Syngeneic Thymocytes Research Articles

Dendritic cells generated with Flt3L and exposed to apoptotic cells lack induction of T cell anergy and Foxp3+ regulatory T cell conversion in vitro

Removal of apoptotic cells, which appear during the steady state, is a pre-requisite to prevent generation of secondary necrotic cells that may lead to autoimmunity. The recognition of apoptotic material by dendritic cells (DCs) has been proposed to convert them into tolerogenic DCs equipped with specialized tolerogenic mechanisms on T cells. However, comparative studies to demonstrate functional alterations of DCs upon exposure to apoptotic cells have not been performed so far. Here we show that immature murine bone marrow-derived DCs generated with GM-CSF (GM-DCs) or Flt3L (FL-DCs) interact with live or apoptotic syngeneic thymocytes. As expected, GM-DCs phagocytose apoptotic but not live cells, FL-DCs only show trogocytosis of membrane parts. Interaction with live or apoptotic thymocytes did not lead to DC maturation. Both GM-DCs and FL-DCs present OVA as protein, peptide and membrane-associated antigens. Interestingly, only GM-DCs were able to induce T cell anergy or convert naïve T cells into FoxP3+ regulatory T cells (Tregs) but FL-DCs did not show either of these effects. Unexpectedly, exposure of immature GM-DCs to live or apoptotic thymocytes did not improve DC functions in both types of in vitro T cell tolerance induction assays. Together, our data suggest that these tolerogenic in vitro measures of immature BM-DCs are not further enhanced by exposure to apoptotic cells and may depend on the generating cytokine.

Heat shock protein-GP96 as an innate sensor of damage and activator of autoreactive NKT and regulatory T cells during liver regeneration

Tissue disintegration after injury leads, in the endoplasmic reticulum (ER), to activation of adaptive pathways known as the ER stress response. It is directed to the correction of unfolded proteins and to the activation of proteasome-dependent ER-associated degradation of the misfolded proteins, but induces also a rapid activation of natural and adaptive immunity, since a ER resident heat shock protein-gp96 acts not only as a molecular chaperone, but also as a strong adjuvant, able to cross-present the antigenic peptides onto MHC class I or MHC class II pathways. Analyzing its potential role in processes of normal growth, in mice subjected to 1/3 partial hepatectomy (pHx) we determined the tissue expression of gp96 protein and mRNA in regenerating liver, thymus and spleen, determining simultaneously the phenotypic profile and spontaneous cytotoxic activity of intrahepatic and splenic mononuclear lymphatic cells (MNLC) against NKT- and NK-cells sensitive targets (syngeneic thymocytes and YAC-1) in wild, perforin and FasL deficient mice. The data have shown that pHx induces fast overexpression of gp96 protein and mRNA in hepatocytes, spleen and thymus, with accumulation of CD3intermediate/NK1.1+/CD69+ cells (liver) and Foxp3+CD4+CD25+ cells (liver and thymus). Simultaneously, intrahepatic MNLC acquired the FasL-dependent cytotoxic potential against NKT-sensitive targets and both, intrahepatic and splenic MNLC, acquired the perforin-dependent cytotoxic potential against NK-sensitive targets, implying that during the disturbance of morphostasis gp96 serves as a natural adjuvant for chaperoning antigenic self peptides into the immune surveillance pathways, resulting in activation of autoreactive NKT and regulatory cells, as well as NK cells. Moreover, cell cycle analysis revealed that G2+M phase of regenerating hepatocytes in PKO mice was translocated from the 1st to the 7th p. o. day, as well as that hepatocytes from FasL deficient mice were arrested in G0/G1 phase.

