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CD4+ Th cells play an important role in the induction and maintenance of specific T cell immunity. Indications for a protective role of CD4+ T cells against HIV-1 infection were found in subjects who were able to control HIV-1 viremia as well as in highly HIV-1-exposed, yet seronegative, individuals. This study describes the identification of an HIV-1-specific Th epitope that exhibits high affinity binding as well as high immunogenicity in the context of at least four different HLA-DR molecules that together cover 50-60% of the Caucasian, Oriental, and Negroid populations. This HIV-1 reverse transcriptase-derived peptide (RT171-190) is highly conserved among different HIV-1 isolates. Importantly, stimulation of PBL cultures from HIV-1 seronegative donors with this peptide resulted in Thl-type lymphocytes capable of efficient recognition of HIV-1-pulsed APCs. Taken together, these data indicate that peptide RT171-190 constitutes an attractive component of vaccines aiming at induction or enhancement of HIV-1-specific T cell immunity.

It has been proposed that the cross-priming of CTL responses in vivo involves the transfer to host APCs of heat shock protein glycoprotein 96-chaperoned antigenic peptides released from the endoplasmic reticulum (ER) of dying or infected cells. We have tested this possibility directly using TAP-deficient cell lines lacking antigenic ER peptides derived from two model Ags, the human adenovirus type 5 early regions E1A and E1B. Although both proteins were well expressed, the cells were not recognized by E1A- or E1B-specific CTLs unless the relevant epitope was either provided exogenously as a synthetic peptide or targeted to the ER in a TAP-independent fashion. Despite the absence of these ER peptides, the TAP1-/- cells were able to efficiently cross-prime E1A- and E1B-specific CTLs following immunization of syngeneic mice. These results indicate that, although purified peptide/glycoprotein 96 complexes are potent immunogens, the mechanism of CTL cross-priming in vivo does not depend upon antigenic peptides in the ER of immunizing cells.

Synthetic peptide-based vaccines have been shown to induce potent protective and therapeutic T cell-mediated immunity in preclinical animal models and are now being evaluated in clinical phase I/II studies for their efficacy against tumors or infectious diseases. However, such vaccines might also specifically tolerize T cells causing enhanced tumor outgrowth, as shown by vaccination with two CTL epitopes derived from the adenovirus type 5 early region 1 (Ad5E1) oncogenes. We now report that modification of the Ad5E1 peptide vaccine either through incorporation of the peptides into liposomes or by ligation of the peptides to lipid tails, another vaccine formulation being tested in the clinic, fails to convert immunosuppression into effective antitumor vaccination. Inclusion of a helper T cell epitope into the vaccine likewise induces enhanced tumor outgrowth and thus does not diminish the capacity of the peptides to tolerize Ad5E1-specific CTL. In contrast, the Ad5E1-derived peptides evoke a strong tumor-protective CTL response when presented on dendritic cells (DC), indicating that the in vivo CTL-tolerizing potential of these peptides is converted to specific immunostimulation when presented on DC. These findings have important implications for the development of peptide-based immune intervention strategies and emphasize the superior nature of Ag-pulsed DC over other peptide-based vaccination protocols as well as the crucial importance of the mode of peptide-Ag delivery in setting the balance between T cell stimulation and tolerization.

Bispecific Abs (BsAb) represent a novel format of immunotherapy, recognizing immune effector cells (e.g., T cells), on the one hand, and target cells (e.g., tumor cells), on the other hand. To be successful, cross-linking of the two cell types is necessary for effector cell activation and subsequent killing of the malignant target cells. We asked the question, whether CTL that were incubated with the BsAb aCD3 x aCD19 and malignant B cells and activated to kill the malignant B cells were still able to eliminate their natural target cells (e.g., virus-infected autologous body cells). To test this, HLA-A*0201-restricted, influenza-specific CTL were incubated with BsAb- and HLA-A*0201-positive B lymphoid tumor cells in combination with HLA-A*0201-positive, virus-infected non-B lymphoid cells as natural target cells. The results showed that even in the presence of BsAb and high amounts of tumor B cells, CTL were still capable of eliminating the virus-infected non-B lymphoid target cells; actually, CTL recognized and eliminated the homologous original target cells preferentially.

A small group of HIV-1-infected subjects who either do not progress to AIDS or progress only slowly have sustained HIV-1-specific CTL responses. It has been suggested that the specificities of these responses differ from the CTL responses of rapid progressors due to recognition of epitopes that are under structural or functional constraints. We have, in this respect, studied the CTL response to reverse transcriptase (RT) in long term survivors (LTS) and in HIV-1-infected individuals who progressed to AIDS within 3 to 6 yr. Both LTS and progressors displayed vigorous RT-specific CTL responses of comparable magnitude during the asymptomatic phase. From each individual at least two CTL lines were obtained from blood samples drawn at different time points during follow-up. A total of 19 CTL lines recognized nine different RT-derived epitopes. CTL obtained from progressors recognized epitopes with a similar degree of amino acid conservation as epitopes targeted by CTL from LTS. Furthermore, five of seven epitopes were recognized by both LTS and progressors. Moreover, one of the epitopes recognized by progressors contained the highly conserved YMDD motif that is essential for RT activity. In conclusion, our data imply that neither the magnitude nor the specificity of HIV-1-specific CTL against RT is a major cause of a more protracted course of disease.

