Syngeneic Spleen Cells Research Articles

Activation of CD4+ lymphocytes by syngeneic brain microvascular smooth muscle cells.

Splenocyte proliferation as measured by [3H]thymidine incorporation was detected when brain microvessel smooth muscle cells (SM) were cocultured with syngeneic spleen cells. This report focuses on the role of different lymphocyte populations in this activation. The central role of CD4+ T cells in the proliferation response has been established by different sets of experiments. The phenotypic characterization of splenic lymphocytes before and after the co-culture showed that the only cell type present in higher number after the co-culture than before is the CD4+ T cell. When CD4+ cells were purified by flow microfluorimetry and co-cultured with SM a strong proliferative response was detected. In contrast, purified CD8+ cells in co-culture with SM cells did not proliferate. The activation of CD4+ cells by SM required direct cell-to-cell contact and could be detected on the fourth day, reaching maximal levels at the 6th and 7th days of the co-culture. The activation is more pronounced in the syngeneic system than under allogeneic conditions and is inhibited by anti-MHC II mAb, but not by anti-MHC II mAb. The finding that vascular smooth muscle cells can activate syngeneic T cells may have important implications concerning the mechanism of induction of vasculitis.

T lymphocyte heterogeneity in the rat: III. Autoreactive T cells are activated by B cells

Evidence has been presented to show that CD4 + autoreactive T cell lines (ATs) 2 2 Abbreviations used: APC, antigen-presenting cell; AT, autoreactive T cell lines; C, complement; KLH, keyhole limpet hemocyanin; LN, lymph nodes; mAb, monoclonal antibody; Mø, macrophages; MBP, myelin basic protein; MPL.LL, MBP-specific cell line; Mops, morphilinopropane sulfonic acid; NRS, normal rat serum; PE, phycoerythrin; RbβR, rabbit anti-rat IgG; Spc, spleen cells; Spc χ, τ-irradiated (2000 rad) spleen cells; SMLR, syngeneic mixed lymphocyte reaction. in the rat require periodic stimulation with syngeneic spleen cells for in vitro proliferation. This proliferation can be blocked by treatment of the stimulator (spleen) cells with mAb to Ia antigens. Although ATs are Ia + and can activate the allogeneic MLR, they fail to be autostimulatory. Fractionation of the spleen cells revealed that ATs can be stimulated with B cells and not by macrophages, although the latter were efficient in several accessory cell functions, including antigen presentation, lectin-dependent T cell activation and allogenic MLR response. Moreover, B cells proliferated and differentiated in response to AT cells. These data are compatible with a model in which ATs respond to hitherto undetermined B cell membrane antigen(s) in association with MHC class II antigens. These results may have important implications in understanding autoimmune responses.

Vaccination with membrane-associated idiotype provides greater and more prolonged protection of animals from tumor challenge than the soluble form of idiotype.

The purpose of this work was to compare the efficacy of immunizing mice with a soluble vs a cell-associated form of a tumor Ag. A murine B cell tumor (2C3), which displays an Id on its cell surface, grows progressively and gives rise to Id-negative tumor variants in nonimmunized animals. We previously reported that the tumor variants arise as a consequence of Id-specific T cell suppression of the Id+ tumor. The Id-specific effector T cells are CD4+, CD8-. Based upon these findings vaccination protocols have been designed and tested to determine whether the expansion of tumor-specific effector T cells would eliminate the Id+ tumors and prevent the subsequent generation of Id- tumor variants in vivo. MHC-restricted T cells typically recognize soluble Ag subsequent to modification by an APC, and APC may ultimately express a processed form of the Ag that is different from that expressed on the surface of the tumor cells. Based upon this assumption, the efficacy of immunizing mice with cell-associated 2C3 Id was compared to immunization with a soluble form of the same Id. Mice were immunized with either irradiated 2C3 cells or syngeneic spleen cells to which 2C3 protein was covalently linked. These immunization protocols provided a complete and lasting protection against a tumor challenge of up to 1 x 10(6) tumor cells. In contrast, most mice hyperimmunized with a soluble form of the Id did not survive this level of tumor challenge in spite of the production of significant levels of anti-Id antibodies. Mice immunized with the soluble form of the Id, which did survive, produced slowly progressing tumors expressing a 1000-fold less of the marker Id. These results illustrate the importance of understanding and properly exploiting a host's natural response to a tumor-specific Ag when designing effective immunization protocols for cancer therapy.

