Thymus-Derived Inhibitor of Lymphocyte Proliferation

A thymus crude factor (TCF) isolated from bovine thymus tissue has been tested for its effects on the proliferation of various murine cells. Specific inhibition in vitro has been found for DNA synthesis in murine T and B lymphocytes which appears not to be based on cytotoxicity. Moreover, TCF, when administered to mice, also interferes with the DNA synthesis in lymphoid tissue in vivo. Our data are suggestive for the presence in TCF of an endogenous ‘chalone-like’ inhibitor of lymphoid cell proliferation in vitro and in vivo.     

Critical role for CXCR4 signaling in progenitor localization and T cell differentiation in the postnatal thymus

T cell differentiation in the thymus depends on sequential interactions between lymphoid progenitors and stromal cells in discrete regions of the cortex. Here we show that CXCL12/CXCR4 signaling is absolutely required for proper localization of early progenitors into the cortex and thus for successful steady state differentiation. All early progenitors in the thymus express CXCR4, and its ligand (CXCL12) is expressed only by stromal cells in the cortex, where early progenitors are found. Early progenitors migrate in response to CXCL12 in vitro, while thymus-specific deletion of CXCR4 in vivo results in failed cortical localization and developmental arrest. These findings indicate a crucial and nonredundant role for CXCR4 in facilitating localization of early lymphoid progenitors to tissue regions of the thymus, where lineage commitment and proliferation are controlled.     

Isolation and characterization of glucocorticoid- and cyclic AMP-induced genes in T lymphocytes.

Glucocorticoids and cyclic AMP exert dramatic effects on the proliferation and viability of murine T lymphocytes through unknown mechanisms. To identify gene products which might be involved in glucocorticoid-induced responses in lymphoid cells, we constructed a lambda cDNA library prepared from murine thymoma WEHI-7TG cells treated for 5 h with glucocorticoids and forskolin. The library was screened with a subtracted cDNA probe enriched for sequences induced by the two drugs, and cDNA clones representing 11 different inducible genes were isolated. The pattern of expression in BALB/c mouse tissues was examined for each cDNA clone. We have identified two clones that hybridized to mRNAs detected exclusively in the thymus. Other clones were identified that demonstrated tissue-specific gene expression in heart, brain, brain and thymus, or lymphoid tissue (spleen and thymus). The kinetics of induction by dexamethasone and forskolin were examined for each gene. The majority of the cDNA clones hybridized to mRNAs that were regulated by glucocorticoids and forskolin, two were regulated only by glucocorticoids, and three hybridized to mRNAs that required both drugs for induction. Inhibition of protein synthesis by cycloheximide resulted in the induction of all mRNAs that were inducible by glucocorticoids. Preliminary sequence analysis of four of the 11 cDNAs suggests that two cDNAs represent previously undescribed genes while two others correspond to the mouse VL30 retrovirus-like element and the mouse homolog of chondroitin sulfate proteoglycan core protein.     

Abelson murine leukemia virus mutants deficient in kinase activity and lymphoid cell transformation.

Abelson murine leukemia virus transforms both lymphoid cells and fibroblasts in vitro and induces a unique type of thymus-dependent lymphoma in vivo. Four fibroblast-transforming strains of Abelson murine leukemia virus were identified, based on the sizes of the Abelson murine leukemia virus-specific phosphoproteins produced by these isolates. Two of these strains, the standard P120- and the P160-producing viruses, transformed lymphoid cells efficiently in vitro and induced Abelson disease in vivo. Two other strains, which synthesized small Abelson murine leukemia virus-specific proteins with molecular weights of 90,000 (P90) and 100,000 (P100), transformed lymphoid cells very poorly both in vitro and in vivo. The reduced oncogenic potentials of these isolates were correlated with a high level of synthesis of fairly unstable P90 and P100. In addition, neither P90 nor P100 functional efficiently in protein kinase assays. The correlation of abnormal metabolism and deficient protein kinase activity with the reduced oncogenic potentials of these virus strains supported a direct role for these proteins and the kinase activity in transformation. Furthermore, these results suggested that the requirements for lymphoid cell transformation and fibroblast transformation are different.     

Studies on the regulation of lymphocyte production in the murine thymus and some effects of a crude thymus extract.

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitiotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G0/G1 cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

Thymus derived inhibitor of lymphocyte proliferation. I. Isolation and assessment of tissue specificity.

