KLH-specific,I-E/C-restricted clones of proliferating T lymphocytes

The recognition of keyhole limpet hemocyanin by a substantial proportion of proliferating clones of murine T lymphocytes was found to be restricted by the I-E/C^k molecule, which is a combinatorial product of genes located in the I-A (Ae) and I-EIC (E) subregions of the murine major histocompatibility complex. The respective roles of the Ae (polymorphic) and E (oligomorphic) gene products in the expression of the structures which are used as restriction elements by these T-cell clones was analyzed by mating parental strains unable to present the antigen and bearing selected Ae and E alleles. Efficient complementation for antigen presentation was found to require the expression by accessory cells of the Ae^k-gene product, whereas all E allelic molecules were functionally equivalent. These results (a) indicate that the immunoregulatory role of I-region gene products, initially described for molecules selected for their limited number of antigenic epitopes, also applies to complex multiepitopic antigens; (b) illustrate the advantage which results from the diversity of the la molecules expressed by accessory cells for the development of potent immune responses; and (c) suggest that a correlation might exist between the degree of polymorphism of a given family of H-2 allelic molecules and their ability to be used as restriction elements for antigen recognition by T lymphocytes.Abbreviations used in this paper FCS fetal calf serum - Hepes N-2-hydroxypiperazine-N-2 ethanesulfonic acid; in polypeptides - G glutamate - L lysine - Ø phenylalanine - KLH keyhole limpet hemocyanin - m. Ab. monoclonal antibodies - T cells thymus-derived cells     

Development and characterization of interleukin-2-independent antigen-specific human T cell clones that produce multiple lymphokines

The development of antigen-specific T lymphocyte lines and clones has greatly facilitated the investigation of T-cell recognition of and response to foreign antigens. In the present study, human antigen-specific helper T cell lines and clones which are completely independent of exogenous interleukin-2 (IL-2) have been developed by cyclic restimulation with the soluble antigen keyhole limpet hemocyanin (KLH) to which the T cell donor had previously been immunized. These T cells uniformly bear the OKT4 phenotype and were shown to require both histocompatible antigen-presenting cells (APC) and antigen for optimal proliferation. The T cell line was composed of a highly antigen-specific and clonable T cell population. Following four cycles of antigen stimulation, limiting dilution cloning analysis showed a Poisson distribution of clonable T cells with a precursor frequency of 0.62, and from 88 to 92% of viable clones were specific for the stimulating antigen. Individual clones were obtained which recognized KLH with either DR 1 (one parental Ia haplotype of the donor) or DR 2 (the other parental Ia haplotype) allogeneic APC, but not both. Following stimulation with KLH, the T cell clones produced IL-2. Peak amounts of IL-2 were assayable in the first 6 to 24 hr after stimulation. In contrast, virtually no IL-2 was detectable in supernatants at 72 to 96 hr, suggesting autoutilization by the proliferating T cells. In addition, some clones were also capable of producing both B cell growth factor and IL-2 following KLH stimulation. These IL-2-independent T cells appeared to be derived from a discrete Leu 8-negative subclass of T4⁺ cells and expressed the full complement of Ia antigen of the donor. Thus, soluble antigen-specific human helper T cell clones have been produced which can be maintained in the absence of exogenous IL-2, elaborate their own growth factors and other immunoregulatory lymphokines, and show fine DR-related restriction to either one or the other parental DR haplotypes in antigen-stimulated proliferative responses.     

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.     

Human helper T-cell clones that recognize different influenza hemagglutinin determinants are restricted by different HLA-D region epitopes