T cell receptor Vβ genes expressed by IgG anti‐DNA autoantibody‐inducing T cells in lupus nephritis: Forbidden receptors and double‐negative T cells

In the (SWR x NZB)F1 (SNF1) model of lupus nephritis, pathogenic variety of IgG anti-DNA autoantibodies are induced by certain T helper (Th) cells that are either CD4+ or CD4-CD8- (double negative; DN) in phenotype. From the spleens of eight SNF1 mice with lupus nephritis, 149 T cell lines were derived and out of these only 25 lines (approximately 17%) were capable of augmenting the production of pathogenic anti-DNA autoantibodies. Herein, we analyzed the T cell receptor (TcR) V beta genes used by 16 such pathogenic autoantibody-inducing Th cell lines. Twelve of the Th lines were CD4+ and among these five lines expressed V beta 8 (8.2 or 8.3). The V beta 8 gene family is contributed by the NZB parent to the SNF1 mice, since it is absent in the SWR parental strain. Three other CD4+ Th lines expressed V beta 4, another was V beta 2+ and one line with poor autoantibody-inducing capability expressed V beta 1. Four autoantibody-inducing Th lines from the SNF1 mice had a DN phenotype and these lines were also autoreactive, proliferating in response to syngeneic spleen cells. Among these DN Th lines, two expressed V beta 6 and one expressed V beta 8.1 TcR. Both of these are forbidden TcR directed against Mls-1a (Mlsa) autoantigens expressed by the SNF1 mice and such autoreactive T cells should have been deleted during thymic ontogeny. Thus, the DN Th cells of non-lpr SNF1 mice are different from the DN cells or MRL-lpr which lack helper activity and do not express forbidden TcR. The spleens of 6 out of 19 nephritic SNF1 animals tested also showed an expansion of forbidden autoreactive TcR+ cells that were mainly DN. Two of these animals expressed high levels of V beta 6 (anti-Mlsa) and V beta 11 (anti-I-E) TcR+ cells, three others had high levels of V beta 11+ cells alone and one animal had an expanded population of V beta 17a+ (anti-I-E) cells. The I-E-reactive TcR again should have been eliminated in the SNF1 thymus, since they express I-E molecules contributed by the NZB parent. The SWR parents of SNF1, are I-E-; moreover, they lack the V beta 11 gene but they express V beta 17a in peripheral T cells. Whereas the NZB parents are I-E+, they lack a functional V beta 17a gene and they delete mature V beta 11+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of experimental allergic encephalomyelitis by MBP-coupled lymphoid cells and by MBP-liposomes: A comparison

In previous experiments, we showed that administration of myelin basic protein (MBP) inserted into phosphatidyl-serine liposomes, to susceptible animals suppressed the clinical manifestations of both acute and chronic-relapsing EAE. In this report we compare the effectiveness of treatment with MBP-liposomes and with MBP-coupled syngeneic spleen cells in EAE protection. Lewis rats treated with 150 μg MBP-liposomes or with 160 μg (35 × 10 6) MBP-coupled spleen cells, given 7 days before and 7 days after encephalitogenic challenge were equally protected against clinical EAE, when compared to untreated controls. In addition to clinical protection, in vitro proliferative responses of lymphocytes from treated rats were significantly reduced, but delayed hypersensitivity (DTH) reactions remained unaffected. Proliferation of lymphocytes from MBP-sensitized donors was inhibited by the addition of spleen cells but not of lymph node cells from treated donors. The inhibitory effect was observed with spleen cells regardless of whether the donors were treated or not, was antigen nonspecific, and localized in a radio-resistant, adherent cell population. Adoptive transfers of spleen cells from treated donors, after a 48-hr in vitro incubation with concanavalin A, showed that the cells from donors treated with MBP-coupled spleen cells, but not with MBP-liposomes, suppressed the disease in recipients, following challenge with MBP-complete Freund's adjuvant (CFA). These results suggest that two distinct mechanisms operate in the protection by MBP-coupled cells and MBP-liposomes, respectively.

