Interactions between MHC-encoded products and cloned T-cells. I. Fine specificity of induction of proliferation and lysis

To study the interactions between T cells and class I MHC products, we developed in vitro a T-cell line reactive to H-2K^b stimulating cells and derived T-cell clones from it. Although the T-cell line could proliferate in the absence of exogeneous T-cell growth factors when stimulated with H-2K^b spleen cells, each of the derived T-cell clones required both H-2K^b stimulating cells and an external source of T-cell growth factor for its propagation. Each of the T-cell clones was also cytolysic for H-2K^b target cells. Such T-cell clones allowed the comparison of the antigenic requirements for proliferation and cytolysis. By using H-2K ^b mutant mice, we found that while the original anti-H-2K^b T-cell line reacted with each of the six mutants tested, the individual T-cell clones could be distinguished in terms of their reactivity pattern. Similar fine specificity patterns were found when H-2K ^b mutant cells were used as stimulating or target cells for any given T-cell clone. Each of the three monoclonal H-2K^b-specific antibodies reacting with different epitopes of the H-2K^b molecule totally inhibited H-2K^b-induced proliferation and lysis by the T-cell clones. Further blocking studies involved use of Fab antibody fragments and definition of their reactivity on cells from the H-2K ^b mutants. We concluded that: (1) blocking with a monoclonal antibody does not prove identity of alloantigens recognized by the T-cells and the antibody; (2) a monoclonal antibody could either block or not block H-2K^b-CTL interactions depending on structural variations of the H-2K^b molecule not affecting the CTL-H-2K^b functional interaction; (3) blocking one type of H-2K^b-T-cell interaction (induction of proliferation) always affects the other type (cytolysis).Abbreviations used in this paper MHC major histocompatibility complex - CTL cytotoxic - T lymphocytes - Th T helper cells - PMA 4-phorbol 12-myristate 13-acetate - Con A Concanavalin A - LPS E. coli lipopolysaccharide - SCA Con A stimulated rat spleen-cells supernatant - SBD B6 anti-DBA/2 mixed lymphocyte culture supernatant - TCGF T-cell growth factors - IL-2 interleukin 2 - mAb monoclonal antibody - FCS fetal calf serum - PBS phosphate buffered saline - C complement     

Dissection of the poly(glu60ala30tyr10) (GAT)-specific T-cell repertoire in H-21 k mice

We examined the antigen recognition of the class II major histocompatibility complex (MHC) of 45 poly(glu60 ala30 tyr10) (GAT)-reactive T-cell clones isolated by limiting dilution cloning of a pool of in vivo-primed and in vitro-restimulated A.TL lymph-node T cells. Each clone expressed the Thy-1.2+, Lyt-1+, Lyt-2-, LFA-1+, Ia-, and H-2Dd+ cell-surface phenotype and exhibited strict specificity for GAT on syngeneic antigen-presenting cells (APCs). The monitoring of the proliferative responses of these clones in the presence or absence of GAT, using APCs from strains with 11 independent H-2 haplotypes, revealed several distinct specificity patterns: (i) most (31 of 45, 73%) T-cell clones recognized GAT in a self-I-Ak-restricted manner; (ii) other alloreactive clones (5 of 45, 11%) were stimulated to proliferate, irrespective of the presence of GAT, in response to allodeterminants expressed on H-2s, H-2d, H-2f or H-2u spleen cells; (iii) a third T-cell clone subset (4 of 45, 9%) was activated by GAT in the context of not only self-I-Ak but also nonself restriction Ia determinants; and (iv) three clones (7%) exhibited a triple specificity, i.e., they recognized GAT in the context of self and nonself Ia determinants and were alloreactive. One of the latter clones responded to GAT in an apparently non-MHC-restricted manner and recognized an I-Ab allodeterminant. These data provide direct evidence that the antigen-specific and alloreactive T-cell repertoires overlap and that the self-MHC restriction of GAT-specific T-cell responses is not absolute in A.TL mice.     

