Thymocyte and macrophage interactions: separation of murine thymocyte subsets and enrichment of syngeneic cell-responding thymocytes by adsorption to macrophage monolayers

The spontaneous binding of murine thymocytes to macrophage monolayers was employed to separate thymocytes into macrophage-unbound and -bound subsets, and the functional reactivities of these two subpopulations were examined. Macrophage-unbound thymocytes were found to be enriched in functional subsets reactive to semi-allogeneic and allogeneic stimulating spleen cells by proliferation in mixed leukocyte culture (MLC). Furthermore, macrophage-unbound thymocytes were frequently found to respond to syngeneic spleen cells. This syngeneic proliferative response showed both memory and specificity upon subsequent restimulation and thus seems to represent a syngeneic mixed leukocyte reaction (SMLR). Syngeneic responding thymocytes were also found to produce interleukin 2 when cultured with syngeneic but not allogeneic stimulator cells. In contrast, macrophage-bound thymocytes showed greatly reduced proliferative responses to allogeneic stimulators and no responses to syngeneic stimulators. The macrophage-bound thymocyte subset was not enriched in detectable suppressive activity; proliferative responses of macrophage-unbound thymocytes to either allogeneic or syngeneic cells were neither suppressed nor enhanced when macrophage-unbound thymocytes were added to the cultures. Thus, the macrophage-unbound subset seems to be enriched in functionally mature thymocytes and the macrophage-bound subset appears to be enriched in functionally immature thymocytes. This functional separation of thymocytes by macrophage adherence also correlated well with thymocyte subpopulations separated by bovine serum albumin density gradients; the low density mature thymocytes showed enhanced responses to both allogeneic and syngeneic stimulators, whereas the high density immature cells were unresponsive. This correlation was supported further by binding studies in which T cell tumor lines derived from C57BL/6 mice were used. ERLD tumor cells, which are similar to cortical immature thymocytes in both enzymatic and surface antigenic markers, were found to bind readily to macrophages, whereas both EL-4 and E male G2 tumor cells, with characteristics of mature thymocytes, bound to macrophages poorly. The binding of thymocytes and ERLD tumor cells to macrophages was not genetically restricted. We speculate that thymocyte binding to macrophages may play a critical role during the functional maturation of thymocytes.     

Regulation of lymphocyte motility by macrophages: characterization of a lymphocyte migration inhibitory factor derived from a macrophage-like cell line

An inhibitory factor on lymphocyte migration was detected using a capillary random migration assay in the culture supernatant of peritoneal exudate macrophages cultured at concentrations greater than 8 x 10(6) cells/ml. After examining different macrophage-like cell lines, J774A.1 cells were found to produce this inhibitory factor, which was termed lymphocyte migration inhibitory factor (LMIF). The inhibitory effect of LMIF on the migration of spleen lymphocytes, thymocytes, and bone marrow cells was determined. The migration of thymocytes was more sensitive to LMIF than was the migration of spleen lymphocytes and bone marrow cells. Interestingly, when the effect of LMIF was tested on the migration of spleen T cells and B cells, T cells were more sensitive than B cells. When the thymocytes were separated by peanut agglutinin into mature and immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes, the migration of mature thymocytes was more sensitive than that of immature thymocytes to the effect of LMIF, suggesting that the greatest effect of LMIF was on the migration of mature T cells. Partial purification of LMIF by ion-exchange and gel-filtration chromatography revealed that it is approximately 14,000 in molecular weight and could exist in either monomeric or dimeric forms. The possible role of this factor in an immune response is discussed.     

Alterations in thymocyte surface markers after in vivo treatment by serum thymic factor.

Three T cell markers (heterologous sheep rosette, autologous rosette, and theta antigen) have been studied in thymocytes after in vivo injection of a single dose of serum thymic factor (Facteur Thymique Sérique: FTS). Within 24 h after such treatment, sheep erythrocyte-binding cells increased 5 to 8 times, while autologous rosette-forming cells decreased by a factor of 3. Using cytotoxicity assays (trypan blue exclusion test and chromium release) it was observed that thymocytes from FTS-treated mice presented a higher sensitivity to antitheta serum and complement than control mice. These results are compatible with the hypothesis that exposure of immature T cells to thymic hormone may induce T cell maturation, as assessed by decreased number of immature autologous rosette forming cells (RFC) and increased number of mature T cells (sheep RFC and theta-positive cells). Facteur Thymique Sérique appears as an interesting probe to define the various intra-thymic cellular compartments of differentiation.     