CTL can play an important role in the defense against tumors. Protective CTL-mediated immunity can be established in animal tumor models after vaccination with synthetic peptides representing CTL epitopes. We now report that immunization with synthetic peptides can also lead to CTL tolerance associated with the inability to reject tumors. B6 tumor cells transformed by the human adenovirus early region 1 (Ad5E1) present an Ad5E1A- and an Ad5E1B-encoded CTL epitope to the immune system. CTL clones directed against either of these epitopes are able to eradicate established Ad5E1-induced tumors, showing that these CTL epitopes are targets of CTL that can mediate tumor regression. Here, we show that protective immunity against Ad5E1-expressing tumor cells can be established by immunization with Ad5E1-transformed cells and with an adenovirus vector containing the Ad5E1 region. Protective immunity, in either case, is associated with specific CTL memory. To test whether vaccination with synthetic peptides leads to protection against Ad5E1-expressing tumor cells, we vaccinated mice s.c. with a low dose of the Ad5E1B peptide. This peptide was chosen because the CTL response against the Ad5E1B-encoded CTL epitope contributes most to the antitumor response in B6 mice after vaccination with Ad5E1-transformed cells. Ad5E1B peptide-vaccinated mice were not protected against the outgrowth of Ad5E1-expressing tumor cells, but instead were no longer able to reject a tumor inoculum that was rejected by nonvaccinated mice. Moreover, the protection induced by tumor cell vaccination against Ad5E1B-expressing tumors was gone when the Ad5E1B-encoded CTL epitope was injected a few days before tumor challenge. This is associated with peptide-induced tolerance of Ad5E1B-specific CTL activity. These findings are relevant for the design of therapeutic approaches against both malignancies and T cell-mediated autoimmune diseases.

The impact of the MHC class I peptide binding stability on the immunogenicity of particular peptide Ags in class I-restricted cytotoxic T lymphocyte responses is not clearly established. Therefore, we have determined the dissociation rate of each peptide from MHC class I at 37 degrees C and compared this to that of a consensus CTL epitope. Newly defined immunogenic peptides formed relatively stable MHC-peptide complexes as shown by their low dissociation rates, whereas nonimmunogenic peptides displayed high dissociation rates. In addition virtually all previously described HLA-A*0201-restricted T cell epitopes showed low dissociation rates. Furthermore, we show that the immunogenicity of HIV-1-derived peptides can be predicted more accurately by their dissociation rate than by the MHC class I binding affinity. Selection of peptides based on affinity and their dissociation rate leads to a more precise identification of candidate CTL epitopes than selection based on affinity alone. These results help to understand why some peptides are recognized by CTL and, along with detailed knowledge of protein processing rules, therefore have important implications for the selection of peptides in peptide-based vaccines.

MHC class II (MHC-II) molecules bind fragments of exogenous Ags in an intracellular endocytotic compartment. In view of divergent data on the MHC-II distribution in different cell lines, it was of interest to localize MHC-II molecules in a natural and the most potent APC type, the dendritic cell (DC). By using immunogold labeling of ultrathin cryosections of cultured mouse spleen DC, we found that MHC-II molecules were present abundantly at the plasma membrane and in intracellular compartments containing internal membrane vesicles and/or membrane sheets. The majority of these compartments was situated late in the endocytotic route, as demonstrated by the late appearance (after a lag of 30 min) of internalized exogenous tracer. These compartments contained the lysosomal enzymes cathepsin D and beta-hexosaminidase, but lacked the late endosomal marker cation-dependent mannose-6-phosphate receptor. We conclude that most of the intracellular MHC-II molecules in cultured spleen DC reside in a compartment with (pre)lysosomal characteristics, resembling the so-called MHC-II-enriched compartments (MIIC), originally described in B cells. We also investigated whether the presence of MHC-II molecules in endocytotic compartments was related to the kinetics of Ag processing and presentation by these cells. Pulse-chase endocytosis experiments with hen egg lysozyme (HEL) as a model Ag showed that activated spleen DC were able to efficiently process and present this Ag to an HEL-specific T hybridoma cell line. However, presentation started only after a lag of 2 h and was maximal after 6 h. The difference in time between the arrival of Ag in proteolytic endocytotic compartments, in particular MIIC, and effective Ag presentation is discussed in the context of DC maturation.