Identification of a T-cell epitope adjacent to neutralization antigenic site 1 of poliovirus type 1.

Proliferative T-cell responses to poliovirus in various strains of mice have been analyzed by using either killed purified virus or capsid protein VP1 synthetic peptides. Following immunization of mice with inactivated poliovirus type 1 (PV1), a specific proliferative response of their lymph node CD4+ T cells was obtained after in vitro stimulation with purified virus. In mice immunized with PV1, PV2, or PV3, a strong cross-reactivity of the T-cell responses was observed after in vitro stimulation with heterologous viruses. By using various strategies, a dominant T-cell epitope was identified in the amino acid 103 to 115 region of capsid polypeptide VP1, close by the C3 neutralization epitope. The T-cell response to VP1 amino acids 103 to 115 is H-2 restricted: H-2d mice are responders, whereas H-2k and H-2b mice do not respond to this T-cell epitope. Immunization of BALB/c (H-2d) mice with the uncoupled p86-115 peptide, which represents VP1 amino acids 86 to 115 and contains both the T-cell epitope and the C3 neutralization epitope, induced poliovirus-specific B- and T-cell responses. Moreover, these mice developed poliovirus neutralizing antibodies.     

Quantitative studies on the precursors of cytotoxic lymphocytes. II. Specificity of precursors responsive to alloantigens and to concanavalin A.

A microculture system was used to examine the specificity of cytotoxic T lymphocytes (CL) derived from single precursors. Our data indicate that clones derived from single precursors of CL (CLP) are specific. Thus, RNC mice (H-2k) simultaneously stimulated by both H-2d and H-2b antigens produce clones which are specific for either H-2b or H-2d but not for both. In lymph node (LN), the frequency of CLP giving rise to clones specific for H-2d and H-2b is 1/480 and 1/860, respectively. Clonal specificity is also maintained when Con A is used to activate the precursors. With a nonspecific assay of CL, it was found that 2.5% of the cells in LN are stimulated by Con A to produce clones of CL. Among the CLP stimulated by Con A, 2.1% of the clones are specific for H-2d alloantigens. An unexpected finding was that 18% of the clones produced by Con A stimulation lysed EL4 tumor cells, which carry H-2b; however, only a small proportion of these clones were specific for the H-2b alloantigen. The antigen on EL4 which accounts for the high response has not been characterized.     

Spleen cells from animals neonatally tolerant to H-2Kk antigens recognize trinitrophenyl-modified H-2Kk spleen cells

A.TL mice injected with (A.AL × A.TL)F₁ cells within 24 hours after birth were rendered tolerant to H-2K^k antigens, as evidenced by acceptance of A.TL skin grafts. When spleen cells from these tolerant animals were cocultured with A.AL stimulator cells, no cytotoxic effector cells were generated in a cell-mediated lympholysis assay. However, when the A.AL stimulator cells were derivatized with trinitrophenol, effector cells that displayed a cytotoxic effect against trinitrophenyl-modified H-2K^k target cells were generated. These data indicate that animals tolerant to H-2 determinants but chimeric to only a minor extent possess cytotoxic precursor cells in sufficient frequency to mount a primary in vitro response against trinitrophenol in the context of an allogeneicH-2K region.     

Analysis of murine major histocompatibility complex class II-restricted T-cell responses to the flavivirus Kunjin by using vaccinia virus expression.

