Fine-specificity of the contact sensitivity to 4-hydroxy-3-nitrophenyl acetyl (NP)

Dorf and colleagues (1-4) found that the contact sensitivity (CS) primed with (4-hydroxy-3-nitrophenyl)acetyl (NP) could be elicited as easily with the iodoanalog (NIP) as with NP when studied in Igh-1b mice but could only be elicited with NP, not NIP, in Igh-1j mice. Since this fine-specificity was parallel to the fine-specificity of anti-NP antibodies in the two types of mice and since anti-NP antibodies of Igh-1b mice are controlled by gene Igh-NPb the authors concluded that CS also was controlled by the Igh-NPb gene. The aim of this study was to confirm their findings with a more quantitative method (5). We confirmed equality of NP and NIP as elicitors of NP-primed CS in Igh-1b mice when the priming antigen was given subcutaneously into non-cyclophosphamide-treated mice (their method). We also found that this priming induced an anti-NP antibody response detectable at the time of challenge. Most experiments were carried out with a method that does not induce a detectable antibody response (pretreatment of mice with 200 mg/kg of cyclophosphamide; application of the sensitizing compound on skin). Since the NP-primed (and NBrP-primed) CS reactions exhibited "expected specificities," the immunizing compound was clearly the most efficient elicitor (relative efficiencies of homologs varied from 2 to 4). The Igh-NPb gene appears not to have a role in "antibody-free" reactions.     

T cell allotypic determinants encoded by genes linked to the immunoglobulin heavy chain locus. I. Establishment of monoclonal antibodies against allotypic determinants

Monoclonal alloantibodies for T cell allotypic determinants were obtained by hybridizing SP-2 tumor cells with BALB/c (H-2d, Igh-1a) spleen cells, which had been repeatedly immunized with Con A-stimulated CB-20 (H-2d, Igh-1b) spleen cells. It was found that these monoclonal anti-CB-20 antibodies detect the new allotypic determinants (distinct from the B cell Igh-C region determinant) expressed only on the augmenting or suppressor T cells. Genetic analysis of these antigenic determinants revealed these antibodies react with the gene products located on the telomeric side chromosome of the Igh variable region gene (Igh-V) cluster. These antibodies when given in vivo caused a modification of antibody production. The antibody activity was absorbed by Con A-stimulated B10.BR (H-2b, Igh-1b), C57BL/6 (H-2b, Igh-1b), CWB (H-2b, Igh-1b), CB-20 (H-2d, Igh-1b), and BAB-14 (H-2d, Igh-1b) spleen cells, but not by Con A-stimulated C3H.SW (H-2b, Igh-1j), BALB/c (H-2d, Igh-1a), A/Sn (H-2a, Igh-1e), and C.AL-20 (H-2d, Igh-1d) spleen cells. In addition, in vivo these monoclonal antibodies modified anti-SRBC antibody production only in Igh-1b allotype-bearing mice. One monoclonal antibody reacted with 4-hydroxy-3-nitrophenyl acetyl- (NP) hapten-specific augmenting T cells, and the other two batches of monoclonal antibodies reacted with NP-specific suppressor T cells of NP-mediated cutaneous responses. A mapping study with these recombinants limits the gene coding for the T cell-specific determinants to a gene within the variable region to the telomeric side of NP-VH and to the centromeric side of prealbumin. This segment is inclusive of all immunoglobulin genes, the region Owen named IgT-C, and a histocompatibility gene (H-Ig).     

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.     

Genetic control of contact photosensitivity to tetrachlorosalicylanilide. II. Igh complex controls the sensitivity induced by photohapten-modified spleen cells but not epidermal cells