Neo-epitopes are required for immunogenicity of the La/SS-B nuclear antigen in the context of late apoptotic cells

Mechanisms responsible for the induction of anti-nuclear autoantibodies (ANA) following exposure of the immune system to an excess of apoptotic cells are incompletely understood. In this study, the immunogenicity of late apoptotic cells expressing heterologous or syngeneic forms of La/SS-B was investigated following subcutaneous administration to A/J mice, a non-autoimmune strain in which the La antigenic system is well understood. Immunization of A/J mice with late apoptotic thymocytes taken from mice transgenic (Tg) for the human La (hLa) nuclear antigen resulted in the production of IgG ANA specific for human and mouse forms of La in the absence of foreign adjuvants. Preparations of phenotypically healthy cells expressing heterologous hLa were also immunogenic. However, hLa Tg late apoptotic cells accelerated and enhanced the apparent heterologous healthy cell-induced anti-La humoral response, while non-Tg late apoptotic cells did not. Subcutaneous administration of late apoptotic cells was insufficient to break existing tolerance to the hLa antigen in hLa Tg mice or to the endogenous mouse La (mLa) antigen in A/J mice immunized with syngeneic thymocytes, indicating a requirement for the presence of heterologous epitopes for anti-La ANA production. Lymph node dendritic cells (DC) but not B cells isolated from non-Tg mice injected with hLa Tg late apoptotic cells presented immunodominant T helper cell epitopes of hLa. These studies support a model in which the generation of neo-T cell epitopes is required for loss of tolerance to nuclear proteins after exposure of the healthy immune system to an excess of cells in late stages of apoptosis.

Increase in hepatic NKT cells in leukocyte cell-derived chemotaxin 2-deficient mice contributes to severe concanavalin A-induced hepatitis.

Leukocyte cell-derived chemotaxin 2 (LECT2) was originally identified for its possible chemotactic activity against human neutrophils in vitro. It is a 16-kDa protein that is preferentially expressed in the liver. Its homologues have been widely identified in many vertebrates. Current evidence suggests that LECT2 may be a multifunctional protein like cytokines. However, the function of LECT2 in vivo remains unclear. To elucidate the role of this protein in vivo, we have generated LECT2-deficient (LECT2(-/-)) mice. We found that the proportion of NKT cells in the liver increased significantly in LECT2(-/-) mice, although those of conventional T cells, NK cells, and other cell types were comparable with those in wild-type mice. Consistent with increased hepatic NKT cell number, the production of IL-4 and IFN-gamma was augmented in LECT2(-/-) mice upon stimulation with alpha-galactosylceramide, which specifically activates Valpha14 NKT cells. In addition, NKT cell-mediated cytotoxic activity against syngeneic thymocytes increased in hepatic mononuclear cells obtained from LECT2(-/-) mice in vitro. Interestingly, the hepatic injury was exacerbated in LECT2(-/-) mice upon treatment with Con A, possibly because of the significantly higher expression of IL-4 and Fas ligand. These results suggest that LECT2 might regulate the homeostasis of NKT cells in the liver and might be involved in the pathogenesis of hepatitis.

Syngeneic pregnancy induces overexpression of natural killer T cells in maternal liver.

Conditions such as stress, infection, autoimmune disease, etc. elevate the number and function of extrathymic T cells that are generated mainly in the liver. As primitive, self-reactive clones of T cells that coexpress receptors of the natural killer (NK) lineage, they mediate cytotoxicity against altered self, malignant and infected cells and have the unique potential to rapidly secrete large amount of T helper 1 (Th1) or Th2 cytokines. To elucidate whether some of these changes occur even during the syngeneic pregnancy, we made phenotypic and functional characterization of mononuclear lymphatic cells (MNLCs) isolated from the liver and spleen of pregnant C57BL/6 mice, testing their cytotoxicity against syngeneic thymocytes as well as against NK- and lymphokine-activated killer (LAK)-sensitive targets. The data have shown that on the sixteenth day of syngeneic pregnancy TCRint, NK1.1+ and IL-2Rbeta+ cells were accumulated in the liver, while the quantities of CD4+ and CD8+ T cells and total number classical NK (NK1.1+CD3- or IL-2Rbeta+CD3-) cells were increased in the spleen. Pregnancy-activated hepatic and splenic MNLCs were more cytotoxic against syngeneic thymocytes, YAC-1 and P815 targets, suggesting that the maternal liver is a main producer of autoreactive NKT clones, which subsequently augment NK- and LAK cell-mediated cytotoxicity in the liver and spleen.