The present paper analyzes the influence of major histocompatibility complex (MHC) class II (Ir) genes on MHC class II-restricted T-cell responses to West Nile virus (WNV) and recombinant vaccinia virus-derived Kunjin virus antigens and identifies the immunodominant Kunjin virus antigens. Generally, mice were primed by intravenous infection with WNV or Kunjin virus, and their CD4+ T cells were stimulated in vitro 14 days later with WNV or Kunjin virus antigens to pulse macrophage or B-cell antigen-presenting cells (APC). WNV-specific in vitro T-cell responses from H-2b mice were higher than those from H-2d, H-2k, and H-2q mice. When recombinant vaccinia virus-derived Kunjin virus antigen preparations were tested in vitro, Kunjin virus-immune T cells of H-2b haplotype responded most strongly to structural (prM, C, E) and membrane-associated nonstructural (NS1) proteins encoded by VKV 1031 and showed weaker responses to cytosolic nonstructural protein NS5 (VKV 1022), whereas the responders of H-2k haplotype responded most strongly to the antigens encoded by VKV 1022 and gave lesser responses to VKV 1031. H-2d T cells gave weaker responses than either H-2b or H-2k cells, with responses to VKV 1031 generally being higher than those to VKV 1022. Responses to VKV 1023 or VKV 1024 encoding all of the NS3 to NS5 gene sequence or to VKV 1023 encoding all of NS3 were weak or absent. Within a given inbred strain, B cells and macrophages differed in their abilities to present recombinant vaccinia virus-derived Kunjin virus antigens, both in terms of magnitude of T-cell responses induced and the particular Kunjin virus protein presented. T cells from different non-MHC genetic backgrounds varied in their requirements of macrophage numbers as APC for maximum reactivity, suggesting that the concentration of class II MHC antigens and other molecules affecting APC-T-cell interaction varied in mice with different genetic backgrounds. Regardless of MHC haplotype, responses to VKV 1024, which encompasses VKV 1023 and VKV 1022, were either absent or lower than those to VKV 1022, possibly reflecting differences in the processing requirements of these two proteins. When mice were primed intravenously with recombinant vaccinia virus and when their CD4+ T cells were stimulated in vitro with native Kunjin virus antigens, VKV 1031 primed more efficiently than Kunjin virus and VKV 1022 primed similarly to Kunjin virus.     

Expression of the I-E target antigen for T-cell killing requires two genes

The H-2Ik region encodes at least two different target antigens for unrestricted T-cell mediated killing. The first is controlled by the I-A region alone and the second depends on a pair of alleles, one located to the left of I-B (presumably in I-A) and the other to the right of I-J (presumably in I-E). Hence, effector cells nominally specific for a product of the I-E region do not kill target cells with the same I-E region as the stimulator unless the I-A region is also shared. Some effectors specific for H-2Ik, such as A.TH anti-A.TL and B10.A(4R) anti-B10.A(2R), cross-react with B10.A(3R) and B10.A(5R) target cells. A product of the H-2b haplotype was shown to complement products of the H-2d or H-2k haplotypes in forming this cross-reactive determinant. The results are consistent with recent biochemical data on the component chains of Ia antigens.     

Virus-specific CD8+ T-cell responses in mice transgenic for a T-cell receptor beta chain selected at random.

The consequences of severely limiting the T-cell receptor (TCR) repertoire available for the response to intranasal infection with an influenza A virus or with Sendai virus have been analyzed by using H-2k mice (TG8.1) transgenic for a TCR beta-chain gene (V beta 8.1D beta 2J beta 2.3C beta 2). Analyzing the prevalence of V beta 8.1+ CD8+ T cells in lymph node cultures from nontransgenic (non-TG) H-2k controls primed with either virus and then stimulated in vitro with the homologous virus or with anti-CD3 epsilon showed that this TCR is not normally selected from the CD8+ T-cell repertoire during these infections. However, the TG8.1 mice cleared both viruses and generated virus-specific effector cytotoxic T lymphocytes (CTL) and memory CTL precursors, though the responses were delayed compared with the non-TG controls. Depletion of the CD4+ T-cell subset had little effect on the course of influenza virus infection but substantially slowed the development of the Sendai virus-specific CTL response and virus elimination in both the TG8.1 and non-TG mice, indicating that CD4+ helpers are promoting the CD8+ T-cell response in the Sendai virus model. Even so, restricting the available T-cell repertoire to lymphocytes expressing a single TCR beta chain still allows sufficient TCR diversity for CD8+ T cells (acting in the presence or absence of the CD4+ subset) to limit infection with an influenza A virus and a parainfluenza type 1 virus.     

