Nature of the cells regulating the production of a migration inhibition factor in C57BL/6 strain mice with low response to Candida albicans antigen

The authors studied cell regulation of macrophage migration inhibition factor (MIF) in response to Candida albicans antigen in C57BL/6 mice. It was revealed that low MIF production in response to this antigen is determined by the existence of specific suppressors which are inhibited by cyclophosphamide. The differences between the mouse lines are accounted for by different activity of suppressor cells of the thymic origin, which is confirmed by the treatment with anti-Thy1-serum and by bone marrow cell transfer.     

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.     

Production of a factor inhibiting macrophage migration to the Candida albicans antigen in mice and their resistance to this microorganism

Resistance to C. albicans, an opportunistic microorganism, has been studied in CBA and C57BL/6 mice, oppositely responsive in the production of the factor inhibiting migration of macrophages to antigen obtained from this fungus. The study has shown that CBA mice, highly responsive in the macrophage migration inhibiting factor, are less resistant to C. albicans, while C57BL/6 mice with low response to this antigen are more resistant to this infective agent. Macrophages play, probably, a certain role in the generalization of the process because not all phagocytized C. albicans cells are digested.     

Suppression of antibody responses in allogeneic mice by products of lymphoid tissue. II. Lack of antigenic specificity and immunogenetic requirements of allogeneic suppressive factor (ASF).

Mice were irradiated, infused with thymocytes and immunized with a variety of antigens, i.e., sheep or horse red blood cells (SRBC or HRBC), diphtheria toxoid (DT) or bovine gamma-globulin (BGG). The spleen cells (T.Spleen cells) were harvested 5 days later and cellfree extracts were prepared. The extracts contained an allogeneic suppressive factor (ASF) that was capable of inhibiting IgM antibody responses of allogeneic or semi-allogeneic unirradiated mice. ASF had to be injected within 24 hr of immunization to be effective and a single injection delayed, rather than abolished, the antibody response at the cellular level. However, daily injections of ASF resulted in persistent suppression of antibody response. ASF activity was antigen nonspecific, i.e., the antigen used to stimulate ASF production did not have to be the same as the antigen used to test for ASF activity. C3H T.Spleen extracts were even immunosuppressive when prepared by exposure to C3BF1 alloantigens only; such extracts suppressed antibody responses of C3BF1 and DBA/2 mice. C3H ASF was removed from extracts after incubation with C3BF1 spleen cells but not after incubation with C3H spleen cells. C3BF1 spleen cells which had been preincubated with C3H ASF were unable to generate antibody-forming cells upon transfer to irradiated C3BF1 host mice. This suggests that the ASF molecule may be or include receptors for alloantigens. The immunogenetic requirements for ASF activity were evaluated by injecting extracts from C3H, C57BL, C3BF and BALB/c T.Spleen cells into C3H, CBA, C57BL, BALB/c, DBA/2, A or C3H.A recipient mice. All extracts tested had ASF activity. However, all allogeneic recipients were not suppressed by the extract material. The suppressive activity of ASF seemed to require two (or more) antigenic differences between donors and recipients of extract material, an H-2K or I antigen difference and a second antigen difference, possibility Ig-1. In the limited numbers of strain combinations tested, T.Spleen extracts suppressed IgM antibody response only if exposed to H-2 and Ig-1 antigens, e.g., BALB/c (H-2d, Ig-1a) ASF suppressed A (H-2a, Ig-1e) but not C3H.A (H-2a, Ig-1a) or DBA/2 (H-2d, Ig-1c). Separate ASF molecules may react with separate antigens on the cell surface, i.e., with H-2 and gammaG2a. Alternatively, one ASF molecule may react with two structurally associated antigens. If the latter is correct, it is conceivable that the beta2-microglobulin which is non-covalently linked to the major component of H-2 molecules expresses allotypic antigens coded for by Ig-1 and beta2-microglobulin is one of the antigens recognized by ASF.     