Loss of a major idiotype (CRIA) after repopulation of irradiated mice.

The normal immune response of A/J mice against arsonate coupled to hemocyanin is characterized by a major recurrent cross-reactive Id, the CRIA. This Id is encoded by a single gene segment combination: VHidcr11-DFL16.1e-JH2 for the H chain and Vkidcr-Jk1 for the L chain. In this report, we show that lethal irradiation of A/J mice followed by reconstitution with autologous or syngeneic lymphoid cells results in loss of major CRIA Id expression in the response to arsonate. Different protocols were performed to repopulate the irradiated mice. First, lethally irradiated A/J mice were reconstituted by the transfer of syngeneic bone marrow cells. Second, A/J mice were lethally irradiated while their hind limbs were partially shielded. Third, lethally irradiated A/J mice received a transfer of syngeneic spleen cells. The three groups of mice produce high titers of antiarsonate antibodies completely devoid of CRIA DH-JH related idiotopes expression. Moreover, a lack of affinity maturation is observed in the secondary antiarsonate response of all irradiated and reconstituted mice. A transfer of syngeneic peritoneal cells or a transfer of primed T cells in irradiated and reconstituted A/J mice do not restore in a significant manner either the recurrent CRIA expression or the affinity maturation of the antiarsonate response. Our data suggest that the choice of this Id is not solely dictated by the Igh locus.

Effects of spleen cells and serum on transfer of immunity to Strongyloides venezuelensis infection in hypothymic (nude) mice

The course of Strongyloides venezuelensis infection in congenitally hypothymic (nu/nu) mice and their heterozygous thymus-bearing littermates (nu/ +) was followed. Unlike the infected nu/+ mice, the nu/nu mice were unable to expel the worms until the end of the observation period (98 days post-infection). In addition, about three times as many eggs were counted at the peak level of infection in faeces of the infected nu/nu mice in comparison with the nu/+ mice. No acquired resistance to rechallenge was observed among the nu/nu mice. Auto-reinfection within the infected nu/nu mice could not be supposed in the present study. The worm expulsion mechanism was generated by nu/nu mice which had been given syngeneic spleen cells from intact +/+ mice. The expulsion of adult worms, as well as the protection against migrating larvae, occurred anamnestically when spleen cells from immune +/+ mice were transferred. The serum transfer, however, only caused a retardation of larval migration. The results support the hypothesis that direct worm immunity and worm expulsion are a T cell-dependent phenomenon.

Immunological regression of metastatic cancer in the liver as a result of “in vivo xenogenization”

We attempted to induce the regression of liver metastatic tumor cells in vivo by the administration to rats of Friend leukemia virus (FV) (in vivo xenogenization). The virus which was used in this experiment, FV, is highly immunogenic and does not normally cause disease in an adult rat. At first, we induced a FV viremia in tumor bearing rats in order to deliver the virus to the site of the tumor cells. FV viremia was induced by injecting 60 mg/kg cyclophosphamide (CY) i.v. after the administration of FV, and by transferring syngeneic bone marrow cells so that FV would be able to infect them and then replicate. In order that the tumor cells which were infected with virus should regress, it was necessary to break down their tolerance to FV antigens. As adoptive immunotherapy we therefore, transferred syngeneic spleen cells from rats which had been immunized with FV to tumor bearing rats. The result of this experiment was that these tumor bearing rats infected with FV which had received either normal syngeneic spleen cells or no spleen cells as controls died from liver metastasis (8 out of 9 rats (89%) and 15 out of 17 (88%) respectively). On the other hand, only 4 out of the 15 (27%) tumor bearing rats which were infected with FV and which received FV-immune spleen cells died from liver metastasis. These sets of data indicate that the in vivo xenogenization of tumor cells are indeed able to induce the regression of metastatic tumor cells.