Cellular and antigenic requirements for production of mixed leukocyte reaction suppressor factor

When murine spleen cells, alloantigen-sensitized previously in vivo, are incubated with spleen cells bearing the sensitizing alloantigens, a supernatant factor is produced that inhibits 3H-thymidine incorporation by responding lymphocytes in the mixed leukocyte reaction. This study evaluates the cellular and antigenic requirements during restimulation for elaboration of this suppressor factor, MLR-TsF. BALB/c spleen cells, sensitized to C57BL/6 (B6) alloantigens in vivo, produced MLR-TsF when cultured with B6 spleen cells in vitro, despite depletion of Sephadex G-10-adherent cells from factor-producing cells, stimulator cells, or from both populations. T cells were not required within the stimulating population, but a requirement for viable stimulator cells was demonstrated when heat-killed or glutaraldehyde-fixed stimulator cells failed to induce MLR-TsF production. The alloantigenic requirements for MLR-TsF production were addressed by 2 approaches. Treatment of stimulator cells with appropriate anti-I region antisera and complement did not affect MLR-TsF production, demonstrating that an absolute requirement for cells expressing I region determinants did not exist. However, spleen cells primed against entire H-2 haplotype differences produced significant quantities of MLR-TsF when they were restimulated with spleen cells homologous to the priming cells in only the I region, in the K and D regions, or in the D region alone. The additive nature of subregion-specific restimulation suggests that distinct subpopulations of K, I, and D region-specific MLR-Ts comprise the MLR-Ts population primed to entire H-2 haplotype differences.     

I-Region genetic restrictions imposed upon the recognition of KLH by murine T-cell clones

Data presented in this paper show that the recognition of keyhole limpet hemocyanin by murine T-cell clones is restricted by products of the I region. These data have been obtained by genetic mapping studies as well as by the use of monoclonal la-specific antibodies which inhibit the ability of antigen-presenting cells to effectively present antigen to such T-cell clones. Use of heterozygous antigen-presenting cells derived from crosses between B6.C-H-2 ^bm12 and B10.A(4R) mice have allowed us to show that both trans-complementing I-A products are used for restriction of recognition of KLH. These data were confirmed using monoclonal Ia antibodies to inhibition recognition of KLH by the same T-cell clones. Thus, we have shown that there exist hybrid molecules formed by free combinatorial association of products encoded within the I-A subregion which restrict the recognition of soluble antigen. Additionally, we have shown that the molecule formed by complementation between the alpha chain encoded within the I-E region and a beta chain encoded within the A region (A_e) can function effectively in presenting KLH to certain murine T-cell clones. These results support the hypothesis that the recognition of individual antigenic epitopes within large multideterminant antigens is under the control of Ir genes.     

Properties of H-2L locus products in allogeneic and H-2 restricted, trinitrophenyl-specific cytotoxic responses.

The role of the recently defined L antigen (a second D region product) in allogeneic and TNP-specific syngeneic primary CML responses has been investigated. The lysis by anti-L specific cytotoxic effector cells was not inhibited when the target cells were pretreated with an antiserum directed against K and D, whereas an antiserum against L completely abrogated this response. Therefore, H-2L products are recognized on the target cell independently of H-2K and H-2D locus products. Both A.SW cells as well as B10 cells were found to respond to Ld alloantigens, in addition to Dd alloantigens when stimulated by cells differing only in the D region. The results of cold target blocking and antiserum inhibition experiments failed to detect cytotoxic cells with specificity of L antigens in association with TNP, under conditions in which TNP-specific effectors to K and D antigens were demonstrable. These findings suggest that there is a more limited involvement of H-2L locus products than the H-2K or H-2D locus products in the induction and specificity of these responses.     