Leukocyte accumulation and changes in extra-renal organs during renal ischemia reperfusion in mice

Multiorgan failure is a life threatening complication in patients with ischemic acute renal failure (ARF). However, little is known about the underlying multiorgan system cellular immunity in ischemic ARF. We therefore studied the dynamics of cells accumulating in the kidneys and other organs in mice and analyzed the characteristics of the accumulated cells. We prepared a unilateral renal ischemia/reperfusion injury (IRI) model in C57BL/6 or C3H/He mice. At 1 to 3 hours after renal ischemia, increased accumulations of neutrophils and intermediate T cells were observed in the clamped kidney, but the same phenomena were also observed in the nonclamped kidney, liver, and spleen. After 24 hours, these cell numbers had returned to preischemic levels, but remained elevated for a longer period in the clamped kidney. The intermediate T cells that accumulated in the kidney and liver in the IRI mice expressed higher Vβ chains specific to forbidden clones than in the control mice. Moreover, the accumulated intermediate T cells in the IRI liver had cytotoxic activity against both tumor cells and syngeneic thymocytes. In the clamped kidney, the accumulated intermediate T cells had less cytotoxic activity against tumor cells; however, the expression of the Fas ligand (FasL) increased, indicating a cell-mediated tissue injury via the Fas/FasL system. Histopathologically, an influx of neutrophils and lymphocytes was observed not only in the clamped kidney but also in the hepatic sinusoids concomitantly with liver dysfunction. These findings indicate that a systemic cellular immune response, including intermediate T cells, affects multiple organs during ischemic ARF, which may play an important role in the development of multiorgan failure. (J Lab Clin Med 2002; 139:269-78)

Augmentation of NKT and NK cell-mediated cytotoxicity by peptidoglycan monomer linked with zinc.

BACKGROUND: Peptidoglycan monomer (PGM), which was originally prepared by biosynthesis from culture fluids of penicillin-treated Brevibacterium divaricatum, is an immunostimulator, the activities of which might be improved by addition of zinc (Zn) to the basic molecule. METHODS: To test the possible cytotoxic effects of this new analogue, we analyzed the ability of PGM-Zn and PGM to change the phenotypic profile of hepatic and splenic mononuclear lymphatic cells and to affect the growth of malignant T-cell line YAC-1 and syngeneic thymocytes. RESULTS: Pretreatment of C57BL/6 mice primarily with PGM-Zn over 6 days (10/mg/kg intraperitoneally) significantly enhanced the proportions of NK1.1high+, CD4-CD8-, CD69+, and CD3intermediate/NK1.1+/IL2R-beta+ (NKT) cells in the liver, and major histocompatibility complex class II+, CD69+, and CD8+ cells in the spleen. Both types of cells were highly cytotoxic against YAC-1 and syngeneic thymocytes, increasing the destruction of YAC-1 by 70% on addition of hepatic cells and by 30% on addition of splenic cells. Destruction of thymocytes increased by 10 and 50%, respectively. CONCLUSION: The results point to PGM-Zn as a potent cytotoxicity-inducing agent, which also generates autoreactive NKT cells.

Intensive expansion of natural killer T cells in the early phase of hepatocyte regeneration after partial hepatectomy in mice and its association with sympathetic nerve activation.

