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.     

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.     

Clonal deletion of self-reactive T cells at the early stage of T cell development in thymus of radiation bone marrow chimeras

Sequential appearance of T cell subpopulations occurs in the thymocytes of irradiated C3H/He mice (H-2k, Mls-1b2a, Thy-1.2) after transplantation with bone marrow cells of AKR/J mice (H-2k, Mls-1a2b, Thy-1.1) (AKR----C3H chimeras). The donor-derived thymocytes of AKR----C3H chimeras on day 14 after bone marrow transplantation (BMT) contained a large number of blastlike CD4+CD8+ cells which represent relatively immature thymocytes, whereas those on day 21 after BMT consisted of small sized CD4+,CD8+ cells which represent a great part in normal thymocytes. To define the developmental stage at which clonal deletion of self-reactive T cells occurs in adult thymus, we followed the fate of V beta 6- or V beta 11-bearing T cells in the donor-derived thymocytes at the early stage of AKR----C3H chimeras. Mature thymocytes expressing high intensity of V beta 6 or V beta 11, which are involved in recognition of Mls-1a or MHC I-E gene products, respectively, were deleted from the donor-derived thymocytes on day 21. Immature thymocytes expressing low intensity of V beta 6 in CD3low thymocyte fraction decreased in proportion, whereas those expressing low intensity of V beta 11 rather increased in proportion in the donor-derived thymocytes of AKR----C3H chimeras from day 14 to day 21 after BMT. These results suggest that the clonal deletion of V beta 6-positive cells occurs just at the stage of immature CD3lowCD4+CD8+ cells, whereas the clonal deletion of V beta 11-positive cells may begin at the transitional stage from CD3lowCD4+CD8+ cells to CD3high single positive cells. Timing of negative selection of thymocytes may vary in distinct T cells capable of recognizing different self-Ag.     

Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients

(AKR x NZB)F1 mice possess the dominant genes, Akv-1, Akv-2, Nzv-1a and Nzv-2a, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F1 thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F1 bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F1 thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system.     

Thymic epithelial genotype influences the production of recombinant leukemogenic retroviruses in mice

By using T1 oligonucleotide fingerprinting and mapping techniques, we analyzed the genomic structure of retroviruses produced by thymocytes and splenocytes of reciprocal bone marrow-and thymus-grafted chimeras. We found that the genetic factor(s) derived from NZB mice that suppresses the development of thymic leukemia in (AKR X NZB)F1 mice also prevents the formation of recombinant leukemogenic viruses and the expression of preleukemic changes in the (AKR X NZB)F1 thymocytes. The NZB mouse gene or genes appeared to exert this suppressive effect by acting on the thymic reticuloepithelial cells and not on the thymic lymphocytes of (AKR X NZB)F1 hybrids. Prospective studies with thymic epithelial grafts from young mice showed that the AKR thymic epithelium could mediate the formation and expression of leukemogenic recombinant viruses and preleukemic changes in thymocytes that lead to the development of thymic leukemia, whereas the (AKR X NZB)F1 thymic epithelium was deficient in this regard. Our results also confirmed a previous observation that during in vivo generation of recombinant leukemogenic viruses, the acquisition of polytropic virus-related sequences in the 3' portion of the p15E gene and the U3 region and in the 5' part of the gp70 gene can occur independently.     

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.     

Development of donor-derived thymic lymphomas after allogeneic bone marrow transplantation in AKR/J mice