Enhanced natural killer (NK) cell activity and NK-sensitive thymic cells in murine muscular dystrophy

Studies have shown that there is an abnormality in the thymus of dystrophic mice with respect to age-dependent thymus weight changes and altered morphology (T. DeKretser and B. Livett, Nature (London), 263, 682, 1976). Recently, others have shown that natural killer (NK) cells can lyse cells of a large, immature, rapidly dividing cell subpopulation within the thymus of normal young (3 weeks of age) mice (M. Hansson, K. Karre, R. Kiessling, J. Roder, B. Anderson, and P. Hayry, J. Immunol., 123, 765, 1979). The NK susceptibility of dystrophic mouse thymocytes as targets was therefore studied. Spleen cells from normal (+/+) and dystrophic ($\text{dy}{}^{\mathrm{2J}}\text{dy}{}^{\mathrm{2J}}$) male C57BL/6J mice 8–10 weeks old were passed over nylon wool and the nonadherent cells were incubated with ⁵¹Cr-labeled YAC-1 lymphoma target cells or thymocytes in a ⁵¹Cr-release assay. Spleen cells from dystrophic mice killed twofold more YAC-1 target cells than did spleen cells from normal mice. Thymocytes from 3- to 4-week-old dystrophic mice were three to four times more susceptible to NK lysis by dystrophic mouse spleen cells as compared with normal mouse spleen cells. Spleen cells from dystrophic mice had the same NK activity against dystrophic and normal mouse thymocytes as targets. Normal mouse spleen cells killed three- to fourfold more dystrophic mouse thymocytes than that of normal mouse thymocytes as targets. Target cellbinding studies revealed that conjugate-forming cells from nylon nonadherent dystrophic mouse spleen cells were found to be two- to fourfold greater than for normal mouse spleen cells using YAC-1 tumor cells as targets. The number of lymphocytes bound per YAC-1 target cell ranged from 2 to 5 for dystrophic mouse spleen cells as compared with 1 to 2 for the normal control group. Using both normal and dystrophic mouse thymocytes as targets, the conjugate-forming cells from dystrophic mouse spleen cells were also found to be twofold greater than in the normal control group. Cold target inhibition studies revealed that the natural killing of dystrophic mouse thymocytes was due to a YAC-1-reactive NK cell. Effector cell depletion studies using monoclonal anti-Thy-1.2 plus complement treatment and plastic petri dish adherence also revealed that the natural killing of dystrophic mouse thymocytes was not due to either T lymphocytes or macrophages. Taken together, these results show an increase in NK-sensitive thymocyte targets in dystrophic mice, in combination with an increase in splenic NK activity.     

The indole alkaloid meleagrin,from the olive tree endophytic fungus Penicillium chrysogenum,as a novel lead for the control of c-Met-dependent breast cancer proliferation,migration and invasion

Fungi of the genus Penicillium produce unique and chemically diverse biologically active secondary metabolites, including indole alkaloids. The role of dysregulated hepatocyte growth factor (HGF) and its receptor, c-Met, in the development and progression of breast carcinoma is documented. The goal of this work is to explore the chemistry and bioactivity of the secondary metabolites of the endophytic Penicillium chrysogenum cultured from the leaf of the olive tree Olea europea, collected in its natural habitat in Egypt. This fungal extract showed good inhibitory activities against the proliferation and migration of several human breast cancer lines. The CH₂Cl₂ extract of P. chrysogenum mycelia was subjected to bioguided chromatographic separation to afford three known indole alkaloids; meleagrin (1), roquefortine C (2) and DHTD (3). Meleagrin inhibited the growth of the human breast cancer cell lines MDA-MB-231, MDA-468, BT-474, SK BR-3, MCF7 and MCF7-dox, while similar treatment doses were found to have no effect on the growth and viability of the non-tumorigenic human mammary epithelial cells MCF10A. Meleagrin also showed excellent ATP competitive c-Met inhibitory activity in Z-Lyte assay, which was further confirmed via molecular docking studies and Western blot analysis. In addition, meleagrin treatment caused a dose-dependent inhibition of HGF-induced cell migration, and invasion of breast cancer cell lines. Meleagrin treatment potently suppressed the invasive triple negative breast tumor cell growth in an orthotopic athymic nude mice model, promoting this unique natural product from hit to a lead rank. The indole alkaloid meleagrin is a novel lead c-Met inhibitory entity useful for the control of c-Met-dependent metastatic and invasive breast malignancies.     