We studied the genetic control of murine contact photosensitivity (CPS)1 to 3,3',4',5-tetrachlorosalicylanilide (TCSA) that was induced by subcutaneous injection of TCSA-photomodified epidermal cells (photoTCSA-EC) and spleen cells (photoTCSA-SC). With regard to the H-2 locus, sensitization with both types of photohaptenated cells showed the same pattern of CPS responses: H-2k and H-2b,d haplotypes were closely associated with low and high responders, respectively. On the other hand, the Igh locus affected the CPS reaction induced by photoTCSA-SC but not -EC; the Igh-1d allotype was related to low responsiveness, while high responders possessed Igh-1a,b. Thus, the photoTCSA-SC sensitization was controlled by H-2 and Igh in a codominant manner. The photoTCSA-SC-induced responses of H-2k but not Igh-1d mice were enhanced by CY pretreatment, suggesting that the mechanisms of low responsiveness in H-2k and Igh-1d mice were different. H-2 identity between donors of photoTCSA-EC and recipients was sufficient for effective sensitization, whereas both H-2 and Igh between donors of photoTCSA-SC and recipients should be identical to obtain maximum sensitization. This further confirmed the involvement of the Igh complex in the genetic control of CPS evoked by photoTCSA-SC. B cells as well as macrophages served as an effective presentation template for the photoTCSA-SC sensitization in the high responder Igh-1a mice, whereas B cells failed in inducing the CPS reaction in the low responder Igh-1d mice. These results suggest that B cells play an essential role in the Igh control phenomenon seen in the photoTCSA-SC sensitization. The present study demonstrated that CPS induced by photohapten-modified cells are differentially regulated by the H-2 and Igh gene loci depending on the cell type used for sensitization.     

T cell subsets in (responder x nonresponder)F1 mice regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine (GAT)

Immune responses to GAT are controlled by H-2-linked Ir genes; soluble GAT stimulates antibody responses in responder mice (H-2b) but not in nonresponder mice (H-2q). In nonresponder mice, soluble GAT stimulates suppressor T cells that preempt function of helper T cells. After immunization with soluble GAT, spleen cells from (responder x nonresponder: H-2b X H-2q)F1 mice develop antibody responses to responder H-2b GAT-M phi but not to nonresponder H-2q GAT-M phi. This failure of immune F1 spleen cells to respond is due to an active suppressor T cell mechanism that is activated by H-2q, but not H-2b, GAT-M phi and involves two regulatory T cell subsets. Suppressor-inducer T cells are immune radiosensitive Lyt-1 +2-, I-A-, I-J+, Qa-1+ cells. Suppressor-effector T cells can be derived from virgin or immune spleens and are radiosensitive Lyt-1-2+, I-A-, I-J+, Qa-1+ cells. This suppressor mechanism can suppress responses of virgin or immune F1 helper T cells and B cells. Helper T cells specific for H-2b GAT-M phi are easily detected in F1 mice after immunization with soluble GAT; helper T cells specific for H-2q GAT-M phi are demonstrated after elimination of the suppressor-inducer and -effector cells. These helper T cells are radioresistant Lyt-1+2-, I-A+, I-J-, Qa-1- cells. These data indicate that the Ir gene defect in responses to GAT is not due to a failure of nonresponder M phi to present GAT and most likely is not due to a defective T cell repertoire, because the relevant helper T cells are primed in F1 mice by soluble GAT and can be demonstrated when suppressor cells are removed. These data are discussed in the context of mechanisms for expression of Ir gene function in responses to GAT, especially the balance between stimulation of helper vs suppressor T cells.     

Genetic control of immune responses to the 18-kDa protein of Mycobacterium leprae. Different TH1 subsets may be involved in proliferative and delayed-type hypersensitivity responses.

In vitro and in vivo responses to the 18-kDa protein of Mycobacterium leprae have been analysed in different strains of mice. Lymphocytes from BALB/cJ (H-2d), BALB.B (H-2b), B10.BR (H-2k), and B10.M (H-2f) mice primed with 18-kDa protein yielded high T cell proliferative responses, while those from C57BL/10J (H-2b) mice yielded lower responses. Both H-2 and non-H-2 genes contributed to the magnitude of responsiveness. F1 mice from high and low responder strains showed high responsiveness to the 18-kDa protein. Supernatants from lymph node cell cultures prepared from 18-kDa protein-immunised BALB/cJ, B10.BR, and C57BL/10J mice contained IL-2 but no IL-4, indicating that activated T cells from both high and low responder mice were of a TH1 phenotype. Cell cultures from low responder C57BL/10J mice produced less IL-2 than those from high responders. The low responsiveness to the 18-kDa protein in proliferative assays might be due to a low frequency of antigen-specific T cells in the C57BL/10J mouse strain. BALB/cJ, C57BL/10J, and F1 (BALB/cJ x B10.BR) mouse strains were tested for in vivo DTH reactions to the 18-kDa protein. All strains, including C57BL/10J, were high DTH responders. Although DTH effector cells and 18-kDa protein-specific proliferative T cells belong to the TH1 subset, our data comparing high and low responder status indicate that distinct TH1 subpopulations are stimulated in response to the 18-kDa protein of M. leprae.     

