Comparative analyses of thymocyte and thymic low-density adherent cell functions.

Thymocytes which have developed in the C3H thymus showed depressed proliferative responses to stimulation with anti-CD3 antibody as compared with those which have developed in the thymus of other strains of mice (i.e. AKR). The present study was conducted to analyze immunological functions of the thymic stromal cell population (low-density adherent cells, LDAC) in the C3H mice using allogeneic bone marrow (BM) chimeras established by BM transplantation in the reciprocal combination of AKR and C3H mice as donor or recipient. The thymic LDAC from C3H mice or the [AKR(donor)-->C3H(recipient)] chimeras contained a high proportion of Mac-1+ cells as compared to AKR mice or the [C3H-->AKR] chimeras. The proportion of Mac-1+ cells paralleled the IL-1- and PGE2-secreting ability of the LDAC cultured either in the presence or absence of LPS and also paralleled the antigen-presenting cell functions of the LDAC. Furthermore, after anti-CD3 stimulation the PGE2 inhibited more profoundly proliferative responses of [AKR-->C3H] or normal C3H thymocytes than those of the [C3H-->AKR] chimera or normal AKR thymocytes. A PGE2 inhibitor, indomethacin, reversed the depressed responses of the thymocytes which had developed in the C3H thymus. These findings suggest that the lower responsiveness of thymocytes from [AKR-->C3H] chimeras to anti-CD3 stimulation may be attributable to large amounts of PGE2 secreted by LDAC and/or to increased sensitivity of thymocytes themselves to PGE2.

Functional and phenotypic analyses of thymic low density adherent cells from murine bone marrow chimeras--influence on thymocyte differentiation.

Previous studies from this laboratory demonstrated a malfunction of the thymus of C3H mice to induce normal level of thymocyte differentiation. Thymocytes developed in the C3H thymus showed depressed proliferative responses to stimulation with anti-CD3 antibody compared with those developed in the other strains. This study was conducted to analyze immunological functions of the thymic stromal cell population in the C3H mice. Using allogeneic bone marrow (BM) chimeras established by reciprocal combination of AKR and C3H mice as donor or recipient, antigen presenting cell (APC) function of low density adherent cells (LDAC) in the thymus was analyzed. The thymic LDAC from C3H mice or [AKR----C3H] BM chimeras where AKR were BM donors and C3H were recipients contained high proportion of Mac-1+ cells as compared to AKR mice or [C3H----AKR] chimeras. The proportion of Mac-1+ cells paralleled the IL-1 secreting ability of the LDAC. Thus, the higher proportion of Mac-1+ cell in the thymus may be responsible for low accessory function observed in C3H thymuses. However, when APC function was analyzed using various T cell hybridomas or a T cell line, the APC functions did not necessarily correlate to the proportions of Mac-1+ cells and amounts of IL-1 produced by the LDAC. When proliferative responses of thymocytes to anti-CD3 stimulation were analyzed in the presence of prostaglandins, PGE-2 inhibited more profoundly the responses of [AKR----C3H] and normal C3H mice than those of [C3H----AKR] and normal AKR mice. Furthermore, a prostaglandin inhibitor, indomethacin, reversed the depressed responses of the former thymocytes which had developed in the C3H thymus. These findings suggest that the hyporesponsiveness of thymocytes from [AKR----C3H] chimeras to anti-CD3 stimulation may be attributable to their increased sensitivity to prostaglandin produced by LDAC.

Analysis of functional sites on a peptide antigen, p43-58, in I-A or I-E-restricted T cell responses.

It has been shown that two different sites (an agretope and an epitope) on a peptide antigen function independently in T cell responses to the antigen. By virtue of these sites, antigens, MHC molecules, and TCRs constitute trimolecule complexes which eventually result in T cell activation. In our previous reports, we have defined that residues 46 and 54 on synthetic peptide composed of residues 43-58 of pigeon cytochrome c (p43-58, AEGFSYTDANKNKGIT) and its analogs function as an agretope and residue 50 as an epitope in both I-Ab and I-Ak-carrying mice. In the present study, to extend our method to the other MHC class II molecules (I-E), we used two peptide antigens, 46D50V54R and 50V54R, which had been prepared by substitution of amino acids at positions, 46, 50 and 54 or 50 and 54 of p43-58 D, V, R or V, R, respectively, and compared the immunogenicity with those of other peptide analogs. The 46D50V54R was shown to be non-immunogenic in I-Ab-carrying mice and the 50V54R was non-immunogenic in I-Ak-carrying mice. In contrast, the 46D50V54R or 50V54R could induce I-E-restricted proliferative responses of T lymphocytes in I-Eb/k- or I-Ek/k-carrying mice, respectively. Furthermore, residues 46 and 54 were shown to function as agretopes and residue 50 as an epitope in the I-E-restricted responses as they did in the I-A-restricted responses, even though some differences were seen between peptide-I-E interaction and peptide-I-A interaction. These agretopes and epitope functioned independently.

