Ia Restriction Specificity of KLH-Specific T Cells from Allogeneic Bone Marrow Chimeras Is Influenced by Histocompatibility at the H-2 and Minor Histocompatibility Loci

Ia restriction specificity involved in T cell proliferative responses to keyhole limpet hemocyanin (KLH) has been analyzed using a variety of allogeneic bone marrow chimeras. The chimeric mice were prepared by reconstituting irradiated AKR, SJL, B10.BR and B10.A(4R) mice with bone marrow cells from B10 mice. When such chimeric mice had first been primed with KLH in complete Freund's adjuvant (CFA), T cells from H-2 incompatible fully allogeneic chimeras showed significantly higher responses to KLH in the presence of antigen-presenting cells (APC) of donor strain (B10) than APC of recipient strain. However, in H-2 subregion compatible chimeras, [B10→B10.A(4R)], which were matched at the H-2D locus and at minor histocompatible loci, the T cells could mount vigorous responses to KLH with antigen-presenting cells (APC) of either donor or recipient type. The same results were obtained as well with chimeras that had been thymectomized after full reconstitution of lymphoid tissues by donor-derived cells. A considerable proportion of KLH-specific T cell hybridomas established from [B10→B10.A(4R)] chimeras exhibited both I-A^b and I-A^k restriction specificities. The present findings indicate that the bias to donor Ia type of antigen specific T cells is determined by donor-derived APC present in the extrathymic environment but that cross-reactivity to the recipient Ia is influenced to some degree by histocompatibility between donor and recipient mice, even though the histocompatible H-2D locus and minor histocompatibility loci seem not to be directly involved in the I-A restricted responses studied herein.     

Study on the possible factors influencing the expression of H-2 restriction specificity and Ir phenotype of antigen-specific proliferative T cells with various types of radiation chimeras

To better understand the factors described previously as influencing the manifestation of H-2 restriction specificity and Ir phenotype of T cells from radiation bone marrow chimeras, we also examined H-2 restriction specificity (Ir phenotype) of antigen (DNP-OVA, (T, G)-A-L, (H, G)-A-L)-specific proliferative T cells generated in various types of H-2 incompatible radiation chimeras prepared under our specific-pathogen-free (SPF) condition. The results indicated the following: (a) T cells generated in F1----parent bone marrow chimeras preferentially manifested host-type H-2 restriction specificity and Ir phenotype, regardless of the radiation dose (8.70 vs 11.59 Gy); (b) T cells recovered from twice-reconstituted F1----(PA----PB) chimeras manifested primary host (PB)-type Ir phenotype; (c) T cells which were recovered from (B10.Thy-1.1 X B10.BR.Thy-1.1)F1----parent (Thy-1.2) bone marrow chimeras and treated with anti-Thy-1.2 plus complement to deplete host-derived T cells still manifested preferentially the restriction specificity for host-type H-2; (d) PA-derived T cells which had differentiated in a fully allogeneic host (PB) environment of (PA + PB)----PB chimeras manifested fully allogeneic host-type Ir phenotype; (e) T cells from F1----parent chimeras that were prepared with 13-day fetal liver cells also manifested host H-2-restricted Ir phenotype; and (f) host preference for Ir phenotype of antigen-specific proliferative T cells was observed even in the case of F1----parent bone marrow chimeras reconstituted with "intact" bone marrow cells. The data suggest that thymic APCs, surviving host T cells or the source of stem cells (adult bone marrow vs 13-day fetal liver), do not necessarily play a significant role in the manifestation of H-2 restriction specificity and Ir phenotype of T cells generated in H-2 incompatible radiation chimeras.     

Cellular basis of the immunohematologic defects observed in short-term semiallogeneic B6C3F1 -- C3H chimeras: evidence for host-versus-graft reaction initiated by radioresistant T cells