Human papillomavirus type 16 (HPV16) is strongly associated with cervical carcinogenesis. The HPV16 E6 and E7 oncoproteins are constitutively expressed in the majority of cervical tumor cells and are, therefore, attractive targets for CTL-mediated immunotherapy. In mice, the outgrowth of a lethal dose of HPV16-induced tumor cells has been prevented by vaccination with a CTL epitope encoded by HPV16 E7, indicating the feasibility of peptide immunization to obtain antitumor CTL responses. In the present study, the immunogenicity of 9 HLA-A*0201-binding peptides encoded by HPV16 E6 and E7 was analyzed in vivo in HLA-A*0201Kb transgenic mice and in vitro in CTL cultures induced from PBMC of HLA-A*0201+ healthy donors. Four peptides with a good binding affinity were immunogenic in HLA-A*0201Kb transgenic mice, and three of them were also highly immunogenic in CTL induction experiments with PBMC of HLA-A*0201+ healthy donors. Human CTL clones specific for these three peptides were capable of lysing the HPV16 E7-containing HLA-A*0201+ cervical carcinoma cell line CaSki. These E7-derived peptides (11-20, YMLDLQPETT; 82-90, LLMGTLGIV; 86-93, TLGIVCPI), therefore, are likely to represent naturally processed human CTL epitopes of HPV16. Additionally, these three HPV16-encoded peptides have the highest affinity of binding to the HLA-A*0201 molecule. In this study, peptides with a lower binding affinity were less immunogenic. Therefore, our data illustrate that the HLA-binding affinity of a peptide has a major impact on its immunogenicity. In conclusion, we have identified immunogenic peptides encoded by HPV16 E6 and E7 that could be used in vaccines for the prevention and treatment of cervical carcinoma.

CTL that lyse melanoma cells were previously isolated from several melanoma patients. Such CTL recognize autologous proteins, indicating the occurrence of autoreactive T cells in melanoma patients. We have now raised CTL, using responding T lymphocytes from healthy donor blood, that lysed not only cells incubated with an HLA-A*0201-binding tyrosinase peptide but also melanoma cells endogenously processing and presenting the epitope. Our results suggest that autoreactive CTL precursors are present in healthy donor blood and can be activated in vitro with synthetic peptides presented on appropriate APCs. Therefore, tissue-specific, autoantigen-derived peptides might be useful in immunotherapy of both poorly and nonimmunogenic tumors.

Mouse embryo cells (C57BL/6, H-2b) transformed by the E1A and E1B genes of adenovirus type 5 (Ad5E1 MEC) are highly immunogenic. Previously, CTL were cloned from mice immunized with Ad5E1 MEC. These CTL clones were capable of tumor eradication in nude mice, and were directed against the Ad5E1A-encoded decapeptide SGPSNTPPEI, presented by the H-2Db MHC molecule. We have now generated Ad5E1 MEC containing a mutated Ad5E1A-encoded epitope. The mutant Ad5E1 MEC induce a strong CTL response when injected into immunocompetent mice. CTL clones generated against mutant Ad5E1-transformed tumor cells recognize an Ad5E1B-encoded epitope (VNIRNCCYI) in the context of H-2Db. Because this epitope is also present on wild-type Ad5E1 MEC, it is concluded that Ad5E1-transformed tumor cells express at least two CTL epitopes. Interestingly, the lysis of Ad5E1 MEC by the Ad5E1B-specific, but not by the Ad5E1A-specific, CTL clones was strongly diminished by the action of the activated ras oncogene. CTL directed against the Ad5E1B-encoded epitope were, like Ad5E1A-specific CTL, able to eradicate large established Ad5E1-induced tumors in B6 nude mice, demonstrating that CTL activity directed against different CTL epitopes expressed by the same tumor can be exploited for immunotherapy of cancer.

The relationship between binding affinity for HLA class I molecules and immunogenicity of discrete peptide epitopes has been analyzed in two different experimental approaches. In the first approach, the immunogenicity of potential epitopes ranging in MHC binding affinity over a 10,000-fold range was analyzed in HLA-A*0201 transgenic mice. In the second approach, the antigenicity of approximately 100 different hepatitis B virus (HBV)-derived potential epitopes, all carrying A*0201 binding motifs, was assessed by using PBL of acute hepatitis patients. In both cases, it was found that an affinity threshold of approximately 500 nM (preferably 50 nM or less) apparently determines the capacity of a peptide epitope to elicit a CTL response. These data correlate well with class I binding affinity measurements of either naturally processed peptides or previously described T cell epitopes. Taken together, these data have important implications for the selection of epitopes for peptide-based vaccines, and also formally demonstrate the crucial role of determinant selection in the shaping of T cell responses. Because in most (but not all) cases, high affinity peptides seem to be immunogenic, our data also suggest that holes in the functional T cell repertoire, if they exist, may be relatively rare.

We have measured the binding affinity for five HLA-A alleles: HLA-A1 (A*0101), A2.1 (A*0201), A3 (A*0301), A11 (A*1101), and A24 (A*2401); of a set of all possible nonamer peptides (n = 240) of human papillomavirus type 16 E6 and E7 proteins. High affinity binding peptides were identified for each of the alleles, thus allowing us to select several candidates for CTL-based vaccines. Moreover, this unbiased set of peptides allowed an evaluation of the predictive value of HLA motifs derived either from the analysis of sequencing of pools of naturally processed peptides or from the binding analysis of polyalanine nonameric peptides that differed in the amino acids (aa) present at the anchor positions. Whereas pool sequencing-derived motifs were present in only 27% of high affinity binders, the more expanded motif, based on analysis of different aa substitutions at the anchor positions, was present in 73% of high affinity binders. Furthermore, it was found that the presence of anchor residues in a peptide was in itself not sufficient to determine binding to MHC class I molecules, because the majority of motif-containing peptides failed to bind to the relevant MHC. Finally, specific HLA motifs were used to predict peptide binders of 8, 10, and 11 aa in length. Several high affinity binding peptides were identified for each of the various peptide lengths, indicating a significant size heterogeneity in peptides capable of high affinity binding to HLA-A molecules.