Deficiency in early development of the thymus-dependent cells in irradiation chimeras attributable to recipient's environment.

Allogeneic bone marrow chimeras were prepared using reciprocal combinations of AKR and C3H mice. When C3H mice were recipients, the number of thymocytes recoverable from such chimeras (C3H recipient chimeras) was small as compared with that from chimeras for which AKR mice were used as recipients (AKR recipient chimeras) regardless of donor strain. The thymocytes from C3H recipient chimeras showed a profound deficiency in generating proliferative responses to stimulation by anti-CD3 mAb (2C11) or anti-TCR (alpha, beta) mAb (H57-597), even though the expression of CD3 and TCR molecules fell within the same range as that in AKR recipient chimeras. Furthermore, after stimulation with immobilized 2C11, the proportion of IL-2R+ cells in the thymocytes from C3H recipient chimeras was much less than that in AKR recipient chimeras. However, no significant difference in proliferative responses to 2C11 plus PMA, in influx of Ca2+ after stimulation with 2C11 or IL-2 production in response to 2C11 plus PMA or PMA plus A23187 was demonstrated between C3H and AKR recipient chimeras. These findings suggest that the thymocytes from C3H recipient chimeras have a deficiency in the signal transduction system as compared with chimeras for which AKR mice are the recipients. The thymic stromal component involved in this difference in the C3H recipient chimeras is discussed.

Modification of the T cell responsiveness to synthetic peptides by substituting amino acids on agretopes.

T cell receptors, major histocompatibility complex molecules, and antigens constitute tri-molecular complexes which induce T cell activation. T cells in I-Ab mice generate proliferative responses to a synthetic peptide composed of residues 43-58 of pigeon cytochrome c (p43-58) and its analogs with substitution at position 50 (50A, 50V, 50L, 50N, 50Q, 50K, and 50M). However, none of these peptides stimulate T cells in I-Ak mice. We substituted two residues at positions 46 and 54 of p43-58(50D), 50V, 50L, 50E, and 50K with two amino acids on agretopes of the I-Ak binding HEL52-61 peptide and immunized I-Ak mice with these newly synthesized peptides: 46D50D54R, 46D50V54R, 46D50L54R, 46D50E54R, and 46D50K54R. Apart from 46D50D54R, these peptides elicited T cell responses in I-Ak mice in an immunogen-specific manner, but did not stimulate those in I-Ab mice. Further, 46D50V54R inhibited competitively the responses of I-Ak restricted T cell hybridomas specific for 46D50E54R. These results demonstrate that the residues at positions 46 and 54 on the peptides act as an agretope and the residue at position 50 acts as an epitope in I-Ak mice, as in I-Ab mice, and provide the possibility of opening up a new method to prepare peptide antigens which induce T cell responses in each murine strain by introducing appropriate amino acids on agretopes.

Donor and recipient specific tolerance in cells from semi-allogeneic, H-2 subregion compatible or fully allogeneic bone marrow chimeras attributable to clonal deletion.

Specificities of tolerance induced in allogeneic bone marrow (BM) chimeras which had been established by injecting allogeneic BM cells pretreated with anti-Thy-1 mAb alone (without complement (C)) were analyzed using Simonsen's splenomegaly assay. Lymphocytes from fully allogeneic, semi-allogeneic and H-2 subregion compatible BM chimeras were specifically unresponsive to donor and recipient antigens (Ag). However, cells from H-2 subregion compatible chimeras initiated as vigorously a GVHR in F1 recipient mice, which were disparate at H-2K and I-A regions, as did spleen cells of donor mice, which were incompatible at the entire H-2 and minor histocompatibility regions of the recipients. The donor cells from such chimeras that initiated these considerable GVHR were either CD4+ or CD8+ T cells. Furthermore, synergistic effects by the CD4+ and CD8+ T lymphocytes were also observed. We found no evidence for a suppressive mechanism(s) in maintenance of the specific tolerance in allogeneic chimeras. Further, when lymphoid cells from these chimeras were adoptively transferred to irradiated mice of the donor strain and maintained for 5 days in the absence of recipient Ag (tolerogen), the adoptively transferred cells were shown to retain their unresponsiveness to the recipient Ag. These results reveal that T lymphocytes from allogeneic BM chimeras prepared by our method had been specifically induced to a tolerant state to both donor and recipient Ag and that the major mechanism of induction and maintenance of long-lasting tolerance is attributable to clonal deletion of both CD4+ and CD8+ T cell subsets rather than to the development of a population of suppressor cells of any sort.