Lethally irradiated C3Hf mice reconstituted with a relatively low dose (2 × 10⁶) of B6C3F₁ bone marrow cells (B6C3F₁ → C3Hf chimeras) frequently manifest immunohematologic deficiencies during the first month following injection of bone marrow cells. They show slow recovery of antibody-forming potential to sheep red blood cells (SRBC) as compared to that observed in syngeneic (C3Hf → C3Hf or B6C3F₁ → B6C3F₁) chimeras. They also show a deficiency of B-cell activity as assessed by antibody response to SRBC following further reconstitution with B6C3F₁-derived thymus cells 1 week after injection of bone marrow cells. A significant fraction of B6C3F₁ → C3Hf chimeras was shown to manifest a sudden loss of cellularity of spleens during the second week following injection of bone marrow cells even though cellularity was restored to the normal level within 1 week. The splenic mononuclear cells recovered from such chimeras almost invariably showed strong cytotoxicity against target cells expressing donor-type specific H-2 antigens (H-2^b) when assessed by ⁵¹Cr-release assay in vitro. The effector cells responsible for the observed anti-donor specific cytotoxicity were shown to be residual host-derived T cells. These results indicate strongly that residual host T cells could develop anti-donor specific cytotoxicity even after exposure to a supralethal dose (1050 R) of radiation and that the immunohematologic disturbances observed in short-term F₁ to parent bone marrow chimeras (B6C3F₁ → C3Hf) were due to host-versus-graft reaction (HVGR) initiated by residual host T cells. The implication of these findings on the radiobiological nature of the residual T cells and the persistence of potentially anti-donor reactive T-cell clones in long-surviving allogeneic bone marrow chimeras was discussed.     

Recipient micro-environment does not dictate the Igh-V restriction specificity of T cell suppressor inducer factor (TsiF) from allogeneic bone marrow chimera in mice

We have ascertained previously from a study of fully allogeneic irradiation chimeras in mice that the H-2 restriction of the suppressor factor (Ly-2 T suppressor factor) is determined by the post-thymic environment protected by the donor cells, rather than by the thymic environment of the recipient. In the present study, we analyzed differentiation influences that determine the Igh restriction specificities of the suppressor inducer T cell factor(s) (TsiF) that are produced by Ly-1+ splenic T cells in fully allogeneic bone marrow chimeras in mice. AKR mice that had been lethally irradiated and reconstituted with B10 marrow cells, [B10----AKR] chimeras, produced Ly-1 TsiF after hyper-immunization with sheep erythrocytes (SRBC) which suppressed antigen--specifically the primary antibody responses to SRBC that were generated in cells of the same Igh-Vb haplotype of donor strain and not those generated in cells of the recipient Igh-Va type. Similar results were obtained when Ly-1 TsiF from [B6----BALB/c] and [BALB/c----B6] chimeras were analyzed. Furthermore, the Ly-1 TsiF from [BALB/c----B6] chimeras suppressed the primary antibody responses of both BALB/c [H-2d, Igh-Va, Igh-Ca] and BAB-14 (H-2d, Igh-Va, Igh-Cb), but not those of CAL-20 (H-2d, Igh-Vd, Igh-Cd). These results demonstrate clearly that the Ly-1 TsiF from allogeneic bone marrow chimeras are donor Igh-V-restricted and are not influenced by the recipient micro-environment, presumably that provided by the thymuses of the recipient mice.     

Antiviral T cell competence and restriction specificity of mixed allogeneic (P1 + P2----P1) irradiation chimeras

Mixed irradiation bone marrow chimeras were prepared by reconstituting lethally irradiated C57BL/10 (B10) or B10.D2 mice with T cell-depleted bone marrow cells of B10 plus B10.D2 origin. These chimeras were healthy and survived well under conventional housing conditions and after experimental laboratory infections. Of a total of 17 chimeras tested, 2 died spontaneously or from the injected virus. Twelve of fifteen chimeras mounted a measurable cytotoxic T cell response to virus. Despite approximately equal percentages of B10 and B10.D2 lymphocytes in chimeras, cytotoxic T cell responses to vaccinia virus and lymphocytic choriomeningitis virus were mediated variably by either syngeneic or allogeneic donor lymphocytes; thus the H-2 type of effector T cells frequently did not correspond to the 50:50 distribution of spleen or peripheral blood lymphocytes. Cytotoxic responses were restricted exclusively to recipient H-2 type. All mixed chimeras examined were able to mount a good IgG response to vesicular stomatitis virus. These results confirm previous data suggesting that such mixed chimeras are healthy and immunocompetent and demonstrate strict recipient-determined restriction specificity of effector T cells; they also suggest that if T help is necessary for induction of virus-specific cytotoxic T cells, it does not require host-restricted interactions between helper T cells and precursor cytotoxic T cells.     

