Opposing regulation of B cell receptor-induced activation of mitogen-activated protein kinases by CD45.

In this study, we examined the contribution made by CD45 to B cell antigen receptor (BCR)-induced activation of mitogen-activated protein kinase (MAPK) family members. We found that CD45 negatively regulated BCR-induced c-Jun NH(2)-terminal kinase (JNK) and p38 activation in immature WEHI-231 cells, whereas in mature BAL-17 cells, CD45 positively regulated JNK and p38 activation and negatively regulated extracellular signal-regulated kinase activity. Furthermore, cooperative action of JNK and p38 dictated BCR-induced inhibition of growth. Thus, CD45 appears to differentially regulate BCR-induced activation of MAPK members, and can exert opposing effects on JNK and p38 in different cellular milieu, controlling the B cell fate.

CD45 is required for CD40-induced inhibition of DNA synthesis and regulation of c-Jun NH2-terminal kinase and p38 in BAL-17 B cells.

Stimulation of B cell antigen receptor (BCR) may induce proliferation, differentiation, or apoptosis, depending upon the maturational stage of the cell and the presence or absence of signals transmitted via coreceptors. One such signal is delivered via CD40; for instance, ligation of CD40 rescues B cells from BCR-induced apoptosis. Here we show that, in contrast to WEHI-231 cells, CD40 ligation did not reverse BCR-induced growth inhibition in the BAL-17 mature B cell line and CD40 ligation itself inhibited proliferation. This inhibitory signaling was not observed in CD45-deficient cells. Further analyses demonstrate that transfection of dominant-negative form of SEK1 or treatment with SB203580 strongly reduced CD40-induced inhibition of BAL-17 proliferation, suggesting a requirement for c-Jun NH2-terminal kinase and p38 in CD40-induced inhibition of proliferation. Interestingly, CD40-initiated activation of c-Jun NH2-terminal kinase and p38 was enhanced and sustained in CD45-deficient cells, and these phenotypes were reversed by transfecting CD45 gene. However, CD40-mediated induction of cell surface molecules was not affected in CD45-deficient cells. Taken collectively, these results suggest that CD45 exerts a decisive effect on selective sets of CD40-mediated signaling pathways, dictating B cell fate.

Src homology region 2 (SH2) domain-containing phosphatase-1 dephosphorylates B cell linker protein/SH2 domain leukocyte protein of 65 kDa and selectively regulates c-Jun NH2-terminal kinase activation in B cells.

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.

CD45 negatively regulates lyn activity by dephosphorylating both positive and negative regulatory tyrosine residues in immature B cells.

Using CD45-deficient clones from the immature B cell line, WEHI-231, we previously demonstrated that CD45 selectively dephosphorylates the Src-family protein tyrosine kinase Lyn and inhibits its kinase activity. To further define the mechanisms of CD45 action on Lyn, we metabolically labeled Lyn from CD45-positive and -negative WEHI-231 cells and analyzed cyanogen bromide fragments by SDS-PAGE analysis. Phosphoamino acid analysis confirmed that Lyn is tyrosine phosphorylated with little serine or threonine phosphorylation. In CD45-negative cells, two bands at 8.2 and 4.1 kDa were phosphorylated in the absence of B cell Ag receptor (BCR) ligation. The 8.2-kDa band corresponded to a fragment containing the positive regulatory site (Tyr397), as assessed by its size and its phosphorylation in an in vitro kinase assay. The 4.1-kDa band was phosphorylated by COOH-terminal Src kinase, suggesting that it contains the COOH-terminal negative regulatory site (Tyr508). CD45 was also shown to dephosphorylate autophosphorylated Lyn in vitro. Thus, CD45 dephosphorylates not only the negative but also the positive regulatory tyrosine residues of Lyn. Furthermore, coimmunoprecipitations using anti-Igalpha Ab demonstrated that Lyn associated with the resting BCR was constitutively phosphorylated and activated in CD45-negative cells. In the parental cells, both regulatory sites were phosphorylated on BCR ligation. Taken collectively, these results suggest that CD45 keeps both BCR-associated and total cytoplasmic pools of Lyn in an inactive state, and a mechanism by which Lyn is activated by relative reduction of CD45 effect may be operative on BCR ligation.

