Induction of allospecific suppressor T cells in vitro.

Incubation of mouse thymic lymphocytes with irradiated allogeneic spleen cells gave rise to suppressor cells. The suppressor activity was assayed by adding the incubated cell mixture to a mixed lymphocyte culture (MLC) in which the responder cells were syngeneic with the sensitized thymocytes and the stimulator cells were syngeneic with the sensitizing spleen cells. Such addition suppressed significantly thymidine incorporation in the mixed lymphocyte reaction (MLR). The suppressor cells were found to carry the θ antigen and to function allospecifically, as shown by cross-testing in three allogeneic combinations. Our data suggest that these cells may originate from immature cortisone-sensitive thymic lymphocytes and also provide some preliminary information concerning their mode of action.     

A model for the autosensitization autoantibody production associated with xenogeneic thymic RNA.

Normal C57BL/6 bone marrow cells cultured for 3 weeks with xenogeneic thymic RNA and syngeneic C57BL/6 antigens (immunoglobulin G or red blood cells) produced anti-immunoglobulin antibody or anti-mouse red blood cell antibody (hemolysin). Addition of both xenogeneic thymic RNA and autoantigens to bone marrow cultures was necessary to elicit autosensitization. Syngeneic thymic RNA would not substitute for xenogeneic RNA. Normal recipients inoculated with syngeneic kidney or spinal cord homogenates and xenogeneic thymic RNA developed albuminuria or motor neuropathies within 10 days. Histologic examination of tissues from these animals revealed immunoglobulin deposits on glomerular or tubular basement membranes or on myelin sheaths. These changes were not observed in tissues from control animals inoculated with only the organ homogenates. Normal mice injected with syngeneic bone marrow cells, which had been autosensitized in vitro against kidney or spinal cord homogenates, also developed albuminuria or motor neuropathies, respectively. These abnormalities were observed only if bone marrow cells had been cultured with both xenogeneic thymic RNA and autoantigens. Histologic examination of tissues from these mice also revealed immunoglobulin deposits in kidney or spinal cord tissues. These results demonstrate that xenogeneic thymic RNA can play important roles in the formation of autoantibodies.     

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells: I. Study of proliferative response

Culture of normal CBA lymphocytes on monolayers of syngeneic thyroid epithelial cells leads to significant thymidine incorporation. The specificity of this model was demonstrated by depletion of the CBA lymphocytes binding to syngeneic thyroid cells and the increase of thymidine uptake after secondary exposure on syngeneic thyroid monolayers. Removal of B cells (by treatment with anti-Ig serum plus complement) or of adherent cells does not modify the proliferative response whereas T-cell depletion strongly diminishes the response. Thus T cells are stimulated to undergo DNA synthesis and are sensitized when exposed to syngeneic thyroid epitelial cells. The nature of the antigens recognized by T cells (native autoantigen, enzyme, or virus-modified autoantigen) is not yet determined. Whether such autoreactive T cells play a role in the onset of experimental autoimmune thyroiditis as regulatory T-cells or cytotoxic effector cells is discussed.     

Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates. Our observations should facilitate efforts to achieve a differentiated functional state of Sertoli and peritubular cells in culture as well as to select secretory proteins for assessing their possible biological role in testicular function.     

In vitro and in vivo studies on the mechanism of experimental autoimmune thyroiditis in guinea pigs

Thyroid explants of inbred strain 13 guinea pigs were grown in a semisynthetic medium containing 0.3 IU of thyroid-stimulating hormone. The monolayer retained the capacity in vitro to form thyroglobulin. Sensitized lymphocytes from animals with autoimmune thyroiditis could specifically lyse these thyroid target cells in vitro in the presence of an appropriate amount of specific antigen. This cytotoxicity was not observed in thyroid epithelial cells which had been incubated (a) with normal lymphocytes or (b) with purified macrophages either from normal animals or from animals with autoimmune thyroiditis. When thyroid cells were incubated with hyperimmune antithyroglobulin serum, cytolysis did not occur, whether or not complement was added. The cytopathic effect of sensitized lymphocytes was further demonstrated to be caused by a soluble cellular product, termed thyroid cytotoxic factor, or TCF, which was released from sensitized lymphocytes under the stimulation of specific antigen, thyroglobulin, and could exert a cytotoxic effect directly on the target cells. Direct cell-to-cell contact was not required in this type of cell-mediated cytolysis.     