Human T-lymphocyte clones (TLCs) were generated against the hemagglutinin (HA) of A/Texas/1/77 influenza virus by limiting dilution. TLCs were then screened for antigen specificity on chemically synthesized peptides representing the HA1 molecule. It has been hypothesized that different T cells that recognize the identical antigenic determinant are controlled by (restricted by) the same class II epitope. Two TLCs, HA1.4 and HA1.7, both recognized the same HA peptide and in proliferation studies exhibited identical restriction patterns. Two other clones, HA 1.9 and HA 2.43, recognized different HA determinants and also had distinct restriction patterns. Proliferation inhibition studies with monoclonal antibodies against human class II molecules demonstrated three unique patterns of blocking with the clones, suggesting that clones may be restricted to a unique class II epitope depending on the HA determinant recognized. These data can be interpreted as supporting the argument that human immune responses to influenza hemagglutinin are under Ir gene control exerted at the level of the viral antigenic determinant recognized in association with particular D-region restricting elements. The determinant selection and clonal deletion theories are compared for their capacity to best explain these findings.Abbreviations used in this paper ³HTdR tritiated methyl thymidine - MHC major histocompatibility complex - HLA human MHC - PBLs peripheral blood lymphocytes - APCs antigen-presenting cells - TLCs T-lymphocyte clones - TCGF T-cell growth factor - MoAbs monoclonal antibodies     

Mixed-Haplotype and Mixed-Isotype Major Histocompatibility Complex Class II Molecules Detected by Murine T-Cell Clones and Their Possible Involvement in Autoimmunity

The recognition of antigen by T lymphocytes (T cells) is restricted by Class I or Class II major histocompatibility complex (MHC) gene products, the phenomenon called “MHC restriction.” MHC restriction is speculated to be one of the major elements for the association of disease susceptibility to MHC haplotypes. Clones of T cells have been shown to be powerful tools for the analysis of such restriction specificity. In this report, I describe unique mixed-isotype Aβ^d/Eα^drestriction molecules detected by T-cell clones in (B6Eα^d× BALB/c)F1 transgenic mice. The restriction specificity of these clones was confirmed by anti-Class II mAb blocking experiments. The ability of spleen cells from Aβ^dand Eα^ddouble transgenic B6 (B6Aβ^dEα^d) mice that express Aβ^d/Eα^dmolecules to present KLH to these clones supported the existence of such unique specificity. I also describe autoreactive as well as KLH-reactive T-cell clones restricted by mixed-haplotype Aβz/Aα^dClass II molecules derived from (NZB × NZW)F1 (B/WF1) mice. The restriction specificity was demonstrated by mAb blocking experiments and by experiments using Class II gene-transfected antigen-presenting cells. It is possible that such unique mixed-isotype and mixed-haplotype Class II molecules are critically involved in autoimmunity. In addition, the detailed methodology for establishing T-cell clones currently employed in my laboratory is described.     

Clonal analysis of antigen-specific interactions between T cells and genetically engineered B cells

In order to investigate T cell-B cell interactions we constructed monoclonal, antigen-specific T- and B-cell populations. The Ia+ B-cell lymphoma A20-2J was transfected with trinitrophenyl (TNP)-specific heavy (mu) and light (kappa) chain Ig genes. A hapten-carrier complex (TNP-keyhole limpet hemocyanin (KLH)) bound to the surface Ig expressed on the transfectant and was presented to carrier-specific T-cell hybridoma clones at markedly low doses of antigen (0.01 microgram/ml) and in an Ia-restricted fashion. Two responses were elicited in the responding T-cell clones: (i) high levels of IL-2 secretion (320 units/ml), and (ii) cytotoxicity directed against the antigen-presenting B cell. This cytotoxicity was inhibited by D-mannose and was directed against innocent bystander cells, unlike cytotoxicity mediated by NK cells or alloreactive cytotoxic T lymphocyte. Helper and cytotoxic functions were often present in different T-cell hybridomas but some clones exhibited both activities. One representative T-cell hybridoma exhibited strong helper function for TNP-primed splenic B cells as detected in a plaque-forming cell assay, but was cytotoxic toward antigen-presenting B cells. Such monoclonal assay systems for studying cognate interactions of heterogeneous T cells and specific antigen-presenting cells will provide us with valuable new approaches for the study of antigen-specific T-cell regulation of B-cell activation in immune responses.     

