Selection of anti-F protein B-cell repertoires in normal mice

The hypothesis that self-tolerance to F protein antigen exclusively concerns T cells was tested by determining the frequencies of B lymphocytes producing anti-F antibodies in bone marrow (BM), spleen and peritoneal exudate (PEC) cells from normal, immune or tolerant animals, and in responder and non-responder mouse strains. Using an ELISA spot assay and lipopolysaccharide stimulation, we found that anti-F frequencies were highest in BM and "naturally activated" large spleen cells, followed by resting spleen and PEC cells. Anti-F specificities were also induced among "natural" Ig-secreting cells of normal individuals. Specific immunization of responder mice doubled the splenic frequencies, while tolerization had no effect. Similar results were obtained in BALB/c and A/J mice, while C57BL/6 contained fewer anti-F B cells in spleen, but not in BM. These results support the notion that self-tolerance to F antigen can primarily be ascribed to T cells, but they also show F-antigen-specific selection of B-cell repertoires.     

IgE-isotype-specific suppressor cells in the mouse: characterization using tetraparental chimera mice of high (DBA/2) and low (SJL) IgE responder embryos

Tetraparental chimera mice were developed by aggregation of IgE high responder (DBA/2) and IgE low responder (SJL) embryos. Anti-dinitrophenyl (DNP) IgE antibody response in such mice (SJL----DBA/2) upon challenge with DNP-keyhole-limpet hemocyanin (KLH) in alum was clearly suppressed, while anti-DNP IgG antibody response was not. High-titer anti-DNP IgE and IgG antibody response developed in F1 hybrid mice of SJL and DBA/2 (SDF1) mice. The experimental results suggest that high IgE antibody production is the dominant trait, and the IgE-specific suppressor gene in SJL mice is autosomal recessive. IgE-specific suppressor T cells in SJL mice actively suppressed IgE antibody formation by DBA/2 immuno-competent cells across the histocompatibility barrier. Hapten-specific B cells and carrier-specific T cells were prepared in SJL----DBA/2 and SDF1 mice by immunization with DNP-KLH or ovalbumin (OA) in alum and transferred to irradiated SDF1 mice followed by challenge with DNP-OA. Hapten-specific B cells and carrier-specific helper T cells clearly developed in SDF1 mice. Recipient mice transferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells showed high-titer anti-DNP IgE and IgG antibody responses. OA-primed SJL----DBA/2 spleen cells cotransferred with DNP-KLH-primed SDF1 spleen cells and OA-primed SDF1 spleen cells completely abolished secondary anti-DNP IgE antibody response. The data suggest that carrier-specific helper T cells for IgE and IgG antibody responses are distinct. The regulatory role of IgE-isotype-specific suppressor cells were considered to be the interference of cooperative cellular interaction between IgE B cells and carrier-specific, IgE-specific helper T cells.     

Responsiveness of T cells to Mutant Major Histocompatibility Complex Class I Antigen

Mechanisms of the activation of T cells responding to major histocompatibility complex (MHC) class I antigen were investigated with special reference to interleukin 1 (IL-1) production from stimulator-type accessory cells. For this purpose, we used mainly fractionated Lyt-2+T cells of C57BL/6 (B6) mice as responder cells and irradiated spleen cells or those deprived of adherent cells of B6.C-H-2bm1 (bm1) mice as stimulator cells. Lyt-2+ T cells of B6 mice proliferated in the presence of irradiated whole spleen cells of bm1 mice but did not to Sephadex G-10 column-passed bm1 spleen cells. The unresponsiveness in the latter case was overcome by the supplement of recombinant IL-1 and/or IL-2 in the culture medium. These interleukins were shown to promote the proliferative response of B6 Lyt-2+ T cells in the presence of stimulator-type T or B cells. Both interleukins also facilitated the generation of cytotoxic T cells from B6 Lyt-2+ cells to H-2Kbm1 antigen in the mixed lymphocyte culture deficient in stimulator-type accessory cells. IL-1 was shown to enhance the expression of IL-2 receptor on the responding Lyt-2+ T cells as assessed by flow cytometry. IL-1 binding to responding T cells were also assayed by means of iodinated IL-1 and was shown to increase significantly on responding Lyt-2+ cells. Overall results indicate that accessory cells might play dual roles in the activation of Lyt-2+ T cells responding to allogeneic MHC class I antigen: direct presentation of the antigen to responder T cells and production of IL-1. Both signals are essentially required for Lyt-2+ T cells responding to allogeneic MHC class I antigen to initiate proliferation and also to differentiate into cytotoxic T cells.     