A 38-kDa Antigen of Mycobacterium tuberculosis Predominantly Induces the Secretion of Interleukin-2, Interferon-gamma and IgG2a Antibodies

In the present study, mice of 3 different haplotypes (H-2d, H-2k and H-2b) were sensitized subcutaneously with heat-killed H37Ra or 38-kDa antigen of Mycobacterium tuberculosis. Lymphocytes obtained from immunized animals were challenged in vitro with 38-kDa antigen in both cases. The dominant pattern of Th1-like lymphokines (IL-2 and IFN-gamma) and preferential production of 38-kDa specific IgG2a-type antibody were observed. It was noted that 38-kDa antigen was recognized permissively by all 3 strains of mice used in the present study. It was interesting to note that C3H/HeJ mice, which express BCG-resistant alleles showed a higher level of proliferative as well as cytokine response as compared to BALB/c and C57BL/6 mice, which bear BCG-susceptible alleles. These results suggest that not only in recall responses but also during the induction as well as expression phase of the immune response mediated by 38-kDa antigen of M. tuberculosis the Th1-like immune response predominates.     

Synthesis and immunochemical study of the antigenic determinant of the H-2Db molecule of the murine major histocompatibility complex

Primary structure of murine class I histocompatibility antigens has been analysed to select possible antigenic determinant. Hexapeptide Leu-Gln-Gln-Leu-Ser-Gly, homologous to the region 95-100 of the H-2Db antigen heavy chain, was synthesised by stepwise elongation of peptide chain beginning from the COOH-terminal Gly. Rabbit anti-hexapeptide antibodies were obtained and shown to interact specifically with purified H-2Db antigen as well as with the native antigen on cell surface. These antibodies bind to lymphocytes of H-2b haplotype (C57BL/6 mice) but not H-2d (BALB/c) or H-2k (CBA). These data suggest that the region 95-100 is responsible for serologic differences between the alleles of H-2 antigens, i.e. it may be a xenotypic as well as an allotypic antigenic determinant. The latter was confirmed by study of interaction of the hexapeptide with allogeneic monoclonal antibodies specific to H-2Db antigen.     

Effects of serotonin on antigen-nonspecific suppressors of the thymus

Study of nonspecific suppressors of the thymus in inhibition of immune response by serotonin is carried out in CBA mice. We have tested a selective effect of 2-deoxyguanosine on antigen nonspecific T suppressors of the thymus. In syngeneic adoptive transfer on the 3rd day after immunization it is established that antigen nonspecific immune response (rosette-forming cells) after serotonin treatment amounts to 71%, in intact animals--37%. When estimating the plague forming cells the antigen nonspecific suppressive thymocytes make up 21.9% in immune mice, 62.5% in immune mice after serotonin treatment, 29.7% in intact animals.     

Genetic aspects of the immunomodulating action of interferon

The data of the study of alpha/beta interferon (IFN) effect in mice of different genotype were presented. CBA mice of H-2k genotype, C57B1/6 mice of H-2b genotype and their hybrid (CBA X C57B1/6) F1 have been used in the experiments. IFN has been injected intraperitoneally in a dose of 100-5000 U/mouse in combination with antigenic stimulation. It was shown that IFN enhanced stem cells migration from bone marrow in CBA, but not in (CBA X C57B1/6)F1 mice. At the same time the splenocytes from CBA mice were more sensitive to inhibition by IFN than splenocytes from C57B1/6 mice. This was found in antibody and immune rosette-formation tests. The effect of IFN on the immune system cells is probably predetermined by the individual genetic characteristics of a mouse strain.     

Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness.

We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells.     

Immunity to leprosy. II. Genetic control of murine T cell proliferative responses to Mycobacterium leprae