Generation of the alloreactive T cell repertoire: K region homology between H-2b T cell precursors and T cell maturation environment is required for the generation of the Kbm6-specific cytotoxic T cell repertoire

The influence of T cell genotype and T cell maturation environment on the generation of the T cell alloreactive repertoire was evaluated in the H-2b cytotoxic T lymphocyte response to Kb mutant determinants expressed by the strain B6-H-2bm6. Specifically, by constructing radiation bone marrow chimeras with B6 or B10 (H-2b) donor cells and B10.BR, B10.A(4R), B10.MBR, and B6.C-H-2bm1 irradiated mice as recipients, it was possible to investigate the major histocompatibility complex (MHC)-encoded gene products of the host environment required for the generation of a bm6-specific H-2b CTL response. The results of such experiments confirmed the previous finding that the alloreactive T cell repertoire is influenced both by T cell MHC genotype and by the MHC gene products of the T cell maturation environment. In addition, the results of the present study further demonstrated that in the chimeric donor and host genetic combinations used, it was both necessary and sufficient that there be a homology of K region-encoded determinants for the generation of a bm6-specific CTL response. Experiments utilizing a mixed responder population of unresponsive B6----B10.D2 spleen cells and responsive Lyt-2 congenic B6.Lyt-2.1 spleen cell suggested that the cellular defect(s) underlying the unresponsiveness of the chimeric cells to bm6-encoded determinants was at the level of the CTL precursor. Together, these findings indicate that an interaction of the K region-encoded gene products of the T cell and its maturation environment play a critical role in the generation of the CTL repertoire specific for bm6 mutant determinants. We discuss here the possibility that this interaction may reflect a requirement that T cells recognize such mutant allodeterminants in association with self restriction elements present on the same mutant K region-encoded molecule.     

Essential requirement of I-A region-identical host bone marrow or bone marrow-derived cells for tumor neutralization by primed L3T4+ T cells

The antitumor activity of Meth A-hyperimmunized BALB/c mouse spleen cells (Meth A-Im-SPL) was assayed by the Winn test in H-2 incompatible bone marrow chimeras in closed colony CD-1 (nu/nu), inbred DDD/1(nu/nu) (H-2s), or inbred BALB/c(nu/nu) (H-2d) mice as recipients. We found that Meth A-Im-SPL suppressed Meth A growth in the chimera nude mice which were reconstituted with bone marrow cells of the H-2d haplotype (i.e., BALB/c, DBA/2 and B10.D2), but not in the chimeras which were reconstituted with bone marrow cells of the H-2a, H-2b, or H-2k haplotype (i.e., B10.A, B10, and B10.BR). These results suggested that H-2 restriction occurred between Meth A-Im-SPL and bone marrow or bone marrow-derived cells in tumor neutralization. Furthermore, Meth A-Im-SPL did not suppress Meth 1 tumors (antigenically distinct from Meth A tumors) in the presence or absence of mitomycin C-treated Meth A in a Winn assay. These results suggested that there is tumor specificity in the "effector phase" as well as in the "induction phase". The phenotype of the effectors in the Meth A-Im-SPL was Thy-1.2+ and L3T4+, because Meth A-Im-SPL lost their antitumor activity with pretreatment with anti-Thy-1.2 monoclonal antibody (mAb) and complement or anti-L3T4 mAb and complement, but not with anti-Lyt-2.2 mAb and complement or complement alone. Positively purified L3T4+ T cells from Meth A-Im-SPL (Meth A-Im-L3T4), obtained by the panning method, suppressed the tumor growth in the chimera nude mice which were reconstituted with bone marrow cells of B10.KEA2 mice (that were I-A region-identical with Meth A-Im-L3T4 cells but not others in H-2) as well as B10.D2 cells (that were fully identical with Meth A-Im-L3T4 cells in H-2). We conclude that Meth A-Im-SPL (L3T4+) neutralized the tumors in collaboration with I-A region-identical host bone marrow or bone marrow-derived cells, and the neutralization was not accompanied by the "bystander effect."     

Limiting dilution analysis of the stem cells for T cell lineage

Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.     