Preparations from bovine thymus tissue were analyzed for their inhibitory effects during in vitro lymphocyte proliferation. The results indicate that these preparations strongly inhibit DNA synthesis in stimulated human peripheral lymphocytes, bone marrow cells, thymocytes and lymphoblastoid cells. This inhibition was dose-dependent and not due to cytotoxicity of the preparations. No inhibition was found of the spontaneous proliferation of HeLa cells and human fibroblasts indicating that the inhibitory effect was specific for proliferating lymphocytes. Control preparations from bovine liver or kidney did not show any suppression in the test systems used.     

Effect of myelopeptides on the synthesis of DNA and total protein in the cells of the lymphoid organs in the mouse

Using double 3H-thymidine/14C-amino acid label, the influence of myelopeptides (MP) on chromosomal DNA and total protein (without histones) synthesis has been studied in vitro in mouse lymphoid organ cells. It has been shown that MP cause a decrease in DNA labelling and a parallel increase in protein labelling in bone marrow cells and have practically no effect on these two parameters in thymus, lymph node and spleen cells. It has been found that MP have only mitogenic effect on the above-mentioned organ cells, but in combination with 2-mercaptoethanol. The data obtained show that MP possess the properties of cell-differentiating factors.     

Systemic administration of recombinant interleukin 2 stimulates in vivo lymphoid cell proliferation in tissues

Recent work in our laboratory has demonstrated that the repeated injections of high doses of recombinant interleukin 2 (IL 2) can dramatically reduce the number of established pulmonary and hepatic metastases and the growth of intradermal tumors in a variety of murine tumor models. We have thus undertaken studies to define the mechanisms underlying these in vivo effects of IL 2. Using an in vivo DNA-labeling technique in which we employed 5-[125I]iodo-2'-deoxyuridine (125IUdR), we examined the in vivo cell proliferation in the tissues of mice treated with IL 2. A proliferation index (PI) was calculated by dividing the raw counts per minute (cpm) of tissues in IL 2-treated mice by the cpm in corresponding tissues of control animals. At an IL 2 dose of 6000 U given i.p. three times a day, the highest 125IUdR incorporation was seen in the lungs, liver, spleen, kidneys, and mesenteric lymph nodes (PI = 6.9, 6.9, 5.1, 7.1, 24.6, respectively, at 5 days). The amount of lymphoid proliferation in these organs was a direct function of the dose of IL 2 administered. Other tissues including thymus, intestines, skin, and hind limb showed no significant increase in 125IUdR uptake even after host treatment with the highest doses of IL 2. Blood and brain demonstrated intermediate incorporation of the radiolabel. Preirradiation of the host largely eliminated the proliferative response to IL 2. Histologic studies of normal and irradiated mice receiving IL 2 corroborated the result of the 125IUdR findings. In normal IL 2-treated mice, large collections of activated lymphoid cells were seen, most prominently in the lungs, liver, and kidneys, whereas markedly decreased lymphoid proliferation was evident histologically in preirradiated mice. A fluorescein-labeled monoclonal antibody directed against the Thy-1.2 surface determinant was used to identify these dividing cells in frozen tissue sections as T lymphoid cells. Activated lymphocytes isolated from the lungs, liver, spleen, and mesenteric lymph nodes of IL 2-treated mice demonstrated significant lysis of a fresh murine sarcoma target in short-term 51Cr-release assays. These studies demonstrate that the systemic administration of recombinant IL 2 causes in vivo activation and proliferation of host lymphoid cells and has important implications for the adoptive immunotherapy of tumors.     

Effects of C-reactive protein on the lymphoid system. II. Inhibition of mixed lymphocyte reactivity and generation of cytotoxic lymphocytes.

C-reactive protein (CRP), an acute phase reactant which increases in concentration during inflammation, has been found to bind to human T cells and to inhibit certain of their functions. In the present study CRP was found to display a binding specificity for theta-bearing cells from mouse peripheral lymphoid tissue but not for thymus cells. CRP inhibited the proliferative response in a similar manner in both murine and human mixed lymphocyte reactions. This inhibition was prevented by the addition of the CRP substrate, pneumococcal C-polysaccharide (CPS), and was not a result of toxicity of CRP for lymphocytes. By contrast the response of spleen lymphocytes to mitogenic Con A concentrations was not altered by CRP. CRP also exerted an inhibitory effect on the in vitro generation of cytolytic T lymphocytes (CL) in mixed lymphocyte reactions of mouse spleen cells. The expression of the cytolytic process by T cells sensitized either in vivo or in mixed lymphocyte cultures was not altered in the presence of CRP. Therefore, CRP appears to influence the inductive phase of the allograft response and perhaps exerts a regulatory effect on cellular immune responsiveness during inflammatory reactions.     