Nitric oxide-donating derivatives of hederacolchiside A1: Synthesis and biological evaluation in vitro and in vivo as potential anticancer agents

A series of nitric oxide (NO) donating derivatives of hederacolchiside A₁ bearing triterpenoid saponin motif were designed, synthesized and evaluated for their anticancer activity. All of the tested furoxan-based NO releasing compounds showed significant proliferation inhibitory activities. Especially compound 6a exhibited strong cytotoxicity (IC₅₀ = 1.6–6.5 μM) against four human tumor cell lines (SMMC-7721, NCI-H460, U251, HCT-116) in vitro and the highest level of NO releasing. Furthermore, compound 6a was revealed low acute toxicity to mice and weak haemolytic activity with potent tumor growth inhibition against mice H22 hepatocellular cells in vivo (51.5%).     

T-cell neoplasm induced by subcutaneous transplantation of a plasmacytoma: characterization of tumor cells.

Thymocytes, isolated 6 days following subcutaneous (sc) transplantation of BALB/c MOPC-315 plasmacytoma into F₁ (BALB/c × C57BL) hybrid mice, when injected sc into normal syngeneic mice, caused the development of a solid sc tumor. The cells of the newly developed tumor were of a mixed population of θ⁺F₁ (BALB/c × C57BL) and θ^? BALB/c cells (approximately 1:1), which represents a new type of mixed T cell-plasma cell neoplasm. Efforts were made to isolate the transformed thymocytes (θ⁺) from the plasmacytoma (θ^?) cells in the new tumor, exploiting differences in their surface properties. Treatment of the mixed tumor cell population with peanut agglutinin (PNA) revealed that only the T tumor cells were agglutinated. The agglutinated cells were recovered after dispersing the clumps with d-galactose (0.15 M) and consisted of 95% θ⁺ cells. The PNA-agglutinated cells were found to induce a similar tumor (85% θ⁺ cells) when injected sc into F₁ (BALB/c × C57BL) mice.     

Enhanced expansion of the thymic CD8+ cell subset as a potential mechanism for the generation of enhanced antitumor cytotoxicity by thymocytes from low-dose melphalan-treated MOPC-315 tumor bearers

Summary We have previously shown that thymocytes from low-dose melphalan (l-phenylalanine mustard)-treated MOPC-315-tumor-bearing mice (melphalan TuB) are able to generate an enhanced level of anti-MOPC-315 cytotoxicity, as compared to thymocytes from untreated MOPC-315-tumor-bearing mice or thymocytes from untreated or low-dose melphalan-treated normal mice, upon in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of recombinant interleukin-2 (rIL-2). Here we show that the generation of enhanced anti-MOPC-315 cytotoxicity by melphalan TuB thymocytes depends on the ability of the thymocytes to proliferate. In addition, the ability of melphalan TuB thymocytes to generate an enhanced level of anti-MOPC-315 cytotoxicity correlated with their ability to proliferate more readily than thymocytes from untreated tumor-bearing mice and thymocytes from untreated or melphalan-treated normal mice in response to stimulation with MOPC-315 tumor cells plus a low concentration of rIL-2. Moreover, although fresh melphalan TuB thymocytes do not contain a higher percentage of phenotypically mature cells (i.e., CD4^–/CD8⁺ or CD4⁺/CD8^–) than do thymocytes from normal mice or untreated tumor-bearing mice, after a 5-day culture with both MOPC-315 tumor cells and a low concentration of rIL-2, cultures of thymocytes from melphalan TuB contained a much higher percentage of CD4^–/CD8⁺ (but not CD4⁺/CD8^–) cells than did cultures of thymocytes from the other two sources. Since CD4^–/CD8⁺ cells were previously shown to be responsible for the exertion of antitumor cytotoxicity by thymocytes stimulated with MOPC-315 in vitro, our results indicate that the enhanced antitumor cytotoxicity exerted by melphalan TuB thymocytes following in vitro stimulation with MOPC-315 tumor cells in the presence of a low concentration of rIL-2 is due, at least in part, to an expansion of the pool of CD4^–/CD8⁺ effector cells.Supported by research grant CA-35 761 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy degreeSupported by career development award CA-01 350 from the National Cancer Institute     