A comparison of H-2 restriction of helper function using T cells from radiation chimeras and nude mice bearing thymus grafts

T cells from nude chimeras and radiation chimeras were tested for ability to cooperate with B cells of the thymus epithelium strain and B cells of the T-stem-cell strain in nude mice of appropriate strain backgrounds and also in irradiated F₁'s together with bone marrow cells of the two relevant strains (i.e., A-nu ← B or A → A × B chimera T cells were tested in A-nu and B-nu and in irradiated A × B F₁ with A bone marrow cells or B bone marrow cells). Whether the donor chimeras were antigen primed or not, all cells, when tested in nudes, provided help for the T-stem-cell type only. In contrast, in most cases, chimera T cells, tested in irradiated F₁'S, provided help for bone marrow cells of both thymus and T-stem-cell strains. These data indicate the involvement of two helper cell populations and an hypothesis to explain their interaction is presented.     

T cell specificity in twice-irradiated F1----parent bone marrow chimeras: failure to detect a role for immigrant marrow-derived cells in imprinting intrathymic H-2 restriction

In an attempt to resolve the issue of whether H-2-restricted T cell specificity is controlled by thymic epithelial cells or by cells of the macrophage/dendritic cell (M phi/DC) lineages, long-term F1----parent chimeras were subjected to secondary irradiation and reconstitution with F1 marrow cells. The rationale was that if F1 M phi/DC enter the thymus only quite slowly after irradiation, as claimed by other investigators, leaving F1----parent chimeras for a period of several months before re-irradiation would ensure that the new wave of T cells generated in the thymus of the chimeras would have no difficulty in making contact with donor-derived F1 M phi/DC. According to the view that M phi/DC rather than epithelial cells control H-2 restriction, the T cells differentiating in these chimeras would be expected to show H-2 restriction to both parental strains. In practice, T cells from twice-irradiated (1000 + 800 rad) chimeras showed strong restriction to host (thymic) H-2 determinants, the degree of restriction to host determinants being as marked as with T cells from once-irradiated chimeras. This finding applied both to T proliferative responses to KLH assayed in vitro and to T helper function for sheep erythrocytes measured in vivo. Preliminary experiments established that the initial dose of irradiation used for preparing the chimeras (1000 rad) resulted in almost total replacement of intrathymic M phi/DC by donor-derived cells within 4 wk of irradiation; M phi/DC were typed by determining their capacity to stimulate mixed-lymphocyte reactions. Collectively, the data imply that, at least under the conditions used, H-2-restricted T cell specificity is controlled by epithelial cells rather than by M phi/DC.     

Thy.1lowCD3− Cells Sorted from Nylon Wool-Passed Bone Marrow Cells Can Augment the H-2 Identical but Not Non-Identical Cytotoxic T Lymphocyte Precursors in Mixed Lymphocyte Cultures

Thy. 1^lowCD3^– cells obtained from nylon wool-passed murine bone marrow (NW-BM) cells by cell sorting did not express CD4, CD8, or T cell receptor-α/β and -γ/δ on their cell surfaces. An extremely limited number of B10.BR (H-2^k) responder lymph node (LN) cells were stimulated with B10. D2 (H-2^d) stimulator spleen cells in cultures containing the minimum required dose of rat T cell growth factor (TCGF). In these cultures, the generation of cytotoxic T lymphocytes (CTL) was very low. B10.BR Thy.1^lowCD3^– NW-BM cells, added to these cultures, could augment the CTL generation vigorously, but neither B10 (H-2^b) nor B10.D2 cells could. When B10 LN cells were used as responder cells in these cultures, B10 Thy. 1^lowCD3^– NW-BM cells could augment the CTL generation, but neither B10.BR nor B10.D2 cells could. Similar findings were obtained when Lyt-2⁺ cells or Thy.1⁺ L3T4^– (CTL precursor) cells sorted from spleen cells were used as responder cells. Both elements, rat-TCGF and Thy.1^low CD3^– NW-BM cells, were essential for this augmentation of the CTL generation in this culture system because neither one alone could augment generation, and rat-TCGF could be replaced by Thy.1⁺ Lyt-2^– helper T (Th) cells sorted from spleen cells. These findings showed that NW-BM cells could augment CTL precursors in a self-major histocompatibility complex (self-MHC)-antigen restricted manner, and further that both NW-BM cells and Th cells had different and independent functions to induce CTL.     