A Mycobacterium leprae-specific gene encoding an immunologically recognized 45 kDa protein

By screening a Mycobacterium leprae lambda gt11 expression library with a serum from an Ethiopian lepromatous leprosy (LL) patient a clone was isolated (LL4) belonging to hybridization group III of a panel of previously isolated M. leprae clones. Members of this hybridization group encode a serologically recognized 45 kDa protein. The complete DNA sequences of the partially overlapping clones LL4 and L1 (hybridization group III) are presented and these revealed the presence of an open reading frame (ORF) predicting a protein with a molecular size of 42 448 Da. Southern hybridizations on total genomic DNA of M. Ieprae, Mycobacterium tuberculosis and eight atypical mycobacteria showed that the LL4 DNA fragment is specific for M. Ieprae DNA even under low-stringency conditions. The M. Ieprae specificity of LL4 DNA was further confirmed by the polymerase chain reaction using four different sets of primers. Western blotting analyses showed that the M. Ieprae 45 kDa protein is frequently recognized by antibodies from leprosy patients and that this recognition is specific since no antibodies could be detected in sera of tuberculosis patients. T-cell proliferation assays also demonstrated T-cell recognition by leprosy patients and healthy contacts of the M. Ieprae 45 kDa protein. The specificity of the LL4 DNA region and the 45 kDa antigen that is encoded by hybridization group III could provide unique tools for the development of M. Ieprae-specific immunological and DNA reagents.     

Functional similarities of AeEα Ia molecules as determined by analysis with T-cell clones

Recognition of A_eE Ia antigens at the functional level was investigated using T-cell clones. The reactivities of an alloreactive and an antigen-reactive clone, both of which recognized A_eE Ia molecules, were compared on a panel of stimulator/antigen-presenting cells of various genotypes. The two clones recognized all tested A _e ^b E ^x Ia molecules, where x is a haplotype capable of expressing an Ia.7-bearing E polypeptide. Ia antigen recognition by either clone could be inhibited by the monoclonal antibody Y-17, which recognizes a combinatorial serologic determinant on certain A_eE molecules. There were no differences in the recognition of Ia by the alloreactive versus the antigen-reactive clone, suggesting that Ia antigens are recognized by the two clones in a fundamentally similar way. The recognition of these various Ia molecules by the two cloned T-cell lines provides evidence that the E polypeptides from H-2 haplotypes k, d, r, and u are functionally indistinguishable.Abreviations MHC major histocompatibility complex - Mb myoglobin - MLR mixed leukocyte reaction - PBS phosphate buffered saline - APC antigen presenting cell - KLH keyhole limpet hemocyanin - GAT poly (glut⁶⁰ alai³⁰ tyr¹⁰)n - (TG)-A—L poly (L-tyr, L-glu)-poly (D, L-ala)—poly L-lys - GLPhe poly (glu⁵⁵ lys³⁶ phe⁹)n     

Establishment and functional characterization of human herpesvirus 6-specific CD4+ human T-cell clones.

In order to clarify the protective immune responses against a newly identified herpesvirus, human herpesvirus 6 (HHV-6), we established HHV-6-specific human T-cell clones and examined their functional properties. Five CD3+CD4+CD8- T-cell clones, which proliferated in response to stimulation with two different strains of HHV-6 in the presence of autologous antigen-presenting cells but not with herpes simplex virus type 1 or human cytomegalovirus, were established from peripheral blood lymphocytes of a healthy individual. The proliferative response of all T-cell clones to HHV-6 antigen was inhibited by addition of anti-HLA-DR monoclonal antibody, indicating that these clones were human leukocyte antigen (HLA) class II DR restricted. Of the five clones, two lysed HHV-6-infected autologous lymphoblasts, but not HHV-6-infected allogeneic cells or natural killer-sensitive K562 cells (group 1); one showed cytotoxicity against HHV-6-infected autologous lymphoblasts as well as HHV-6-infected allogeneic cells and K562 cells (group 2); and the remaining two showed no cytotoxic activity (group 3). The cytotoxic activity of group 1 was inhibited by addition of anti-HLA-DR monoclonal antibody to the culture, whereas this monoclonal antibody had no effect on the cytotoxicity of group 2 and did not induce the cytotoxicity of group 3. Perforin, which is one of the mediators of cytotoxicity, was abundantly expressed in group 1 and 2 clones. Moreover, all groups of clones produced gamma interferon after culture with antigen-presenting cells followed by HHV-6 antigen stimulation. These results suggest that HHV-6-specific CD4+ T cells have heterogeneous functions.     