Requirement of PEST domain tyrosine phosphatase PEP in B cell antigen receptor-induced growth arrest and apoptosis.

Signaling events leading to B cell growth or apoptosis are beginning to be unravelled, but detailed information is still lacking. To identify signaling molecules involved in B cell antigen receptor (BCR)-initiated pathways, we used the immature B cell line, WEHI-231, to investigate protein tyrosine phosphatases (PTP) whose expression was modulated by BCR ligation. Among the PTP cloned by reverse transcription-PCR, mRNA expression of the proline-, glutamic acid-, serine- and threonine-rich (PEST) domain phosphatase (PEP) was selectively elevated 3.1-fold within 3 h after anti-IgM antibody stimulation. In contrast, expression of another PEST domain phosphatase, PTP-PEST, was unaffected. Western blot analysis revealed that 71% of PEP was located in the cytosolic fraction, while 29% was in the membrane fraction. To examine the direct contribution made by PEP to BCR-initiated signal transduction, we transfected an antisense PEP cDNA into WEHI-231 cells. Two stable clones were established in which PEP expression was reduced by 34% and 47%, respectively. Strikingly, BCR-mediated inhibition of DNA synthesis was significantly rescued in the clones, and G1 phase cell cycle arrest and apoptosis were almost completely ablated. Considered collectively, these results indicate that PEP is a positive, crucial regulator in determining B cell fate triggered by BCR engagement.

SHP-1 is involved in neuronal differentiation of P19 embryonic carcinoma cells.

Accumulating evidence suggests that tyrosine phosphorylation plays an important role in the development of the central nervous system and in the differentiation of neuronal cells. To identify protein tyrosine phosphatases (PTPs) that might regulate signaling events leading to neuronal cell differentiation, we cloned PTP genes from the murine P19 embryonic carcinoma cell line and examined the change of their expression during differentiation. P19 cells are known to be pluripotent and the aggregate formation and subsequent replating in the presence of retinoic acid (RA) induce growth arrest and neuronal differentiation. The results demonstrated that among several PTP genes expressed in P19 cells, a cytosolic Src homology region 2 domain-containing PTP, SHP-1, is expressed highly in undifferentiated P19 cells, but is reduced to an undetectable level at day 3 after replating in the presence of RA. Further, SHP-1 was tyrosine-phosphorylated and activated at day 1 after replating. When ectopic SHP-1 was constitutively expressed, P19 cells continued to proliferate and failed to differentiate upon stimulation with RA. Collectively, these results suggest that the regulated expression and activity of SHP-1 may be involved in the neuronal differentiation of P19 cells.

Hematopoietic cell phosphatase, SHP-1, is constitutively associated with the SH2 domain-containing leukocyte protein, SLP-76, in B cells.

Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1; previously named HCP, PTP1C, SH-PTP1, and SHP) is a cytosolic protein tyrosine phosphatase that contains two SH2 domains. Recent data have demonstrated that the gene encoding SHP-1 is mutated in motheaten (mc) and viable motheaten (mc') mice resulting in autoimmune disease. More recently, SHP-1 has been shown to negatively regulate B cell antigen receptor (BCR)-initiated signaling. To elucidate potential mechanisms of SHP-1 action in BCR signal transduction, we studied proteins that interact with SHP-1 in B cells. Both anti-SHP-1 antibody and the two SH2 domains of SHP-1 expressed as glutathione S-transferase fusion proteins precipitated at least three phosphoproteins of approximately 75, 110, and 150 kD upon anti-immunoglobulin M stimulation of the WEHI-231 immature B cell line. Binding of SHP-1 to the 75- and 110-kD proteins appeared to be mediated mainly by the NH2-terminal SH2 domain of SHP-1, whereas both the NH2- and COOH-terminal SH2 domains are required for maximal binding to the 150-kD protein. Immunoprecipitation and Western blot analysis revealed that the SHP-1-associated 75-kD protein is the hematopoietic cell-specific, SH2-containing protein SLP-76. Further, this protein-protein association was constitutively observed and stable during the early phase of BCR signaling. However, significant tyrosine phosphorylation of SLP-76 as well as of SHP-1 was observed after BCR ligation. Constitutive association of SHP-1 with SLP-76 could also be detected in normal splenic B cells. Collectively, these results suggest possible mechanisms by which SHP-1 may modulate signals delivered by BCR engagement.