AKR/J gene(s) unlinked to H-2 determines dominant inheritance of lymphocyte hyporesponsiveness to acetylcholine receptor

Mice with the H-2b major histocompatibility complex haplotype are high immune responders to nicotinic acetylcholine receptors (AChR), whereas mice with the H-2k haplotype are generally low responders. F1 progeny of C57BL/6 (H-2b) mice crossed with mice of most H-2k strains are high responders to AChR in standard conditions of testing helper T cell proliferation in vitro (4 X 10(5) lymph node cells/microwell, 1 wk after primary challenge in vivo). In contrast, the F1 progeny of AKR/J (H-2k) crossed with high responder (H-2b) strains (B6, A.BY, or C3H.SW) were all hyporesponsive to AChR when lymphocytes were tested at 4 X 10(5) cells/well. However, at a density of 1 X 10(6) or greater/well, a high level of antigen-specific responsiveness was demonstrable in the F1 hybrid lymphocytes. A shift from low to high responsiveness to AChR at high cell densities was observed also in the H-2b strain AKR.B6. Other strains previously demonstrated to be low responders to AChR did not become responsive to AChR when lymphocyte numbers were increased to 1.4 X 10(6)/well. The N2 generation yielded by backcrossing (AKR X B6)F1 mice to AKR/J were all low responders, whereas N2 progeny derived by backcrossing F1 to B6 were high or low responders in a ratio of approximately 1:1 (independent of their H-2 phenotype). Results consistent with this observation were obtained in (AKR X B6) F2 mice. These data suggest that at least one AKR/J gene outside of the H-2 complex exerts a hyporesponsive influence on the I-A-dependent helper T cell response to AChR in H-2b 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.     

Cross-induction of predominant NPb idiotypic antibodies with derivatives of (4-hydroxy-3-nitrophenyl) acetyl

Immunization of C57BL/6 mice with bovine gamma-globulin (BGG) conjugated with (4-hydroxy-3-nitrophenyl) acetyl (NP) induced a population of anti-NP antibodies that bear predominantly lambda light chain, exhibit heteroclitic affinity for heterologous NP derivatives, and share NPb idiotype. The present study analyzes the idiotypes of antibodies induced with BGG conjugated with the iodo-, bromo-, or nitro-NP derivatives (NIP, NBrP, and NNP). NIP-BGG, NBrP-BGG, and NNP-BGG, induce specific antibodies bearing NPb idiotype in C57BL/6 or C57BL/10 congenic mice, but not in many other inbred strains. Furthermore, the quantity of NPb idiotype in immune sera from various mouse strains immunized with NIP-BGG, NBrP-BGG, and NNP-BGG was similar to that in sera from mice immunized with NP-BGG. Anti-idiotypic antisera against C57BL anti-NP, anti-NIP, or anti-NBrP antibodies exhibit extensive idiotype binding of specifically purified B6 anti-NP, -NIP, -NBrP, and -NNP antibodies. These purified antibodies contain a high percentage (greater than 70%) of lambda-chain-bearing molecules. The data indicate that an extensively shared repertoire composed of predominantly lambda-bearing NPb-positive idiotypic antibodies is used in response to NP and its derivatives in C57BL mice.     

Synthesis of 5-(4-hydroxy-3-methylphenyl) -5- (substituted phenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone derivatives with anti-viral activity

A series of N1-nicotinoyl-3- (4-hydroxy-3-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their in vitro anti-viral activity. Among the compounds, the electron withdrawing group substituted analogues 5-(4-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4, 5-dihydro-1H-1- pyrazolyl-4-pyridylmethanone (4b), 5-(2-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyri- dylmethanone (4i), 5-(4-fluorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro- 1H-1-pyrazolyl-4-pyridylmethanone (4h) and 5-(2,6-dichlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro- 1H-1-pyrazolyl-4-pyridyl methanone (4j) were the most promising and the halogeno function appeared to be essential for antiviral activity.     