Episialin (MUC1) is a mucin-like glycoprotein abundantly expressed on most carcinoma cells. As a result of its extended and rigid structure, it reduces intercellular adhesion. We investigated whether this antiadhesion function allows tumor cells expressing high levels of episialin to escape from immune recognition. To test this hypothesis, we transfected episialin-negative (episialin-) melanoma cells (A375) with the MUC1 cDNA-encoding episialin. The results demonstrated that episialin-positive (episialin+) melanoma cells were significantly less susceptible to lysis than episialin- melanoma cells by both alloantigen or rIL-2-stimulated cytotoxic effector cells. In addition, cold target inhibition experiments with episialin+ and episialin- cells clearly demonstrated preferential lysis of episialin- cells. Furthermore, antibody blocking studies showed that lysis of episialin+, but not of episialin-, melanoma cells was predominantly dependent on the leukocyte function-associated Ag-1/intracellular adhesion molecule adhesion route, suggesting that episialin+ target cells adhere less efficiently to effector cells than episialin- target cells. This notion was supported by the observation that conjugate formation of the effector cells with episialin+ target cells was significantly impaired. From these results we conclude that over-expression of episialin as found on many tumor cells may indeed affect efficient lysis by cytotoxic lymphocytes and thus may contribute to escape from immune surveillance.

CD27 is a lymphocyte-specific member of a recently identified receptor family with at least 10 members that includes the receptors for nerve growth factor and TNF, CD40, and Fas. Several members of this family play a role in cell differentiation, proliferation, and survival. Within the amino terminal ligand binding domain of these receptors, repeat motifs have been identified. These repeats contain many cysteine residues in a conserved pattern, characteristic of this family. We have isolated and characterized the human CD27 gene to gain insight into the evolution of this type of receptor domain. The gene was localized on chromosome 12, band 12p13. Sequence analysis showed no correlation between the intron/exon organization and the subdivision of the protein into distinct domains. Structural information for the cysteine-rich domain is contained within three exons. In addition, the splice sites in the CD27 gene are located in a different position from those in the related nerve growth factor receptor gene. However, a comparison of the splice sites within the regions encoding the respective ligand-binding domains of the CD27 and nerve growth factor receptor genes identifies the archetypal cysteine-rich building blocks, from which the members of this family may have arisen during the course of evolution. From this observation, we propose a new organization of the repeat motifs.

C57BL/6 (B6, H-2b) mice are CTL responders to both Sendai virus and Moloney leukemia virus. In the former response the H-2Kb class I MHC molecule is used as CTL restriction element, in the latter response the H-2Db molecule. B6 dendritic cells (DC) are superior in the presentation of Sendai virus Ag to CTL in comparison with B6 normal spleen cells. Con A blasts have even less capacity to present viral Ag than NSC, and LPS blasts show an intermediate capacity to present viral Ag. H-2Kb mutant bm1 mice do not generate a CTL response to Sendai virus, but respond to Moloney leukemia virus, as demonstrated by undetectable CTL precursors to Sendai virus and a normal CTL precursor frequency to Moloney virus. Compared to B6 mice, other H-2Kb mutant mice show decreased Sendai virus-specific CTL precursor frequencies in a hierarchy reflecting the response in bulk culture. The Sendai virus-specific CTL response defect of bm1 mice was not restored by highly potent Sendai virus-infected DC as APC for in vivo priming and/or in vitro restimulation. In mirror image to H-2Kb mutant bm1 mice, H-2Db mutant bm14 mice do not generate a CTL response to Moloney virus, but respond normally to Sendai virus. This specific CTL response defect was restored by syngeneic Moloney virus-infected DC for in vitro restimulation. This response was Kb restricted indicating that the Dbm14 molecule remained largely defective and that a dormant Kb repertoire was aroused after optimal Ag presentation by DC. In conclusion, DC very effectively present viral Ag to CTL. However, their capacity to restore MHC class I determined specific CTL response defects probably requires at least some ability of a particular MHC class I/virus combination to associate and thus form an immunogenic complex.

The B6.C-H-2bm12 (bm12), H-2 I-Ab mutant, originated in the C57BL/6 (B6, H-2b) strain, is a nonresponder to the male antigen H-Y in both T cell proliferation and cytotoxic T lymphocyte (CTL) responses. Defective H-Y presentation by bm12-adherent cells, as a cause of the CTL nonresponsiveness, can be excluded, because 1) CTL from primed responder/nonresponder F1 female mice (B6/bm12 F1) were activated by H-Y antigen on antigen-presenting cells (apc) from either parent; 2) T cells from primed nonresponder bm12 females did not generate CTL to H-Y presented by responder B6 apc, but conversely, CTL from B6 females could be activated by antigen on bm12 apc; and 3) adherent cell-depletion experiments indicated that male adherent cells are necessary for generation of optimal H-Y-specific CTL responses, male adherent bm12 cells being equally as efficient as male adherent B6 cells in presentation to F1 T cells. The need for T helper cell activation by H-Y-presenting adherent cells during secondary in vitro restimulation can be circumvented by interleukin 2 (IL 2), because IL 2 completely restored the H-Y-specific CTL response of B6/bm12 F1 cells in cultures depleted of adherent cells. However, addition of IL 2 during in vitro restimulation of bm12 cells did not result in an H-Y-specific CTL response, indicating that H-Y-specific, I-A-restricted T helper cells are probably needed in vivo during priming for the generation of sufficient numbers of Db-restricted CTL memory cells. The data are compatible with the concept that H-Y antigen is presented in the context of the I-A alpha, beta molecule on the surface of adherent cells to T helper cells and in the context of the Db molecule on the surface of nonadherent as well as adherent cells to CTL precursors. The most likely explanation for the difference in H-Y response between B6 and bm12 include positive selection of the responder phenotype by the I-Ab alpha, beta molecule of the B6 strain or generation of suppressor T cells in the bm12 strain.

