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.

Transforming activity of the RL-akt gene, a c-akt gene activated by long terminal repeat insertion in murine leukemia RL(male symbol)1 cells.

The unique antigen peptide pRL1 on BALB/c radiation-induced leukemia RL(male symbol)1 cells is derived from the normally untranslated 5' region of the mouse c-akt gene. Insertion of an endogenous long terminal repeat into the first coding exon of the gene resulted in the enhanced production of an altered akt protein, RL-akt, and creation of the tumor rejection antigen peptide pRL1. In this study, we constructed an RL-akt-expressing vector to investigate the transforming ability and anti-apoptotic activity of RK-akt in NIH/3T3 cells. RL-akt-expressing clones formed more colonies than did c-akt-expressing clones in soft agar and exhibited increased saturation density, a lower serum requirement for growth, and tumorigenicity on athymic nude mice. Immunoblot analysis of subcellular protein distribution showed that a considerable proportion of RL-akt was distributed in the membrane fraction. Thus, RL-akt expressed in NIH/3T3 cells appeared to behave like the v-akt oncoprotein. Furthermore, the RL-akt gene conferred resistance to the apoptosis induced by the calcium ionophore A23187 and by ultraviolet irradiation of NIH/3T3 cells. These findings indicate that the RL-akt gene is able to transform cells and exerts an anti-apoptotic effect on recipient cells, thereby implicating the gene in leukemogenesis of RL(male symbol)1 cells.

Expression of multiple unique rejection antigens on murine leukemia BALB/c RLmale symbol1 and the role of dominant Akt antigen for tumor escape.

Using the pRL1a Ag-loss RLmale symbol1 tumor variant cell line RM2-1, we demonstrated the presence of tumor Ags other than pRL1a that were recognized by CTLs on RLmale symbol1 cells. Semiallogeneic CB6F1 or syngeneic BALB/c CTLs generated against RM2-1 lysed RM2-1 and RLmale symbol1 cells to a similar extent, but no killing was observed with any other tumor or normal cells examined. Clonal analysis and sensitization with reversed phase-HPLC fractions revealed that there were Dd- and Ld-binding peptides recognized by RM2-1 CTLs. Lysis by bulk CTLs stimulated against RLmale symbol1 and limiting dilution analysis suggested that the pRL1a peptide was dominantly recognized to the RM2-1 peptides by CTLs on RLmale symbol1 cells. The rejection response against the parental RLmale symbol1 tumor was much less than that against RM2-1 cells in either CB6F1 or BALB/c mice, suggesting that the presence of altered Akt molecules from which the dominant pRL1a peptide was derived inhibited the rejection response against RLmale symbol1. Depletion of CD4 T cells caused the regression of RLmale symbol1 at the doses in which the tumor grew in untreated mice. The generation of pRL1a CTLs was inhibited in RLmale symbol1-bearing mice. Thus, immunoregulatory CD4 T cells were most likely activated by the altered Akt molecules and inhibited the efficient generation of CTLs against the dominant pRL1a Ag in RLmale symbol1.

Isolation of MHC class I-restricted tumor antigen peptide and its precursors associated with heat shock proteins hsp70, hsp90, and gp96.

We have previously demonstrated that vaccination with heat shock proteins hsp70, hsp90, and gp96 elicits specific immunity against the tumor from which the hsps were purified. Although the association of tumor Ag peptides with these hsps have been suggested, the identification of the peptides or their precursors stripped from the hsps remained to be resolved. We show in this report that an Ld-restricted cytotoxic T lymphocyte epitope of a mouse leukemia RLmale symbol1 and its precursors are associated with the chaperones hsp90 and hsp70 in the cytosol and gp96 in the lumen of the endoplasmic reticulum. Hsp70 was associated with only final sized octamer, while hsp90 was found to associate with the octamer and two distinct precursor peptides. The gp96 was associated with the octamer and one of the two precursors. Thus, each of the hsps bound a distinct set of peptides. Our results have demonstrated for the first time that the hsps associate not only with final sized tumor Ag peptide but also with its precursors. The implication of this evidence is also discussed in terms of the roles of hsps in MHC class I Ag processing/presentation.

Possible involvement of autoreactive CD4 T cells in generation of cytotoxic T lymphocytes on in vitro stimulation with H-2 class I-binding tumor antigen peptide.