A novel activity on thymocytes cells exerted by the rattlesnake (Crotalus durissus cumanensis) venom

Introduction: The thymus is active mainly during the neonatal and pre-adolescent periods. Objective: To test naïve thymocytes proliferation and monocytes stimulation. Materials and methods: We collected fresh thymus tissue from neonate mice after surgery. Suspension cells were coated onto Ficoll-Hypaque support. The obtained cells (thymocytes) were cultured measuring the proliferation of naïve T cells stimulated by Crotalus durissus cumanensis (Cdc) venom at sub-lethal doses (20 ng). Then, we supplemented the wells with AlamarBlue™ and incubated them for 5 h to test their proliferation. Mononuclear cells from mice peripheral blood were collected and layered onto the support of the Ficoll-Hypaque solution. We added the thymocytes actively dividing (25 x 10⁵ cells) from cultures stimulated with Cdc venom at 20 ng/well to cultured monocytes freshly obtained from the Ficoll-Hypaque separation. Both cell populations were incubated for 36 h until monocytes matured to macrophages.Results: The naïve thymocytes rapidly proliferated after stimulation with the Cdc venom (NTCdc) and these successively induced the maturation and function of monocytes progenitor cells to mature macrophages, which ingested Chinese ink. Conclusions: The naïve thymocytes proliferated by stimulation with the Cdc venom and subsequently the NT/Cdc induced the rapid maturation and function of monocytes progenitor cells becoming mature macrophages with their phenotypic characteristics.     

Synthesis and evaluation of 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-purine as new EGFR inhibitors

In present study, we described the synthesis and biological evaluation of a new class of EGFR inhibitors containing 2,9-disubstituted 8-phenylthio/phenylsulfinyl-9H-purine scaffold. Thirty-one compounds were synthesized. Among them, compound C9 displayed the IC₅₀ of 29.4?nM against HCC827 cell line and 1.9?nM against EGFR^L858R. Compound C12 showed moderate inhibitory activity against EGFR^{L858R/T790M/C797S} (IC₅₀?=?114?nM). Western bolt assay suggested that compound C9 significantly inhibited EGFR phosphorylation. In vivo test, compound C9 remarkably exhibited inhibitory effect on tumor growth at 5.0?mg/kg by oral administration in established nude mouse HCC827 xenograft model. These results indicate that the 2,9-disubstituted 8-phenylsulfinyl/phenylsulfinyl-9H-purine derivatives can act as potent EGFR(L858R) inhibitors and effective anticancer agents. Additionally, optimization of compound C12 may result in discovering the fourth-generation EGFR-TKIs.     

Structure-Plant Phytotoxic Activity Relationship of 7,7′-epoxylignanes, (+)- and (−)-verrucosin: Simplification on the aromatic ring substituents

The synthesized 7-aryl derivatives of (7R,7′S,8S,8′S)-(+)-verrucosin were applied to growth inhibitory activity test against ryegrass at 1 mM. 7-(3-Ethoxy-4-hydroxyphenyl) derivative 12 and 7-(2-hydroxyphenyl) derivative 4 showed comparable activity to those of (+)-verrucosin against the root (−95%) and the shoot (−60%), respectively. The growth inhibitory activity test against lettuce using synthesized 7-aryl derivatives of (7S,7′R,8R,8′R)-(−)-verrucosin at 1 mM showed that the activities of 7-(3-hydroxyphenyl) derivative 20 and 7-(3-ethoxy-4-hydroxyphenyl) derivative 28 are similar to that of (−)-verrucosin against the root (−95%). Against the shoot, 7-(3-hydroxyphenyl) derivative 20 showed higher activity (−80%) than that of (−)-verrucosin (−60%). As the next step, (7S,7′R,8R,8′R)-7-(3-hydroxyphenyl)-7′-aryl-(−)-verrucosin derivatives, in which the most effective 3-hydroxyphenyl group is employed as 7-aromatic ring, were synthesized for the assay against lettuce. In this experiment, 7′-(2-hydroxyphenyl) derivative 37 and 7′-(3-hydroxyphenyl) derivative 38 showed similar activity to that of derivative 20. The effect of 7- and 7′-aryl structures of 7,7′-epoxylignanes on the plant growth inhibitory activity was clarified. The 7- and 7′-aryl structures were simplified to show comparable activity to or higher activity than that of (−)-verrucosin. The plant growth inhibitory activity of a nutmeg component, (+)-fragransin C_3b, was estimated as −80% inhibition at 1 mM against ryegrass roots.     