Small cellular lung carcinoma (SCLC) cell lines are susceptible to lysis by NK cells. SCLC, normally negative for MHC class I Ag, were rendered positive for HLA-A and -B Ag by two methods: treatment with IFN-gamma or transfection with HLA class I genes. Exposure to IFN-gamma induced high levels of class I Ag and reduced susceptibility to NK-mediated lysis. However, transfection with either HLA-A2, HLA-B27, or HLA-B27 with beta 2m did not result in reduced susceptibility to NK cells. These transfectants expressed amounts of HLA class I Ag comparable to those in IFN-gamma-treated, untransfected cells. Transfection with the beta 2m gene or plasmid alone neither influenced levels of surface class I Ag nor resulted in reduced susceptibility to lysis by NK cells. Thus, the effects of IFN-gamma on NK susceptibility can be dissociated from the induction of class I Ag.

We have studied the effect of mutations in H-2Kb on recognition of Moloney virus antigens by Kb-restricted Moloney virus-specific cytotoxic T lymphocytes (CTL) generated from H-2Db-mutant B6.C-H- 2bm13 ( bm13 ) mice. On the basis of the bm13 virus-specific recognition pattern of a series of Kb-mutant virus-infected (V+) cells, these Kb mutants could be divided into two groups that either shared the relevant H-2K restriction specificities with H-2Kb ( bm3 , bm5 , bm6 , and bm11 ) or lacked them completely (bm1) and/or almost completely ( bm8 ). In the study presented in this report, we concentrated on the alloreactive CTL included in the repertoire of bm13 Moloney-specific CTL. These CTL lysed noninfected (V-) allogeneic target cells of many H-2 types, including all Kb-mutant cells tested (bm1, bm3 , bm5 , bm6 , bm8 , and bm11 ). Recognition of V- cells of two Kb mutants, bm3 and bm11 , which share a common amino acid substitution at position 77 of the H-2K molecule with an additional change at position 89 in bm3 , was compared with recognition of the other Kb-mutant V- target cells in cold target inhibition studies. These studies showed that bm3 and bm11 cells share a determinant or determinants on the H-2K molecule recognized by alloreactive CTL among the Moloney virus-specific CTL population not present on the other Kb-mutant cells; lysis of both target cells was inhibited only by bm3 and bm11 V- cells. On the other hand, lysis of bm3 V+ target cells by bm13 virus-specific CTL was inhibited by all Kb-mutant V+ cells ( bm3 , bm5 , bm6 , and bm11 ) that were killed virus specifically by bm13 CTL. Thus, common determinants on the H-2K molecule of these Kb-mutant V+ cells are recognized by Kb-restricted Moloney virus-specific CTL. From the difference in inhibition pattern of the lytic reaction against bm3 V+ vs bm3 V- cells, we conclude that determinants on the same H-2K molecule recognized by Moloney virus-specific CTL and allocross -reactive effector cells included in the Moloney virus-specific population are not identical.

The objective of this study was to elucidate the mechanism responsible for inhibition as well as induction of cytolytic activity in cytotoxic T lymphocytes (CTL) by cluster-defined 3 (T3) (CD3) monoclonal antibodies (mAb). A series of isotype heavy chain switch variants (murine IgG1, IgG2a, IgG2b, IgA, and IgE) of a single CD3 mAb was used in the analysis of effects of CD3 mAb on the cytolytic activity of an allospecific CTL clone. The results obtained indicate that the inhibition of cytolytic activity of CTL by CD3 mAb is not caused by interference with events that occur after conjugate formation, such as programming for lysis and the delivery of the lethal hit, but by inhibition of specific recognition of the target cell by the CTL. We also present evidence that induction of nonspecific CTL activity by CD3 mAb is only achieved by direct binding of the Fc receptor to the CD3 mAb. This observation allowed us to use the CD3 mAb-induced nonspecific cytolytic activity of CTL as a sensitive assay for detection of Fc receptors for various heavy chain isotypes. So far, evidence was found for human Fc receptors reactive with murine IgG1, IgG2a, and IgG2b, but not for murine IgA or IgE. The combined results indicate a similar role for Fc receptors in induction of cytolytic activity of CTL by CD3 mAb as well as in CD3 mAb-induced proliferative responses of T cells.