Selective regulation of Lyn tyrosine kinase by CD45 in immature B cells.

It has been well established that protein-tyrosine phosphatase CD45 is critically involved in the regulation of initial tyrosine phosphorylation and effector functions of T and B cells. However, the signaling pathway governed by CD45 is not completely understood. In B cells, it has not been unequivocally resolved as to which protein-tyrosine kinases (PTKs) associated with B cell antigen receptor are regulated by CD45 in intact cells. As a first step toward the elucidation of CD45-initiated signaling events, we have tried to identify physiological substrates for CD45 by analyzing PTK activity in CD45-deficient clones recently generated from the immature B cell line WEHI-231. The results clearly demonstrated that among PTKs examined (Lyn, Lck, and Syk), only Lyn kinase is dysregulated in the absence of CD45 such that without B cell antigen receptor ligation, Lyn is hyperphosphorylated and activated in CD45-negative clones. Thus, Lyn seems to be a selective in vivo substrate for CD45 in immature B cells.

Antigen receptor-initiated growth inhibition is blocked in CD45-loss variants of a mature B lymphoma, with limited effects on apoptosis.

Role of CD45 in B cell antigen receptor (BcR)-mediated signaling events in mature B cells was examined using BAL-17 and its CD45-negative clones. In the CD45-negative clones, BcR stimulation induced tyrosine phosphorylation almost identical to the parental cells, with a few exceptions of reduced phosphorylation, especially of a protein of about 60 kDa. BcR-induced calcium responses were reduced in the CD45-negative clones, but the kinetics were similar to the parent. BcR stimulation led to growth inhibition in the parental cells, but signals for growth inhibition were completely blocked in the CD45-negative clones. Interestingly, the same stimulation induced low, but significant levels of apoptosis both in the parent and in the CD45-negative clones. Thus, in mature BAL-17 cells, CD45 subtly mediate early signaling events (tyrosine phosphorylation and Ca2+ mobilization), and is absolutely required for the signaling pathway leading to growth regulation, but has limited effects on apoptosis.

Assignment of the human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ) gene to chromosome band 7q31.3.

Human protein tyrosine phosphatase, receptor-type, zeta (PTPRZ; also denoted HPTP zeta or RPTP beta) has a large extracellular region with the N-terminal carbonic anhydrase-like domain and a cytoplasmic region with two tandemly located protein tyrosine phosphatase domains. One of the characteristics of PTPRZ is its preferential expression in the central nervous system. We localized the human PTPRZ gene to chromosome band 7q31.3 by somatic cell hybrid mapping and fluorescence in situ hybridization.

Developmental regulation of gene expression for the MPTP delta isoforms in the central nervous system and the immune system.

MPTP delta is a murine transmembrane protein tyrosine phosphatase which has three isoforms, types A-C, differing in the structure of the extracellular regions. In this study, we examined MPTP delta isoform expression in the brain and the immune system at discrete developmental or differentiation stages. RT-PCR analysis demonstrated that another isoform, type D, is transcribed from the MPTP delta gene. In the brain, only type D was expressed until postnatal day 7 (P7), but after P14, all four isoforms were detected. In contrast, the spleen, thymus and all the hematopoietic cell lines examined express only types B and C isoforms. An in situ hybridization study showed that MPTP delta mRNA is diffusely expressed throughout the spleen, but its expression in the thymus is restricted to the medullary region.

Negative regulation of apoptotic death in immature B cells by CD45.