When C57BL/6 mice were partially hepatectomized (PHx), severe lymphocytosis was induced in the liver in the early phase of hepatocyte regeneration (4 to 12 hours after PHx). A major lymphocyte subset expanding in this organ was estimated to be natural killer 1.1(+) (NK1.1(+)) intermediate CD3 (CD3(int)) cells (i.e., NKT cells). CD3(int) cells are extrathymic T cells generated in situ in the liver. These changes were suppressed when mice with PHx were pretreated with a beta-adrenergicD antagonist (i.e., beta-blocker), propranolol (PPL). This might have been caused by sympathetic nerve stimulation during hepatocyte regeneration. An alpha-blocker showed a similar effect, although the magnitude of suppression was lower than that of the beta-blocker. We previously showed that NK and NKT cells express surface beta-adrenergic receptors and are activated in number by sympathetic nerve stimulation. In the present study, NK cytotoxicity mediated by liver lymphocytes obtained from mice with PHx decreased, whereas NKT cytotoxicity against syngeneic thymocytes increased. Purified CD3(int) cells were also found to be able to mediate NKT cytotoxicity against regenerating hepatocytes. These results suggest that sympathetic nerve stimulation after PHx results in subsequent activation of NKT cells and that these NKT cells might be associated with immunologic surveillance during hepatocyte regeneration.

Differentiation of Forbidden T Cell Clones and Granulocytes in the Parenchymal Space of the Liver in Mice Treated with Estrogen

Estrogen was administered to B6 (NK1.1+strain), BALB/c (Mls-1b2a, Vβ3+cells being forbidden clone), or (B6 × BALB/c) F1mice (1 mg/mouse). On days 3 and 10, the number of cells yielded by the liver doubled, whereas that yielded by the thymus decreased prominently. The numbers of cells in the spleen, bone marrow, and blood were unchanged. c-kit+stem cells, which give rise to multilineage cells, were present in the liver and bone marrow. The proportion of such c-kit+cells in the liver increased while that in the bone marrow decreased on day 3. Therefore, the absolute number of c-kit+stem cells increased severalfold in the liver and clusters of lymphoid cells became visible in the parenchymal space. At that time, the expression of recombination activating gene-1 and -2 mRNAs became prominent. Reflecting these phenomena, the number and proportion of IL-2Rβ+CD3intcells (i.e., primordial T cells) increased in the liver on days 3 and 10. An increase in the number or proportion of such CD3intcells was seen even in the thymus and uterus. In parallel with the increase of CD3intcells, the proportion of granulocytes also increased in various organs on day 3. Forbidden clones were present in either the NK1.1+or the NK1.1−subset of CD3intcells in (B6 × BALB/c) F1mice treated with estrogen and liver mononuclear cells in such mice acquired potent cytotoxicity against syngeneic thymocytes. These results reveal that estrogen has the ability to potentiate the generation of self-reactive T cells and granulocytes in the liver and other organs.

The costimulatory and differentiating activity of soluble class I MHC antigens for an autologous thymocyte population

Combined cultivation of macrophages with syngeneic thymocytes resulted in accumulation of soluble H-2Kk antigens in culture medium. Incubation of intact autologous thymocytes with these soluble class I MHC molecules was shown to induce functional maturation of thymocytes assayed in local graft-vs-host reaction. Similar thymocyte costimulating activity was detected for the papain-solubilized purified H-2Kk antigens. Soluble class I antigens were shown to costimulate IL2 production by thymocytes in response to submitogenic doses of exogenous IL2 and to increase PHA-induced thymocyte proliferation. Soluble class I molecules were shown to increase the level of expression of function-associated membrane antigens, H-2Kk, CD8 and CD4, and to trigger thymocyte differentiation. The expression of I-Ak antigens remained invariable. It was also shown that soluble autologous class I molecules may function as direct amplifiers of thymocyte proliferation in autologous, but not allogeneic, mixed leukocyte reactions. It is concluded that soluble MHC class I molecules are capable of triggering functional and phenotype differentiation of syngeneic thymocytes and acting as restricted coaccessory molecules when thymocyte activation is induced by a submitogenic dose of different stimuli.

Involvement of 4F2 antigen expressed on the MHC-negative target cells in the recognition of murine CD3+CD4-CD8- alpha beta (V alpha 4/V beta 2) T cells.