Host Response to Myeloma: Effect of Syngeneic Spleen Cells on the Growth and Functionof MOPC 104E Myeloma in Vitro

The effect of coculturing nonadherent and plastic adherent cells from the spleen with MOPC 104E KI81 for short (24 h) and long term (7 days) was investigated. In both culture systems, the effect of the spleen cells on the secretion of IgM by plaque-forming cells assay and growth of the plasmacytoma by cell counts and flow cytometry was measured. In these studies, a low effector:target (E:T) ratio which did not produce cytotoxicity to MOPC 104E cells was used. We observed that while nonadherent spleen cells from normal or MOPC 104E-primed mice inhibited secretion of IgM by the MOPC 104E cells, they stimulated the proliferation of MOPC 104E cells two times faster than MOPC 104E cells cultured alone. Plastic adherent cells from the spleens of normal mice or MOPC 104E-primed mice also inhibited secretion of IgM by the tumor cells as measured by plaque formation, and stimulated proliferation of MOPC 104E in 24 h coculture. Plastic adherent cells from normal nonprimed mice initially stimulated the myeloma to grow, but by 24 h, a large fraction of the population was in the G1 or possibly resting state. The effect of nonadherent and plastic adherent cells on the stem cell activity of MOPC 104E was also tested in 7-day colony-forming assays. Nonadherent cells had no effect on colony-forming units or plaque formation. Plastic adherent cells from normal spleen cells inhibited plaque formation by 68% but had no effect on colony formation. However, plastic adherent cells from spleens of mice primed in vivo with MOPC 104E tumor cells suppressed plaque formation by 98% and also reduced colony formation. The results showed that inhibition by macrophages of IgM production by MOPC 104E cells is independent of cell proliferation. The adherent macrophages from both normal and in vivo-primed spleen cells were Mac.1 positive after 7 days of coculture with MOPC 104E cells. However, the density of Mac.1 was greater on primed macrophages.

Tumor growth in vivo selects for resistance to tumor necrosis factor.

The relationship between in vivo tumor growth and resistance to TNF in WEHI-164 cells has been examined. When a highly TNF-sensitive clone of WEHI-164 was grown in vivo in syngeneic mice it became resistant to rTNF such that a 4 to 5 log higher concentration of TNF was required to produce tumor lysis in vitro. When compared with an in vitro selected TNF-resistant variant, the in vivo selected line was significantly more tumorigenic. The resistant phenotype of both the in vivo and in vitro selected variants was stable in culture and both selected lines were also resistant to lysis by syngeneic spleen cells with natural cytotoxic activity. The parental clone and the two variants were equally sensitive to lysis by allo-CTL and expressed similar levels of MHC class I Ag. Resistance to TNF in the two variants was not a function of de novo production of TNF measured as supernatant TNF activity or TNF mRNA expression. These studies are the first to demonstrate that in vivo tumor growth results in resistance to TNF and therefore may have direct relevance to the efficacy of TNF in the treatment of human neoplasms.