Analysis of immunological tolerance to major histocompatibility complex antigens. I. High frequencies of tolerogen-specific cytotoxic T lymphocyte precursors in mice neonatally tolerized to class I major histocompatibility complex antigens

Injection of (CBA X A)F1 cells into neonatal CBA mice rendered them tolerant to skin grafts of (CBA X A)F1 origin. Limiting dilution analysis revealed a very low frequency of tolerogen-inducible cytotoxic T lymphocyte precursors (CTL-P) in spleens of tolerant mice. Two in vitro procedures allowed, however, the induction of tolerogen-specific CTL-P of high frequencies in tolerant mice: (a) the "by-pass" activation of spleen cells from tolerant mice by concanavalin A under short-term bulk culture conditions followed by culture of limiting numbers of activated responder cells, and (b) absorption of spleen cells from tolerant mice on monolayers of tolerogen-activated T cells from normal syngeneic mice. Furthermore, spleen cells from tolerant mice, recently challenged with a tolerogen-bearing skin graft, specifically suppressed the activation of tolerogen-reactive splenic CTL-P from normal CBA mice under limiting dilution conditions. These data confirm the presence of tolerogen-specific CTL-P of high frequency in tolerant mice and suggest their functional inactivation through a suppressive mechanism.     

Selection of the T cell repertoire during ontogeny: Limiting dilution analysis

The relative frequencies of cytotoxic T lymphocyte precursors (CTL-P) specific for minor histocompatibility antigens that are restricted to either k or b major histocompatibility complex antigens in normal B6 mice and in B6 → (B6 CBA)F₁ radiation chimeras were estimated. The ratio of k-restricted to b-restricted CTL-P determined for the radiation chimeras served as a base value to which the ratio of k: b-restricted CTL from normal B6 mice can be compared. The frequencies of allorestricted CTL-P in B6 mice were determined after the B6 cells had been depleted of k-reactive cells by filtration through irradiated B6.H?2^k mice. Lymphocytes from immunized animals were used to obtain frequency estimates of CTL-P specific for minor H antigens. The cultures contained nu/nu spleen cells syngeneic to the responder, but no exogenous interleukin 2 (T cell growth factor) since a high degree of nonspecific killing was found in IL2-supplemented cultures. It was found that the CTL-P frequency of B6 mice to BALB.B antigens in the lymph node cells of normal or immune B6 mice were 1 in 300000 and 1 in 8000, respectively. In B6→ (B6 × CBA)F₁ radiation chimeras, the ratio of BALB.B/BALB.K-specific CTL-P was 3 :1s ratio was 17 :1 in normal B6 mice. Twenty-five percent of CTL clones from normal B6 mice lysing BALB.K target also lysed BALB.B target whereas less than 1% of CTL clones lytic on BALB.K target from the chimeras showed this cross-reaction The results are compatible with the idea of complete positive selection of self MHC-restricted CTL-P in the absence of priming effects by foreign antigen.     

Microheterogeneity in regulatory circuits for T-cell alloresponsiveness.