Cross-linking of membrane IgM receptor on B cells induces tyrosine phosphorylation within 1 min. This biochemical alteration triggers a cascade of signaling events which ultimately leads to activation in mature B cells but growth arrest and cell death by apoptosis in immature B cells. To study the mechanisms underlying the bifurcation of signals, we chose to examine the role of receptor-type protein tyrosine phosphatase (PTP) CD45 using CD45- clones isolated from an immature B cell line WEHI-231. Here we report that in CD45- clones, tyrosine phosphorylation was constitutively induced but not enhanced by anti-IgM stimulation and anti-IgM-induced Ca2+ flux was slightly delayed but evidently prolonged. Further, the degree of growth arrest and DNA fragmentation induced by anti-IgM antibody was more evident in CD45- clones than the parental cells. These results indicate that initial alterations in signaling are effectively transduced into effector signals and that IgM receptor-mediated growth arrest and apoptosis in immature B cells are negatively regulated by CD45.

Extensive N nucleotide addition in junctional region of T cell receptor V gamma 5 genes rearranged in fetal liver-derived thymocytes in radiation chimera mice.

The V gamma 5 chain of T cell receptor gamma delta is preferentially expressed by murine fetal thymocytes. The fetal V gamma 5 gene is known to be homogeneous and lacks N nucleotide addition. We previously reported that V gamma 5+ cells were detected among donor-derived thymocytes from irradiated C3H/He mice soon after reconstitution with AKR/J fetal liver (FL) cells, but that only a few V gamma 5+ were detected among donor-derived thymocytes from irradiated C3H/He mice reconstituted with adult bone marrow (ABM) cells. The results suggest that preferential use of V gamma 5 is determined at the level of T cell precursor generation. In the present report, we analyzed whether the junctional region of FL-derived V gamma 5 genes in the FL chimeras is of fetal type. Sequencing analysis showed that V gamma 5 genes from FL chimeras contained extensive N nucleotide addition and were diverse both in nucleotide sequences and deduced amino acid sequences. V gamma 5 genes from donor-derived thymocytes of ABM chimeras, which by polymerase chain reaction were revealed to be less frequent than those of FL chimeras, also showed extensive N addition. Furthermore, the terminal deoxynucleotidyl transferase (TdT) gene, which encodes an enzyme that adds N nucleotides into the junctional region, was expressed at high level in the donor-derived thymocytes of FL chimeras and normal 8-week-old AKR/J thymocytes but at low level in day 17 fetal AKR/J thymocytes. Our results suggest that the preferential rearrangement of the V gamma 5 gene is determined at the level of T cell precursor generation but the homogeneous V gamma 5 sequence of fetal type is generated only in the fetal thymic microenvironment where TdT gene expression is low or absent. The nomenclature of murine TcR gamma chains is according to Reilly et al. (Nature 1986. 321: 878). The relationship between the different nomenclature systems is summarized in Takagaki et al. (J. Immunol. 1989. 141: 2112).

Chromosomal assignment of the gene for protein tyrosine phosphatase HPTP delta.

Protein tyrosine phosphatase (PTP) negatively regulates the effect of protein tyrosine kinases and is implicated in the regulation of a variety of biological phenomena such as cell activation, differentiation and neoplastic transformation. To gain insight into the role of PTPs, we cloned the human receptor-type PTP gene and assigned the chromosome harboring the gene for HPTP delta by using DNAs from human-mouse hybrid cell lines and by fluorescence in situ hybridization. The results clearly demonstrated that HPTP delta gene maps to human chromosome 9p24.

Induction of CD45 isoform switch in murine B cells by antigen receptor stimulation and by phorbol myristate acetate and ionomycin.