Synergy among rat T cells in the proliferative response to alloantigen.

A synergistic interaction in the proliferative response to alloantigen is described for mixtures of rat thymus and lymph node cells. The optimal conditions for synergy are quantitatively defined. Regression analysis of the slope of the dose-response curve has been utilized to estimate the degree of interaction in thymus-lymph node cell mixtures. The slope of the response of cell mixtures was noted to be significantly greater than the slope for the response of lymph node cells alone. Irradiation was shown to have a differential effect on the response of thymus and lymph node cells in mixtures. Irradiated thymus cells retained the capacity for synergy in mixtures, whereas irradiated lymph node cells did not. Additional studies have demonstrated that both de novo protein synthesis and specific antigen recognition by both responding cell populations in mixtures was required for maximal synergy. These studies demonstrate that synergy cannot be explained as an artifact of altered cell density in vitro. They establish that thymus cells and lymph node cells represent distinct subsets which manifest qualitatively different functions in the proliferative response to alloantigen. Thymus cells can respond directly to alloantigen by proliferation but also have the capacity to amplify the proliferative response of lymph node cells—a capacity which is resistant to X irradiation but requires recognition of alloantigen and de novo protein synthesis. Lymph node cells may similarly respond by proliferation to alloantigen but lack the amplifier activity of thymus cells. Synergy for rat lymphoid cells, like mouse lymphoid cells, has been shown to involve an interaction of thymus-derived lymphocytes.     

General similarities and differences in the postradiation death of various animal species and of man

It was found in various animal species and man that an ordered internucleosome fragmentation of DNA is characteristic of lymphoid cells dying in the interphase. Both in vivo and in vitro, the postirradiation DNA degradation in thymocytes of rodents and piglets preceded the increase in the permeability of their plasma membrane. The in vivo kinetics of death of lymphoid cells from the thymus and spleen is similar in rodents and piglets. Rat thymocytes died in vitro earlier than thymocytes of piglets, calves and man which was evidently associated with a worse adaptive capacity of the latter to cultivation conditions.     

Thymic humoral factor gamma 2: purification and amino acid sequence of an immunoregulatory peptide from calf thymus

Thymic humoral factor gamma 2 (THF-gamma 2), an octapeptide essential for immune regulation, was purified from calf thymus. The purification of THF-gamma 2, monitored in vitro and in vivo in mouse splenocyte proliferation assays, was achieved by gel filtration of low molecular weight thymus extracts followed by ion-exchange chromatography and sequential reversed-phase high-performance liquid chromatography. The process yielded 5 micrograms of THF-gamma 2/1000 kg of thymus tissue. The concentration of THF-gamma 2 required for augmentation of lymphocyte proliferation and interleukin 2 production was 5 ng/mL in vitro and 10 ng/kg per mouse in vivo. THF-gamma 2 has the amino acid sequence Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. The proposed structure has been confirmed because a peptide was synthesized on the basis of this sequence that showed activity identical with that of the biological molecule. It shows no homology to the amino acid sequence of other thymic hormones nor is it part of any peptide or protein of known sequence. THF-gamma 2 retains essentially all of the biological activity of the thymus extract from which it is derived.     

A soluble 61-kDa protein is associated with inhibition of lectin-induced proliferation and IL-2 synthesis

An inhibitor of lectin-induced splenocyte proliferation from serum of normal chickens has been characterized. This suppressive factor, found in both serum and plasma and at concentrations as low as 3%, causes a 50% inhibition in proliferative responses to T-cell lectins of autologous and heterologous lymphoid cells. The inhibitor in serum also dramatically suppresses murine IL-2 synthesis, proliferation of murine spleen cells stimulated with PHA, and synthesis of DNA in xenogeneic-transformed mammalian lymphoblastoid cell lines. Serum does not block binding of the lectin to lymphoid cells and the suppressive activity cannot be overcome by any dose of lectin. The inhibitor of DNA synthesis is destroyed by pepsin. NH4(2)SO4 (50%) and TCA (15%) treatments both precipitate the suppressor factor, which further indicates that the suppressive factor is a protein. A 330-fold purification of the inhibitory protein from serum was obtained when boiled serum was passed over a Sepharose 6B and then a DEAE-Sephacel column which was washed at pH 5.0 and eluted with 0.2 M NaCl. SDS-PAGE with silver staining revealed a nonreduced protein with an apparent molecular weight of 61 kDa. Less than 2 micrograms of the protein thus obtained caused a 50% inhibition in the proliferation of chicken lymphoid cells to Con A. The inhibitor of DNA synthesis is therefore not cytotoxic, does not bind to Con A or to mannose or glucose residues on lymphocytes, is acid and heat stable, and is associated with a protein that has a molecular weight of 61 kDa. Since such low concentrations of this naturally occurring, proteinaceous, immunosuppressive factor cause substantial inhibition of IL-2 synthesis and proliferative activity of T cells, this protein may be a very important immunomodulator.     