Mixed leucocyte cultures of responder spleen cells from individual B10 mice, challenged with irradiated allogeneic B10.D2 or B10.BR spleen cells, were used to generate discrete pools of cytotoxic T lymphocytes (CTL) with which to immunize (B10 x B10.D2)F1 or (B10 x B10.BR)F1 animals. Aliquots of the original donor B10 spleen cells were stored at -70 degrees. Eighteen days after immunization of the F1 animals, spleen and serum preparations from these mice were tested, in reciprocal fashion, for their ability to affect the development of CTL from the thawed donor B10 spleen lymphocytes in fresh cultures challenged with either the original or a third party allogeneic stimulus. Evidence for individual specific suppression by F1-T lymphocytes or by F1-serum (antibody) molecules was obtained. By priming (B10 x B10.D2)F1 mice with B10 lymphoid cells the F1 animals can also be shown to resist the otherwise lethal GvHD induced by sublethal whole body irradiation followed by intravenous challenge with B10 lymphoid cells. Using F1 mice primed and subsequently challenged with lymphocytes prepared from individual donors, self-preference in the regulation of GvHD was also seen. A similar fine specificity of regulatory cells was observed using suppressor cells from individual mice rendered neonatally tolerant of histocompatible cells (by inoculation of F1 hybrid cells within 24 hr of birth). These findings suggest that the mouse T cell alloreceptor repertoire is subject to a process of somatic diversification during normal ontogenesis. By examining the regulation of alloresponsiveness in T lymphocytes of B10.Br origin which differentiate from the appropriate stem cells in a B10 or B10.Br host, we have uncovered evidence that the T-cell alloreceptor repertoire is, in part at least, determined by the host MHC environment.     

A simple method for the quantitation of specific cytotoxic precursors to synrgeneic tumors

Cytotoxic T lymphocytes (CTLs) to EL4, a syngeneic lymphocytic leukemia of C57BL/6 (B6) (H-2^b) mice, were obtained by culturing normal B6 spleen cells with irradiated EL4 tumor cells for 4 days in conical bottom mitrotiter trays. A much lower, though significant, amount of cytotoxicity towards EL4 was observed in B6 spleen cell cultures not stimulated with EL4. The cytotoxic effector cells were shown to be Thy-1⁺ and their cytolytic activity to EL4 tumor cells was inhibited by an anti-H-2^b serum. Cold target competition studies suggest that the B6 anti-EL4 CTLs could discriminate between EL4 tumor cells and a second H-2^b lymphoma, C1498. The converse experiment yielded similar results. Culture conditions limiting for CTL precursors (CLPs) to EL4 were attained by including interleukin 2 (IL2), a lymphokine obtained by stimulating murine or rat spleen cells with concanavalin A, in the culture medium. In the presence of IL2, the CLP frequencies in B6 spleen cells to EL4 and C1498 in antigen-stimulated cultures were 113 and 231 per 10⁶ responder cells, respectively. In non-antigen stimulated B6 spleen cell cultures, the apparent CLP frequencies to EL4 and C1498 were 6 and 33 per 10⁶ responder cells, respectively. In agreement with the cold target competition studies using CTLs generated in the absence of IL2, we found that the CTL clones produced in cultures stimulated with EL4 and C1498 in the presence of IL2 were specific for the stimulating antigen. The specificity of the CTL clones obtained from cultures stimulated with IL2 in the absence of antigen appears to be different from those derived from antigen-stimulated cultures. The potential applications and limitations of this culture system are discussed.     

Allosuppression of B cells in vitro by graft-vs.-host reaction-derived T cells is caused by cytotoxic T lymphocytes

