (CDC) titer, and hemagglutination (HA) titer were lower than x1. (E) Histological examination of the rejected heart xenograft revealed a large area of myocytolysis without interstitial cellular infiltration. (F) In vitro experiments showed that rat complement attacked guinea pig erythrocytes (Egp) via the alternative pathway. These findings indicate that rejection in this discordant xenograft model of guinea pig-to-rat was caused by primary activation of complement via the alternative pathway.

The role of CD8+ and CD4+ cells in islet allograft rejection.

The requirements of CD8+ and CD4+ cells for islet graft rejection in combinations with different histoincompatibilities were investigated by in vivo administration of anti-Lyt-2.2 (CD8) mAb, anti-L3T4 (CD4) mAb, or both to recipient mice. In B10.AQR----B10.A (H-2K-incompatible) and B10.A(5R)----B10.A (H-2K- and IA-incompatible) combinations, administration of either anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb completely blocked islet graft rejection, indicating that neither CD8+ cells nor CD4+ cells alone were capable of mediating rejection, and that collaboration of CD8+ cells and CD4+ cells was necessary. On the other hand, in the BALB/c----B6 (H-2- and non-H-2-incompatible) combination, administration of anti-Lyt-2.2 (CD8) or anti-L3T4 (CD4) mAb resulted in rejection of most of the grafts, although survival was prolonged significantly, and administration of both anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb together completely blocked rejection. These results suggested that either CD8+ or CD4+ cells were capable of mediating rejection, but that rejection was maximal in the presence of both T cell subsets. Immunohistochemical analyses showed marked depletion of CD8+ cells and CD4+ cells in grafted islets as well as spleens when anti-Lyt-2.2 (CD8) and anti-L3T4 (CD4) mAb, respectively, were injected.

Variable expression on lung cancer cell lines of HLA-A2-binding MAGE-3 peptide recognized by cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTL) specific for HLA-A2-binding MAGE-3 peptide (FLWGPRALV) were generated by repetitive stimulation of PBMC with the peptide in the presence of EBV-transformed B blasts and IL-2. Using these CTL, we investigated the expression of the HLA-A2-binding MAGE-3 peptide on lung cancer cell lines. Of 14 cell lines investigated, 1-87, PC-9, OU-LC-KI, 11-18 and LK87 were derived from HLA-A2 positive patients. But cytofluorometry analysis showed that 1-87, PC-9 and OU-LC-KI, but not 11-18 or LK87 expressed the HLA-A2 antigen. All five cell lines expressed MAGE-3 gene mRNA. Twelve of thirteen CTL lines from two HLA-A2 positive donors showed no cytotoxicity against any of the 14 lung cancer cell lines. CTL line TI-1 showed cytotoxicity against 1-87 but not against any of the other cell lines. Treatment of 1-87 with IFN-gamma greatly augmented the cytotoxicity of TI-1 and induced it in the other 12 CTL lines, confirming the expression of the peptide on 1-87. No cytotoxicity was induced by IFN-gamma treatment of PC-9 or OU-LC-KI. However, PC-9 and OU-LC-KI pulsed with the peptide were killed efficiently by all of the CTL lines, suggesting no expression of the peptide on those cells. A low level of cytotoxicity was induced on 11-18 but not LK87 by IFN-gamma treatment, although expression of the HLA-A2 antigen was not observed by cytofluorometry. These findings showed that expression of the HLA-A2-binding MAGE-3 peptide recognized by CTL was variable on lung cancer cell lines.

Roles of CD8+ and CD4+ cells on lethal graft-versus-host disease in nude mice.

Roles of CD8+ and CD4+ cells on lethal graft-versus-host disease (GVHD) were investigated. Injection of spleen cells from C57BL/6 (B6) female mice into (BALB/c x B6)F1 nu/nu female mice caused subacute lethal GVHD (survival: 10-50 days). Injection of anti-Lyt-2.2 (CD8) monoclonal antibody (mAb) on days zero, four and 14 into recipient mice prolonged their survival for at least the 200-day observation period. Injection of anti-L3T4 (CD4) mAb also prolonged survival of the mice for more than 70 days, but they eventually died by 150 days. Pretreatment of the donor B6 spleen cells with anti-Lyt-2.2 (CD8) mAb and complement (C) prevented the development of GVHD, and their pretreatment with anti-L3T4 (CD4) mAb and C markedly prolonged the survival of recipient mice. Injection of a mixture of donor spleen cells pretreated with anti-Lyt-2.2 (CD8) mAb and C and those pretreated with anti-L3T4 (CD4) mAb and C induced subacute lethal GVHD. Injection of anti-L3T4 (CD4) mAb, but not anti-Lyt-2.2 (CD8) mAb on days five, nine and 14 prolonged survival of the recipient mice. These results indicated that the collaboration of CD8+ cells and CD4+ cells was necessary for induction of subacute lethal GVHD. CD4+ cells but not CD8+ cells were involved in mediating subacute GVHD from the onset of the disease. CD8+ cells were, however, capable of inducing late-onset lethal GVHD. Direct phenotyping of T cells in the recipient mice revealed that the CD4+ cells were incapable of repopulating without CD8+ cells, but that CD8+ cells were capable of repopulating without CD4+ cells.

Inhibition by CsA and FK506 of the in vitro proliferative response of gamma delta T cells on stimulation with anti-TCR delta monoclonal antibody.

Tg.Tla'-3-1 mice are transgenic C3H/He mice with a Tlaa-3 gene derived from A mice. We demonstrated that spleen cells from Tg.Tlaa-3-1 mice, but not C3H/He mice, showed a proliferative response on stimulation with immobilized anti-TCR delta monoclonal antibody (mAb 3A10). Thus, Tg.Tlaa-3-1 mice can be useful for analysis of the function of gamma delta T cells. Using spleen cells from Tg.Tlaa-3-1 mice, we showed that cyclosporin A (CsA) and FK506 inhibited the in vitro proliferative response of gamma delta T cells on stimulation with anti-TCR delta mAb. The dose-dependent inhibitory effect of CsA and FK506 on proliferation of gamma delta T cells on stimulation with anti-TCR delta mAb was similar to that on proliferation of alpha beta T cells on stimulation with anti-TCF beta mAb. Inhibition by CsA and FK506 of proliferation of gamma delta T cells was not reversed by addition of rIL 2 (recombinant interleukin 2) during culture. The proliferative response of gamma delta T cells among spleen cells from TCR alpha beta-depleted Tg.Tlaa-3-1 mice was also inhibited by CsA and FK506, suggesting that the inhibition directly affected gamma delta T cells without mediation by alpha beta T cells.

[Analysis of tumor rejection antigen peptides recognized by specific CTL].

Tumor rejection antigens, recognized by cytotoxic T lymphocytes (CTL), have been identified in several tumors. In malignant melanoma MAGE-1 and -3 antigen peptides, recognized by specific CTL, were defined. Tyrosinase, gp100 and Melan A/MART-1, normally expressed in the melanosome, were also shown to be recognized by specific CTL. In murine tumors, three antigenic peptides were identified. These are P1A in mastocytoma P815, MUT1 in murine lung carcinoma (3LL) derived from connexin 37, and pRL1 in murine leukemia RL male 1 derived from c-akt proto-oncogene. Analysis of the tumor rejection antigen peptides will elucidate the molecular nature of the tumor rejection antigen and facilitate their therapeutic use as tumor vaccine.