Recognition of class II molecules by human T cells

The HLA-D region of individuals with the DRw11, w52, DQw3 haplotype encodes multiple molecular products of three distinct subregions, DR, DP, and DQ. Since each molecule can carry multiple stimulatory epitopes, the repertoire of allogeneic T-cell responses to determinants of this haplotype can be quite large. In the present experiments, alloreactive cloned T-cell lines recognized six distinct epitopes associated with DRw11, DRw52, DQw3 haplotypes. Panel studies established that three epitopes were DRwll-like and three were DRw52-like. Blocking with monoclonal antibodies showed that two DRw11-like epitopes were carried by DR-subregion products and one DRwll-like epitope was carried by DQ-subregion molecules. DRw52-like epitopes were detected on separate DR subregion-encoded molecules. One of them carried both DRwl1-and DRw52-like epitopes, the other carried two of the DRw52-like epitopes. These epitopes, which represent functional units that trigger T-cell responses, can be detected at the present time only with the methods used in this report. Conventional allogeneic T-cell responses represent the summation of responses to multiple epitopes encoded by different D-subregion genes.     

Immunocytochemical localization and immunochemical characterization of an insulin-related peptide in the pancreas of the urodele amphibian,Ambystoma mexicanum

Summary The pancreas of the axolotl, Ambystoma mexicanum, was investigated by immunocytochemical methods for the presence of immunoreactivity to a number of antisera raised against mammalian insulins. All anti-insulin antisera tested revealed substantial amounts of reaction products confined solely to the aldehyde-fuchsinophilic B cells of the endocrine pancreas. The reactive cell population was detected by use of one polyclonal antiserum against bovine insulin and eight different monoclonal antibodies against insulins from various mammalian species. Six of these antibody clones have known specificity to sub-regions of the insulin molecule. Additionally, fractions of an ethanol-HCl extract of pancreatic tissue from Ambystoma was studied in both conventional dot-blot tests by means of the same panel of antibodies and a two-site sandwich time-resolved immunofluorometric assay for human insulin involving two of the monoclonal antibodies. These experiments support the immunocytochemical observations by demonstrating the existence of an insulin-related peptide with a great deal of structural resemblance to mammalian insulins and displaying antigenic determinants in common at least with the amino acid residues A_8–10 and B_26–30. In conclusion, we interpret the findings as indicating that the immunocytochemically revealed tissue bound antigen in the Ambystoma pancreatic B-cells may be a peptide related to human insulin.Supported in part by SNF grant 11-5082 (G.N.H.). The authors are indebted to Dr. P. Rosenkilde for the gift of the Ambystoma material     

Characteristics and functions of Sendai virus-specific T-cell clones

Several murine Sendai virus-specific T-cell clones were characterized in vitro and in vivo. All T-cell clones were phenotypically Thy-1.2+, and most clones were Lyt-1+,2-; one T-cell clone was Lyt-1-,2-. Some of the clones proliferated in response to antigen presented on I region-compatible stimulator cells. Proliferation could be inhibited by monoclonal antibodies directed against class II antigens. Clones which proliferated in response to antigen secreted lymphokines which could be identified as Interleukin 2 and Interleukin 3. All of the clones tested in vivo induced a delayed-type hypersensitivity response in syngeneic mice challenged with antigens. Depending on the experimental conditions chosen, Interleukin 2-producing clones as well as non-Interleukin 2-producing clones mediated help for stimulation of cytolytic T lymphocytes.     

Isolation of CD4and CD8T-Cell Clones from Mice Immunized with Synthetic Peptides on Splenic Dendritic Cells

Splenic dendritic cells (DC) express high levels of MHC, co-stimulator, and adhesion molecules and have been shown to be extremely potent antigen-presenting cells for both CD4⁺and CD8⁺T-cell responses. Previous studies have shown that murine DC can be loaded with exogenous antigens and used to prime CD4⁺, Class II-restricted T-cell responsesin vivo.This article describes protocols for immunization using DC loaded with peptides bound to Class I or Class II molecules and for the derivation and characterization of CD4⁺and CD8⁺antigen-specific T-cell lines and clones. The rationale for using DC as antigen-presenting cells is discussed, together with the advantages and disadvantages of these cells compared with more conventional methods of immunization.     