T cell proliferative responses to Mycobacterium leprae were measured after immunization of mice at the base of the tail with antigen and challenging lymphocytes from draining lymph nodes in culture with M. leprae. This T cell response to M. leprae has been compared in 18 inbred strains of mice. C57BL/10J mice were identified as low responder mice. The congenic strains B10.M and B10.Q were found to be high responders, whereas B10.BR and B10.P were low responders. F1 (B10.M X C57BL/10J) and F1 (B10.Q X C57BL/10J) hybrid mice were found to be low responders, similar to the C57BL/10J parent, indicating that the low responsive trait is dominant. Whereas B10.BR mice were shown to be low responders to M. leprae, B10.AKM and B10.A(2R) were clearly high responders, indicating that the H-2D region influences the magnitude of the T cell proliferative response. Gene complementation within the H-2 region was evident. Genes outside the H-2 region were also shown to influence the response to M. leprae. C3H/HeN were shown to be high responder mice, whereas other H-2k strains, BALB.K, CBA/N, and B10.BR, were low responders. Gene loci that influence the T cell proliferation assay have been discussed and were compared to known background genes which may be important for the growth of intracellular parasites. Because mycobacteria are intracellular parasites for antigen-presenting cells, genes that affect bacterial growth in these cells will also influence subsequent immune responses of the host.     

Lung tumor-reactive cytotoxic lymphocytes generated in mixed lymphocyte reaction between C3HfeB/HeN (H-2kb) and C3H/HeN (H-2k) strain mice.

The tumor-associated transplantation antigen expressed by several transplacentally induced lung tumors of C3HfeB/HeN mice (H-2kb haplotype) has previously been shown to exist as a normal tissue alloantigen in mice of known H-2k and H-2a haplotypes. This antigen is not expressed in normal tissues of C3HfeB/HeN mice but is expressed in C3H/HeN mice, the strain from which the C3HfeB/HeN mice were originally derived. The present study indicates that spleen cells from C3HfeB/HeN and C3H/HeN mice respond reciprocally in the mixed lymphocyte reaction. Cytotoxic T lymphocytes specific for the tumor-associated alloantigen can be readily generated in mixed lymphocyte reactions in which spleen cells from C3HfeB/HeN mice are reacted with x-irradiated spleen cells from C3H/HeN or A strain mice. These cells are effective in suppressing the growth of the C3HfeB/HeN-derived lung tumor 85 in x-irradiated syngeneic recipients.     

In vivo-induced suppression of T cell proliferation: the relationship between the specificity of induction and control

We have previously shown that sc immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and to peptide 8. In contrast, immunization of B10.BR (H-2k) mice either with TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous Ag. In the present article , we report that in the B10.BR (H-2k) strain, ip prepriming with (TMVP) 7 days prior to sc immunization with peptide 8 causes a drastic reduction in the in vitro proliferative response of peptide 8-specific T cells while no such effect is seen in the congenic C57BL/10 (H-2b) strain. This suppression of T cell responsiveness can be transferred with TMVP-primed spleen cells. Moreover, deleting T cells from the transferred spleen cells abrogates the suppressive effect. In both H-2 haplotypes, ip prepriming with peptide 8 causes suppression of the proliferative T cell response induced by subsequent immunization with peptide 8. This prepriming has no effect on the response to TMVP immunization of B10.BR mice but does effect the response of C57BL/10 mice. Using various synthetic peptides to analyze the specificity of the suppression, we have determined that (1) T cells involved in the suppression of the proliferative T cell response to a single peptide determinant do not suppress the proliferative T cell response to other determinants on the protein antigen and (2) these T cells with suppressor function, and proliferating T cells which are ultimately regulated, can exhibit specificity for the same epitope. These studies suggest that there may exist fundamental differences as to how T cells which participate in suppression an proliferating T cells (which include mainly T helper cells) recognize protein antigens.     

H-2 effects on cell-cell interactions in the response to single non-H2 alloantigens. IV. Variations in the proliferative response to H-Y and H-3

Individual mice were tested for their proliferation T-cell response to H-Y- and H-3-incompatible stimulator cells in secondary mixed lymphocyte culture. Responders expressing the H-2b haplotype were restricted in their response to stimulators presenting H-Y and H-3 in the context of H-2b. Lymphocytes from individual B10 females proliferated in response to H-Y presented with I-Ab and Db. The ratio of I-Ab/Db-restricted responses varied between individual responders, indicating significant qualitative variation between genetically identical responders. The majority of the proliferative response in all tested mice was restricted to the entire H-2b haplotype suggesting complementation of I-Ab- and Db-region genes in presenting the H-Y antigen. Similar observations were made in the response of individual B10.LP mice to the H-3 antigen. H-3-specific, proliferating T cells were restricted to H-3 antigen presented with KbAb and Db with significant variation between individuals in proliferative response to H-3 plus KbAb and Db. In contrast to the response to H-Y, the proliferative response to H-3 plus H-2b could be accounted for by the summation of the proliferative responses to H-3 plus KbAb and Db. These observations demonstrate that the proliferative response to non-H-2H antigens in the context of I-region determinants is not a sine qua non for the T-cell response to these antigens. Further, the individual qualitative and quantitative variation observed with individual genetically identical mice has strong implications for our knowledge of intrastrain variation in immune responsiveness and the characterization of inbred strains for immune responsiveness.     

Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.     

A requirement for helper T cells in the induction of delayed-type hypersensitivity

This paper describes a model system for studying the role of helper T cells in the induction of delayed-type hypersensitivity (DTH). Cyclophosphamide- (CP) treated mice sensitized with antigen 3 days later develop high levels of delayed-type immunity; however, DTH cannot be demonstrated in mice that are sensitized with antigen 1 day after drug treatment. The inability to respond to antigen 1 day after CP treatment can be restored if either normal or low-dose primed spleen cells are transferred at the time of sensitization. Although irradiated (1500 rad) normal spleen cells are unable to restore DTH, such treatment has no effect on the primed spleen cell population. The lymphocytes responsible for restoring the DTH response were identified as T cells, in that treatment with anti-Thy-1.2 serum and C abrogated their effect. Furthermore, restoration of the DTH response was dependent on the presence of antigen at the time of lymphocyte transfer; irradiated primed cells could not transfer DTH alone. The DTH effector cells in reconstituted mice were identified as originating from the host and not from the transferred cell population. This was accomplished by using anti-H-2 serum to identify the source of the DTH effector cells after transferring parental (H-2b) irradiated primed spleen cells into CP-treated F1 mice (H-2b,k). Thus, the irradiated transferred cells are behaving as helper T cells and promoting the development of DTH effector cells in the host.     

Inhibition of the HCV Core Protein on the Immune Response to HBV Surface Antigen and on HBV Gene Expression and Replication In Vivo

The hepatitis C virus (HCV) core protein is a multifunctional protein that can interfere with the induction of an immune response. It has been reported that the HCV core protein inhibits HBV replication in vitro. In this study, we test the effect of the HCV core gene on the priming of the immune response to hepatitis B surface antigen (HBsAg) and on the replication of HBV in vivo. Our results showed that the full-length HCV core gene inhibits the induction of an immune response to the heterogeneous antigen, HBsAg, at the site of inoculation when HCV core (pC191) and HBsAg (pHBsAg) expression plasmids are co-administered as DNA vaccines into BALB/c mice. The observed interference effect of the HCV core occurs in the priming stage and is limited to the DNA form of the HBsAg antigen, but not to the protein form. The HCV core reduces the protective effect of the HBsAg when the HBsAg and the HCV core are co-administered as vaccines in an HBV hydrodynamic mouse model because the HCV core induces immune tolerance to the heterogeneous HBsAg DNA antigen. These results suggest that HCV core may play an important role in viral persistence by the attenuation of host immune responses to different antigens. We further tested whether the HCV core interfered with the priming of the immune response in hepatocytes via the hydrodynamic co-injection of an HBV replication-competent plasmid and an HCV core plasmid. The HCV core inhibited HBV replication and antigen expression in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, the mouse models of acute and chronic hepatitis B virus infections. Thus, the HCV core inhibits the induction of a specific immune response to an HBsAg DNA vaccine. However, HCV C also interferes with HBV gene expression and replication in vivo, as observed in patients with coinfection.     