AKR leukemogenesis: identification and biological significance of thymic lymphoma receptors for AKR retroviruses.

We have previously demonstrated that in vitro cell lines of mouse thymic lymphomas express surface receptors specific for the retrovirus that induced them. This study extends these observations to an analysis of receptor-bearing cells in the preleukemic and leukemic phases of spontaneous AKR thymic lymphomagenesis. AKR mice regularly begin expressing N-tropic retroviruses (as assayed on NIH fibroblasts by the XC plaque assay) in several tissues early in life; thymic lymphocytes also express these viruses, but are not autonomously transformed. Later thymic lymphomas emerge which are capable of metastasizing in the host of origin or transplanting leukemias into syngeneic hosts. Just prior to the appearance of thymic lymphomas, these mice also begin producing xenotropic retroviruses [as assayed in xenogeneic (For example, mink) fibroblasts], and concomitant with the appearance of the leukemias is the appearance of "recombinant" retroviruses which cause mink fibroblast foci (MCF); these viruses express elements of both N- and X-tropic virus envelopes and N-tropic viral gene products in their cores. Spontaneous AKR leukemias also produce other retroviruses which do not cause XC plaques or mink fibroblast foci; these are called SL viruses. The subject of this study was to test whether in vivo thymocytes in the preleukemic and leukemic periods also bear receptors specific for N-tropic, recombinant MCF and SL AKR retroviruses. We demonstrated that each spontaneous thymic lymphoma does bear receptors that bind viruses produced by the lymphomas and MCF-247 to a high degree and that bind N-ecotropic AKR retroviruses less well. Thymic lymphocytes predominating in the preleukemic period do not express detectable levels of receptors for either of the viruses. In some mice, receptor-positive cells co-exist with receptor-negative cells; only the receptor-positive cells are capable of transplanting leukemia to syngeneic hosts. We conclude that the presence of specific cell surface receptors for lymphoma cell-produced and recombinant AKR retroviruses is a marker for leukemia in these hosts.     

Increased mitogenic reactivity of normal spleen cells to T lectins induced by thymus humoral factor (THF)

When normal spleen cells were incubated for 24 hr in medium containing thymic humoral factor (THF) and then stimulated by phytohemagglutinin (PHA) or concanavalin A (Con A), a significant increase in the mitogenic reactivity of these cells was observed. When stimulation to T lectins was performed simultaneously with THF, a strong inhibition in cell reactivity was found. It seems that these opposite effects of THF on cell reactivity to T lectins are determined by the sequence of events which lead to maturation of lymphoid cells. Thymic humoral factor does not modify the response of cells to B mitogen lipopolysaccharide (LPS), thus suggesting that this maturative effect on lymphoid cells is exerted on T lymphocytes only.     

Sensitization of T lymphocytes in vitro by syngeneic macrophages fed with tumor antigens.

We investigated the interaction between T lymphocytes and macrophages in the vitro sensitization of lymphocytes against tumor cells. Spleen cells were sensitized in vitro by syngeneic peritoneal macrophages that had been fed with cell-free antigen preparation of syngeneic tumor cells. The sensitized T lymphocytes acquired specific cytotoxic cells. The sensitized T lymphocytes acquired specific cytotoxic activity in vitro and the capacity to inhingeneic fibroblasts, or the antigen preparation by itself were not able to sensitize the lymphocytes against the tumor.     

Experimental autoimmune orchitis: in vitro induction of an autoimmune disease.