Soluble expression of recombinant human secondary lymphoid chemokine (SLC) in E. coli and research on its in vitro and in vivo bioactivity

Secondary lymphoid tissue chemokine (SLC) is a CC chemokine that plays an important role in leukocytes homing to lymphoid tissues. The ability of SLC to co-localize both T cells and dendritic cells formed the rationale to evaluate its utility in cancer immunotherapy. The in vivo antitumor effect of murine SLC (mSLC) has been well documented, but little is known about that of human SLC (hSLC). To investigate the antitumor efficiency in vivo of hSLC, the hSLC gene was artificially synthesized and induced to express as a soluble form in Escherichia coli. After purification, the purity of the recombinant human SLC (rhSLC) protein was above 95% by SDS-PAGE analysis. The K(d) of rhSLC binding to peripheral blood lymphocytes (PBLs) was 0.2186 +/- 0.02675 microM as assessed by FACS, and the maximal chemotactic index of rhSLC was 9.49 at 100 nM as assessed by in vitro chemotaxis assay. Then genomic sequences of hSLC and mSLC, and of human CCR7 (hCCR7) and murine CCR7 (mCCR7), the receptor for SLC, were aligned. It was found that hSLC and mSLC share 70.72% identity and hCCR7 and mCCR7share 86.77% identity. Furthermore, we found that rhSLC could chemoattract murine peripheral blood mononuclear cells (PBMCs) in vitro. On the basis of these facts, immune competent mice inoculated with S180 sarcoma cells were chosen as an in vivo model. Intratumoral injections of rhSLC inhibited tumor growth and increased survival. These findings suggest that, despite its incapability to bind to either human or murine CXCR3, which is related to angiostasis, rhSLC can induce an antitumor response in vivo by another route. This report proves that rhSLC has a potent tumor-inhibition ability that makes it a promising candidate agent in cancer immunotherapy.     

Selective cell surface expression of thymus leukemia antigen during S phase of the cell cycle.

The expression of the thymus leukemia antigen (TL) was studied on a murine leukemia cell line (ASL-1W) grown in vitro and separated into cell cycle phases by velocity sedimentation or growing synchronously in culture. The expression of TL was determined qualitatively by direct cytotoxicity and quantitatively by a modified inhibition of cytotoxicity assay. TL expression was found to vary with DNA synthesis. The hypothesis that expression is coordinately regulated with DNA synthesis, and the relationship of this to the restricted expression of TL on rapidly dividing cells is disucssed.     

Deoxyribonucleic acid synthesis in mammalian nuclei. Incorporation of deoxyribonucleotides and chain-terminating nucleotide analogues

The properties of a nuclear preparation from rat liver and thymus are described. (1) Nearest-neighbour analysis after incorporation of (32)P-labelled nucleotide residues from dATP, dCTP, dGTP, dTTP and arabinofuranosyl analogues of CTP and ATP shows template-dependent DNA synthesis. (2) Where primer termini are limiting, incorporation of arabinofuranosyl analogues of AMP and CMP residues proceeds to a limit indicating that both of these analogues are DNA chain terminators. (3) No large differences have been found between the priming potentialities or the intrinsic DNA polymerase activities of nuclei from resting or regenerating liver and the relationship of this DNA synthesis in vitro to DNA replication or repair in vivo is briefly discussed.     

The murine Kupffer cell. II. Accessory cell function in in vitro primary antibody responses, mitogen-induced proliferation, and stimulation of mixed lymphocyte responses

The ability of murine Kupffer cells to function in several in vitro immunologic systems was investigated. These cells have been shown previously to function as accessory cells in antigen-stimulated T cell proliferation in response to protein antigens. In the present study it has been demonstrated that murine Kupffer cells also are competent as accessory cells in in vitro primary antibody responses to TNP-KLH and for T cell proliferative responses to concanavalin A. In addition, murine Kupffer cells were found to be potent stimulators of mixed lymphocyte responses. These studies extend previous observations by demonstrating that Kupffer cells are competent accessory cells in several distinct in vitro correlates of in vivo immune responses. The role of Kupffer cells in in vivo immune responses, particularly those to enterically derived antigens, may require re-evaluation in the light of these findings.