Ronald Palacios

IL3-dependent mouse clones that express B-220 surface antigen, contain ig genes in germ-line configuration, and generate B lymphocytes in vivo

The continuously proliferating clones L/B AgA2, CB/Bm 7, Ba/C1, and Bc/Bm 11 were established from bone marrow of MRL/LPR, CBA/J, and BALB/c mice. These clones carry the B cell lineage surface antigen B-220 but not antigens normally expressed on mature B lymphocytes, myeloid cells, or T lymphocytes. Their immunoglobulin mu heavy chain and kappa light chain genes are in germ-line configuration. The G418 resistance gene was introduced into each clone with a retrovirus vector and then used as a selective marker for the progeny of transfected cells. Clones L/B AgA2, CB/Bm 7, and Bc/Bm 11, but not Ba/C1, could develop into antibody-secreting cells after in vivo transfer. None gave rise to cells responsive to polyclonal T cell activators, nor did any differentiate into cells that could develop into granulocyte/macrophage-colony-forming cells in vitro. All grew in interleukin 3 but not in other cytokines. We conclude that clones L/B AgA2, CB/Bm 7, and Bc/Bm 11 are early precursors of B lymphocytes.

A novel disulfide-linked heterodimer on pre—T cells consists of the T cell receptor β chain and a 33 kd glycoprotein

We describe a novel signal-transducing protein complex, which consists of the T cell receptor (TCR) beta chain that is disulfide linked to a 33 kd glycoprotein and noncovalently associated with proteins of the CD3 complex on the surface of the pre-T cell line SCB.29. This 33 kd glycoprotein, provisionally designated gp33, represents neither of the known TCR chains and has escaped previous detection because it labels poorly by surface iodination. This glycoprotein is absent from the surface of mature T cell lines. A TCR beta complex with identical molecular masses before and after reduction can be immunoprecipitated from surface-iodinated large thymocytes of TCR alpha-deficient mice. The novel gp33-TCR beta complex may be entirely or partly responsible for control of early T cell development exerted by the TCR beta protein.

T cell receptor (TCR) beta chain homodimers on the surface of immature but not mature alpha, gamma, delta chain deficient T cell lines.

Transfected T cell receptor (TCR) beta chain genes are expressed as homodimers on the surface of immature (Sci/ET27F) but not on mature (58 alpha‐beta‐) T cell lines which lack TCR alpha, gamma and delta chains. The homodimer on Sci/ET27F cells is tightly bound to CD3 delta and CD3 epsilon while the association with CD3 gamma and CD3 zeta proteins is rather weak. Crosslinking of the TCR beta homodimers resulted in a strong and rapid calcium flux. In 58 alpha‐beta‐ T cells the beta TCR chain could be easily visualized intracellularly but was not transported to the cell surface. The Scid cell lines considerably facilitate the molecular analysis of early differentiation events in the thymus which are likely to be regulated by the beta TCR homodimer.

Recombinant interleukin 4/BSF-1 promotes growth and differentiation of intrathymic T cell precursors from fetal mice in vitro.

Recombinant mouse interleukin 4/BSF‐1 (rIL4/BSF‐1) together with phorbol myristate acetate (PMA) promotes growth of one out of approximately four intrathymic T cell precursors from fetal mice (14‐15 days gestation). This response is not inhibited by even high concentrations of monoclonal antibody against the receptor for interleukin 2. Fetal thymocytes activated by rIL4/BSF‐1 plus PMA give rise to cytolytic T cells after 7‐21 days of culture. All the proliferating cells are Thy1+, some of them express Lyt2 but none has detectable L3T4 T cell differentiation antigens nor T cell antigen receptor (F23.1) on the cell membrane as assessed by immunofluorescence staining and flow fluorocytometry analysis. It is concluded that rIL4/BSF‐1 exerts both growth and differentiation activities on normal intrathymic T cell precursors. The results provide evidence for an alternative growth factor to interleukin 2 involved in proliferation of T cell precursors. These findings open new and direct ways of studying cellular and molecular events during the differentiation of normal intrathymic T cell precursors in vitro and extend the spectrum of target cells for IL4/BSF‐1.

Prethymic and Intrathymic Mouse T-Cell Progenitors. Growth Requirements and Analysis of the Expression of Genes Encoding TCR/T3 Components and other T-Cell-Specific Molecules

During mouse development, precursor cells from fetal liver colonize the thymus at d II of gestation {Moore & Owen 1967. Owen & Ralf 1970). In postnatal life, Ihe bone marrow becomes the main source of T-cell progenitors. As early as 1952, Kaplan & Brown reported that thigh-shielding during sytemic X-ray irradiation not only conferred hemopoietic protection but also promoted regeneration of the thymus. That the thymus continuously receives immigrant bone marrow precursor cells has been shown in studies using thymus grafts (Metcalf & Walkonig-Vaartaga 1964, Dukor et al. 1965, Schlesinger & Hurvitz 1968). Thymus-repopulation assays suggest that 0.03% to 0.25% of the bone marrow cells would have the capacity lo repopulate the thymus following intravenous (I.V.) transfer into X-ray irradiated mice (Basch et al. 1978. Boersma et al. 1981, Greiner et al. 1978. Lepault et al. 1983). Recent studies suggest that marrow Tceil progenitors begin to enter the thymus of lethally irradiated mice 2 d after their I.V. transfer and that the T-cell progenitors do not enter the thymus in a single wave but enter this organ over a period of at least 3 d (Mulder et al. 1988). (For a review on migration of T-cell precursors to the thymus. see Scollay et al. (1986)).