H-2K molecules positively select V beta 17a+ CD4(-)8+ T cells in bone marrow and thymic chimeras

Population size of V beta 17a brightly positive cells among CD4(-)8+ thymocytes was analyzed in thymic chimeras as well as bone marrow (BM) chimeras in which SWR/J mice were used as BM donors and various strains of mice including H-2Kb mutant (bm) mice as recipients. It was shown that the proportion of V beta 17a+ CD4(-)8+ thymocytes was determined by H-2K molecules expressed on thymic epithelial cells. The highest proportion was observed in Ks and Kb thymuses, the intermediate proportion in Ks/q and Kk, and the lowest in Kq thymuses. Fine analysis of the H-2Kbm molecules involved in the positive selection revealed that the region important to the selection was located on the beta-pleated floor of antigen recognition site. According to the three-dimensional class I structure, this site appears not to be directly accessible to the T cell antigen receptor. Thus, the present finding suggests that the substitutions of amino acids at this site alter the shape and charge of the peptide binding site and eventually influence the positive selection of the V beta 17a+ T cell repertoire during differentiation.     

Intrathymic T cell differentiation in radiation bone marrow chimeras and its role in T cell emigration to the spleen. An immunohistochemical study

Immunohistochemical studies were made on the regeneration of T cells of host- and donor-type in the thymus and spleen of radiation bone marrow chimeras by using B10- and B10.BR-Thy-1 congenic mice. Both the thymic cortex and the medulla were first repopulated with thymocytes of irradiated host origin, restoring the normal histologic appearance by days 11 to 14, regardless of the H-2 compatibility between the donor and the host. In Thy-1 congenic chimeras, thymocytes of donor bone marrow origin, less than 100 cells in one thymic lobe, were first recognized at day 7, when the thymus involuted to the smallest size after the irradiation. The thymocytes of donor-type then proliferated exponentially, showing a slightly faster rate when higher doses of bone marrow cells were used for reconstitution, reaching a level of 100 million by day 17 and completely replacing the cortical thymocytes of host origin by day 21. The replacement of cortical thymocytes started from the subcapsular layer in a sporadic manner. The replacement of medullary thymocytes from host- to donor-type occurred gradually between days 21 and 35, after the replacement in the cortex was completed. In the spleen, about 1 million survived cells were recovered at day 3 after the irradiation, and approximately 60% of them were shown to be host-type T cells that were observed in the white pulp areas. The host-type T cells in the spleen increased gradually after day 10, due to the influx of host-type T cells from the regenerating thymus. Thus a pronounced increase of T cells of host-type was immunohistochemically observed in the splenic white pulp between days 21 and 28, when thymocytes of host-type were present mainly in the thymic medulla. These host-type T cells were shown to persist in the spleen for a long time, as long as 420 days after the treatment. Phenotypically, they were predominantly Lyt-1+2+ when examined at day 28, but 5 mo later, they were about 50% Lyt-1+2+ and 50% Lyt-1+2-. Donor-type T cells in the spleen began to appear at about day 14 in chimeras that were transplanted with a larger dose of bone marrow cells, whereas this was slightly delayed in those grafted with a smaller dose of bone marrow cells, starting at about day 28.(ABSTRACT TRUNCATED AT 400 WORDS)     

Predominant T cell receptor gene elements in TNP-specific cytotoxic T cells.

H-2b class I-restricted, TNP-specific CTL clones were obtained by limiting dilution cloning of either short term polyclonal CTL lines or spleen cells of TNP-immunized mice directly ex vivo. Sequence analyses of mRNA coding for TCR alpha- and beta-chains of 11 clones derived from CTL lines from individual C57BL/6 mice revealed that all of them expressed unique but clearly nonrandom receptor structures. Five alpha-chains (45%) employed V alpha 10 gene elements, and four of those (36%) were associated with J beta 2.6-expressing beta-chains. The alpha-chains from these four TCR, moreover, contained an acidic amino acid in position 93 of their N or J region-determined sequences. Clones isolated directly from spleen cells carried these types of receptors at lower frequency, 27% V alpha 10 and 19% J beta 2.6, indicating that bulk in vitro cultivation on Ag leads to selection for these particular receptors. However, even in TNP-specific CTL cloned directly ex vivo, V alpha 10 usage was increased about fivefold over that in Ag-independently activated T cells in H-2b mice (4 to 5%). The selection for V alpha 10/J beta 2.6-expressing cells was obtained repeatedly in other TNP-specific CTL lines from C57BL/6 mice but not in FITC-specific CTL from the same strain or in TNP-specific CTL lines from B10.BR (H-2k) or B10.D2 (H-2d) mice. We conclude from this (a) that the selection for V alpha 10/J beta 2.6+ T cells is driven by the complementarity of these receptors to a combination of TNP and MHC epitopes and (b) that predominant receptor structures reflect the existence of a surprisingly limited number of "T cell-relevant" hapten determinants on the surface of covalently TNP-modified cells.     