Experimental autoimmune orchitis (EAO) can be induced in vitro. Normal lymph node lymphocytes cultured with autologous dissociated testis cells form rosette-like aggregates and later undergo blast transformation and proliferation. These stimulated lymphocytes cause in vivo EAO lesions, when injectd into syngeneic recipients. Moreover, their autoimmune reactivity can be monitored by an in vitro cytostasis assay. Density gradient analysis of the early lymphocyte-testis cultures reveals that the autoimmune reactive lymphocytes are enriched in the rosette populations. It therefore appears that testicular self-antigens are recognized by clonally preformed autologous lymphocytes.     

Spent media from immature seminiferous tubules and Sertoli cells inhibit adult rat Leydig cell aromatase activity

Adult rat Leydig cell aromatase activity is stimulated 2.5 fold by LH or dbcAMP. Spent media prepared from seminiferous tubules or Sertoli cells of immature rats depress both the basal and the LH stimulated estradiol syntheses (25 and 20% decreases, respectively). These inhibitory effects are further enhanced when FSH is added to the culture medium of seminiferous tubules or Sertoli cells. Rat serum as well as culture media from other cell lines are ineffective while seminiferous tubule media from other immature animals (mouse, guinea-pig, calf) inhibit the aromatase activity. This Sertoli cell factor is a heat stable protein (molecular weight greater than 10 kDa), different from the LHRH-like Sertoli cell compound, which acts on the aromatase activity at a step beyond the adenylate cyclase.     

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.     

Human autoantibodies to spermatogenic antigens and Sertoli cells

Immunofluorescence staining using human autoantibodies is a simple and reliable method for investigation of meiotic and post-meiotic cells. Patients suffering from autoimmune diseases often produce circulating autoantibodies to antigens of germ cells and Sertoli cells. Four hundred human autoimmune sera were screened by indirect immunofluorescence on mouse seminiferous tubule cells. Autoantibodies of several specificities were found: one group reacted with organelles of meiotic prophase spermatocytes or spermatozoa. Included in this group were autoantibodies to synaptonemal complexes, sex vesicle, acrosome, and sperm tail. A second group of autoantibodies was found to stain different spermatogenic cell types uniformly, such as round spermatids or Sertoli cells.     

Experimental allergic encephalomyelitis: clinical disease and enhanced cellular transfer in the absence of lymphocyte proliferative responses against syngeneic MBP

Experimental allergic encephalomyelitis (EAE) was induced in Lewis rats using several different immunization protocols, and draining lymph node cells from these animals were assayed for proliferation against heterologous, homologous, and syngeneic MBP, and syngeneic spinal cord. Proliferative responses were largely stimulated by nonsyngeneic antigenic determinants and correlated better with the antigen used to induce EAE than with signs of autoimmune disease. Lymph node cells from rats immunized with either guinea pig spinal cord or syngeneic MBP did not proliferate measurably when restimulated in vitro with syngeneic MBP, yet lymphoid cells from these animals were enhanced in their capacity to transfer EAE following in vitro stimulation with syngeneic MBP.     

Evaluation of cholesteryl ester transfer in the seminiferous tubule cells of immature rats in vivo and in vitro