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.

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 αβ or γδ T-cell receptor (Tcr) increases steadily with age. The anatomical site(s) where cells arise are unkown. 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 BW100.129. 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 Tcrd 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.

Both cloned interleukin 2 and purified interleukin 1 are required for optimal growth of purified L3T4+ and LyT 2+ lymphocytes initiated by concanavalin A

Concanavalin A (Con A), cloned interleukin 2 (IL-2), purified interleukin 1 (IL-1) or two different crude preparations containing IL-1 activity alone, did not induce proliferation of rigorously accessory cell (AC)-depleted splenic L3T4+ or Lyt 2+ lymphocytes. Con A together with saturating concentrations of cloned IL-2 (100 U/ml) promoted less than 40% of the proliferative responses observed in AC-supplemented L3T4+ and Lyt 2+ T-cell cultures. The three preparations of IL-1 used supported minimal proliferation of Con A-treated purified L3T4+ or Lyt 2+ lymphocytes. However, all these IL-1 preparations promoted significant growth of the T-cell populations if AC (1%) were included in the cultures. Cloned IL-2 combined with purified IL-1 promoted proliferation of Con A-treated L3T4+ and Lyt 2+ lymphocytes achieving approximately 75% of the responses observed in AC-supplemented T-cell cultures. The additive effect of IL-1 was apparent in the presence of saturating concentrations of cloned IL-2. Finally, Con A alone induced a detectable number of both L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors as determined with the anti-mouse IL-2 receptor antibody 7D4 by immunofluorescence and FACS analysis. Purified IL-1 neither induced detectable number of L3T4+ or Lyt 2+ T cells to express IL-2 receptors nor increased the number of Con A-treated T cells bearing IL-2 receptors. We have interpreted these findings to indicate the following: Con A alone is sufficient to induce highly purified L3T4+ and Lyt 2+ lymphocytes to express IL-2 receptors. Cloned IL-2 and purified IL-1 are required for optimal growth of L3T4+ and Lyt 2+ lymphocytes and these cytokines together efficiently replace AC in growth of T cells initiated by Con A. IL-1 alone does not replace AC in Con A-induced activation of mouse T cells. IL-1 exerts potentiation on IL-2-driven growth of Con A-treated L3T4+ and Lyt 2+ lymphocytes. The additive activity of IL-1 on growth of normal T cells is not due to increased production of IL-2 in the cultures or induction of normal T cells to expression of IL-2 receptors by IL-1. We propose that IL-1 optimizes the action and/or interaction of IL-2 with its receptors on the T-cell membrane (by, i.e., increasing affinity of the IL-2 receptor for its ligand and/or stabilizing the IL-2 receptor).

Splenocytes and bone marrow cells from T-cell deficient Nu/Nu mice secrete interleukin 3 activity after stimulation in vitro.

We show here that the combination of Concanavalin A (Con A), phorbol myristate acetate (PMA), and Ionomycin (Iono) reproducibly stimulated splenocytes from Nu/Nu mice and bone marrow cells from both normal and Nu/Nu mice to secrete interleukin 3 (IL-3) in vitro. IL-3 was measured by its property of supporting the growth of four different clones known to grow only in IL-3. None of the agents indicated above nor several other types of stimuli tested could induce the cells to secrete IL-3 activity. IL-3 activity from induced cells of either tissue was detected after 24 hr of culture, peaked at 48 hr and either declined by 72-96 hr of culture (bone marrow cells) or remained relatively constant through the 4-day culture period (splenocytes). The cells participating in the production of IL-3 activity in Nu/Nu spleen were THY1+, L3T4-, LyT2-, B-220-, J11d-, Ia-, and those in the marrow from either normal or Nu/Nu mice were THY1+, J11d+, L3T4-, LyT2-, B-220-, Ia-. Finally, we present evidence that Ia-positive cells negatively regulate the production of IL-3 activity by both splenocytes and marrow cells. We conclude that Nu/Nu splenocytes and bone marrow cells from both normal and Nu/Nu mice can secrete IL-3 activity after proper stimulation in vitro and that such property is negatively regulated by Ia-positive cells.

Thymic Epithelial Cells Induce in Vitro Differentiation of PRO-TLymphocyte Clones into TCR α, β/CD3+ and TCRγ, δ/CD3+ Cells

In the intact thymus, it is difficult to determine the molecular requirements for the induction of T-cell differentiation and to assess the roles of the various types of cells in the thymic environment (e.g. epithelial cells, macrophages, dendritic cells). Furthermore, precursor-product relationships can only be inferred indirectly.