Biomedical subjects

J Samaridis

Publications and source records attributed to J Samaridis.

9 recordsLinked to original sources

PubMed1992

Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro.

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.

Animals↗

PubMed1991

Thymus colonization in the developing mouse embryo.

We have directly followed the formation of and the thymus colonization by pro-T lymphocytes in the developing C57BL/6 mouse embryo by using the monoclonal antibody JORO 37-5 specific for pro-T lymphocytes, immunofluorescence staining and flow fluorocytometry or microscopy analysis. The results show that JORO 37-5+ cells begin to appear in the liver at day 9 of gestation. These JORO 37-5+ cells migrate to and colonize the thymus 1 day later, where they expand vigorously during the next 4-5 days and, subsequently, switch off expression of JORO 37-5 as they further differentiate into mature thymocytes.

Animals↗

PubMed1991

Development of lymphocytes in interleukin 7-transgenic mice.

We have developed and established mouse transgenic lines in which the mouse interleukin 7 gene was targeted for expression in the lymphoid cell compartment. Northern blot analysis indicate that the transgene is expressed in bone marrow (BM), spleen and thymus, but not in kidney, liver, brain or heart. Both the frequency and absolute numbers of B cell precursors and mature B lymphocytes are increased in the BM and spleen of the transgenic mice. Although there is no expansion of the pro-T lymphocyte population in the BM, the number of all major subsets of thymocytes and peripheral T lymphocytes is increased in the majority of the transgenic mice analyzed. The B and T cell lymphocytes in the transgenic mice are functionally competent. In contrast, the number of granulocytes and macrophages in the BM of transgenic mice is similar to that in control non-transgenic littermates. Our results indicate that interleukin 7 plays an important role in vivo in the development of B and T lymphocytes.

Animals↗

PubMed1991

Rearrangement patterns of T-cell receptor genes in the spleen of athymic (nu/nu) young mice.

Although the athymic nude mouse is grossly deficient in peripheral T cells, the number of lymphocytes bearing T-cell markers (L3T4, LyT2) and the alpha beta or gamma delta T-cell receptor (Tcr) increases steadily with age. The anatomical site(s) where these cells arise are unknown. Splenocytes from 3-5-week-old C57BL/6 (nu/nu) mice contain 2%-5% Pro-T cell progenitors identified with the Joro 37-5 and Joro 75 antibodies, but not mature T cells. To study Tcr gene rearrangement outside the thymus, we fused splenocytes from 3-5-week-old C57BL/6 nude mice with the T-cell lymphoma BW 100.129. Of 22 hybrids that grew stably in culture, four had Tcrd-VD1-D2-J1, two had Tcrd-VD2-J1, and seven had Tcrd-D1-D2 types of rearrangement. Eight hybrids had rearranged the Tcrg-2 gene cluster, but none had rearranged Tcrg-1, -3, or -4. None of the hybrids had rearranged the Tcrb gene cluster and 13 contained DJ rearrangements at the Igh locus. We conclude that the spleen is one of the extrathymic sites where T-cell progenitors can rearranged Tcrd and Tcrg genes. However, there was no evidence for Tcrb gene rearrangements in this organ. Furthermore, the analysis of this limited number of hybrids suggests that extrathymic Tcr gene rearrangements seem to be distinct and much less diverse than those found in the developing thymocytes.

Animals↗

PubMed1990

Identification and characterization of pro-T lymphocytes and lineage-uncommitted lymphocyte precursors from mice with three novel surface markers.

The study of prethymic stages of T cell development has been limited because specific markers for mouse pro-T lymphocytes were not available. We developed a panel of rat monoclonal antibodies (mAbs) that bind to our pro-T lymphocyte clones obtained from bone marrow of young adult mice and the thymus of 14-d-old embryos. The mAbs, called Joro 30-8, Joro 37-5, and Joro 75, were found to bind to all pro-T clones tested but not to cell lines representing later stages of T cell development, B lymphocyte, or myeloid lineages. We determined the frequency and tissue distribution in normal and immunodeficient mouse strains as well as the ontogeny in liver and thymus of cells positive for these mAbs. The results were consistent with the pattern of reactivity observed with cell lines. We isolated Joro 30-8+, Joro 37-5+, and Joro 75+ bone marrow cells by cell sorter and found that: (a) phenotypically, they are Thy-1+, CD4-, CD8-, CD3-, B-220-, IgM-, F4/80-, and PgP-1+; (b) they grew in response to the combination of interleukin 3 (IL-3) + IL-4 or IL-3 + IL-4 + IL-6; and (c) Joro 37-5+ and Joro 75+ marrow cells gave rise to mature T lymphocytes but not to B lymphocytes, while Joro 30-8+ marrow cells generated both T and B lymphocytes after 8-12 wk of transfer into severe combined immunodeficient (Scid) mice. In normal mice subjected to 600 rad of irradiation to induce a wave of thymus recolonization, we found by flow fluorocytometry analysis that Joro+ cells entered the thymus 2 d after irradiation, expanded during the next 4 d, and underwent further differentiation, and from day 8 up to day 21, post-irradiation Joro+ cells were no longer detectable in the thymuses. Immunohistochemical analysis of normal thymus shows the presence of very few Joro 30-8+, Joro 37-5+, and Joro 75+ lymphoid cells in the subcapsular area and outer cortex but not in the medulla. The kinetic analysis of tissue sections from thymuses at various days post-irradiation suggests that Joro+ cells enter the thymus via blood vessels through the subcapsular and outer cortex areas; subsequently, these cells seem to migrate to the inner cortex without reaching the medulla, and give rise to Joro- thymocytes.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Nonrandom rearrangement of T cell receptor J alpha genes in bone marrow T cell differentiation cultures.