In C57BL/6 (B6, H-2b) mice, the secondary in vitro CTL response against Moloney leukemia virus is restricted and regulated by the H-2Db locus. B6.C-H- 2bm13 ( bm13 ) mice, however, carrying a mutation at the Db locus, show an increased H-2Kb-restricted CTL response without a demonstrable CTL component restricted by the mutant Dbm13 molecule (D----K shift). These purely Kb-restricted bm13 virus-specific CTL were incubated with a series of Kb mutant virus-infected target cells to study the effect of the mutations at the target cell level. Of six Kb-mutant virus-infected target cells tested, bm1 cells were not recognized and bm8 cells were recognized only marginally by bm13 virus-specific CTL, whereas bm3 , bm5 , bm6 , and bm11 cells were fully recognized. Thus, the bm3 , bm5 , bm6 , and bm11 Kb mutants fully share the relevant H-2K restriction specificities with H-2Kb, whereas the bm1 mutant totally and the bm8 mutant almost completely lack these specificities. This result differs markedly from the restriction site relationships among B6 and these Kb mutants in other antigenic systems. The most striking example concerns the bm11 mutant, which is fully recognized by Moloney-specific CTL, but not at all by Sendai, minor H (H-3.1, H-4.2), and sulfhydryl hapten-specific CTL. Monoclonal anti-H-2Kb antibody B8-3-24 inhibited virus-specific lysis by bm13 CTL of all Kb virus-infected mutant target cells to which this antibody binds. Lysis of bm5 and bm11 but not of bm3 target cells was inhibited, in line with the fact that B8-3-24 antibody does not bind bm3 . On the other hand, not only bm5 and bm11 but also bm3 virus-infected target cells blocked virus-specific lysis to the same extent as syngeneic bm13 target cells. Therefore, bm13 virus-specific CTL populations do not recognize the discrete cluster alteration in the Kbm3 molecule, as identified by antibody B8-3-24. The bm1 and the bm8 mutations, which have structural alterations in completely different sites of the Kb molecule, show complete or almost complete loss, respectively, of Kb-Moloney restriction sites. This finding supports the notion that these virus-specific CTL recognize conformational determinants rather than linear amino acid sequences.

Normal human E rosette-forming, Fc-IgG receptor-bearing cells (so-called T gamma cells) were separated into two functionally different subpopulations. Both subpopulations bind the monoclonal antibody OKM1 (directed against an antigen present also on monocytes and granulocytes). The first subpopulation accounts for about 70% of the total T gamma cell population, does not bind OKT3 (a monoclonal antibody directed against an antigen present on most T lymphocytes), and displays strong killer (K) cell and natural killer (NK) cell activity. The second subpopulation accounts for about 30% of the total T gamma population, binds both OKT3 and OKM1 (confirmed with a double-labeling assay), and displays low K and NK cell activity. Each subset contained less than 10% null cells. Comparison of T gamma cell populations obtained by adherence to a monolayer of IgG-coated human erythrocytes or by rosette formation with these cells revealed that pure T gamma cells with normal killer cell and natural killer cell activity were best obtained with the monolayer technique. Comparison of the enzymic and functional profile of T gamma cells, monocytes and granulocytes, as well as changes during culture of these cells in vitro, failed to indicate a relationship between T gamma cells and cells of the myelomonocytic lineage.

Two class I MHC mutant mouse strains, bm14 and bm13, differ from the strain of origin B6 in one and three amino acids in the alpha 1 and alpha 2 domains of the H-2Db molecule, respectively. These alterations result in specific failure to generate a CTL (Tc) response to the male-specific Ag H-Y. Immunization and/or restimulation in vitro with syngeneic male dendritic cells (DC), expressing very high levels of class I MHC molecules, restored the H-Y-specific Tc response of bm14 but not of bm13 mice. Serologically Db determinants were lost in normal spleen cells of both mutants, because FACS analysis showed a decreased binding of Db domain-specific mAb. Although bm13 DC show a higher fluorescence than bm13 normal spleen cells it is still strongly reduced (30 to 50%) in comparison with B6 DC. Surprisingly, bm14 DC show an equally very strong binding compared with B6 DC with these mAb. The quantitative expression of class I molecules on APC thus appears to be a major determinant in the regulation of Tc responses. In addition, immunization with DC markedly influenced the target cell specificity of the ensuing Tc response. The combined data clearly demonstrate that besides the highly efficient class II-restricted presentation of Ag to Th, shown previously, DC are also superior in the presentation of Ag in the context of class I molecules to Tc. bm14 DC are capable of directly activating H-Y-specific Lyt-2+ Tc memory cells without the need for L3T4+ Th. These biologic effects of DC can at least in part be explained by their very high class I MHC expression. Moreover, these results reiterate that class I MHC Db mutants and different APC can be used to study the contribution of specific class I domains to Tc recognition and restriction specificity.

We have used a panel of human HLA-B7-specific CTL clones to identify an HLA-B7 gene (JY150) transfected into human M1 fibroblasts (M1/B7). Only a subset of the CTL clones recognized the M1/B7 cells, whereas all CTL clones recognized the donor of the B7 gene, the cell line JY (HLA-A2,2;B7,7;Cw-,-;DR4,w6). Analysis of the fine specificity of these CTL clones was performed by testing the reactivity on M1 cells transfected with an HLA-B27K gene and on a panel of cell lines typed for HLA-B7 subtypes (variants). These results, combined with one-dimensional IEF analysis of the M1/B7 cells and the B7 subtypes, indicated that the differential recognition by the CTL clones of the transfected gene was not caused by aberrant expression of the gene itself or due to the absence of critical accessory molecules on the M1 fibroblast cells. Our data suggest that the widely used HLA-B7 reference cell line JY is not homozygous at the HLA-B locus, but contains two different B7 alleles encoding the B7.2 and B7.4 subtypes.