The transplantation of bone marrow cells from BALB/c (but not C57BL/6 and C3H/HeN) mice was observed to lead to the development of thymic lymphomas (leukemias) in AKR/J mice. Two leukemic cell lines, CAK1.3 and CAK4.4, were established from the primary culture of two thymic lymphoma, and surface phenotypes of these cell lines found to be H-2d and Thy-1.2+, indicating that these lymphoma cells are derived from BALB/c donor bone marrow cells. Further analyses of surface markers revealed that CAK1.3 is L3T4+ Lyt2+ IL2R-, whereas CAK4.4 is L3T4- Lyt2- IL2R+. Both CAK1.3 and CAK4.4 were transplantable into BALB/c but not AKR/J mice, further indicating that these cells are of BALB/c bone marrow donor origin. The cells were found to produce XC+-ecotropic viruses, but xenotropic and mink cell focus-forming viruses were undetectable. Inasmuch as thymic lymphomas are derived from bone marrow cells of leukemia-resistant BALB/c strain of mice under the allogeneic environment of leukemia-prone AKR/J mice, this animal model may serve as a useful tool not only for the analysis of leukemic relapse after bone marrow transplantation but also for elucidation of the mechanism of leukemogenesis.     

Sequential changes of thymocyte surface antigens with presence or absence of graft-vs-host reaction following allogeneic bone marrow transplantation

Lethally irradiated AKR mice were reconstituted with C57BL/6 bone marrow cells. Though the allogeneic marrow transplantation protected AKR recipients from acute irradiation deaths, the mice given unmanipulated marrow developed severe GVHR disease, and 80% died within 50 days. The thymus and spleen from the recipient mice, following recovery of body weight between the 10th and 20th days, gradually involuted and became miniscule after Day 30. Thymocytes from recipients were found to be entirely of donor cell type by Day 15. Thereafter, however, as the graft versus host reaction (GVHR) developed, changes in sensitivity of the thymocytes to four different alloantisera directed toward donor histocompatibility antigens (H-2^b, Thy 1.2, Lyt 1.2, and Lyt 2.2) were observed and these changes were associated with changes in antigen expression or quantity of Thy 1 antigens on the thymocytes. A different pattern of changes was observed in antigen expression on thymocytes in mice given B6 marrow cells that had been pretreated with anti-Thy 1 serum which prevented initiation of graft-vs-host disease and in the mice which received marrow not so treated and which regularly led to graft-vs-host disease. By contrast, the amount of H-2 antigen on the thymocytes from chimeras with or without GVHR was elevated equally. The mechanisms of these changes are discussed.     

Experimental Models for Prevention of Graft-versus-Host Reaction in Bone Marrow Transfusion

Induction and suppression of splenomegaly and cytotoxicity against C57BL/6 cells were studied in (AKR × C57BL/6) F1 hybrid adult mice after the transfer of AKR lymphoid and bone marrow cells. 1) Splenomegaly and cytotoxicity were dissociated in the developmental stages of the graft-versus-host reaction. When lymphoid and bone marrow cells of normal AKR mice were injected into F1 recipients, splenomegaly was prominent on days 5 and 7, but cytotoxicity of spleen cells was not detected. Splenomegaly became less prominent but the cytotoxicity became detectable on day 14 after the injection. 2) Cytotoxic activity of spleen cells of F1 recipients was suppressed by the treatment of AKR donors with C57BL/6 lymphoid cells in Freund's complete adjuvant. Splenomegaly, however, was substantially enhanced by such a treatment of the donors. On the other hand, induction of the cytotoxic activity was facilitated by the treatment of donors with C57BL/6 skin grafts. 3) F1 hybrid mice could be protected from the graft-versus-host reaction by the injection of AKR anti-C57BL/6 serum or pretreatment of AKR donors with sonicated cellular antigens of C57BL/6.     

Experimental models for prevention of graft-versus-host reaction in bone marrow transfusion. I. Selective suppression and augmentation of splenomegaly and cytotoxicity.