Cross-reactive idiotypic determinants on antibodies and antigen-specific helper T cell continuous lines

Anti-idiotypic antibodies produced in C57BL/6 mice (H-2b, Igh-1b) against (T,G)-A--L-specific antibodies of C3H.SW mice (H-2b, Igh-1j) were used to probe (T,G)-A--L-specific helper T cell lines and clones for the expression of idiotypic determinants on the cell surface of the monoclonal functional T cells. By using the fluorescence-activated cell sorter (FACS II), anti-idiotypic sera of individual mice that specifically bind C3H.SW anti-(T,G)-A--L antibodies were shown to stain significantly cells of the E-9M(+) continuous T helper line originated from C3H.SW (T,G)-A--L "educated" T cells. The same antisera did not react with a helper T cell line of C3H.SW origin specific to human gamma-globulin. They also did not stain a (T,G)-A--L-specific helper T cell line derived from CWB (H-2b, Igh-1b) mice, which differ from C3H.SW mice only in their heavy chain allotypes. Thus, the expression of the idiotypic determinants on the T cell lines appears to be antigen-specific and linked to the heavy chain allotypic marker as shown for the specific antibodies. Different clones derived from the E-9 M(+) line were tested their reactivity with the individual anti-idiotypic sera. All clones but one (1.11) were stained significantly. The clones were tested for their biologic activity and all of them except clone 1.11 were found to exert helper activity specific to (T,G)-A--L. Thus, individual anti-idiotypic sera against C3H.SW anti-(T,G)-A--L antibodies recognize cross-reactive idiotypic structures on the surface of antigen-specific monoclonal helper T cells.     

A recombinant malaria protein that can induce Th1 and CD8+ T cell responses without antibody formation.

Inbred strains of mice were immunized with p190-3, a 38-kDa recombinant protein derived from p190, a major merozoite surface Ag of the malaria parasite Plasmodium falciparum. Ag-specific proliferative T cell responses were obtained in H-2b, H-2d, and H-2k mouse strains. Surprisingly, mice of the H-2b haplotype (e.g., C57BL/6) did not give a measurable antibody response to the recombinant protein administered in Freund's adjuvant, but CD8+/CD4- as well as CD4+/CD8- T cells specific for p190-3 could be obtained after in vivo priming and in vitro selection with Ag. Distinct epitopes of p190-3 recognized by the CD8+ and CD4+ T cells from C57BL/6 mice were identified. The CD8+ T cells could kill H-2b APC in the presence of the appropriate epitope-containing peptide. The p190-3-specific CD4+ cells isolated from C57BL/6 mice were of the Th1 type. In contrast, Th2 cells, but no CD8+ T cells were present in a p190-3-specific line from BALB/c mice, which give good antibody responses to p190-3.     

Co-dominant restriction by a mixed-haplotype I-A molecule (alpha k beta b) for the lysozyme peptide 52-61 in H-2k x H-2b F1 mice

Helper (CD4+) T lymphocytes recognize protein Ag as peptides associated to MHC class II molecules. The polymorphism of class II alpha- and beta-chains has a major influence on the nature of the peptides presented to CD4+ T lymphocytes. For instance, T cell responses in H-2k and H-2b mice are directed at different epitopes of the hen egg lysozyme (HEL) molecule. The current studies were undertaken with the aim of defining the role of mixed haplotype I-A (alpha k beta b and alpha b beta k) molecules in T cell responses to HEL in (H-2k x H-2b)F1 mice, as well as the nature of the immunogenic peptides of HEL recognized in the context of I-A alpha k beta b and I-A alpha b beta k. A series of HEL-reactive T cell lines and hybridomas derived from MHC class II heterozygous (C57BL/6 x C3H F1) mice were established. Their responsiveness to HEL and synthetic HEL peptides was analyzed with the use of L cells transfected with either I-A alpha k beta b or I-A alpha b beta k as APC. Out of 28 clonal T cell hybridomas tested, 13 (46%) only responded to HEL presented by I-A alpha k beta b, 11 (40%) by I-A alpha b beta k (and to a minor extent I-A alpha k beta k), only 4 (14%) were primarily restricted by I-Ak, and none by I-Ab. All the I-A alpha k beta b-restricted T cell hybridomas responded to the HEL peptide 46-61 and to its shorter fragment 52-61, even at concentrations as low as 0.3 nM. As this determinant has been previously defined as immunodominant for I-Ak but not for I-Ab mice, these results suggest a role for the I-A alpha k chain in the selection and immunodominance of HEL 52-61 in H-2k mice. The fine specificity of I-A alpha k beta b-restricted T cell hybridomas for a series of different HEL peptides around the sequence 52 to 61 suggests that peptide 52-61 binds to I-A alpha k beta b with higher affinity than to I-A alpha k beta k. The peptides recognized in the context of I-A alpha b beta k and I-A alpha k beta k were not identified.     