We demonstrated that RL male 1-specific cytotoxic T lymphocytes (CTL) were generated in spleen cells from the tumor-rejected CB6F1 mice on in vitro stimulation with Ld-binding pRL1a peptide. We showed that CD4 T cells and antigen-presenting cells were necessary for CTL generation. Furthermore, CTL generation was inhibited by the addition of anti-I-Ad mAb, but not anti-I-Ek/d mAb, to the culture. However, no binding of the pRL1a peptide to the I-Ad molecule could be demonstrated. No inhibition by the pRL1a peptide of I-Ad-restricted IL-2 production by ovalbumin (OVA)-specific CD4 T cell hybridoma DO-11.10 was observed on stimulation with the specific OVA peptide. Moreover, no specific proliferative response or IL-2 production was observed with CD4 T cells in spleen cells from the tumor-rejected mice on stimulation with a range of mitomycin C-treated RL male 1 cells, or with RL male 1 lysate or the pRL1a peptide. Rather, the activation of autoreactive CD4 T cells and the IL-2 production from them were observed. CD4 T cells from CB6F1 mice that had rejected other tumors such as EL4 or MOPC-70A also helped the generation of pRL1a-specific CTL. These findings suggested the involvement of autoreactive CD4 T cells in CTL generation against the pRL1a peptide.

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.

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.

Diversity of epitopes recognized by cytotoxic T lymphocytes that are specific for rejection antigen peptide pRL1a presented on BALB/c leukemia RL Male 1.

Cytotoxic T lymphocytes (CTL) generated in (BALB/c x C57BL/6)F1 (CB6F1) and BALB/c spleen cells stimulated with BALB/c radiation-leukemia RL Male 1 cells or pRL1a (IPGLPLSL) peptide itself recognized pRL1a on RL Male 1 in association with Ld. We first studied pRL1a peptide residues used for binding to the Ld molecule by examining the inhibition by variant peptides with single Ala substitutions at each position (P) of recognition of P815 target cells sensitized with Ld-binding p2Ca (LSPFPFDL) peptide for BALB/c anti-p2Ca CTL. The results showed that Leu at P8 is predominantly involved in the binding and Pro at P2 is partially involved. Substitution of Gly to Ala at P3 increased binding. We then investigated the epitope residues recognized by four pRL1a-specific CTL clones by examining their cytotoxicity against the P815 target sensitized with variant pRL1a peptides. Recognition by clone Y-16 involved predominantly Leu at P4 and P6, and also Pro at P5 and Ser at P7, and partially Ile at P1. Recognition by clone U-41 involved predominantly Ile at P1 and Leu at P6, and partially Gly at P3, Leu at P4, Pro at P5 and Ser at P7. Recognition by clone P-2 involved predominantly Leu at P4 and P6, and Ser at P7, with no partial involvement of other substitutions being observed. Finally, recognition by clone B-24 predominantly involved all residues, except Gly at P3, which was partially involved. TCR V beta genes utilized by those CTL clones were different. The findings show that tumor antigen peptide pRL1a generates a wide repertoire of CTL clones that differ in TCR V beta usage and in the intrapeptide epitope residues they recognize.

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.

Double-cleavage production of the CTL epitope by proteasomes and PA28: role of the flanking region.

BACKGROUND: Proteasomes are known to produce major histocompatibility complex (MHC) class I ligands from endogenous antigens, and the gamma-interferon-inducible proteasome activator PA28 has been thought to play an important role in the generation of immunodominant MHC ligands by proteasomes. Several attempts have been made to show that proteasomes have the ability to yield cytotoxic T lymphocyte (CTL) epitopes effectively from model polypeptides derived from viral and intracellular proteins in vitro, but their antigen processing mechanism is poorly understood. RESULTS: Proteasomes produce the tumour rejection antigen precursor peptide pRL1b (SIIPGLPLSL), but not pRL1a (IPGLPLSL), bound to the H-2Ld molecule, from synthetic peptides covering the CTL epitope. This double cleavage production of pRL1b by proteasomes seemed to depend on the length of the flanking regions adjacent to either end of the CTL epitope, in which their successive deletions caused the almost complete prevention of pRL1b excision. The newly identified PA28 collaborates with proteasomes for efficient production of pRL1b, by promoting not only single cleavage of all susceptible peptides, but also dual cleavage in some peptides harboring certain characteristic lengths. CONCLUSION: The flanking regions outside pRL1b of suitable length appear to be essential for the correct CTL epitope production, possibly functioning as anchors to trap target peptides for proteasomal degradation. We propose a novel mechanism for dual-cleavage excision of immunodominant epitopes by proteasomes and PA28.

Production of murine leukemia RLmale1 rejection antigen peptide pRL1a by proteolysis of natural precursor pRL1b.