Induction and suppression of splenomegaly and cytotoxicity against C57BL/L cells were studied in (AKR X C57BL/6) F1 hybrid adult mice after the transfer of AKR lymphoid and bone marrow cells. 1) Splenomegaly and cytotoxicity were dissociated in the developmental stages of the graft-versus-host reaction. When lymphoid and bone marrow cells of normal AKR mice were injected into F1 recipients, splenomegaly was prominent on days 5 and 7, but cytotoxicity of spleen cells was not detected. Splenomegaly became less prominent but the cytotoxicity became detectable on day 14 after the injection. 2) Cytotoxic activity of spleen cells of F1 recipients was suppressed by the treatment of AKR donors with C57BL/6 lymphoid cells in Freund's complete adjuvant. Splenomegaly, however, was substantially enhanced by such a treatment of the donors. On the other hand, induction of the cytotoxic activity was facilitated by the treatment of donors with C57BL/6 skin grafts. 3) F1 hybrid mice could be protected from the graft-versus-host reaction by the injection of AKR anti-C57BL/6 serum or pretreatment of AKR donors with sonicated cellular antigens of C57BL/6.     

Bone marrow-derived cells are essential for intrathymic deletion of self-reactive T cells in both the host- and donor-derived thymocytes of fully allogeneic bone marrow chimeras

The fate of self-reactive T cells was examined in both the host- and donor-derived thymocytes of fully allogeneic bone marrow (BM) chimeras of two strain combinations of AKR/J (H-2k, IE+, Thy-1.1, Mls-1a2b) and C57BL/6 (H-2b, IE-, Thy-1.2, Mls-1b2b). Sequential appearance of host- and donor-derived T cells occurred in the thymus of both AKR----B6 and B6----AKR chimeras in which 5 x 10(6) of T cell-depleted BM cells were used to reconstitute recipients lethally irradiated with 950 rad. Thymocytes bearing V beta 6 high, which recognize MHC class II IE-binding Ag encoded by Mls-1a allele, were detected in neither host- nor donor-derived thymocytes of AKR-B6 chimeras in which Mls-1a and IE were expressed only by the BM-derived cells. Thymocytes bearing V beta 11high capable of recognizing IE were also deleted in the host- and donor-derived thymocytes of the AKR----B6 chimeras. One million of BM cells were inadequate to deletion of the B beta 6high and V beta 11high T cells in the host-derived thymocytes of these chimeras. On the other hand, significant number of V beta 6high and V beta 11high thymocytes were detected in both the host- and donor-derived thymocytes in B6----AKR chimeras where sufficient dose of IE- stem cells were used to reconstitute irradiated Mls-1aIE+ recipients. These results suggest that clonal deletion of the host- and donor-reactive T cells in both the host- and donor-derived thymocytes is an important mechanism for the induction of transplantation tolerance in allogeneic BM chimeras and that BM-derived APC may be essential for the intrathymic elimination of both the host- and donor-reactive T cells in the BM chimeras.     

c-fos expression interferes with thymus development in transgenic mice

To study the function of the proto-oncogene c-fos in hematopoietic tissues, transgenic mice were generated that express c-fos from the H2-Kb promoter in several organs. These H2-c-fos mice have enlarged spleens and hyperplastic thymuses containing an increased number of thymic epithelial cells. The exogenous c-fos expression specifically affects T cell development in the thymus, thereby increasing the fraction of mature thymocytes. Results obtained with bone marrow radiation chimeras suggest that the altered distribution of T cell subsets is not a direct effect of c-fos expression within the T cell lineage. No changes in the proportion of hematopoietic cell lineages are seen in the spleen, and these mice do not develop lymphoid malignancies. B and T cell function, however, is impaired, and H2-c-fos mice are immune deficient. It appears that c-fos specifically stimulates the proliferation of thymic epithelial cells, and may thus indirectly affect T cell development.     

Strain difference in the radiosensitivity of immunocompetent cells and its influence on the residual host-vs-graft reaction in lethally irradiated mice grafted with semiallogeneic bone marrow