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV-1) and the human antibody response directed to p17 in HIV-1 infection. Three large peptides covering residues 12-29, 53-87 and 87-115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53-87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti-rp17 antibodies had relatively high affinities (K_A = 1.9 × 10⁵?1.4 × 10⁸ M^?1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (K_A = 2.0 × 10⁵?1.8 × 10⁷ M^?1). Four HIV-1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV-1-infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.     

Antigen-specific helper T cells recognize determinants encoded in the H-2D region of the H-2 gene complex

Observations have frequently been interpreted as showing that the helper T cells which collaborate with alloantigen-specific cytotoxic T-cell precursors can only recognize antigens encoded in the I region of the H-2 gene complex. An experimental system is described here that allows analysis of the recognition repertoire of these helper cells. CBA helper T-cell precursors can be primed in vitro to antigens encoded in the H-2 ^b gene complex. These helpers can then be tested for the existence of a subset of helper cells which recognize antigens encoded in the D region of H-2 ^b haplotype. CBA thymocytes were used as a source of cytotoxic T-cell precursors that respond poorly in the absence of exogeneous helper activity. The source of alloantigen was varied by using irradiated spleen cells from various (BALB/c × recombinant)F₁ hybrid mice as stimulator cells. When the stimulator cell bears BALB/c determinants recognized by the cytotoxic T-cell precursor and also bears only the D region antigens of the H-2 ^b haplotype, an anti-BALB/c cytotoxic response is generated only if the anti-H-2^b helper population contains cells able to recognize H-2D^b. A positive cytotoxic response was obtained, indicating that helper cells are not limited to recognition of I region antigens and can efficiently recognize antigens encoded in the D region of the H-2 gene complex. This was confirmed by the demonstration of helpers specific for H-2D^d. We were unable to detect any evidence for Ia-restricted recognition of the H-2D alloantigens, suggesting that, as for cytotoxic T lymphocytes (CTL), helper cell recognition of class I alloantigens is an unrestricted event.     

Role of the major histocompatibility complex in T cell activation of B cell subpopulations: antigen-specific and H-2-restricted monoclonal TH cells activate Lyb-5+ B cells through an antigen-nonspecific and H-2-unrestricted effector pathway

Monoclonal T helper (TH) cell populations were employed to study the mechanism of activation of the Lyb-5+ B cell subpopulation in T cell-dependent antibody responses in vitro. It was demonstrated that monoclonal T cell populations were sufficient to help rigorously T-depleted unprimed (B + accessory) cells for direct plaque-forming cell responses to trinitrophenyl- (TNP) conjugated keyhole limpet hemocyanin (KLH). The activation of several lines of cloned (H-2b X H-2k)F1 TH cells was antigen (KLH) specific and H-2 restricted. Individual clones were restricted to H -2b, H-2k, or unique (H-2b X H-2k)F1 encoded determinants. Under the experimental conditions employed, responses mediated by cloned TH cells were found to result in the activation of the Lyb-5+ B cell subpopulation. The activation of Lyb-5+ B cells by cloned TH cells did not require covalent linkage of carrier and hapten, and responses could be stimulated in the presence of free KLH plus TNP conjugated to an irrelevant carrier. The H-2 restriction of TH cell function was shown to reflect a requirement for T cell recognition of determinants expressed by accessory cells, whereas no requirement existed for restricted T cell recognition of B cells. These findings suggest that the help provided by monoclonal TH cells, once activated, was both antigen nonspecific and H-2 unrestricted. Consistent with this interpretation, it was found that the supernatant of antigen-stimulated TH cells provided antigen-nonspecific help to T-depleted spleen cells. Thus, these results demonstrate that the activation of Lyb-5+ B cells by antigen-specific and H-2-restricted monoclonal TH cell populations is itself antigen nonspecific and H-2 unrestricted.     