An acute graft-vs.-host reaction (GVHR) was induced by i.v. injection of 10(8) lymphoid cells from C57BL/10 (B10) donors (H-2b/b) into adult non-irradiated (B10 X DBA/2)F1 mice (H-2b/d). Previous experiments have established that spleen cells obtained from such GVHF1 mice suppress the primary antibody response of normal F1 spleen cells to sheep red blood cells. This type of suppression was termed "allosuppression", and it was shown to be mediated by Ly-2+ T cells of donor origin that react against H-2 antigens of the host. It was unclear, however, whether the actual mechanism of allosuppression was due to suppressive factors generated by donor T cells or whether the latter killed the F1 B cells. Here, we show that for their suppressive effect GVHF1 spleen cells need direct cell contact with F1 spleen cells; no suppressive effect was measured in a double-chamber culture system in which the GVHF1 spleen cells were separated from the responding normal F1 spleen cells by a cell-tight membrane filter. The suppressive effect only affected cells expressing the appropriate H-2 class I alloantigen; in mixed cultures of irradiated F1 spleen cells and GVHF1 spleen cells with third-party B cells the antibody response of the third-party B cells was not suppressed. GVHF1 spleen cells showed cytotoxic T lymphocyte (CTL) activity specific for the allogeneic F1 H-2 antigen. The suppressive effect of the GVHF1 spleen cells did not differ from that exerted by cloned CTL specific for MHC class I alloantigens; cloned CTL suppressed the primary antibody response of murine spleen cells without affecting the response of third-party B cells added to the cultures. The combined findings show that "allosuppression" in vitro is not due to suppression of F1B cells, but to a direct killing of these cells by alloreactive CTL.     

Genetic control of the spontaneous activation of CD4+ Th cells in systemic lupus erythematosus-prone (NZB x NZW) F1 mice

The F(1) hybrid of autoimmune hemolytic anemia-prone NZB and nonautoimmune NZW strains of mice has been studied as a murine model of systemic lupus erythematosus. Both NZB and F(1) hybrid mice show age-dependent spontaneous activation of peripheral CD4(+) T cells as reflected by the elevated frequencies of CD4(+) T cells positive for CD69 early activation marker. Both strains also show age-dependent abnormal decrease of the frequencies of CD62L(+) naive CD4(+) T cells and/or NTA260(+) memory CD4(+) T cells in the spleen. We studied the multigenic control of these abnormal features of peripheral CD4(+) T cells in (NZB x NZW) F(1) x NZW backcross mice by quantitative trait loci mapping and by association rule analysis. The abnormally elevated frequencies of CD69(+)CD4(+) T cells and decreased frequencies of CD62L(+) naive and/or NTA260(+) memory CD4(+) T cells were under the common genetic control, in which the interaction between MHC and a hitherto unknown locus, designated Sta-1 (spontaneous T-cell activation) on chromosome 12, plays a major role. The allelic effects of these loci likely predispose CD4(+) T cells to the loss of self-tolerance, and are responsible for the accelerated autoimmune phenotypes of (NZB x NZW) F(1) hybrid mice.     

Genetic control of eosinophilia. Analysis of production and response to eosinophil-differentiating factor in strains of mice infected with Trichinella spiralis.

Bone marrow cultures were established from mice undergoing parasitic eosinophilia after infection with Trichinella spiralis. In the presence of eosinophil-differentiation factor (EDF/IL-5) eosinophil precursor cells differentiated and could be identified and counted after a 7-day in vitro culture period. The EDF-bone marrow assay system was used to determine differences in bone marrow eosinophil precursor capacity between a number of inbred strains of mice. Bone marrow cultures from high peripheral eosinophil-response phenotype strains of mice (NIH, SWR & SJL) contained significantly greater numbers of eosinophil precursor cells than the low response strain C57BL/10. All congenic strains of mice with the B10 background, i.e. C57BL/10, B10.S, B10.BR and B10.G were found to have low eosinophil precursor capacity. Bone marrow cultures obtained from F1 hybrids (NIH x C57/BL10, SJL x C57/BL10 and SWR x C57BL/10) demonstrated high precursor numbers, indicating that low responsiveness is inherited as a recessive characteristic. When spleen cells from T. spiralis-infected, high and low responder strains of mice were stimulated in vitro with concanavalin A (Con A) or with parasite antigen, it was found that low responder phenotype strains produced quantities of two eosinophilopoietic lymphokines EDF and IL3, which were similar to, if not greater than high responder strains. This suggests that bone marrow precursor capacity and not T cell lymphokine release is an important limiting factor in determining strain-dependent eosinophilia.     