Demyelination and early remyelination in experimental allergic encephalomyelitis passively ransferred with myelin basic protein-sensitized lymphocytes in the Lewis rat

Histological studies were performed on Lewis rats with experimental allergic encephalomyelitis (EAE) passively transferred by myelin basic protein (MBP)-sensitized syngeneic spleen cells in order to determine the relationship between demyelination and neurological signs. Neither imflammation nor demyelination was present on the day prior to the onset of neurological signs but both were present in the spinal roots and spinal cord on the day of onset of tail weakness (4 days after passive transfer). Demyelination and the neurological signs both increased over the next 48 h. There was evidence that the caudal roots were more severely affected than the rostral roots. The peripheral nerves were spared. Demyelination in the spinal cord was concentrated in the dorsal root entry and ventral root exit zones. The initial stages of repair of demyelinated spinal root fibres by Schwann cells were observed on the earliest day that clinical recovery commenced (day 7). At this time some demyelinated fibres were closely associated with debris-free Schwann cells, and occasional fibres were completely invested by 1–2 layers of Schwann cell cytoplasm. Remyelination (compact myelin lamellae formation) by Schwann cells was first observed in the spinal roots on day 9. By the time of complete clinical recovery (day 11) the majority of affected spinal root fibres had thin new myelin sheaths. Repair of central nervous system myelin by oligodendrocytes was slower than peripheral nervous system myelin repair. Investment of demyelinated spinal cord axons by oligodendrocytes was observed on day 9, and remyelination by these cells was seen on day 10. We conclude that the neurological signs of passively induced MBP-EAE can be accounted for by demyelination of the lumbar, sacral and coccygeal spinal roots and spinal cord root entry and exit zones, and that the subsequent clinical recovery can be explained by investment and remyelination of demyelinated peripheral and central nervous system fibres by Schwan cells and oligodendrocytes respectively.

Effect of IFN‐γ treatment and in vivo passage of murine tumor cell lines on their sensitivity to lymphokine‐activated killef (LAK) cell lysis in vitro; association with H‐2 expression on the target cells

Interferon-gamma (IFN-gamma) treatment or in vivo passage of the murine YAC-1 lymphoma resulted in reduced sensitivity to in vitro lysis by syngeneic murine spleen cells cultured in rIL-2 (LAK-cells). IFN-gamma treatment also rendered the murine B16 melanoma less sensitive to lysis by syngeneic LAK cells, whereas in vivo passage did not alter LAK sensitivity. The reduction in sensitivity to lysis correlated with enhanced expression of cell surface H-2 on the target cells. The possible role of H-2 was studied with a beta 2-microglobulin-deficient, and thus H-2-deficient, variant of the YAC-1 lymphoma. This variant line remained H-2 negative even after IFN-gamma treatment or in vivo passage, and was highly sensitive to LAK-cell-mediated lysis, even after IFN-gamma treatment or in vivo passage. The present results are discussed in relation to IFN-gamma and in vivo induced modulation of MHC class-1 molecules on target cells and the possible consequences for interaction with activated as well as "natural" effector cells.

The Effect of T Cell Depletion from Spleen Cell Allografts on Graft-versus-Host Disease and Long-Term Immune Reconstitution in H-2 Haplotype-Identical Murine Combinations

Graft-versus-host disease (GVHD) and states of immune reconstitution in allogeneic chimera mice across minor histocompatibility antigens were analyzed in excess of 9 months after injecting AKR/JSea (AKR) spleen cells into irradiated C3H/HeSlc (OH) mice.When T cell-depleted AKR spleen cells were used as inoculum cells, neither graft failure nor GVHD was seen for 9 months postgrafting in the OH mice irradiated with 660 rad or more.In an AKR - C3H (850 rad) model, Thy1.1 + or L3T4 + T cell depletion from donor AKR spleen cells abolished both acute and chronic GVHD in lethally irradiated C3H mice.Lyt2 + T cell depletion, however, resulted in acute and chronic GVHD in more than half of the recipient OH mice.Moreover, actual existence of donor(AKR)-type T cells with L3T4 phenotype, but not Lyt2 phenotype, was always observed in the spleen of the OH mice suffering from acute GVHD. In addition, the C3H mice that were irradiated with 850 rad, grafted with AKR spleen cells depleted of Lyt2.1 + T cells, escaped from acute GVHD and survived for more than 10 mo postgrafting, showed impaired activities of immune responses such as delayed footpad reaction to sheep red blood cells, antibody production tested by IgM plaque forming cells and reactivity to an intracellular bacterium Listeria monocytogenes as compared with the C3H mice reconstituted with syngeneic C3H spleen cells or Thy1.1 + or L3T4 + T cell-depleted AKR spleen cells. These results suggest that L3T4 + T cells, rather than Lyt2 + T cells, contained in the grafted cells not only cause acute GVHD but also a long-term immunodeficient state (chronic GVHD) in recipient mice in the H-2-identical murine combinations examined here.