Alloantigenic sites on class I major histocompatibility complex antigens: 61-69 region in the first domain of the H-2Kb molecule induces specific antibody and T cell responses

The most polymorphic residues in the first domain of class I major histocompatibility complex (MHC) molecules are in the 61-69 region. We have chosen the H-2Kb molecule for determining the role of this region in the induction of alloimmune responses. A synthetic peptide, Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly corresponding to this region was synthesized. T cells enriched from the lymph nodes of allostrain mice that were previously primed with H-2Kb containing cells or with the synthetic peptide in complete Freund's adjuvant undergo extensive in vitro proliferation in response to the synthetic (61-69)H-2Kb peptide. The response was dependent on the presentation of the (61-69)H-2Kb peptide by the syngeneic antigen-presenting cells and was blocked by anti-class II MHC monoclonal antibodies. This peptide fragment of class I MHC molecule activates only helper/inducer type T cells that are involved in the primary responses but not the effector cytotoxic T cells. When coupled to a carrier protein, (61-69)H-2Kb peptide induced antibodies in allostrain mice that bind to intact H-2Kb molecule. No antibodies or T cell responses could be induced in syngeneic H-2b mice. The antigenic site on the H-2Kb molecule recognized by two H-2Kb-specific monoclonal antibodies B8 X 3 X 24 and Y-25 was also mapped in the 61-69 region by direct binding to the synthetic peptide. Therefore the 61-69 region on the H-2Kb molecule represents the first defined sequence on a class I molecule that is directly involved in the induction of alloimmune responses.     

Heterogeneity in the response of T cells to antigens presented by B lymphoma cells

Proliferation of antigen-specific T-cell populations was induced in cultures stimulated with antigen and a suitable source of antigen-presenting cells. Soluble (keyhole limpet hemocyanin) and particulate (horse red blood cells) antigens were presented by irradiated spleen cells and by a variety of B-lymphoma-cell lines, providing support for antigen-specific H-2-restricted T-cell responses. A marked heterogeneity was demonstrated, however, in the capacity of T-cell lines to proliferate in response to antigen presented by the B-lymphoma cells. T-cell populations were prepared from the lymph nodes of antigen-primed mice and restimulated in vitro in the presence of antigen and irradiated spleen cells. During the first six in vitro restimulations, these T-cell populations maintained the capacity to respond to antigen presented either by irradiated spleen cells or by B-lymphoma cells. Continued growth of these T-cell populations, again in the presence of antigen and irradiated spleen cells, resulted in the generation of T-cell lines which had lost the ability to respond to antigen presented by B-lymphoma cells. These lines however, fully retained the capacity to proliferate in the presence of antigen and irradiated spleen cells. T-cell clones derived from one of these lines were also unable to respond to antigen presented by B-lymphoma cells but again proliferated in the presence of antigen and irradiated spleen cells. Supernatants containing high levels of IL-1, IL-2, or IL-3 activity failed to reconstituted the antigen-specific response of T-cell lines which had lost the capacity to respond to antigen presented by B-lymphoma cells. Furthermore, titrated numbers of irradiated spleen cells, while having the capacity to support T-cell proliferation themselves, failed to synergize with B-lymphoma cells in the support of antigen-specific T-cell proliferation. Thus we have defined populations of antigen-specific, H-2-restricted T cells which do not recognize antigen presented by B-lymphoma cells and can therefore discriminate between different antigen-presenting cell types.     

Role of the H-2I region in the generation of an antiviral cytotoxic T-cell response in vitro

The participation of H-2I gene products in generating virus-specific proliferative and/or cytotoxic T-lymphocyte (CTL) responses was investigated. Spleen cells from mice infected with vaccinia virus were restimulated secondarily in vitro with syngeneic virus-infected peritoneal exudate cells (PEC) and then restimulated in tertiary cultures with virus-infected PEC from syngeneic and partially histoincompatible strains of mice. Based on the finding that comparable proliferative responses resulted when stimulating the responding cells were histocompatible at the H-2K, I, or D region of the major histocompatibility complex (MHC), the additively enhanced, but not potentiated, proliferation caused by identity at two or three H-2 regions was analyzed. Enhancement of proliferation followed when the H-2K/D components plus virus and the H-2I components plus virus were either on the same, or alternatively on two, stimulating cells. This suggests that H-2K, D, and I plus virus trigger distinct T-cell subsets. A virus-specific CTL response was generated in vitro when spleen cells from virus-primed mice and even unprimed mice were stimulated with cells sharing only H-2K and/or H-2D of the MHC. Identity at the H-2I region did not stimulate a CTL response, nor did it influence the magnitude of the $\text{K}\text{D}$ restricted response. Nevertheless, the presence of anti-Ia antiserum in cultures of syngeneic stimulators and responders inhibited the cytotoxic response to a great extent. Therefore, H-2I region products seem to participate in the generation of virus-specific CTL in vitro.     