Interleukin 3 augments the murine primary cytolytic T lymphocyte response to allogeneic tumor cells

The primary anti-H-2k allospecific cytolytic T lymphocyte (CTL) response by BALB/c (H-2d) spleen cells in vitro to x-irradiated RDM4 (H-2k) tumor cells is weak. This response has been shown to be augmented by CTL helper factor (CHF), a factor present in supernatants of spleen cells cultured with Sendai virus (SC-CM). Conditioned medium from WEHI-3 cells (WEHI-CM) also contains activity that augments the BALB/c anti- RDM4 CTL response. Attempts to separate the CHF activity from interleukin 3 (IL 3), also present in WEHI-CM, were unsuccessful. Purified IL 3 was then tested, and was found to increase the BALB/c anti- RDM4 CTL response by five- to 10-fold. IL 3 is apparently the only material in WEHI-CM that is active in this assay. The response is apparently a classical CTL response because: 1) the effector cells are sensitive to monoclonal anti-Thy-1.2 antibody plus C; 2) the response is dependent on antigen stimulation, and it peaks on day 5 or 6 of culture; and 3) the effector cells are specific for H-2k targets. IL 3 must be added very early during the in vitro culture period for maximal augmentation of the response, consistent with possible action of IL 3 as a differentiation factor.     

Genetic regulation of the antibody response to H-2Db alloantigens in mice. IV. Antigens that activate helper T cells for IgG, coded for by the non-H-2 genome

When B10.A(5R) mice are immunized with congenic C57BL/10 cells only 2-ME-sensitive antibodies (IgM type) are found directed against H-2Db. To obtain 2-ME-resistant antibodies (IgG type) 5R mice must be immunized with noncongenic cells (e.g., A.BY); in this case non-H-2 cell surface antigens will activate helper T cells to induce anti-Db IgG antibody production by B cells. An attempt was made to define helper antigens that activate helper T cells. Neither N-2 antigens of seven H-2Db recombinant strains nor a limited set of non-H-2 cell surface antigens were able to serve as helper antigens. By using individual backcross mice as antigen, one helper antigen was found on the background of strain A under the conditions used, whereas other backgrounds may carry more than one antigen. The helper antigen is dominantly expressed in F1 mice and has to be presented on the same cell as H-2Db to induce the switch from IgM to IgG.     

Haplotype-Specific Interactions of Non-H-2-Linked Genetic Factors Controlling the Mouse C4 and Slp Protein Levels

The influence of non-H-2 linked genes on the plasma levels of the H-2 S-region encoded proteins C4, Slp, and factor B was tested in Recombinant Inbred (RI) strains. The A X B and B X A RI strains exhibit a continuous range of C4 and Slp levels from very high to very low which reach beyond the levels of their parental strains, C57BL/6J and A/J, indicating involvement of several trans-regulatory (non-H-2-linked) genes. Only limited variation in levels of factor B has been found. No linkage relationship could be established for the trans-regulatory genes, because more than one gene is involved. A complex interaction of H-2 haplotype, genetic background, sex, and possibly maternal effect in determining the C4 and Slp protein plasma levels has been observed. The H-2-dependent sex effect is evident, because males have higher C4 levels than females in RI strains with H-2b but not with H-2a haplotype. This sex effect is also background dependent, because it is present in the H-2b congenic strain on A background (A.BY) but not in C57BL/10 and C57BL/6 (both H-2b). Mice from RI strains with H-2b haplotype have in general higher C4 levels than mice with H-2a haplotype.     

Immunosuppressive factor(s) specific for L-glutamic acid50-L-tyrosine50 (GT). I. Production, characterization, and lack of H-2 restriction for activity in recipient strain.

The random synthetic copolymer of L-glutamic acid50-L-tyrosine50 (GT) fails to elicit a GT-specific antibody response in all inbred strains of mice tested. Preimmunization with GT specifically inhibits a GT-MBSA response in certain H2d,k,s, but not other, H-2a,b,q, nonresponder mice. This unresponsiveness is mediated by GT-specific suppressor T cells. Extracts prepared from lymphoid cells of GT-primed suppressor haplotype mice inhibit the development of primary GT-specific antibody responses to GT-MBSA in normal syngeneic mice. Nonsuppressor haplotype mice do not produce GT-specific suppressor factor. The GT-suppressive extract has affinity for antigen and a m.w. of less than 50,000 daltons, thus, resembling antigen-specific immunosuppressive factors already described. However, the GT-suppressive extract does not appear to have H-2 restrictions since it works across allogeneic barriers. Evidence is presented that two genes are required for factor-mediated suppression.