A striking difference in radiosensitivity was noted between C3H/He (H-2k) and C57BL/6J (H-2b) strain mice when assessed by primary anti-SRBC PFC response of intact animals and primary cell-mediated lympholysis (CML) response of spleen cells to allogeneic cells in vitro, the C3H strain being more radioresistant. On the other hand, when C3H and B6 mice were exposed to 6.62 to 10.40 grays (Gy) of x-rays and then were transplanted with 2 X 10(6) bone marrow cells from B6C3F1 (H-2b/k) donor mice within 3 hr or at 24 hr after radiation exposure, the early mortality caused by residual host-vs-graft (HVG) reaction was much higher when C3H mice were used as recipients. Furthermore, the proportion of surviving animals manifesting host-type lymphohemopoiesis, i.e., host-type revertants, was much higher in B6C3F1 to C3H than in B6C3F1 to B6 combination. Spleen cells from such host-type revertants manifested strong anti-donor reactivity when assessed by mixed lymphocyte reaction (MLR) and/or CML in vitro. Increase of radiation doses to the recipients to 10.40 Gy resulted in 100% survival and 100% donor-type lymphohemopoiesis in both groups of chimeras. These results indicate strongly that a genetic difference in radiosensitivity of immune system of the recipients can greatly influence the magnitude of residual HVG reactions observed in hybrid to parental strain bone marrow transplantation in mice.     

Thymic epithelium is programmed to induce preleukemic changes in retrovirus expression and thymocyte differentiation in leukemia susceptible mice: studies on bone marrow and thymic chimeras

In the leukemia-prone AKR thymus, ecotropic and xenotropic-related viruses are expressed that generate leukemogenic recombinant viruses before the onset of leukemia. We have shown previously that (AKR X NZB)F1 hybrid mice do not develop leukemia because they severely restrict the expression of these retroviruses in their thymuses. The thymic microenvironment of the (AKR X NZB)F1 mice appeared to be of particular importance in determining this restriction, which was specified by an NZB-derived genetic influence. In the present study we analyze reciprocal thymus graft and irradiation bone marrow chimeras to establish that this influence is exerted by thymic reticuloepithelial cells. Prospective studies with thymic epithelial grafts from young mice show that the AKR thymic epithelium can simultaneously induce the amplified expression of retroviral genes, and changes in patterns of thymocyte differentiation that precede the development of leukemia, whereas the (AKR X NZB)F1 thymic epithelium is deficient in this regard.     

Interactions of bone marrow cells from young and old mice with syngeneic and allogeneic thymic tissue

Age-related changes manifested in MHC-linked recognition of bone marrow (BM) cells by the thymic stroma were studied in vitro model of thymus-BM chimeras. Fetal thymuses (FT) depleted of self-lymphocytes were colonized with BM cells from syngeneic and allogeneic donor mice. When cells from young (3-month-old) or old (24-month-old) donors syngeneic to the stroma were seeded in a mixture with cells of allogeneic young origins (C57BL/6J-Thy1.2 and ARK/J-Thy1.1 seeded onto C57BL/6J FT), the syngeneic cells showed an age-related developmental advantage. Accordingly, cells from the old syngeneic mice manifested a significantly reduced capacity to compete with allogeneic cells when compared with the young syngeneic cells. When allogeneic BM cells from young or old mice were seeded onto the thymic stroma in a mixture with BM cells from young donors syngeneic to that stroma (BALB/c-Thy1.2 mixed with C57BL/Ka-Thy1.1 seeded onto C57BL/6J or C57BL/Ka FT), the Thy1+ cells which developed were mainly of syngeneic origin. The age of the allogeneic cells had no significant effect on the results. However, when old allogeneic cells were mixed with old syngeneic cells, the developmental advantage of the syngeneic cells was not manifested. When seeding of allogeneic cells was followed 1 day later by seeding of syngeneic cells, the syngeneic advantage was eliminated, suggesting that the MHC-linked competition began during the first 24 hr of contact with the thymic tissue. When BM-derived thmocytes grown in FT explants were transferred onto second FT recipient explants of the same genotype as the first ones, the syngeneic advantage was abolished, suggesting either that the thymic microenvironment was modified as a result of colonization or that it induced a change in the BM cells. In this respect, the young allogeneic BM-derived thymocytes showed a significant advantage when compared with the old cells. Thus, the MHC-linked syngeneic preference in the early development of BM cells is also manifested in aging mice, yet at a level that is significantly reduced compared with that seen in the young mice.     