T cells of an unique phenotype (CD3+CD4-CD8- alpha beta TCR+) develop in vitro from the hematopoietic progenitors, the majority of which carry homologous alpha beta TCR (V alpha 4J alpha 28/V beta 2D beta 1.1J beta 2.6). In the present study, antigen corresponding to the particular alpha beta TCR was investigated, taking advantage of the fact that growth of T cell hybridomas was arrested by their TCR stimulation. Results indicated that both syngeneic and allogeneic thymocytes, particularly from newborn mice, could specifically inhibit the proliferation of a T cell hybridoma with the V alpha 4/V beta 2 TCR (15H1.2). Proliferation of neither TCR-missing hybridoma subclones nor those with unrelated alpha beta TCRs was affected at all. It was also found that embryonal carcinoma (EC) cells without classical MHC antigens could specifically inhibit the proliferation of 15H1.2 cells. We then raised a mAb, 14.37, against an EC line, OTF9, that could interfere with the ability of them to inhibit the growth of 15H1.2. Pretreatment of 15H1.2 cells with anti-V beta 2 and OTF9 cells with 14.37 mAb respectively completely abrogated the growth inhibition of 15H1.2 by OTF9. The 14.37 antigen was a 120 kDa heterodimer glycoprotein consisting of 85 and 36 kDa proteins. In normal lymphoid tissues, the expression of 14.37 antigen exhibited an apparently inverse relationship with that of class I MHC antigens. Thus, ontogenically it was strongly expressed in fetal and newborn mice, rather rapidly declined thereafter, and remained at very low levels in adult organs. In a given stage, the distribution of 14.37 antigen and class I MHC was rather exclusive to each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Human growth hormone promotes engraftment of murine or human T cells in severe combined immunodeficient mice.

Growth hormone (GH) has previously been implicated in T-cell development, and here we test its efficacy in promoting T-lymphocyte engraftment in mice with severe combined immune deficiency (SCID). SCID mice receiving syngeneic thymocytes and treated with recombinant human GH (rhGH) had significantly better T-cell engraftment in their lymph nodes. Human T-cell engraftment was also strongly promoted by rhGH in SCID recipients receiving human peripheral blood lymphocytes. Additionally, although mature human cells have not been thought to enter the murine thymus, human T cells were detected in the SCID thymus after rhGH treatment. Thus, rhGH can be used to optimize long-term peripheral T-cell engraftment in these human-mouse chimeras and may also be useful clinically in treating T-cell deficiencies.

Expression and function of intercellular adhesion molecule 1 (ICAM-1) on rat thymic macrophages in culture

The expression of intercellular adhesion molecule 1 (ICAM-1) was studied on freshly isolated rat thymic macrophages (TMF) and after their cultivation in serum-free medium using monoclonal antibody (mAb) 1A 29 and a streptavidin-biotin immunoperoxidase technique. ICAM-1 was expressed on about 80% of freshly isolated TMF. Upon cultivation, the percentage of ICAM-1+ TMF decreased to about 30-40% in 12-day-old culture. Using double immunofluorescence staining it was found that ICAM-1 was expressed both on cortical (R-MC 40+) and CMZ/medullary (R-MC 43+) macrophage subsets. ICAM-1 was up-regulated on TMF in culture by recombinant IFN-gamma, IL1 and TNF-alpha and was down-regulated by dexamethasone. Syngeneic thymocytes bound to cultivated TMF in a rosette form at both 37 degrees C and 4 degrees C. IFN-gamma treatment did not increase the binding formation. The binding between thymocytes and IFN-gamma-stimulated TMF at 37 degrees C was inhibited by pretreatment of TMF with anti-ICAM-1 mAb or pretreatment of thymocytes with anti-LFA-1 mAb, indicating that ICAM-1 on TMF is one of the ligands involved in TMF/thymocyte adhesion and subsequent direct cell-cell communication.