Genetic control of the humoral response to an H-2 public specificity.

The humoral response of mice to an H-2 public specificity, termed H-2. '28' was found to be under genetic control. The genes determining this specificity were mapped to both the H-2K and H-2D regions, suggesting possible structural homologies between the products determined by these two regions. Genetic analyses indicated that a single non-H-2-linked gene regulates the anti-H-2. '28' response. In a backcross study, no linkage was detected between this putative H-2. '28' Ir gene and either the V H region or the Ly-2 locus to which the K-light chain locus is linked. Thus, a regulatory rather than a structural genetic locus seems a more likely basis for differences in response to this antigen. Our data further indicate that control of the humoral response to H-2. '28' is determinant specific since responses of the same backcross mice to other K and D alloantigens were not found to be subject to the same control.     

Identification of an immunodominant epitope within the phosphoprotein of rabies virus that is recognized by both class I- and class II-restricted T cells.

Immunization of H-2k mice with live rabies virus induces cytolytic T lymphocytes to the phosphoprotein of rabies virus. The antigenic determinant responsible for stimulating this class I-restricted cytolytic response was mapped to 50 amino acids (residues 180 to 229) of the phosphoprotein by using vaccinia virus recombinants expressing either the full-length phosphoprotein or C-terminal truncations of the phosphoprotein. The epitope was more finely mapped to residues 191 to 206 by using synthetic peptides. Several CD4+, class II-restricted T-cell lines were isolated from splenocytes of H-2k mice immunized with the vaccinia virus-rabies virus phosphoprotein recombinant virus. These lines were specifically stimulated by the phosphoprotein, and in addition, each line proliferated and released lymphokines in response to the same synthetic peptide shown to stimulate phosphoprotein-specific, class I-restricted cytolytic T cells.     

Non-specific peptide size effects in the recognition by site-specific T-cell clones. Demonstration with a T site of myoglobin.

Six regions (T sites) of myoglobin (Mb) were found by a comprehensive synthetic strategy to stimulate Mb-primed lymph-node cells. To define precisely the N-terminal boundary of the immunodominant T site (residues 107-120) with site-specific T-cell clones and to determine the effects of peptide size on their stimulation, two sets of peptides were employed. In one set, the peptides were elongated to the left from His-113 by one-residue increments of the Mb sequence. The other set represented an identical stepwise elongation by one-residue increments of the Mb sequence, but which were extended by additional unrelated ('nonsense') residues to a uniform size of 14 residues. Examination of the proliferative responses of eight T-cell clones, derived from Mb-primed DBA/2 (H-2d) or SJL (H-2s) mice, revealed a dramatic non-specific size requirement. In every clone, the longer nonsense-extended peptides achieved maximum stimulating activity at a lower optimum peptide dose than its natural-sequence, but shorter, analogue. In addition, slight (one-residue) differences in the N-terminal boundaries among the clones was observed. Thus, the fine specificity of each clone was mapped to the region from residue 111 or 112 to about residue 120 of Mb, which coincides with the site of B-cell recognition and resides in a small discrete surface region of the protein chain.     

Identification of T-helper epitopes in the VP1 capsid protein of poliovirus.