Suppressive activity of long-term myelin basic protein-specific SJL T cell lines

Myelin basic protein (MBP)-specific T cell lines derived from SJL mice lose the ability to transfer adoptively experimental allergic encephalomyelitis (EAE) after 5–6 restimulations with antigen in vitro. In order to test whether such lines were suppressive, non-encephalitogenic T cell lines were co-cultured with a freshly derived encephalitogenic T cell line. Following co-culture in the presence of MBP and irradiated syngeneic spleen cells the mixture was tranferred adoptively to syngenic recipients. Severe EA was observed in recipients of the encephalitogenic cell line alone but not in animals which received the co-culture. A co-culture period was required as mixing the encephalitogenic and non-encephalitogenic T cell lines just prior to transfer was without effect. Not all non-encephalitogenic cell lines were found to be suppressive. Culture fluids from the suppressive, but not the non-suppressive lines were found to inhibit MBP-driven proliferation of T cell clones and encephalitogenic lines in vitro. Nineteen of 55 MBP-specific T cell clones derived from suppressive lines were found to elaborate the supppressive supernatant activity. The suppressive effect was not antigen-specific since the same culture supernatants inhibited proliferation of an ovalbumin-specific SJL T cell clone. The suppressive effect became apparent only after T cell lines had lost encephalitogenicity and was not mediated by tumor necrosis factor, lymphotoxin or prostaglandin.

Establishment of a Novel Method to Induce Tolerance in Adult Mice across Fully Allogeneic (Entire H-2 plus Multiminor Histocompatibility) Antigen Barriers, Using Supralethal Irradiation Followed by Injection of Syngeneic Bone Marrow Cells plus (Donor X Recipient) F1 Spleen Cells

A novel method was established which can regularly induce profound tolerance in miceacross entire H-2 plus multiminor histocompatibility (H) antigen (fully allogeneic) barriers. When recipient AKR/J Sea (AKR; H-2 k) or C3H/He Slc (C3H; H-2 k) mice were irradiated with 900 rad followed 1 day later by injection of 1 × 10 7 T cell-depleted syngeneic bone marrow cells plus 5 × 10 7 viable, but not mitomycin C-treated, C57BL/6 Slc(B6) × AKR (or C3H) F1 spleen cells via intravenous (i.v.) route, a specific tolerant state was induced against B6 (H-2 b) antigens. In the tolerant C3H mice, the EL-4 tumor, which originates from B6, was accepted in a tolerogen-specific manner. Moreover, B6 skin grafts were permanently accepted in most of the tolerized AKR and C3H mice. Immunological parameters, including cytotoxic T lymphocyte (CTL) activity and the mixed lymphocyte reaction (MLR), were almost completely suppressed in the tolerant mice. An assay for chimerism using a direct immunofluorescence method revealed that the tolerant AKR mice were chimeric for the first 5 weeks after tolerance induction but not definitely chimeric thereafter. In the tolerant AKR mice, strong suppressor cells were not detected. This method could be used in order to investigate mechanisms of tolerance to allogeneicantigens in future experiments.