Distinct patterns of lymphokine requirement for the proliferation of various subpopulations of activated thymocytes in a single cell assay

The frequency and capacity for clonal expansion of several murine thymocyte subpopulations responsive to various IL (fetal day 15, and adult CD4-8-, CD4+8- and CD4-8+) were investigated using a single-cell limiting-dilution cell culture system without filler cells. This assay requires the presence of PMA and ionomycin. The main conclusions of these studies are the following: 1) IL-4 is a better growth factor than IL-2 for immature thymocytes (fetal day 15 or adult CD4-8-). 2) IL-2 is a better growth factor than IL-4 for mature phenotype thymocytes (CD4+8- and CD4-8+). 3) IL-4 is a relatively poor growth factor for adult CD4-CD8- thymocytes and CD4+CD8- thymocytes, while it induced strong responses in fetal day 15 and CD4-8+ thymocytes. 4) IL-6 enhanced the response of CD4+8- thymocytes to either IL-2 or IL-4. 5) Cortisone-resistant thymocytes grown initially with IL-4 and then switched to IL-2 showed a significant decrease in cloning efficiency. No inhibitory effect was observed when cells were cultured first with IL-2 and then switched to IL-4. 6) Finally, supernatant from Con-A stimulated rat spleen cells induced maximal growth of all adult thymocyte populations tested, suggesting that unidentified thymocyte growth factor(s) remain to be characterized. These results indicate that the maturational stage of thymocytes determines their requirements for activation and proliferation.     

The immunocompetence of murine stromal cell-associated thymocytes

Thymocyte subpopulations that associate in vivo with distinct nonlymphoid cells of the thymus have been isolated, and their immunocompetence was analyzed. Previous studies have indicated that greater than 95% of such cells bear a surface antigen phenotype representative of immature thymocytes, and are among the earliest thymic compartments repopulated by bone marrow-derived cells after lethal and sub-lethal irradiation. Stromal cell-associated thymocytes may be activated in vivo because they proliferate well in vitro with no additional stimulus, and show little increase in proliferation with the addition of T cell mitogens or allogeneic spleen cells. Stromal cell-associated T cells contain cytotoxic T lymphocyte (CTL) precursors that are indistinguishable from mature peripheral T cells by the parameters of self tolerance, alloreactivity, H-2 restriction, and stringency of self H-2 preference. CTL precursor frequencies and the cytotoxic activity of cells further separated on the basis of high levels of Thy-1 expression argue against the possibility that stromal cell-associated CTL activity is due solely to contaminating mature lymphocytes. Our data suggest that stromal cell-associated thymocytes represent an intermediate subpopulation of thymocytes that is functionally mature and that expresses an immature surface phenotype. Furthermore, the imposition of self tolerance and MHC restriction specificity appears to be tightly associated with the acquisition of immunocompetence in these thymocyte subpopulations.     

Inhibition of erythroid cell growth by allogeneic murine lymphocytes. Evidence for a synergism between lymph node cells and thymocytes