Poliovirus-specific T lymphocytes were isolated from virus-immunized mice of different H-2 haplotypes. Immunological characterization of this population indicates that the effector population involved in the observed poliovirus-specific proliferative response was that of CD4-positive T-helper cells. Proliferative responses also were induced within these T-lymphocyte populations upon stimulation with either purified VP1 capsid protein or VP1 synthetic peptides. By using these synthetic peptides, several T-helper epitopes were identified. Generally, proliferative responses were observed in three regions of VP1. Two regions spanning VP1 residues 86 to 120 and 201 to 241 were recognized by T lymphocytes from BALB/c (H-2d), C57BL/6 (H-2b), and C3H/HeJ (H-2k) backgrounds. Analyses using synthetic peptides of nonoverlapping sequences indicated that the region spanning residues 201 to 241 may contain several T epitopes and may account for the strong proliferative response observed. In addition, for two of the three haplotypes examined, T epitopes were observed within residues 7 to 24 of VP1. Additional epitopes which appeared to be restricted to specific H-2 backgrounds were identified. T epitopes within VP1 that are common between different strains of mice appeared to lie within previously identified neutralizing antigenic sites in poliovirus.     

Nature of T lymphocyte recognition of macrophage-associated antigens. III. Definition of the antigenic regions of human fibrinopeptide B involved in guinea pig T-cell responses

The clonal diversity of guinea pig T-lymphocyte responses to the 14-amino-acid peptide antigen human fibrinopeptide B (hFPB, Bβ1–14) and sequential hFPB³ homologs (Bβ5–14 and 7–14) was examined using bromodeoxyuridine (BUdR) and light elimination of T cell responses. PPD and hFPB-immune strain 2 guinea pig T cells and macrophages were stimulated in a first culture with PPD, Bβ1–14, 5–14, or 7–14; BUdR was added on the second day and the cultures exposed to light on the third day. The BUdR and light-treated T cells recovered from the first culture were restimulated in a second culture containing fresh stimulator macrophages and PPD, Bβ1–14, 5–14, and 7–14. BUdR and light-treated T cells initially stimulated with Bβ1–14 in the first culture showed no responsiveness to Bβ1–14, 5–14, or 7–14 in the second culture. BUdR and light-treatment of T cells initially stimulated with Bβ5–14 eliminated 70 to 80% of the subsequent response to Bβ1–14 and all of the responsiveness to Bβ7–14. Similar treatment of T cells stimulated with Bβ7–14 reduced responsiveness to Bβ1–14 by 50 to 60% and to Bβ5–14 by 60 to 70%. These observations indicate that T-cell responses are directed against three antigenic regions in the hFPB molecule; the major region defined by the carboxy-terminal sequence including residues 7 to 14, a second minor antigenic region including residues 5 and 6, and a third minor region including the amino terminal residues 1 to 4. Results are discussed with respect to the regions of the hFPB molecules that are recognized by antigen-binding T-cell receptors and the regions which interact with stimulator macrophages.     

Genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin.

The immune response to a synthetic peptide, H3 HA1(305-328), representing the C'-terminal 24 amino acid residues of the HA1 chain of the hemagglutinin of the H3 subtype of influenza virus is controlled by genes in the I region of the major histocompatibility complex. Mice of the H-2d haplotype are high responders and produce antibody for several months after a single injection of peptide without carrier. Mice of the H-2b, H-2k, and H-2q haplotypes are low antibody responders. Investigation of recombinant and congenic mouse strains revealed that high responsiveness requires the genes that encode the I-Ed molecule. Immunoassays, involving direct binding to analogs of this peptide and inhibition by both these analogs and synthetic epitopes, were used to analyze the specificity of the polyclonal response. In BALB/c mice, the primary antibody response is directed principally against the antigenic site 314-LKLAT-318, whereas the secondary response after a boost is predominantly directed to a distinct site, 320-MRNVPEKQT-328. The T-cell response to the peptide H3 HA1(305-328), as measured by antigen-induced proliferation of primed T cells in vitro, is also I-Ed restricted in high-responder H-2d mice and is directed against an antigenic site that does not require the four C-terminal residues unique to the H3 influenza subtype. A different epitope appears to be recognized by T cells from CBA (H-2k) mice, which proliferate to a moderate extent on exposure to the peptide but, nevertheless, do not provide help for an antibody response.