In this study, we demonstrated that NH2-terminal Ser and Ile residues of pRL1b (SI-pRL1a) (SIIPGLPLSL) are not involved in the recognition by RLmale 1-specific cytotoxic T lymphocyte. The sensitization activity observed with pRL1b (SI-pRL1a) was not greater than that of peptides substituted with irrelevant amino acids at these positions. In serum-free medium, pRLla retained sensitization activity, but pRL1b (SI-pRL1a) did not. Furthermore, addition of bestatin to serum-containing medium blocked sensitization by pRL1b (SI-pRL1a). On the other hand, the addition of captopril enhanced it, probably by inhibiting the degradation of pRL1a by ACE. pRL1a-D peptide with D-Ile in place of the L-Ile residue of pRL1a (IPGLPLSL) showed sensitization, but SI-pRLla-2,3D peptide, which has D-Iles in place of the L-Ile residues of pRLlb (SI-pRL1a), and which was not cleaved between the two D-Iles, did not. The findings suggest that pRL1a is the antigenic peptide bound to L(d) molecules and pRL1b (SI-pRL1a) peptide is its natural precursor, which generates pRL1a via proteolysis.

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.

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.

Requirement of CD4+ T cells and antigen-presenting cells for primary in vitro generation of CD8+ cytotoxic T cells against Ld-binding self-peptide p2Ca.

We investigated the cellular requirement for primary in vitro generation of cytotoxic T-lymphocytes (CTL) in BALB/c spleen cells against Ld-binding self-peptide p2Ca. Depletion of CD4+ T-cells in vitro by pretreatment with anti-CD4 monoclonal antibody (mAb) and complement or in vivo by administration of anti-CD4 mAb abrogated generation of CTL. Depletion of adherent cells by passing spleen cells through a nylon wool (NW) column also abrogated generation of CTL. Addition of peritoneal exudate cells (PEC) to spleen cells passed through the NW column restored CTL generation. These findings indicate that both CD4+ T-cells and antigen-presenting cells (APC) were necessary for CTL generation. Treatment of PEC with paraformaldehyde (PFA), but not mitomycin-C (MMC) abrogated their ability to restore CTL generation when mixed with spleen cells from the NW column, suggesting that an endocytic pathway could be involved in presentation of p2Ca on APC.

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.

Characterization of mouse monoclonal antibody B1.4 reactive with human invasive bladder cancer and some other malignant tumors but not with normal urinary epithelium.

Immunohistochemical analysis by indirect immunoperoxidase staining demonstrated that monoclonal antibody (mAb) B1.4 derived from a mouse immunized with a bladder cancer cell line EJ-1 was reactive with a high proportion of high-grade and invasive bladder tumors, but not with the majority of low-grade and superficial bladder tumors, or normal urinary epithelium. Among 71 primary bladder tumors classified by pathological grading, positive stainings were observed in 1 of 34 tumors (3%) of grade 1, 8 of 20 tumors (40%) of grade 2 and 14 of 17 tumors (82%) of grade 3. When the tumors were classified by pathological staging, positive stainings were observed in only 8 of 54 (15%) superficial tumors of stages Ta and T1, but in 15 of 17 (88%) invasive tumors of stages T2 and T3. mAb B1.4 showed restricted positive stainings with normal tissues including renal glomerulus, vascular endothelium, squamous epithelium of esophagus, glandular epithelium of prostate, and epithelium of pancreatic acinar gland and minute duct, while positive stainings were observed in a range of tumor tissues other than bladder tumor. Mixed hemadsorption assays with a panel of cell cultures showed also that the antigen recognized by mAb B1.4 was expressed on a range of tumor cell lines. These findings suggest that the antigen recognized by mAb B1.4 may appear after malignant transformation, and be an indicator of malignant potential of bladder cancer.

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 of B6RV2 leukemia growth by immunization with purified unique antigen.

Monoclonal antibody (mAb) NU7-99 reacted with only B6RV2 cells, not with 28 other leukemia cell lines, fibroblasts or normal tissues. Biochemical analyses of the unique antigen on B6RV2 cells that reacted with NU7-99 mAb indicated its relationship to xenotropic murine leukemia virus gp70. The antigen that reacted with NU7-99 mAb was extracted from the surface of B6RV2 cells with n-butanol and purified by ion-exchange chromatography and affinity chromatography. Growth of B6RV2 tumors in semi-syngeneic mice was inhibited by immunization of the mice with a purified preparation of this unique antigen.

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.