Murine lymphoid cells from thymus and lymph nodes were tested for synergistic response in a graft-vs-host test. The test is based on the principle that allogeneic lymphocytes inhibit erythroid cell proliferation in the spleens of irradiated mice infused with syngeneic bone marrow cells.I was observed that mixtures of thymocytes and lymph node cells from the same parental strain yielded graft-vs-host responses in irradiated F₁-hybrids higher than expected by summing the responses of the two cell populations tested separately. A similar synergistic response was obtained using mixtures of thymocytes and lymph node cells obtained from the two parental strains of the hybrid, whereas such an effect was not detected using mixtures of lymph node cells or mixtures of thymocytes from the two parental strains. Nor could synergy be demonstrated between parental strain lymph node cells and thymocytes syngeneic with the bone marrow target cells. Thymocytes obtained from one parental strain which were injected into its irradiated F₁-hybrid transformed into a population of sensitized cells in the spleens of the recipients. This transformation was suppressed by the simultaneous injection of lymph node cells from the second parental strain. Since there is a synergistic immune response by such cell mixtures it is concluded that thymocytes may enhance the graft-vs-host response of lymph node cells. Parental strain thymocytes and lymph node cells, the latter being specifically immunologically tolerant to the bone marrow target cells, failed to give a synergistic response indicating that thymocytes do not transform unresponsive lymphocytes into responsive, but rather enhance the reactivity of existing, specifically responsive cells.The results thus show that thymocytes may enhance the response of lymph node cells in this specific graft-vs-host assay.     

Inhibitors from Carob (Ceratonia siliqua L.): II. Effect on Growth Induced by Indoleacetic Acid or Gibberellins A(1), A(4), A(5), and A(7)

Two inhibitory fractions (B₁ and C) from extracts of immature fruit of carob were tested for their ability to inhibit the action of indoleacetic acid (IAA) in three bioassays. There was no reduction of IAA-induced reactions in the Avena curvature test, abscission of debladed coleus petioles, or growth of cucumber hypocotyls. The highest ratio of inhibitor to IAA was 10,000 times greater than the ratio necessary to inhibit by 50% the growth caused by an equivalent amount of gibberellin A₃ in pea seedlings. At the highest concentration used, fraction C alone caused curvature of Avena coleoptiles. The inhibitory fractions appeared to enhance the effect of IAA in the cucumber test.     

Histone Deacetylase 3 Is Required for Efficient T Cell Development

Hdac3 is a key target for Hdac inhibitors that are efficacious in cutaneous T cell lymphoma. Moreover, the regulation of chromatin structure is critical as thymocytes transition from an immature cell with open chromatin to a mature T cell with tightly condensed chromatin. To define the phenotypes controlled by Hdac3 during T cell development, we conditionally deleted Hdac3 using the Lck-Cre transgene. This strategy inactivated Hdac3 in the double-negative stages of thymocyte development and caused a significant impairment at the CD8 immature single-positive (ISP) stage and the CD4/CD8 double-positive stage, with few mature CD4⁺ or CD8⁺ single-positive cells being produced. When Hdac3^−/− mice were crossed with Bcl-xL-, Bcl2-, or TCRβ-expressing transgenic mice, a modest level of complementation was found. However, when the null mice were crossed with mice expressing a fully rearranged T cell receptor αβ transgene, normal levels of CD4 single-positive cells were produced. Thus, Hdac3 is required for the efficient transit from double-negative stage 4 through positive selection.     

Somatic inactivation of Tp53 in hematopoietic stem cells or thymocytes predisposes mice to thymic lymphomas with clonal translocations

TP53 protects cells from transformation by responding to stresses including aneuploidy and DNA double-strand breaks (DSBs). TP53 induces apoptosis of lymphocytes with persistent DSBs at antigen receptor loci and other genomic loci to prevent these lesions from generating oncogenic translocations. Despite this critical function of TP53, germline Tp53^−/− mice succumb to immature T-cell (thymic) lymphomas that exhibit aneuploidy and lack clonal translocations. However, Tp53^−/− mice occasionally develop B lineage lymphomas and Tp53 deletion in pro-B cells causes lymphomas with oncogenic immunoglobulin (Ig) locus translocations. In addition, human lymphoid cancers with somatic TP53 inactivation often harbor oncogenic IG or T-cell receptor (TCR) locus translocations. To determine whether somatic Tp53 inactivation unmasks translocations or alters the frequency of B lineage tumors in mice, we generated and analyzed mice with conditional Tp53 deletion initiating in hematopoietic stem cells (HSCs) or in lineage-committed thymocytes. Median tumor-free survival of each strain was similar to the lifespan of Tp53^−/− mice. Mice with HSC deletion of Tp53 predominantly succumbed to thymic lymphomas with clonal translocations not involving Tcr loci; however, these mice occasionally developed mature B-cell lymphomas that harbored clonal Ig translocations. Deletion of Tp53 in thymocytes caused thymic lymphomas with aneuploidy and/or clonal translocations, including oncogenic Tcr locus translocations. Our data demonstrate that the developmental stage of Tp53 inactivation affects karyotypes of lymphoid malignancies in mice where somatic deletion of Tp53 initiating in thymocytes is sufficient to cause thymic lymphomas with oncogenic translocations.     