Allogeneic bone marrow transplantation in conventional mice: I. Effect of antibiotic therapy on long term survival of allogeneic chimeras.

In the present communication the beneficial effect of long term antimicrobial treatment with poorly absorbable antiboitics on the survival of allogeneic bone marrow chimeras was investigated. The combination of C57Bl mice as bone marrow donors and CBA/CA mice as irradiated recipients (800 rad) was used because of their strong histoincompatibility on the H-2 loci. All allografted recipients received 10 X 10(6) bone marrow cells. The majority of the recipients, which were rendered gnotobiotic by an antimicrobial treatment, achieved stable long term chimerism. In contrast, the conventional chimeras died from secondary disease within 9 weeks after transplantation. As early as 14 days after allogeneic bone marrow grafting the gnotobiotic recipients tolerated the reassociation with a conventional microflora without a change in the rate of mortality. Bone marrow cells (8 X 10(6) i.v.) and spleen cells (2 X 10(6) i.v.) collected from allogeneic chimeras failed to induce graft-versus-host-reaction (GVH) in a second lethally irradiated host. The data indicate, that the high rate of mortality in murine allogeneic bone marrow chimeras results from delayed GVH-reaction and systemic infection. The marrow graft, once established seems to exert tolerance against the allogeneic host. The pathogenesis of the systemic infection has not yet been worked out. It is assumed that it originates from bacteremia, induced by radiation dependent lesions of the epithelial integrity and defected lymphatic tissue in the gut.     

An absence of T cells in murine bone marrow allografts leads to an increased susceptibility to rejection by natural killer cells and T cells

The mechanisms behind the increased incidence of marrow graft failure in recipients that receive allogeneic marrow depleted of T cells were studied. Recipient mice were lethally irradiated and challenged with bone marrow cells (BMC) from C.B-17 +/+ (+/+) donors. Radioisotope 125IUdR incorporation was assessed 5 to 7 days after transfer to determine the extent of engraftment. Some groups received BMC in which the T cells were removed by treatment with antibody and C. In addition, some groups received BMC from T cell-deficient C.B-17 scid/scid (SCID) mice to determine the postulated need for donor T cells in hematopoiesis and engraftment. In a model system that distinguishes between possible host NK cell and radioresistant T cell-mediated rejection of marrow allografts, it was determined that the absence of donor T cells in a marrow graft does not affect engraftment in syngeneic recipients. However, both host NK cell and radioresistant T cell rejection was markedly enhanced when SCID BMC or BMC from C.B-17 +/+ donors that had T cells removed by antibody and complement were infused into irradiated allogeneic recipients. Furthermore, the addition of alloreactive thymocytes as a source of T cells could abrogate this increased susceptibility of the BMC to host rejection mechanisms. As determined by histology and 59Fe uptake, the addition of thymocytes resulted in enhanced erythropoiesis. These results suggest that the increased incidence of marrow graft failure when BMC depleted of T cells are used is a result of active rejection by host effector cells and that the adverse effect of marrow T cell depletion can be reversed by the addition of thymocytes.     

Genetically determined resistance to foreign bone marrow transplantation in mice: characterization of the effector cells