Differences in the effect of indomethacin and preincubation on the lymphoproliferative response to concanavalin A of spleen cells from low responder C57BL/6 and high responder BALB/c mice

In vivo treatment with 100 micrograms of indomethacin each 48 h for 2 weeks enhanced the proliferative response to concanavalin A (Con A) of spleen cells from mice of the C57BL/6 (B6) strain, low responder to T cell mitogens, but did not modify the response of spleen cells from mice of the high responder strain BALB/c (C). The enhancing effect of in vivo indomethacin treatment was more marked in cultures of B6 splenocytes stimulated with high, moderately supraoptimal doses of Con A than in cultures stimulated with optimal mitogen doses. Addition of indomethacin to cultures of spleen cells from untreated donors induced greater increase of the lymphoproliferative response of cells from low responder B6 than from high responder C mice. The enhancing effect of indomethacin added in vitro was observed in cultures stimulated by optimal but not by supraoptimal doses of Con A. The addition of indomethacin did not enhance the response of B6 spleen lymphocytes depleted of adherent cells. Preincubation for 24 h prior to mitogen stimulation increased the response to high Con A doses of spleen cells from low responder B6 mice whereas this procedure did not enhance lymphocyte proliferation in cultures of spleen cells from high responder C mice. Supplementation with indomethacin in vitro combined with preincubation induced additive enhancing effects on DNA synthesis by B6 spleen lymphocytes, suggesting that each treatment acts through different mechanism(s). The results indicated that spleen cells from low responder B6 strain mice are more sensitive than cells from high responder C mice to the potentiating effect of indomethacin and preincubation on the proliferative response to Con A. These observations suggest that mechanisms sensitive to indomethacin and to preincubation contribute to the depression of mitogen induced DNA synthesis in low responder B6 mice.     

Evidence for a T suppressor cell-inducing antigenic determinant shared by ADJ-PC-5 plasmacytoma and syngeneic BALB/c spleen cells

The BALB/c plasmacytoma ADJ-PC-5 induces in early stages of tumorigenesis specific T suppressor (Ts) cells apparently as the first reaction of the immune system towards the growing tumor. In attempts to apply the concept of early induction of Ts cells in neoplasia for the induction of tolerance to allografts we have tried to induce Ts cells against alloantigens by injecting allogeneic spleen cells in doses increasing exponentially from 10 to 10(5) cells/mouse. Experiments using various strain combinations failed to demonstrate induction of specific Ts cells by this procedure. To explain the apparent discrepancy between these and the tumor data the capacity of the ADJ-PC-5 tumor cells to induce Ts cells in allogeneic combinations was analyzed. ADJ-PC-5 cells are capable of inducing specific Ts cells in CBA/J mice. These Ts cells specifically suppress the induction of a primary cytotoxic T cell response of CBA/J spleen cells against BALB/c, BALB.B and (BALB/c X C57BL/6)F1 cells but not one against B10.D2 or C57BL/10 target cells. The ADJ-PC-5-induced CBA/J Ts cells have been further analyzed using a panel of different target or responder cells. From these experiments the following conclusions can be drawn: the CBA/J Ts cells are specific for an antigen which is shared by ADJ-PC-5 and normal BALB/c spleen cells. The antigen in question is not coded for by the H-2d major histocompatibility complex. The data suggest that the Ts cell-inducing antigen on ADJ-PC-5 tumor cells is not a tumor associated neoantigen but a self antigen also present on normal BALB/c lymphoid cells.     