In this study, we examined whether CD45 isoform can be switched in murine mature B cells and what signals are responsible for the process. Stimulation of murine splenic B cells with lipopolysaccharide did not reduce the expression of CD45RA-, B-, and C-exon-dependent epitopes or a CD45 common epitope, but rather enhanced the expression. Stimulation with goat antimouse IgM antibody did not significantly reduce CD45 expression but caused a partial reduction in the expression of CD45RA-, B-, and C-exon-dependent epitopes. Phorbol myristate acetate (PMA) alone did not significantly alter the expression of CD45 but the combination of PMA and ionomycin induced a strong reduction in the expression of CD45RA-, B-, and C-exon-dependent epitopes without affecting the level of CD45 common epitope expression. Reverse transcription and polymerase chain reaction analysis demonstrated that CD45 isoform switch induced by anti-IgM or PMA plus ionomycin is indeed mediated by alternative splicing of A-, B-, and C-exon-derived mRNA. These results suggest that CD45 isoform of murine mature B cells can be switched by antigen receptor-mediated signals, and the process seems to be regulated at least in part by protein kinase C activation and mobilization of calcium ions.

Appearance of TCR-alpha beta+ CD4-CD8- skin intraepithelial lymphocytes in radiation bone marrow chimeras.

Thymocytes expressing homogeneous TCR V gamma 5/V delta 1 gene without N nucleotide addition (canonical sequence) have been reported to be positively selected in fetal thymus. Because skin intraepithelial lymphocytes (s-IEL) also express canonical sequence, it is proposed that s-IEL are generated by an epidermis-specific homing of these positive selected fetal V gamma 5/V delta 1-bearing thymocytes. We have reported that positive selection of V gamma 5+ thymocytes is not seen in AKR/J-->C3H/He bone marrow chimeras. In this report, we analyzed the phenotype of donor-derived s-IEL from AKR/J-->C3H/He bone marrow chimeras that appeared in the absence of positive selection of V gamma 5-bearing thymocytes. We found interestingly that almost all donor-derived s-IEL in the bone marrow chimeras expressed TCR-alpha beta lack expression of both CD4 and CD8. Furthermore, TCR-alpha beta+ s-IEL were detected in adult-thymectomized bone marrow chimeras, indicating that the TCR-alpha beta+ s-IEL are extrathymically derived. All these results suggest that the s-IEL expressing TCR-alpha beta appear extrathymically in the absence of positive selection of V gamma 5/V delta 1-bearing thymocytes. Our results may also explain the contradicting result between human s-IEL, which are TCR-alpha beta+ CD4- CD8- and murine s-IEL, and suggest the importance of extrathymic T cell differentiation pathway of the TCR-alpha beta lineage in immune surveillance.

MPTP delta, a putative murine homolog of HPTP delta, is expressed in specialized regions of the brain and in the B-cell lineage.

Protein tyrosine phosphatases (PTPs), together with protein tyrosine kinases (PTKs), are involved in the regulation of cell activation, growth, and differentiation. To further elucidate the fine tuning of cell growth and differentiation through tyrosine phosphorylation, we tried to isolate mouse receptor-type PTP (RPTP) cDNA clones by screening mouse brain cDNA libraries with mouse CD45 PTP domain probes under reduced-stringency conditions. Characterization of isolated cDNA clones for RPTP showed that the cytoplasmic region contains two tandem repeats of PTP domain of about 230 amino acids with intrinsic phosphatase activity. The extracellular region was composed of immunoglobulin (Ig)-like domains and fibronectin type III (FN-III)-like domains. The gene was highly homologous to human PTP delta (HPTP delta) and thus was named MPTP delta (murine counterpart of HPTP delta). The MPTP delta gene appeared to generate at least three species of mRNA, which differ in the composition of the extracellular domain: type A, one Ig-like and four FN-III-like domains; type B, one Ig-like and eight FN-III-like domains; and type C, three Ig-like and eight FN-III-like domains. Interestingly, the 5' untranslated region and the leader peptide of types A and B were completely different from those of type C. Northern (RNA) blot analysis demonstrated that brain, kidney, and heart cells express three mRNA species of about 7 kb. Antibody directed against part of the extracellular domain of type A MPTP delta recognized a 210-kDa protein in brain and kidney lysates. In situ hybridization of brain samples revealed that MPTP delta mRNA is present in the hippocampus, thalamic reticular nucleus, and piriform cortex, where some Src family PTKs have been also demonstrated to exist. Although MPTP delta mRNA was not detected in lymphoid tissues, all of the pre-B-cell lines tested and one of three B-cell lines tested expressed MPTP delta mRNA, whereas antibody-producing B-cell hybridomas and T-cell and macrophage lines did not. Finally, the MPTP delta locus was tightly linked to the brown (b) locus on mouse chromosome 4.