Sertoli cells and germ cells are separated from the interstitial blood capillaries by an extracellular matrix and the peritubular cells, which constitute a barrier to the movement of plasma lipoproteins. The present study was undertaken to evaluate in vivo and in vitro the high density lipoprotein (HDL) cholesteryl ester transfer from plasma to seminiferous tubule cells in the testis of 30-day-old rats. Firstly, the transfer of HDL cholesteryl oleate from plasma to testicular compartments was evaluated and, secondly, the role of apolipoproteins A-I and E in the uptake of cholesteryl ester by Sertoli cells was investigated. At 2 h after the administration of HDL reconstituted with [3H]cholesteryl ester, dimyristoyl phosphatidylcholine and apolipoproteins, the tissue space in the interstitial cells (740 +/- 60 microliters g-1 cell protein) was fourfold higher than that in the seminiferous tubule cells (170 +/- 10 microliters g-1). Sertoli cells were isolated and incubated with [3H]cholesteryl ester HDL reconstituted with apolipoprotein A-I or E to evaluate the mechanisms of cholesteryl ester influx. At the same apolipoprotein concentration (50 micrograms apolipoprotein ml-1 medium), the uptake of [3H]cholesteryl oleate from phospholipid-apolipoprotein E vesicles was twofold higher than that with phospholipid-apolipoprotein A-I vesicles. The presence of heparin reduced the uptake of cholesteryl ester from apolipoprotein E vesicles but not with apolipoprotein A-I vesicles, indicating that uptake of apolipoprotein A-I vesicles via a secretion of apolipoprotein E by the cells themselves was not involved. These results demonstrate that plasma lipoprotein cholesterol is able to cross the testis lamina propria and that Sertoli cells take up cholesteryl ester for seminiferous tubule cell metabolism mainly via an apolipoprotein E pathway.     

Cytotoxicity of autosensitized lymphocytes restricted to the H-2K end of targets

Lymphocytes from rodents cultured on syngeneic fibroblasts become cytotoxic against syngeneic but not against allogeneic target cells. We investigated whether known antigens are involved in the phenomenon and the data indicate that H-2 antigens must be shared between sensitizing fibroblasts and responder lymphocytes to generate autocytotoxic cells. Furthermore, the cytotoxicity of autosensitized lymphocytes is restricted to target cells identical with respect to theK and/orI regions. F₁ hybrid lymphocytes cultured on parental fibroblasts develop cytotoxicity towards sensitizing cells. In contrast, parental lymphocytes cultured on F₁ hybrid fibroblasts will not damage the F₁ cells, although they are cytotoxic against both syngeneic and allogeneic parental cells. In addition, parental or F₁ hybrid lymphocytes cultured on parental fibroblasts are not cytotoxic against F₁ hybrid target cells. Fibroblasts heterozygous for theK end only, are also resistant to the cytotoxic action of such lymphocytes. Thus it seems that H-2 antigens, specifically theK end, antigens have a significant role in the phenomenon of autosensitization.     

T lymphocyte subpopulations and lymphocytotoxic antibodies in patients with autoimmune haemolytic anaemia

Evaluation of T lymphocyte subpopulations was performed on peripheral blood of patients affected by idiopathic or associated autoimmune haemolytic anaemia. A marked reduction of absolute number of T gamma and T mu cells was observed in 11 of 16 patients; a decrease of both OKT4+ and OKT8+ cells was found in 8 of 10 patients. Circulating cytotoxic antibodies against autologous and allogenic T lymphocytes and/or thymocytes were found in almost all the cases. T lymphocyte subsets depletion, probably connected to antibodies against T lymphocytes and their thymic precursors, could play a role in autoimmunity because of T3+/T4+ cell depletion.     

Antigen-specific augmentation factor involved in murine delayed-type footpad reaction. IV. Effect of delayed-type hypersensitivity augmentation factor on in vitro induction of DTH

We found an antigen-specific factor capable of augmenting delayed-type hypersensitivity (DTH) in the serum of mice sensitized with heterologous erythrocytes to induce a delayed footpad reaction (DFR), or in the culture supernatant of the mixture of sensitized T cells and specific antigens. This factor (DTH augmentation factor; DAF) was confirmed to augment DTH in transferred recipients. In this paper, such an activity of DAF was further investigated using the system with in vitro induction and local transfer of DTH. DAF also augmented the primary in vitro induction of DTH, when spleen cells from mice transferred with the DAF-containing serum 12 hr previously or spleen cells incubated with the DAF-containing serum on ice for 2 hr were cultured with heterologous erythrocytes. DAF acted on the induction phase of DTH and augmented a typical DTH which was dependent on Thy-1-positive T cells. DAF showed antigen specificity, but was not assigned to conventional immunoglobulin. The activity of DAF was detected when nylon-wool nonadherent cells were incubated with DAF prior to the culture of those cells and antigens, but not detected when only nylon-wool adherent cells were incubated with DAF. Thus, DAF exerted its effect through binding to acceptor cells which were included in nylon-wool nonadherent spleen cells from normal mice.     