T-cell recognition and antigen presentation of myoglobin. Protein recognition by site-specific T-cell clones is influenced by amino acid substitutions outside the site.

Six T-cell clones from SJL mice were prepared from T-cell lines that were obtained by passage with synthetic myoglobin (Mb) peptide 107-120. In addition, a T-cell clone, specific to this region of Mb, was isolated from a Mb-passaged T-cell culture. The proliferative responses of these clones to Mb variants from 14 different species were studied. It was found, as expected, that amino acid replacements within the site affected its recognition by the T-cell clones. In addition to these effects, the T-cell recognition site, like the sites recognized by antibodies, was also influenced by substitutions of residues that are close to site residues in three-dimensional structure but are otherwise distant in sequence. This is noteworthy in view of the fact that six of the clones were selected with a free peptide, and thus the environmental residues are clearly not part of the 'contact' residues of the site. These findings are discussed in relation to the presentation of the antigen and are interpreted as indicating that Mb is presented in its intact form to the T-cells in vitro.     

Polymorphism detection and sequence analysis of human T-cell receptor Vα-chain-encoding gene segments

The T-cell receptor (Tcr) provides specificity for antigen recognition by its variable domain, primarily consisting of two germline encoded variable (V) region gene segments. Thus it has been suggested that inherited polymorphisms in the TCRV gene segments could contribute to differential immune responsiveness (e.g., autoimmunity) in human populations. In the present study, we have sought potentially functional polymorphisms in the germline TCRAV gene segments. Using denaturing gradient gel electrophoresis on polymerase chain reaction (PCR)-amplified products from the pooled DNA of many individuals, we identified polymorphisms in the TCRAV2S1, AV4S1, AV7S1, and AV8S1 gene segments. A complete DNA sequence analysis of these PCR products identified polymorphisms that affected amino acids in the predicted antigen-binding regions of the Tcr chain, as well as polymorphisms in the introns. Genotype analysis of all nine DNA point mutations showed a 5%–50% range (averaging 35%) of minor allele frequencies, often resulting in individuals homozygous for the alternate allele forms. All possible haplotype combinations of the amino acid-affecting polymorphisms were found, indicating that in human populations there are a large number of different germline haplotypes encoding V gene segment alleles. These TCRAV coding region polymorphisms provide the rationale for, and allow the direct testing of, hypotheses concerning inherited polymorphisms within the T-cell receptor genes that may contribute to autoimmune susceptibility.The nucleotide sequence data reported in this paper have been submitted to the Genbank nucleotide sequence database and have been assigned the accession numbers L11159–L11162.     

Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.     

The ability of cytotoxic T cells to recognize HLA-A2.1 or HLA-B7 antigens expressed on murine cells correlates with their epitope specificity

Two groups of human and murine cytotoxic T lymphocyte (CTL) clones specific for human leukocyte antigen (HLA)-A2 or -B7 can be distinguished based on their ability to kill murine transfectants expressing these molecules. The clones which do not recognize murine transfectants exhibited greatly reduced conjugate formation with these cells, indicating that the inability to lyse these cells occurs in recognition and binding. No systematic differences in inhibitory titer between the two types of CTL clones were seen with anti-CD8 (Lyt-2), anti-LFA-1, or monoclonal antibodies against HLA class I molecules. However, blocking with anti-HLA class I monoclonal antibodies suggested that different CTL clones recognized spatially separate epitopes on HLA-A2 and -B7. In addition, a correlation between the inability to recognize murine transfectants and fine specificity was seen. Eight of nine clones which did not lyse murine transfectants also failed to recognize human cells expressing HLA-A2.2 or -A2.3. In contrast only 5 of 12 clones which lysed transfectants failed to recognize the variant molecules. Analogous data were obtained with human CTL clones raised against HLA-A2.1. These findings suggest that CTL clones that do not lyse murine cells expressing appropriate antigens recognize epitopes that have been altered or lost as a consequence of expression on the murine cell surface. It is suggested that the loss of HLA-associated epitopes on the murine cell surface may be due to differences between mouse and human cells in the processing or presentation of class I-associated peptides.