Synthesis of 5-(4-hydroxy-3-methylphenyl)-5-(substituted phenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone derivatives with anti-viral activity

A series of N¹-nicotinoyl-3- (4-hydroxy-3-methyl phenyl)-5-(substituted phenyl)-2-pyrazolines were synthesized by the reaction between isoniazid (INH) and chalcones and were tested for their in vitro anti-viral activity. Among the compounds, the electron withdrawing group substituted analogues 5-(4-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4, 5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone (4b), 5-(2-chlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone (4i), 5-(4-fluorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridylmethanone (4h) and 5-(2,6-dichlorophenyl)-3-(4-hydroxy-3-methylphenyl)-4,5-dihydro-1H-1-pyrazolyl-4-pyridyl methanone (4j) were the most promising and the halogeno function appeared to be essential for antiviral activity.     

Characterization of the antibody response against the type II collagen induced by anti-idiotypic antibody.

We reported that rabbit anti-idiotypic antibody (Ab2) against mAb, termed 1-5 (Ab1) and reactive with human type II collagen (CII) induced antibody response to CII in DBA/1J mice susceptible to collagen-induced arthritis. In the present study, we further characterized the anti-CII antibody response elicited by Ab2 with respect to epitope specificity, putative genetic background, and IgG subclass. Most of anti-CII antibodies (polyclonal Ab3) derived from Ab2-immunized mice were of the IgG1 subclass. We purified polyclonal Ab3, using a CII-coupled immunoadsorbent column and we developed monoclonal Ab3 from Ab2-immunized mice. Both purified polyclonal Ab3 and two monoclonal Ab3s specifically reacted with a selected epitope on CII, recognized by Ab1. The anti-CII antibody response stimulated by Ab2 was observed in DBA/1J (H-2q, Igh-1c) and DBA/2 (H-2q, Igh-1c) mice, but not in the BALB/c (H-2d, Igh-1a) and C57BL/6 (H-2b, Igh-1b) strains, thereby suggesting that the anti-CII antibody response elicited by Ab2 is controlled by the Igh gene.     

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.     

Cytotoxic T-lymphocyte tolerance to minor H-43a alloantigen is induced exclusively in the context of the self major histocompatibility complex class I H-2Kb molecules

We elucidated previously that cytotoxic T lymphocyte precursors (CTLp) against H-43a allo-antigen, which we had discovered as a new mouse minor H antigen, were primed in H-43b mice only in the context of self H-2Kb restriction element, and that anti-H-43a CTLp tolerance was induced in H-43b mice by injection with H-43a spleen cells (SC) from H-43 congenic mice, i.e., under the condition of disparity at only the H-43 locus. The present study attempted to determine whether the H-2Kb restriction element for anti-H-43a CTLp priming is also implicated in the induction of anti-H-43a CTLp tolerance. For this purpose, we used a newly established H-43b C3W (H-2k) strain which is H-43 congenic to H-43a C3H/HeN. When (C3W X B10.MBR)F1 (H-43b, H-2Kk/b, Ik/k, Dk/q) mice were injected with H-43a-bearing (C3H/HeN X B10.AKM)F1 (H-43a/b;H-2Kk/k,Ik/k,Dk/q)SC, their selfH-2Kb-restricted anti-H-43a CTLp were were primed (cross-priming). By contrast, injection of H-43a-bearing (C3H/HeN X B10.MBR)F1 (H-43a/b; H-2Kk/b,Ik/k, Dk/q)SC, which differ from (C3H/HeN x B10.AKM) F1 SC solely at H-2K and possess H-2Kb molecules, did not prime but specifically inactivated the anti-H-43a CTLp of (C3W x B10.MBR)F1 mice. These results indicate clearly that anti-H-43a CTLp tolerance is induced exclusively in the context of the H-2Kb element expressed on the antigenic H-43a SC.     