Perturbation of the idiotypic network: II. Transfer of suppression to neonatal mice

The cellular mechanisms which are responsible for idiotype perturbation induced by repeated stimulation with either allogeneic mixed lymphocyte culture-generated syngeneic blasts or allogeneically stimulated syngeneic spleen cells were investigated as described in the preceding article. Using the splenic fragment culture system, the precursor frequencies of T and B cells for anti-phosphorylcholine (PC) antibodies and T15 idiotypic antibodies were determined in allogeneically challenged mice. Adoptive transfers of T cells to neonatal BALB/c mice induced suppression of the T15 + anti-PC responses. In addition, the effect of immunization with internal image-bearing anti-idiotopes on the level of anti-PC antibodies and T15 idiotype was determined. Results from this study demonstrated (i) a decrease in the precursor frequency in the PC-specific and idiotype-specific B cell repertoire; (ii) a decrease in the precursor frequency of T helper cells, which recognize idiotypes and anti-idiotypes; (iii) the possibility to transfer T15 suppression to neonatal mice; and (iv) the possibility to restore T15 dominance by anti-idiotypic antibody immunization. These data indicate that both the T and B compartments are involved in the maintenance of suppression induced by repeated exposure to alloantigen-sensitized syngeneic cells. Collectively these findings show that a nonspecific general suppression induced by allohyperimmunization can perturb the T15 + anti-PC response.

Frequencies and Interactions of Regulatory T Cells. I. The Balance between Help and Suppression Regulates the Primary Immune Response to Keyhole Limpet Hemocy

Frequencies and efficiencies of regulatory T cells from non-immunized mice were estimatedin several assay systems differing from each other in cellular composition and antigen dose (NIP-KLH). The NIP-specific and total IgM responses were quantified. Using 10 4 syngeneic B cells and 50 υg/ml NIP-KLH, helper (Th) cells from 5 day immune donors were detected in frequencies of 1:3000-1:4000 in lymphnode and spleen T cells, with an efficiency of 70-90 ng IgM/Th cell in C57B1/6 mice. In non-immune spleen T cells, Th cells were observed in frequencies of 1:16,000-1:38,000, with comparable efficiency, but these Th cells appeared suppressed at increased T cell doses. Polyclonal activation led to the appearance of multiple independently regulated populations of Th cells with similar efficiencies. In the presence of 10 5 syngeneic spleen cells, treated once with anti-Thy-1 antibody and complement and 50 υg/ml NIP-KLH, suppressor activity was observed in the same T cell population. Similar to help, suppression fluctuated with increasing T cell numbers. Using 1 × 10 6 spleen cells and 50 υg/ml NIP-KLH as assay system, T cells enhanced the responses. Again, several independently regulated populations were observed, with efficiencies slightly higher than those of the above-described Th cells. By maintaining the cellular components of the assay system constant (10 4 B cells) and titrating the antigen, Th cell frequencies showed little variation up to 100 υg/ml NIP-KLH and were always suppressed at higher T cell numbers. At 200 υg/ml NIP-KLH, the frequency was increased to 1:2000, and not suppressed, i.e., was identical to the frequency observed in mice immunized 5 days previously. Efficiencies increased with increasing doses of antigen. The results strongly indicate that regulatory T cell function shows ≪plasticity≫, in the sensethat the appearance and the frequencies of helping and suppressing T cell populations highly depend on the micro-environment present in culture.

Infectious and noninfectious tolerance are blocked by a monoclonal antibody to T-suppressor factor

Two forms of hapten-specific unresponsiveness have been demonstrated following intravenous (iv) injection of hapten-conjugated syngeneic spleen cell based on the nature of the antigen-presenting cell (APC): I-J +, I-A − APC have been shown to induce T-suppressor cells (Ts cells) which are demonstrated upon adoptive transfer, while I-J −, I-A + APC induce a nontransferable tolerance. In this paper we report that a monoclonal antibody specific for T-suppressor effector cells and factors (14-12) can block the Ts cells induced by I-J +, I-A − APCs and the tolerance induced by I-J −, I-A + APCs. In addition, it sufficiently overcomes suppression such that injection of TNP-spl iv induces immunity rather than suppression. We show that the I-A +, I-J − TNP-spl, which induce nontransferable tolerance upon iv injection, are the cells which induce immunity in 14-12-treated recipients. These results demonstrate that injection of I-J −, I-A + APC does not lead to clonal deletion and the tolerance induced by the iv injection of both I-J +, I-A − and I-J −, I-A + APC Operate via Ts Cells.