T-Cell Subpopulations in the Bone Marrow and Spleen from Thymectomized, Irradiated Mice Reconstituted with Normal or Nude Bone Marrow Cells

Adult thymectomized and lethally irradiated (AtxXB) BALB/c mice, reconstituted with either nude or normal bone marrow (BM) cells, have been studied to evaluate numbers and mitogen responsiveness of T cells and T-cell progenitors in the BM and the spleen. Although mice reconstituted with nu/nu cells contained significant higher numbers of Thy 1.2-positive cells in the BM than mice reconstituted with normal BM cells, smaller in vitro mitogen responses were generally given by the former mice. However, spleen and BM cells of both groups responded to phytohaemagglutinin and to concanavalin A. On the basis of comparisons with responses of BM and spleen cells from normal and ATx mice, anti-Thy 1.2 treatment of cells, and 1 g velocity sedimentation experiments, it is suggested that most mitogen responders in the BM of ATxXB mice are Thy 1.2-negative, pre-thymic T-cell precursors and, accordingly, that these cells are able to differentiate to some extent in the absence of the thymus.     

CD4 T cell priming in dendritic cell-deficient mice

Bone marrow (BM) chimeras (BMC) generated from mice carrying a null (-/-) mutation in the relB gene of the NF-kappaB family represent an ideal model for in vivo studies on the role of dendritic cells (DC) in the adaptive immune response. The spleen and lymph nodes (LN) of relB(-/-) BMC contain a small number of residual DC, mainly CD8alpha(+), that fail to up-regulate MHC class II and co-stimulatory molecules after stimulation in vitro. Moreover, residual spleen DC of relB(-/-) BMC have a 4-fold decrease in the ability to uptake and process soluble model antigen, ovalbumin (OVA), and failed to prime CD4 and CD8 T cells in vitro and in vivo. In addition, they also failed to present OVA peptide to OT-II transgenic T lymphocytes at a normal 1:10 (stimulator:responder) cell ratio. In spite of these multiple DC defects, relB(-/-) BMC immunized with plasmid DNA targeted to the spleen as the site of immune induction develop a specific CD4(+) T cell response comparable to that of relB competent mice. These data demonstrate that CD4( +) T cells can be primed in the absence of functional DC and suggest that relB may gauge the T cell response in vivo.     

Requirement of parental T lymphocytes for the in vitro induction of F1 hybrid anti-parent cytotoxicity.

Primary F₁ anti-parent cell-mediated lympholysis (CML) is a specific response generated in mixed cultures by F₁ T lymphocytes with the aid of macrophage-like cells of responder and stimulator type. A model was described in which the induction of this response required T lymphocytes among stimulators, as judged by the lack of induction following T cell depletion of parental cells by (a) separation on nylon wool columns, (b) treatment with anti-Thy-1 antiserum and complement, (c) treatment of donor mice with rabbit anti-mouse thymocyte serum, or (d) congenital absence of the thymus in nu/nu donors. The parental T lymphocytes required were relatively mature, as indicated by resistance to cortisone, persistence after adult thymectomy, and sensitivity to xenogeneic anti-thymocyte antiserum. T-deprived cell populations retained the ability to induce allogeneic CML. Parental lymphoma cells, spleen cells stimulated by mitogens for T or B lymphocytes, normal cells of central and peripheral lymphoid organs, and peritoneal exudate cells, all served to a varying extent as targets for anti-parent cytotoxic effectors (CTL), but there was no close correlation with their ability to serve as stimulators. Spleen or peritoneal exudate cells of athymic mice were capable of serving as “antigenic” targets for specific F₁ anti-parent CTL, and yet failed to induced the response. Thus, the role of parental T cells in the induction of F₁ anti-parent cytotoxicity was not that of bearing the “antigen”. It was concluded that irradiated parental T lymphocytes provided back stimulation to F₁ hybrid responders, either directly or indirectly via the activation of macrophage-like cells. Under certain circumstances, however, the requirement for parental T cells can be circumvented.     