Mice of most strains show a genetically determined ability to reject a variety of foreign marrow grafts even after lethal irradiation. The phenomenon is both host strain and donor marrow graft-dependent. To characterize the effector cell responsible for graft rejection, attempts were made to 1) determine to what morphologic subclass it belongs; 2) determine its life span; and 3) establish whether genetically different host environments influence the functioning of the effector cell. Mice of the 129/J strain (normally nonresistant), C57BL/6 strain (made non-resistant), and the homozygous mutants of C57BL/6, i.e., C57BL/6 (bg/bg), were recipients of C57BL/6 marrow or spleen cells. After lethal irradiation, hosts were given marrow or spleen cells from normal, strongly resistant C57BL/6 donors pretreated with a) 950 R whole body irradiation or b) twice daily injections for 4 days of the cell cycle toxic drug hydroxyurea followed by 950 R. In other cases, hosts were recipients of the lymphoid cell-rich fraction of marrow from irradiated C57BL/6 donors or adherent cells taken from cultures of marrow cells of unirradiated C57BL/6 donors. Three hours after receiving C57BL/6 marrow or spleen cells, irradiated hosts were given allogeneic DBA/2 marrow (always strongly rejected by C57BL/6 mice and always accepted by 129/J strain mice). Seven days later, host spleens were removed and the numbers of microscopic colonies were counted from subserial sections. The results demonstrate that 1) mice either normally or rendered nonresistant to a marrow allograft can be made to develop resistance by the administration of either whole spleen cells or marrow lymphoid cells from lethally irradiated strongly resistant donors; 2) adherent cells from cultures of marrow from strongly resistant mice are ineffective in conferring resistance; 3) the cell effective in conferring resistance has a life span greater than 4 but less than 7 days; and 4) the effector cell can function in genetically different environments of nonresistant strains.     

Similarity and difference in the mechanisms of neonatally induced tolerance and cyclophosphamide-induced tolerance in mice

The mechanisms of cyclophosphamide (CP)-induced tolerance were investigated by comparing with those of neonatally induced tolerance. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were given i.v. either AKR/J Sea (AKR; H-2k, Mls-1a) or (AKR x C3H)F1 (AKC3F1; H-2k, Mls-1a/b) spleen cells and treated i.p. with CP 2 days later, a long-lasting skin allograft tolerance to AKR was induced in each case without any signs of graft-vs-host disease (GVHD). However, typical signs of GVHD were observed in the C3H mice neonatally tolerized with AKR spleen cells, but not in those tolerized with AKC3F1 spleen cells. The expression of TCR V beta 6, which is strongly correlated with the reactivity to Mls-1a Ag (of donor AKR origin), in the periphery was quite different between the two types of tolerant C3H mice. Namely, in the lymph nodes of the C3H mice tolerized with AKR spleen cells and CP, only CD4(+)-V beta 6+, but not CD8(+)-V beta 6+, T cells selectively disappeared, whereas both of them were abrogated in the lymph nodes of the C3H mice neonatally tolerized of AKR. By contrast, in the thymus of the two types of tolerant C3H mice, both CD4+CD8- and CD4-CD8+ single-positive thymocytes expressing TCR V beta 6 were clonally deleted, suggesting that the thymic involvement was the same in each type of tolerance. These results suggest that the preferential disappearance of the CD4(+)-V beta 6+ T cells (of host origin) and the effector T cells of GVHD (of donor origin) occurred only in the periphery of the C3H mice tolerized with AKR spleen cells plus CP and was attributable to the destruction of Ag-stimulated T cells by the CP treatment. In contrast, the intrathymic clonal deletion of immature V beta 6+ T cells was a common mechanism for both of the tolerance induction systems.     

Stimulation by normal and leukemic mouse sera of colony formation in vitro by mouse bone marrow cells

Using a modification of the agar gel method for bone marrow culture, serum from various strains of mice has been tested for colony stimulating activity. Ninety percent of sera from AKR mice with spontaneous or transplanted lymphoid leukemia and 40–50% of sera from normal or preleukemic AKR mice stimulated colony formation by C57B1 bone marrow cells. Sera from 6% of C3H and 30% of C57B1 mice stimulated similar colony formation. The incidence of sera with colony stimulating activity rose with increasing age. All colonies were initially mainly granulocytic in nature but later became pure populations of mononuclear cells. Bone marrow cells exhibited considerable variation in their responsiveness to stimulation by mouse serum. Increasing the serum dose increased the number and size of bone marrow cell colonies and with optimal serum doses, 1 in 1000 bone marrow cells formed a cell colony. Preincubation of cells with active serum did not stimulate colony formation by washed bone marrow cells. The active factor in serum was filterable, non-dialysable and heat and ether labile.