Regulation of lipopolysaccharide- and IL-4-induced immunoglobulin heavy chain gene activation: differential roles for CD45 and Lyb-2.

We have demonstrated that CD45, a receptor-type protein tyrosine phosphatase, selectively regulates IgG production at the generative phase of precursors of IgG producers whereas Lyb-2 regulates IgG1 production induced by IL-4 in lipopolysaccharide (LPS)-activated B cells by acting on the generation of IgG1 precursor cells. These results point to an interesting possibility that both CD45 and Lyb-2 mediate a critical regulatory step(s) in IgG class switching. The present study was conducted to examine this possibility by elucidating the molecular mechanisms whereby CD45 and Lyb-2 control IgG synthesis in B cells activated by LPS and IL-4. Northern blot analysis showed that steady-state levels of C gamma 3, C gamma 2b and C gamma 1, but not Cmu, mRNA in LPS-activated B cells were reduced approximately 3- to 5-fold by CD45 mAb, and that the C gamma 1 mRNA level in B cells activated by LPS and IL-4 was significantly decreased by Lyb-2 mAb and CD45 mAb. Further, CD45 mAb inhibited expression of germline gamma 2b and gamma 3 transcripts induced by LPS and germline gamma 1 transcript expression induced by IL-4 plus LPS, but caused no inhibition in IL-4-induced germline gamma 1 transcript expression. In contrast, Lyb-2 mAb did not exert any inhibitory effect on the generation of germline gamma 1 transcripts induced by IL-4 and LPS or by IL-4 alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Deletion of self-reactive T cells in the donor-derived T cells but not in the host-derived T cells in fully allogeneic radiation chimeras. Mls-reactive T cells in allogeneic radiation chimeras.

Kinetics of T cells bearing V beta 6 capable of recognizing Mls-1a were examined in the thymus and peripheral lymphoid organs of two allogeneic bone marrow chimeras; AKR/J(H-2k, Thy1.1,Mls-1a)----C3H/He(H-2k, Thy1.2,Mls-1b) and AKR/J----C57BL/6(H-2b,Thy1.2, Mls-1b). Sequential appearance of host- and donor-derived T cells occurred in the thymus and the peripheral lymphoid organs of both AKR----C3H and AKR----B6 chimeras. The first cells to repopulate the thymus were Thy1.2+ host-derived radioresistant cells, which were synchronized in their development. The host-derived cells in thymus of AKR----B6 chimeras differentiate more rapidly than those in AKR----C3H chimeras. An almost complete replacement from host-derived cells to donor-derived cells occurred by day 21 after reconstitution in AKR----C3H and AKR----B6 chimeras. In the donor-derived thymocytes, none of CD4- or CD8-single positive thymocytes expressed high density of V beta 6 in either AKR----C3H or AKR----B6 chimeras, whereas the host-derived thymocytes in AKR----B6 chimeras contained an appreciable number of CD4-single positive thymocytes bearing V beta 6. In the peripheral lymphoid organs, T cells bearing V beta 6 were virtually abolished in Thy1.1+ cell pool of both AKR----C3H and AKR----B6 chimeras. While V beta 6+ T cells of host-origin were detected in the peripheral lymphoid organs in AKR----B6 chimeras. These result indicated that the donor-derived mature T cells showed deletion of V beta 6 in the thymus and the peripheral lymphoid organs in both AKR----C3H and AKR----B6 chimeras, whereas lack of V beta 6 deletion was observed in the host-derived mature T cells in the AKR----B6 chimeras. These results suggested that the host-derived thymocytes may likely to escape undergoing a negative selection against donor-phenotype in the radiation bone marrow chimeras.