Effects of cholinergic agonists on the proliferation and protein synthesis in a cultured thymic epithelial cell line

Thymic epithelial cells appear to release the humoral factors endowing precursors of T cells (thymus-dependent lymphocytes) with the capacity to differentiate and maturate into relatively mature T cells. We have separated the polypeptide fractions containing these factors from the culture supernatant of thymic epithelial cell line. Thymus is reported to be innervated by autonomic nervous system from the prenatal to the pubertal period. But the physiological significance of the nervous system in this lymphoid organ remains obscure. And the modulator of the epithelial cell functions, namely, production and release of the bioactive polypeptides have never been clarified. We show here that acetylcholine (Ach) or carbamylcholine (Cch) enhanced the proliferation of thymic epithelial cells from the TAD3 cell line at preconfluent state, and the protein synthetic activity at confluent state. This phenomenon was completely suppressed by the pretreatment of alpha-bungarotoxin (alpha-BTx). These results suggest that nicotinic Ach-receptors exist on the epithelial cell surface membrane and that the differentiation and maturation of thymic lymphocytes are indirectly regulated by the activated functions of thymic epithelial cells stimulated with cholinergic agonists.     

Thymus-derived lymphocyte-dependent rejection of syngeneic papovavirus (SV40) and methylcholanthrene tumors in inbred hamsters.

Treatment of specifically sensitized MHA hamster lymphoid cells with rabbit antisera specific for hamster thymus-derived lymphocytes, in the presence of C, eliminated those cells capable of inhibiting the growth of syngeneic SV40 and methylcholanthrene tumors in vivo. Thymectomized, lethally-irradiated, bone marrow-reconstituted hamsters, shown to be devoid to T cell function, were, after attempted specific sensitization to the two syngeneic tumor cell lines, unable to reject either tumor by direct challenge in vivo. In addition, lymphocytes from such animals were incapable of inhibiting the growth of either tumor cell line in normal syngeneic recepients in the tumor cell neutralization assay. These data strongly support the conclusion that specifically sensitized thymus-derived lymphocytes are required for the rejection of syngeneic SV40 and methylcholanthrene tumors in inbred hamsters.     

Induction of graft versus leukemia effects by cell-mediated lymphokine-activated immunotherapy after syngeneic bone marrow transplantation in murine B cell leukemia

 The feasibility of inducing graft versus leukemia (GVL) effects with allogeneic T cells in recipients of autologous bone marrow transplantation (BMT) was studied in a murine model (BCL 1) of human B cell leukemia/lymphoma. Allogeneic cell therapy, induced by infusion with peripheral blood lymphocytes, a mixture of allogeneic spleen and lymph node cells and allogeneic activated cell therapy, induced by in vitro recombinant-interleukin-2(rIL-2)-activated allogeneic bone marrow cells in tumor-bearing mice, prevented disease development in adoptive BALB/c recipients. Concomitant in vivo activation of allogeneic lymphocytes with rIL-2 suppressed even more effectively the development of leukemia in secondary adoptive recipients of spleen cells obtained from treated mice. In contrast, in vivo administration of rIL-2 after syngeneic BMT, with or without equal numbers of syngeneic lymphocytes, led to disease development in secondary recipients. Our data suggest that effective cell therapy can be achieved after SBMT by allogeneic but not syngeneic lymphocytes and that anti-leukemic effects induced by allogeneic lymphocytes can be further enhanced by in vitro or in vivo activation of allogeneic effector cells with rIL-2. Therefore, cell therapy by allogeneic lymphocytes following autologous BMT could become an effective method for inducing GVL-like effects on minimal residual disease provided that graft versus host disease can be prevented or adequately controlled. Received: 14 May 1996 / Accepted: 6 August 1996