Two monoclonal antibodies (CLB-CD 27/1 and CLB-CD 27/2) were raised against a novel determinant on human T lymphocytes. One of these antibodies, CLB-CD 27/1 (clone 9F4), was grouped by the Third International Workshop and Conference on Human Leucocyte Differentiation Antigens together with three other monoclonal antibodies (VIT 14, OKT 18A, and S152) in the new cluster CD27. In this paper we show that antibodies belonging to this cluster recognize an antigen present on a large subset of peripheral T lymphocytes and most medullary thymocytes. At least two different nonoverlapping epitopes were identified with directly labeled monoclonal antibodies. Immunoprecipitation studies indicate that the target antigen of CD27 antibodies is a polypeptide of 55 kDa, which appears in the form of a disulfide-linked homodimer on the T lymphocyte membrane (Tp55). Stimulation of T cells via the T3/T cell antigen-receptor complex, with either phytohemagglutinin or CD3 monoclonal antibodies, resulted in a fivefold increase in the membrane expression of Tp55, whereas activation by phorbol myristate acetate caused a marked down-regulation. Moreover, an additional molecule of 32 kDa was precipitated from the membrane of activated but not of resting T cells. Addition of CD27 antibodies to cultures stimulated with either phytohemagglutinin or CD3 monoclonal antibody led to enhanced proliferation, whereas no effect was observed in phorbol myristate acetate or interleukin 2-stimulated cultures. The possible role of the Tp55 antigen in T cell activation is discussed.

The pathologic symptoms in F1 mice with chronic graft-vs-host disease (GVHD) (GVH F1) strongly resemble those of systemic lupus erythematosus (SLE). Mice with SLE-like GVHD do not produce antibodies to a number of non-self and self antigens. This finding is inconsistent with the widely accepted view that the (auto)-antibody formation in SLE is polyclonal in the sense that B cells are triggered at random, i.e., irrespective of their specificity. In the present study, therefore, we performed a systematic study of the kinetics of total IgM- and IgG-secreting splenic B cells and tested their specificities. The total IgM-secreting B cell population was increased only in the first week after the initiation of SLE-like GVHD; it seemed to reflect a random, but self-limited, polyclonal B cell stimulation. In contrast, the total number of IgG-secreting cells in the GVH F1 mice was increased to a much higher extent than that of the IgM-secreting cells and remained increased. At no time during GVHD was there an increase in the number of plaque-forming cells (PFC) spontaneously secreting IgG antibodies to non-self antigens. The GVH reaction (GVHR) did, however, lead to the appearance of PFC that secreted IgG antibodies to DNA. Similarly, the GVH F1 mice showed high serum titers of antibodies to self antigens characteristic of SLE and to endogenous viruses, but during the entire observation period they failed to develop serum antibodies to non-self antigens and insulin. Hence, the enhanced production of Ig, especially that of IgG, that occurs in SLE-like GVHD is not a random process, because it requires the presence of antigen, or signal 1. The data support our hypothesis that only certain kinds of self antigen, such as DNA and cell membrane epitopes, can cross-link the Ig receptors on the corresponding B cells and thus provide an adequate signal 1. Given the increase in help, or signal 2, in chronic GVHD, only the B cell clones that simultaneously receive an adequate signal 1 seem to be driven into clonal proliferation and IgG secretion.

A combined virologic and immunologic study was done in mice that were either naturally or deliberately infected with C-type RNA viruses. In about 60% of B10. A mice, natural infection was accompanied by detectable anti-VEA antibodies in the serum. The absence of such antibodies was a reliable indicator of the absence of virus in B10 mice. Deliberately infected young adult B10 mice failed to eliminate the virus completely, despite high titers of anti-VEA antibodies. B10 mice exposed to B tropic viruses at birth made anti-VEA antibodies but failed to eliminate the virus. By contrast, some NIH Swiss mice exposed to N-tropic virus at birth made anti-VEA antibodies and eliminated the virus.

The induction of a graft-vs-host reaction in (BALB/c X A)F1 mice by i.v. injection with BALB/c lymphoid cells leads to a lymphoid hyperplasia that may progress to malignant lymphoma. In the present paper, the following aspects of graft-vs-host-reaction lymphomagenesis were studied: 1) the cellular requirements for the induction of lymphomas, 2) their cellular origin, and 3) the role of murine leukemia viruses. The development of graft-vs-host-reaction lymphomas was found to be mediated by donor T cells and to require the presence of histoincompatibility between donor and host. Histologically, the vast majority of these lymphomas were either of follicular center cell or of immunoblastic type, whereas immunoperoxidase studies showed that they were virtually all B cell derived. Most of the lymphomas were of host origin. In the DNA of approximately 80% of the lymphomas, integrated murine leukemia virus proviruses were detected. In the B cell lymphoma DNA, integrated ecotropic proviruses prevailed, but recombinant murine leukemia virus and/or deleted murine leukemia virus genomes were also detected in some tumor DNA.