Thyroid-specific T cells in the normal Wistar rat: II. T cell clones interact with cloned Wistar rat thyroid cells and provide direct evidence for autoantigen presentation by thyroid epithelial cells

Strains of rat differ in their susceptibility to experimental autoimmune thyroiditis (EAT). We recently observed that the normal Wistar rat has lymph node (LN) T cells which recognize the newly available cloned Wistar thyroid cell line (WRT) and/or rat thyroglobulin (rTg). We have now cloned thyroid-specific T cells and characterized their interaction with the WRT target cell. Twenty-three T cell clones were tested for their reactivity to syngeneic thymocytes, WRT cells alone, or WRT cells with thymocytes. All the clones were of the CD4+ CD8− phenotype. Seven of 23 T cell clones proliferated in the presense of WRT cells alone or with the combination of WRT cells and thymocytes, exhibiting stimulation indices of 1.5 to 5. In all but one of the T cell clones responding to WRT cells alone was there no evidence that the additional presence of thymocytes supplied a stronger “second” proliferative signal than the WRT cells. These WRT-reactive clones which were able to be more extensively characterized were MHC class II restricted, secreted rat interferon (IFN)-γ in response to WRT cell exposure, and one clone showed cross-reactivity with rTg antigen. Induction of WRT cell MHC class II antigen by prior treatment with IFN-γ failed to further enhance the WRT cell-induced T cell proliferation. These data provide the first evidence for direct antigen presentation by thyroid epithelial cells (TECs) in the absence of other antigen-presenting cells. Further-more, they provide evidence that TECs are able to provide the appropriate “second” signals required for T cell activation and successful autoantigen presentation.

The effects of interleukin 2 on early and late thymocyte differentitaion in foeatl thymus organ culture

The effects of interleukin 2 (IL-2) on in vitro T cell development in organ cultures of day 14 foetal mouse thymus were investigated. Over a 12 day culture period IL-2 was found to inhibit the development of each of the major thymocyte subpopulations, defined by CD4 and CD8 expression. Since each subpopulation was reduced in number, a common precursor to these subsets was thought to be inhibited by IL-2. Indeed, subsequent analyses of subsets of CD4- CD8- cells, characterized by expression of HSA, Pgp-1, and IL-2R, demonstrated that CD4- CD8- cells expressing IL-2R and their postulated progeny were reduced in IL-2-treated cultures. The number of cells reputed to be the immediate precursor of CD4- CD8- IL-2R+ cells, i.e. CD4- CD8- Pgp-1+IL-3R- cells, was not change by treatment with IL-2. Interestingly, however, numbers of the most immature subset of CD4- CD8- cells, identified as having the HSA1oPgp-1+IL-2R- and CD3- phenotype, increased in IL-2-treated cultures and was the only population of cells to do so. We also document the failure to detect any LAK cell activity against syngeneic thymocytes in IL-2-treated cultures and therefore hypothesize that the inhibition of T cell differentiation in these cultures is due to an arrest in differentiation of CD4- CD8- cells bearing the IL-2R.

Interaction between thymocytes and thymus-derived macrophages: II. Engulfment of thymocytes by macrophages

A high percentage (80–90%) of immature thymocytes were engulfed by syngeneic thymus-derived macrophages (TDMφ) following cocultivation for 3 days. Elimination occurred via internalization of thymocytes by the macrophages. We unequivocally demonstrated the presence of many live thymocytes inside the TDMφ by means of specific staining. Mature PNA − thymocytes were phagocytized to a lower degree than immature thymocytes, and T splenocytes were not eliminated at all. Bone marrow-derived macrophages internalized immature thymocytes to a degree similar to TDMφ. Since thymocyte survival was not at all affected by Mφ culture supernatants alone, we conclude that cell to cell contact is necessary for thymocyte elimination. To identify the surface molecules which participate in internalization of thymocytes by the macrophages, both cell types were pretreated with a variety of agents. Treatment of thymocytes with tunicamycin (N-glycosylation inhibitor) and anti-Lyt-2 mAb decreased their elimination by Mφ. Similarly, treatment of Mφ with neuraminidase, trypsin, and anti-Ia mAb markedly suppressed their capacity to engulf thymocytes. On the other hand, thymocyte elimination was unaffected by (1) cell cultivation in syngeneic serum rather than heterologous serum; (2) use of allogeneic rather than syngeneic thymocytes; and (3) use of X-irradiated Mφ and LPS-activated Mφ rather than nontreated Mφ.