Increased expression of Ia antigens on B cells after neonatal induction of lymphoid chimerism in mice: role of interleukin 4

BALB/c mice rendered chimeric at birth by injection of 10(8) (A/J X BALB/c)F1 spleen cells develop a lupus-like autoimmune disease linked to the activation of donor B cells by host T cells. As in vitro studies previously indicated that interleukin 4 (IL4) was a mediator of the interactions between T and B cells, we analyzed the intensity of Ia antigen expression on B cells of chimeric mice. Flow cytometric analysis with anti-Ia monoclonal antibodies (mAb) revealed that B cells from spleens and lymph nodes of 2-week-old chimeric BALB/c mice displayed a two- to threefold increase in membrane Ia antigen expression, this increase still being present in spleens of 30-week-old animals. An increase in Ia antigen expression was also found in the small number of donor B cells detected in spleens and lymph nodes of chimeric mice. IL4 was the major stimulus leading to increased B cell Ia antigen expression, as this phenomenon was substantially prevented by in vivo treatment of chimeric mice with the anti-IL4 11B11 mAb. In vitro experiments revealed that host splenic T cells of chimeric mice, while unable to generate anti-donor cytotoxic T lymphocytes, secreted significant amounts of IL 4 when stimulated in mixed lymphocyte cultures (MLC) with donor alloantigens. This IL4 secretion led to an increased expression of Ia antigens on donor-type F1 B cells present in MLC. No significant increase in Ia antigen expression was found on syngeneic BALB/c B cells co-cultured with T cells from chimeric mice unless A/J B cells were added to the cultures. Taken together, these findings indicate that increased Ia antigen expression on donor B cells is induced by IL4 secreted by anti-donor T cells. IL4 released in this setting also leads to increased Ia antigen expression on host B cells through a bystander effect.     

Genetic control of mast cell development in bone marrow cultures. Strain-dependent variation in cultures from inbred mice.

A comparison was made of the capacity of bone marrow cells (BM) from genetically distinct strains of mice to develop into mast cells under defined conditions of in vitro culture. In the presence of conditioned media derived from ConA treated spleen cells from normal or Trichinella spiralis-infected mice, mast cell development occurred readily. After 21 days of culture mast cells comprised more than 90% of the total cell population. BM taken from certain strains of mice (SWR and NIH) produced large numbers of mast cells, total cell numbers increasing between 2 and 5 fold; other strains (C57BL/10 [B10] B10 congenics) produced relatively few mast cells, total cell numbers not increasing above the starting concentration or declining during culture. The genetic factors determining the strain-response phenotype (no. of mast cells in culture) were predominantly associated with the background genome. No significant differences in response were noted between the B10 congenic strains B10 [H-2b], B10.G [H-2q] or B10.BR [H-2k], which differ only at the MHC, whereas major differences were seen between B10.G and the other H-2q strains [SWR and NIH]. Response phenotype was not inherited as a simple dominant trait; F1 progeny of high x low responder strains were intermediate between the parental values. The expression of genetic influences upon mast cell response phenotype appears to be at both the level of mast cell precursor cells, as determined from limiting dilution assays of BM from high, low and F1 (high x low) strains, and at the level of mast cell proliferation, as determined by repeated sub-culture of mast cells from these strains.     

Macrophages but not B cells from aged mice are defective in stimulating autoreactive T cells in vitro

In the present study the effect of aging on the capacity of Ia+ cells to stimulate autoreactive T cells in the syngeneic mixed lymphocyte reaction (SMLR) was investigated. Using young CD4+ T cells as responders, it was observed that unseparated whole spleen cells from aged mice had normal stimulatory activity comparable to that of young spleen cells. Interestingly, however, when purified splenic adherent cells (SAC) enriched for macrophages or splenic B cells were used as stimulators, aged SAC but not aged B cells were found to be defective in stimulating autoreactive T cells. This defect in aged SAC was not due to decreased expression of Ia antigens since the percentage of Ia+ SAC and density of Ia antigen expression was similar in both young and old mice. Also, the B cells from aged mice expressed normal levels of Ia antigens. Aged SAC, when mixed with young SAC could also actively suppress the normal SMLR. However, this suppression was not due to increased prostaglandin production but was found to be associated with interleukin-1 (IL-1) regulation, inasmuch as addition of exogenous IL-1 could completely reconstitute the defective stimulatory activity of aged SAC and also abolished the suppressor activity of the SAC. Aged mice also demonstrated an intrinsic defect in the CD4+ T cells responding in the SMLR. Together, our studies on the SMLR demonstrate an age-related defect in responder autoreactive T cells and in stimulator splenic macrophages but not in the stimulatory activity of B cells.     