A patient and his parents, deficient for lymphocyte function associated antigen-1 (LFA-1) and Mo1 (OKM1), were studied with respect to leukocyte surface marker expression and functional properties. The patient had a history of severe recurrent bacterial infections. Two siblings had already died of bacterial infections. The patient's granulocytes, monocytes, and lymphocytes expressed low but detectable amounts (less than or equal to 10%) of LFA-1 and Mo1. Intracellularly, LFA-1 and Mo1 (OKM1) were detectable and LFA-1 expression was enhanced on patient T cells stimulated with phytohemagglutinin. Granulocytes and monocytes of both the patient's parents expressed markedly decreased amounts of LFA-1 and Mo1. Lymphocytes of the mother expressed 40 to 60% of the amount of LFA-1 expressed on control lymphocytes, but his father's lymphocytes showed a normal LFA-1 expression. Granulocytes of the patient and of his deceased sister showed normal phagocytosis, but they had a dysfunction in the activation of the oxidative metabolism. Functional activities mediated by patient T cells were all normal. Moreover, all lymphocyte functions, including killer (K), natural killer (NK), cytotoxic T cell activity, helper activity for in vitro immunoglobulin (Ig) production by normal B cells, and PHA-induced proliferation were inhibitable by anti-LFA-1 monoclonal antibodies. K and NK activity mediated by patient leukocytes was 100-fold more sensitive to the inhibiting effect of anti-LFA-1 antibody than K and NK activity of normal donor leukocytes. Thus, although the amount of LFA-1 expressed was strongly reduced, it was still sufficient and required for the functional activity exhibited by patient T cells. The major functional defect observed with leukocytes of the patient and his father was an apparent B cell defect. B cells of the father and of the patient failed to produce Ig in the pokeweed mitogen (PWM)-driven system. The B cells of patient and of his father only produced Ig when cultured with T cells of the father, and not with normal donor T cells or T cells of the mother, in the presence of exogenous interleukin 2 (IL 2). In addition, the father's B cells produced Ig when cocultivated with patient T cells in the IL 2-driven system. This restriction of helper T cell activity is noteworthy because PWM- and IL 2-driven Ig synthesis by normal lymphocytes show no histocompatibility requirements between cooperating T and non-T cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of the major histocompatibility complex of the mouse (H-2 complex) on the antibody response against murine leukemia virus (MuLV) was investigated after 3 different ways of virus presentation (milk transmission of virus, spontaneous virus activation, and immunization with inactivated virus). The antibody response against MuLV was measured in the sera of H-2 congenic C57BL V+ sublines (V+ denotes positive for milk-transmitted MuLV), (B10.AV+ X C57BL)F1 mice, (C57BL X AKR)F1 mice and of C57BL animals after immunization with inactivated AKR virus. The pattern of immune responsiveness of the different C57BL strains to MuLV was independent of virus replication and was similar for the 3 ways of virus presentation. Serum antibody levels were controlled by 2 genes within the H-2 complex. The first gene (Ir-MuLV-1) was located in the I-A region and was complemented by a second gene (Ir-MuLV-2), which was situated in the regions to the right of the I-B region. Presence of 2 responder alleles (Ir-MuLV-1+,2+) led to an optimal antibody response (H-2b haplotype). Animals that only expressed a responder allele in the I-A region (Ir-MuLV-1+,2-) were intermediate responders. Animals lacking a responder allele in the I-A region (Ir-MuLV-1-,2+ or Ir-MuLV-1-,2-) were low responders.

The expanded T cell populations of 10 patients with either T gamma lymphocytosis (five patients) or proven chronic T cell malignancy (five patients) were analyzed with respect to functional activity in vitro, including proliferative responses to mitogens, cytotoxic activity (killer [K] and natural killer [NK] cell activity), and regulatory activity on pokeweed mitogen- (PWM) induced immunoglobulin (Ig) synthesis (help and suppression) in comparison with marker phenotypes. In each of the five patients with T gamma lymphocytosis, only one out of three functionally distinct cell types was found: T gamma-K cells, T gamma-S cells, or T gamma-NK/K cells, which mediated K-cell activity, suppressive activity, and both NK and K cell activity, respectively. An expanded T gamma-K cell population was demonstrated in three patients with neutropenia with or without recurrent infections. T gamma-S cells were found in a patient with severe hypogammaglobulinemia, and T gamma-NK/K cells in one patient with asymptomatic lymphocytosis. T gamma-K and T gamma-S cells had a similar surface-marker profile (E+ or E-, Fc gamma+, OKT1-3+4-8+I1-M1-), whereas that of T gamma-NK/K cells was different (E+, Fc gamma+, OKT1-3-4-8-I1+M1+). Longitudinal studies of three untreated patients with T gamma-K lymphocytosis showed that the abnormalities were persistent but not progressive. In contrast, five patients with chronic T cell malignancy (two with T-CLL, two with cutaneous T cell lymphoma [CTCL], and one with T-PLL) all had progressive disease. The neoplastic cells in these cases were E+, Fc gamma-OKT1+4+6- with variable expression of the OKT3 and OKT8 markers. The only functional activity observed in these cells was suppressive activity by OKT3-4+8- cells from a patient with CTCL.