TCR J alpha genes span a distance of approximately 65 kb on mouse chromosome 14. Due to the existence of 50 to 100 discrete J genes, a potential for great diversity exists within the V-J-C alpha gene products and within the ultimate repertoire of alpha beta TCR. We have prepared hybridomas from an in vitro system that supports T cell differentiation among bone marrow cells. We have examined the J alpha genes among these cells and categorized rearrangements according to their location within the J alpha locus. It was found that alpha rearrangements were always present among the hybridomas bearing beta gene rearrangements. When two bone marrow-derived alpha-bearing chromosomes could be demonstrated in these hybridomas, both were always rearranged and rearrangements on homologous chromosomes were shown to reside in similar regions of the J alpha locus. Most surprisingly, when hybridomas were categorized by the culture from which they derived, cells from the same culture (designated as a set) demonstrated a skewing of alpha rearrangements to restricted segments of J alpha genes. In one hybridoma, rearrangements on homologous chromosomes involved J alpha genes that were either identical or situated within a 1-kb segment of DNA. The skewing within sets could not be due to clonal identity between hybridomas as the beta and gamma rearrangements in all hybridomas were different. Results suggested that skewing of J alpha gene rearrangements occurred during the course of T cell development in vitro. Should the same situation occur in vivo, the number of distinct TCR J alpha sequences available for expression in early development may be far less than that predicted by gene number alone.

Animals↗

PubMed1989

Thymic epithelial cells induce in vitro differentiation of PRO-T lymphocyte clones into TCR alpha,beta/T3+ and TCR gamma,delta/T3+ cells.

PRO-T lymphocyte clones, which have the T cell receptor (TCR) alpha, beta, gamma and delta genes in germline configuration and heterogeneous T cell precursors freshly isolated from bone marrow of athymic nude mice, gave rise to single positive L3T4+ TCR alpha,beta+ and double negative (L3T4-LyT2-) TCR alpha,beta+ or TCR gamma,delta+ cells, but not to any cells expressing LyT2, when co-cultured with the thymic epithelial clone ET. The T cell progenitors were able to develop into cells expressing LyT2 only when cocultured with heterogeneous thymic epithelial cell preparations. The progeny of the induced PRO-T clones included cells bearing V beta 8, V beta 17 and V gamma 3 gene family products. The presence of cells expressing a TCR gamma, delta/T3 receptor complex in the cultures was also documented by the expression of RNA transcripts from the TCR delta and TCR gamma genes by induced PRO-T cells. The TCR/T3+ cells generated in the cultures expressed functionally competent T cell receptor complexes. Our results show that: (i) the same PRO-T clone can give rise to all major subsets of thymocytes upon interaction with the appropriate thymic epithelial cells; (ii) both TCR alpha,beta+ and TCR gamma,delta+ cells may originate from a common T cell progenitor; (iii) L3T4+ TCR alpha, beta+ and L3T4-LyT2- TCR alpha,beta+ cells do not necessarily pass through a L3T4+LyT2+ intermediate stage of development; and (iv) different types of thymic epithelial cells play an essential role in the differentiation of PRO-T cells into either L3T4+ TCR alpha,beta+ L3T4-LyT2- TCR alpha,beta+ or L3T4+LyT2+ and LyT2+ TCR alpha, beta+ cells in vitro. Finally, we have attempted to integrate our results and those of others in a suggested model of T cell development within the thymus.

Animals↗

PubMed1989

Immature and advanced patterns of T cell receptor gene rearrangement among lymphocytes in splenic culture.

Bulk populations and 39 hybridomas from splenic Con A cultures were analyzed for rearrangements among TCR genes: alpha, beta, gamma, and delta. Patterns were categorized to reveal general rules governing gene rearrangement within the activated adult peripheral population. Many patterns of gene rearrangement were consistent with previous studies of T cell lines. Additional points of interest were the following: 1) A large proportion of Con A-stimulated splenic cells bore no TCR gene rearrangements. 2) One splenic hybridoma exhibited an unusual gene pattern, with rearrangements, at alpha and beta, but not J gamma 1 or J gamma 2 loci. 3) Multiple gamma rearrangements were noted other than V1.2-J2 and V2-J1. 4) One hybridoma exhibited TCR gene rearrangements typical of day 14 to 15 fetal thymocytes, as well as rearrangements at immunoglobulin gene loci. 5) Among hybridomas with J alpha rearrangements, homologous chromosomes exhibited rearrangements at similar positions along the J alpha locus.

Aging↗

PubMed1988

Progression of rearrangements at T cell receptor beta and gamma gene loci during athymic differentiation of bone marrow cells in vitro.

A previously described in vitro system that supports T cell differentiation from bone marrow (BM) precursors was analyzed for T cell receptor gene rearrangement and expression. Cultured populations from days (d.) 0, 3, 6, and 12-13 were fused with BW5147. Only hybridomas from d.6 and d.12-13 bore BM-derived rearrangements. The rearrangement complexity was higher on d.12 than d.6 in that patterns consistent with D beta 1-J beta 1 and D beta 2-germ-line configurations decreased while V-D-J beta 2 and V gamma 2-J gamma 1 joining became dominant. Northern blots of d.13 BM cultures revealed gamma, alpha, and beta (1.0 and 1.3 kb) transcripts. Parallels between patterns in BM cultures and thymus ontogeny were recognized.

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