Interaction between thymocytes and thymus-derived macrophages: I. Surface components participating in mutual recognition

Incubation of C57BL/6 thymus-derived macrophages (TDMφ) with syngeneic thymocytes resulted in binding of thymocytes to macrophages and rosette formation. Up to 60% of the TDMφ formed rosettes with thymocytes after 6 hr of interaction at 4 °C. Rosette formation of the immature PNA + thymocyte fraction was up to fivefold higher than that of PNA − and cortisone-resistant thymocytes. Pretreatment of PNA − thymocytes with neuraminidase enhanced thymocyte binding to macrophages up to sevenfold, whereas a marked reduction of rosette formation was seen following (1) incubation of thymocytes with tunicamycin; (2) incubation of macrophages with 20 m M d-galactose, GLCNaC, or GalNaC; (3) treatment of macrophages or thymocytes with trypsin; (4) treatment of macrophages with anti-1-A b mAb and its F(ab′) 2 fragment; (5) treatment of thymocytes with anti-Lyt-2.2 mAb; and (6) addition of EDTA and EGTA to the interacted two cell populations.

Control of helper-T-cell proliferation by recognition of la and Mac-1 antigens on phagocytic cells of the thymic reticulum

Phagocytic cells of the thymic reticulum (P-TR) have been previously described as being Iapositive, Mac-1-positive accessory cells which pursue a close relationship with thymocytes. They form rosettes with thymocytes, and these rosettes are inhibited by antibody directed against the complement receptor type 3 CR3 (anti-Mac-1). P-TR induce the proliferation of syngeneic thymocytes. In the present paper, we show that thymocytes enriched in mature medullary type are induced to proliferate in coculture with syngeneic P-TR, while the cortical type does not. After 5 days of culture, 85% of the thymocytes are of helper L3T4 +Lyt-2 − phenotype. As previously shown by others for syngeneic reactions, antibodies directed against related class II antigens (anti-I-A and anti-I-E) block this helper-T-cell syngeneic proliferation. A new finding is the blockage of helper-T-cell proliferation by anti-Mac-1 as well as with anti-LFA-1 antibodies, showing that accessory molecules may be as important as specific recognition of class II antigen molecules in the control of thymocyte proliferation and hence in thymocyte selection. Mac-1, like LFA-1, belongs to a novel family of differentiation antigens involved in cell interactions. The blockage of cell recognition and interaction between P-TR and thymocytes by either anti-Ia or anti-Mac-1 during the early induction phase of the syngeneic response leads to its inhibition. We demonstrate that P-TR/thymocyte interaction stimulates the enhanced expression of IL-2 receptors on thymocytes, a step which is necessary for helper-T-cell proliferation. The mechanism of syngeneic proliferation inhibition by anti-Ia, anti-Mac-1, and LFA-1 antibodies may be the prevention of IL-2 receptor expression on thymocytes, and/or the inhibition of IL-2 secretion. Although this is an in vitro model, which may not totally reflect in situ situation, our results indicate that thymic accessory cells may participate in a positive selection process which leads to helper-T-cell proliferation.

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma.

BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct 51Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1+ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.

Role of cells sensitive to anti-mouse brain serum in the regulation of CFU-S proliferation

CFU-S differentiation and regeneration kinetics in the spleen and femur was studied after treatment of bone marrow cells with RAMB serum. The effect of thymocytes on the rate of CFU-S regeneration was also investigated. It was found that CFU-S regeneration in the spleen was similar in RAMBS-treated and intact cell populations on days 4-14 after transplantation. On the contrary, the rate of CFU-S regeneration in the femur was slower in RAMBS-treated than in intact bone marrow cells. However, the growth rate in the femur could be restored to the normal level by the administration of freshly isolated syngeneic thymocytes to mice pre-injected with RAMBS-treated CFU-S population. The treatment of bone marrow suspension with RAMB serum did not affect the differentiation of spleen colonies. It is suggested that RAMBS eliminates cell population regulating CFU-S proliferation, without affecting its differentiation.