Immature CD4+CD8+ Thymocytes Are Preferentially Infected by Measles Virus in Human Thymic Organ Cultures

Cells of the human immune system are important target cells for measles virus (MeV) infection and infection of these cells may contribute to the immunologic abnormalities and immune suppression that characterize measles. The thymus is the site for production of naïve T lymphocytes and is infected during measles. To determine which populations of thymocytes are susceptible to MeV infection and whether strains of MeV differ in their ability to infect thymocytes, we used ex vivo human thymus organ cultures to assess the relative susceptibility of different subpopulations of thymocytes to infection with wild type and vaccine strains of MeV. Thymocytes were susceptible to MeV infection with the most replication in immature CD4⁺CD8⁺ double positive cells. Susceptibility correlated with the level of expression of the MeV receptor CD150. Wild type strains of MeV infected thymocytes more efficiently than the Edmonston vaccine strain. Thymus cultures from children ≥3 years of age were less susceptible to MeV infection than cultures from children 5 to 15 months of age. Resistance in one 7 year-old child was associated with production of interferon-gamma suggesting that vaccination may result in MeV-specific memory T cells in the thymus. We conclude that immature thymocytes are susceptible to MeV infection and thymocyte infection may contribute to the immunologic abnormalities associated with measles.     

Presence of an enlarged pool of MOPC-315-specific cytotoxic T lymphocyte precursors in the thymuses of mice that eradicated a large MOPC-315 tumor as a consequence of low-dose melphalan therapy

Summary We have previously shown that, as a consequence of low-dose melphalan (l-phenylalanine mustard) treatment, thymocytes from mice bearing a large, day-10 MOPC-315 tumor, but not thymocytes from normal mice, acquire the ability to generate an enhanced level of antitumor cytotoxicity upon in vitro stimulation with MOPC-315 tumor cells plus low concentrations (9.0–90 IU/ml) of exogenous interleukin-2 (IL-2) [23]. Here we show that the time interval between tumor inoculation and low-dose melphalan therapy as well as the magnitude of tumor burden at the time of the chemotherapy are important for the ability of the drug to render thymocytes more responsive to in vitro stimulation with MOPC-315 tumor cells plus low concentrations of recombinant IL-2 (rIL-2). Specifically, the chemotherapy was found to be effective in enhancing the thymic antitumor reactivity only if the mice bore a large, late-stage tumor. Comparison of thymocytes from untreated mice bearing a large, late-stage tumor to thymocytes from normal mice revealed that tumor-bearer thymocytes contained approximately a three-fold higher frequency of cytotoxic T lymphocyte precursors (CTLp) for MOPC-315-associated antigens. Following curative low-dose melphalan therapy of mice bearing a large, latestage MOPC-315 tumor, the frequency of CTLp for MOPC-315-associated antigens increased further, reaching a level approximately tenfold higher than that found among thymocytes of normal mice. At the same time, the frequency of CTLp for an antigenically unrelated allogeneic tumor (EL4) as well as the overall percentage of mature cells was not increased. The cells responsible for the exertion of the enhanced antitumor cytotoxicity following in vitro stimulation of thymocytes from mice treated with low-dose melphalan when they have a large, latestage MOPC-315 tumor are of the CD8⁺/CD4^– phenotype. Thus, the enhanced level of antitumor cytotoxicity generated by thymocytes from mice that are treated with low-dose melphalan when they have a large, late-stage MOPC-315 tumor is due, at least in part, to the presence of an enlarged pool of CTLp specific for MOPC-315-associated antigens, which mature into CD8⁺/CD4^– effector cells upon stimulation with MOPC-315 tumor cells plus low concentrations of rIL-2.supported by Research Grant CA-35761 from the National Cancer Institute and Research Grant EY-05945 from the National Eye Institute. This work is in partial fulfillment of the requirements for the Doctor of Philosophy degree of M. M. B. M. B. M. was supported by a Career Development Award CA-01350 from the National Cancer Institute.