Generation of cytotoxic T cells specific for minor histocompatibility antigens by cross challenge in vitro with H-2 disparate adherent cells

Although it is well known that an H-2-restricted cytotoxic T cell response to minor histocompatibility antigens (MIHA) can be primed in vivo with H-2 disparate spleen cells, it has not been previously possible to induce cytotoxic T lymphocyte (CTL) precursors (CTLp) in vitro by this type of challenge. In this work, we demonstrate that the inability to cross challenge in vitro is due to the existence of inhibitory effects that can be obviated by cell fractionation, and to insufficient priming in vivo. BALB/c CTLp (H-2d) that have been repeatedly primed in vivo with B10.D2 can be challenged in vitro with C57BL10/J (H-2b) or B10.BR (H-2k)-adherent cells to generate CTL able to lyse B10.D2 (H-2d) target cells. The H-2 restriction properties of the cross-challenged CTL specific for MIHA were analyzed by using the technique of cold target competition. Within the limits of detection in bulk cultures, the entire response appeared to be H-2 unrestricted, whether the cross challenge was with intact C57BL10/J-adherent cells, or with membrane fragments of C57BL10/J presented by BALB/c adherent cells. The frequency of CTLp responsive to cross challenge was analyzed by limiting dilution, with cold target competition at each cell number to establish the restriction properties of the MIHA-specific CTL induced. We were able to detect two subsets of H-2-unrestricted CTLp responsive to intact C57BL10/J-adherent cells; one present at high frequency (1/250 T cells) and subject to suppressive effects at high cell number, and a second present at lower frequency (1/9800 T cells). There appeared to be a relatively infrequent subset of H-2-restricted CTLp as well (1/52,500 T cells). The frequency of CTLp responsive to cross challenge is of comparable magnitude to the frequency of H-2-restricted CTLp responsive to H-2-matched cells bearing MIHA. These observations are discussed in relationship to immunodominance and clonal dominance effects in the response to MIHA.     

Functional requirements of (Phe,G)-A--L-specific T-cell clones of (H-2 b x H-2 kF1 origin

T-cell clones specific for the synthetic polypeptide antigen poly(LPhe, LGlu)-poly(DLAla)--poly(LLys) of (C57BL/6 x C3H/HeJ)F₁ origin were tested for their biological activities. One group of clones was restricted in its proliferative response to the H-2 ^b haplotype, the second to the H-2 ^k haplotype, and the third to the F₁ unique Ia determinants. All the clones which proliferated in response to antigen secreted interleukin-2 (IL-2) following stimulation. The H-2 restriction of the IL-2 secretion was the same as that of the proliferation. Two of the clones tested, C.6 and C.10, could provide help to B cells in antibody production. However, the genetic restriction profile of the helper activity was less stringent than that for the proliferative response. Thus, C.6, which proliferated in the presence of F₁ antigen-presenting cells only, could help B cells and accessory cells of C3H/HeJ. C.10, which was restricted in its proliferative response to the H-2 ^b haplotype, could collaborate with B cells and accessory cells of the H-2 ^k haplotype as well. The antibody response of both clones was restricted to the parental or F₁ strains.Abbreviations used in this paper (T, G)-A-L poly-(LTyr, LGlu)poly(DLAla)--poly(LLys) - (Phe, G)-A--L poly(LPhe, LGlu)-poly(DLAla)--poly(LLys) - APC antigen-presenting cells - Con A concanavalin A - FCS fetal calf serum - IL-2 interleukin-2     

Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.     

Influence of a Small Number of Mature T Cells in Donor Bone Marrow Inocula on Reconstitution of Lymphoid Tissues and Negative Selection of a T Cell Repertoire in the Recipient