Functional analysis of cloned macrophage hybridomas. V. Induction of suppressor T cell responses

It has been suggested that macrophage-like accessory cells are involved in suppressor T cell (Ts) induction. To further analyze this issue, we obtained several cloned macrophage hybridoma cell lines by somatic cell fusion of the macrophage tumor P388D1 of DBA/2 (H-2d) origin with splenic adherent cells of CKB mice (H-2k). Several cloned lines displayed the serological and functional characteristics of macrophages. We evaluated the ability of these hybridomas to induce third order or effector Ts (Ts3) to suppress the contact sensitivity response against the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). In contrast to the parental P388D1 and two other macrophage hybridomas, one macrophage hybridoma clone, termed 63, when conjugated with NP, induced Ts3, which suppressed contact sensitivity responses against NP but not DNFB, showing that the Ts3 were antigen specific. Macrophage hybridoma 63 could specifically induce Ts3 activity in either H-2k, H-2d, or H-2k/H-2d heterozygous hosts. Thus, macrophage hybridoma 63 functionally expressed major histocompatibility complex-related restricting determinants, and the fusion with cells from a H-2k macrophage donor caused a functional complementation of H-2d-related, Ts-inducing elements. The genetic restriction governing induction of Ts3 was controlled by genes that mapped to I-J region. Furthermore, NP-conjugated macrophage hybridoma 63 could serve as a target for elicitation of suppressor responses after administration of I-Jk, but not I-Jb, restricted suppressor factor. The data suggest that macrophage hybridomas represent a means to dissect heterogeneity within the macrophage population. The data also imply that the I-J determinants expressed on macrophages represent a ligand for the antigen receptor of Ts.     

Functional analysis of cloned macrophage hybridomas. IV. Induction and inhibition of mixed lymphocyte responses

A series of macrophage (M phi) hybridomas were generated by fusion of drug-marked P388D1 (H-2d) tumor cells with CKB (H-2k) splenic adherent cells. The ability of this panel of cloned M phi hybridomas expressing various levels of surface Ia antigens to induce allogeneic mixed lymphocytes responses (MLR) was examined. All MLR stimulatory M phi hybridomas expressed surface Ia antigens. However, some Ia+ and all Ia- M phi hybridomas were unable to induce vigorous MLR responses. Furthermore, even after induction of surface Ia antigen expression with Con A supernatants (Con A Sn) or purified interferon-gamma, the nonstimulatory M phi hybridomas remained ineffective at inducing strong MLR proliferative responses. Furthermore, addition of the latter M phi hybridoma clones (both with and without Con A Sn treatment) to conventional MLR cultures resulted in inhibition of MLR responses. The series of inhibitory M phi hybridomas secreted normal levels of IL 1 upon stimulation with lipopolysaccharide. After surface Ia induction with Con A Sn, the inhibitory M phi hybridomas could stimulate secretion of IL 2 and expression of IL 2 receptors. Moreover, although they inhibited conventional MLR responses, IL 2 production and IL 2 receptor expression were not significantly inhibited. Addition of these M phi hybridomas 24 to 48 hr after initiation of MLR response also inhibited MLR proliferation. The results indicated that the group of inhibitory M phi hybridomas can inhibit MLR responses after IL 2 secretion and acquisition of IL 2 receptors. Finally, this inhibitory activity has been maintained during 1 yr of continuous in vitro culture, and the hybridomas represent a stable "homogeneous" subpopulation of inhibitory macrophages. Thus, the inhibitory phenotype appears to reflect arrest at a distinct differentiation stage.