Genetic regulation of delayed-type hypersensitivity responses to poly (Tyr,Glu)-poly(DLAla)--poly(Lys): expression of the genetic defect in the induction and manifestation phases in H-2s and H-2f mice.

The genetic defect of H-2s and H-2s non-responder mouse strains in both the induction and manifestation phases of delayed-type hypersensitivity (DTH) responses to poly(LTyr,LGlu)-poly(DLAla)--poly(LLys)[(T,G)-A--L] was analysed. Utilizing an in vitro system to activate DTH effector T cells, we observed that non-adherent T cells of (H-2f X H-2b) F1 or (H-2s X H-2b)F1 responder mice, could not be activated on antigen bearing adherent cells of H-2f or H-2s haplotypes. On the other hand, these T cells were effectively sensitized on adherent cells derived from either F1 or parental (H-2b) responder mice. These results indicate that in these mouse strains the genetic defect, in the induction phase of DTH, is expressed at the level of the antigen presenting cell. In subsequent experiments, we were able to "correct' the non-responsiveness of H-2s recipients by transfer of educated and irradiated (H-2s X H-2b)F1 T cells together with normal F1 adherent cells. Normal non-adherent and nylon wool enriched T cells failed to restore these responses. Similarly, antigen-pulsed F1 irradiated peritoneal exudate cells could stimulate DTH responses in SJL recipients of (SJL X C57BL/6)F1 (T,G)-A--L educated cells. The genetic defect of H-2s mice in the manifestation phase of the DTH reaction is thus also expressed on the antigen presenting cell.     

Severe Graft-Versus-Host Disease in SCID Mice is Associated with a Decrease of Selective Donor Cell TCR Vβ Specificities and Increased Expression of IFN-γ and IL-4

Differences in T-cell selection and severity of graft-versus-host (GVH) disease were observed in immunodeficient C.B-17 SCID (SCID) mice after injection of allogeneic T lymphocytes from CBA/J or C57Bl/6 (B6) mice. Infiltrating donor cells were analysed in bone marrow (BM), liver and spleen of newborn recipients and 5 days post-engraftment the number of B6 cells significantly exceeded that of CBA/J cells in these organs. At that time, cells in BM of B6 and CBA/J injected recipients were augmented in intracellular IL-4, IL-10, and TNF-α, whereas only cells in B6 treated BM were increased in IFN-γ, and both treated groups of mice had up-regulated endogenous MHC class I and class II expression in the three organs. Already on day 5, and more pronounced day 10, B6 treated SCIDs had a relative decrease of four different TCR-Vβ specificities among donor cells, whereas CBA/J injected mice had an abnormal expansion of Vβ14⁺ donor T cells 10 days post injection. At the same time, the total cell contents of BM and spleen of B6 injected mice were substantially decreased, and this was paralleled by signs of severe GVHD; whereas SCIDs treated with CBA/J exhibited much milder symptoms. Moreover, adult SCID mice injected with Vβ2, 4, 8 and 14 depleted B6 T cells showed an increased percentage of infiltrating donor cells and an enhanced decrease in BM cell content compared to SCIDs treated with total B6 T cell repertoire. In vitro , the Vβ2, 4, 8 and 14 depleted population was more responsive to SCID spleen stimulators. Thus, a disturbed immunoregulation among donor T cells, caused by multiple changes in the TCR repertoire, may be responsible for inducing the severe GVHD.