Allo-chimerism and clonal elimination of self antigen (Ag) (Ia + Mls-1^a) reactive Vβ6+ T cells were analyzed and compared between allogeneic bone marrow (BM) chimeras reconstituted with BM cells which had been treated with anti-Thy-1 monoclonal antibody (mAb) plus complement (C) (T^– chimeras) and BM chimeras which had been reconstituted with BM cells pretreated with anti-Thy-1 mAb alone (T⁺ chimeras). When lethally irradiated AKR (Mls-1^a) mice were reconstituted with BM cells from B10 or B10 H-2 congenic mice, both T⁺ and T^– chimeras were entirely free of signs of graft-versus-host reaction (GVHR). However, complete replacement of the AKR lymphoid tissues by donor BM cells was accomplished at an early stage in T⁺ chimeras but not in T^– chimeras. On the other hand, clonal elimination of Vβ6⁺ T cells reactive to the recipient Ag (Mls-1^a) was abolished in T⁺ chimeras but successfully induced in T^– chimeras. The Vβ6⁺ T cells not eliminated in T⁺ chimeras showed depressed responses against Mls-1^a antigens. The findings herein demonstrate that T cells which contaminate a BM inoculum survive in recipient mice after treatment with anti-Thy-1 mAb without C in vitro followed by BMT. The surviving T cells have been estimated to represent fewer than 0.5% of the BM cells inoculated. These cells appear to accelerate the full replacement of recipient lymphoid tissues by donor cells. Furthermore, the T cells which survive in the marrow inoculum influence eventually the development of a tolerant state in the T cell repertoire of the donor.     

Host-determined T cell fine specificity for self-H-2 in radiation bone-marrow chimeras of standard C57BL/6 (H-2b) mutantHz1 (H-2ba), and F1 mice

Reciprocal radiation bone-marrow chimeras were produced between the standard C57BL/6 (=B6) and the mutant B6.C-H-2 ^ba (=Hz1) strain. When infected with vaccinia virus, these chimeras, as well as an (Hz1 × B6)= Hz1 chimera, produced cytotoxic cells that killed vaccinia-infected H-2K^kH-2D^b target cells but failed to kill virus-infected H-2K^bH-2D^d cells. Virus-infected (Hz1 × B6)F₁ B6 chimeras, however, killed both types of target. These experiments demonstrate strict T-cell specificity capable of differentiating between two molecules that apparently differ by a single amino acid substitution.     

Cytotoxic T lymphocyte response to minor H-42a alloantigen in H-42b mice: clonal inactivation of the precursor cytotoxic T lymphocytes by veto-like spleen cells that express the H-42a antigen

When (B10.BR X CWB)F1 (BWF1; H-2k/b) mice carrying the H-42b allele at the minor H-42 locus were injected with H-42a C3H.SW (CSW; H-2b) or C3H (H-2k) spleen cells (SC), self-H-2Kb restricted anti-H-42a pCTL in the BWF1 recipients were primed and differentiated to anti-H-42a CTL after in vitro stimulation with (B10.BR X CSW)F1 (BSF1; H-2k/b, H-42b/a) SC. In contrast, anti-H-42a pCTL in H-42b mice were inactivated by injection with H-42-congenic H-42a SC, and stable anti-H-42a CTL tolerance was induced. Preference of H-2Kb restriction of anti-H-42a CTL was strict, and self-H-2Kb-restricted anti-H-42a CTL did not lyse target cells carrying H-42a antigen in the context of H-2Kbm1. Involvement of suppressor cells in the anti-H-42a CTL tolerance was ruled out by the present cell transfer study and the previous cell-mixing in vitro study. Notably, treatment with anti-Thy-1.2 antibody (Ab) plus complement (C) wiped out the ability of CSW SC in the priming of anti-H-42a pCTL of BWF1 mice but left that of C3H SC unaffected, and injection of the anti-Thy-1.2 Ab plus C-treated CSW SC induced anti-H-42a CTL tolerance in the BWF1 recipients. Furthermore, H-42a/b, I-Ab/bm12 [CSW X B6.CH-2bm12 (bm12)]F1 SC could not prime anti-H-42a pCTL in H-42b, I-Ab (CWB X B6)F1 recipients, whereas H-42a/b, I-Ab (CSW X B6)F1 SC primed anti-H-42a pCTL in H-42b, I-Ab/bm12 (CWB X bm12)F1 recipients. The unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC was sometimes corrected by immunization of H-42b female mice with H-42-congenic H-42a male SC. Taking all of the results together, we propose the following. Unresponsiveness of anti-H-42a pCTL in H-42b mice to H-42-congenic H-42a SC is caused by "veto cells" contained in the antigenic H-42a SC. Anti-H-42a pCTL in the H-42b recipients directly interacting with H-42-congenic H-42a SC, which bear H-42a antigen and H-2Kb restriction element, are inactivated or vetoed.(ABSTRACT TRUNCATED AT 400 WORDS)