Mode of antigen presentation required for triggering of T cell response: Analysis by use of azobenzenearsonate-tyrosine derivatives as antigens and L cells transfected with I-A k genes as antigen presenting cells

Our previous study demonstrated (1) that the presence of charged groups (amino and carboxyl groups) at the α-carbon of tyrosine is essential for activation of azobenzenearsonate- l-tryosine (ABA- l-Tyr specific T cells, and (2) that T cells recognizes ABA- l-Tyr in association with macromolecules on syngeneic spleen cells used as antigen presenting cells (APC). The present study was undertaken to confirm that the macromolecules on APC are Ia molecules, by using L cells transfected with Aβ k and Aα k genes as APC. I-A k restricted ABA- l-Tyr specific cloned T cells, and T hybridoma cells were activated by ABA- l-Tyr in the presence of the L cell transfectants, of which expression of I-A k molecules had been proven by specific binding of anti-I-A k monoclonal antibody (MAb) 10.2.16 on the cell surface. The pattern of responses of I-A k restricted ABA- l-Tyr specific T cells to various ABA-Tyr derivatives presented by the I-A k expressing L cell transfectants was similar to the pattern obtained by using H-2 k spleen cells as APC. Thus, ABA- l-Tyr and ABA-Tyr derivatives, which have both amino and carboxyl groups at the α-carbon of Tyr, presented by the L cell transfectants triggered good response of I-A k restricted ABA- l-Tyr specific T cells. By contrast, ABA-Tyr derivatives, which lack the amino or carboxyl group, or both groups, at the α-carbon of Tyr, presented by the L cell transfectants could not activate the ABA- l-Tyr specific T cells at all. Furthermore, anti-I-A k MAb, but not anti-I-E k MAb, inhibited completely the response of I-A k restricted ABA- l-Tyr specific T cells to ABA- l-Tyr presented by the L cell transfectants. These results indicate strongly that the macromolecules on APC which associate with ABA- l-Tyr are Aβ kAα k gene products, i.e., I-A k molecules.

Down-regulation of cytotoxic T lymphocyte development by a minor stimulating locus-induced suppressor cascade that involves Lyt-1+ suppressor T cells, IA- macrophages, and their factors.

Down-regulation of the development of CTL has been studied in mice both in vivo and in vitro. To generate CTL to hapten-altered self Ag in vivo, an immunization protocol has been used in which the host's Th cells are stimulated by a minor locus histocompatibility Ag (Mlsd) and its precursor CTL are activated by trinitrophenylated syngeneic spleen cells. Injecting the H-2 compatible Mls-disparate spleen cells along with the TNP-coupled self cells into the hind paws causes TNP-self specific CTL to appear in popliteal lymph nodes within 5 days. We have previously reported that inducing Ts cells by i.v. injecting Mlsd-bearing cells prevents in vivo generation of TNP-self specific CTL after immunization in this way. Here the induced Ts cell as well as the mechanism by which it functions have been further examined. The suppression was seen to extend to allogeneic as well as TNP-self Ag, provided the Mlsd-tolerized animal was reexposed to Mlsd-bearing cells at the time of immunization for CTL. By transferring the Mlsd-induced suppression adoptively we have learned that the splenic suppressor cell bears Thy-1.2 as well as Lyt-1.1 Ag and inhibits the generation of CTL at the afferent limb. In addition, Mlsd-induced PEC of Mlsd-tolerized mice, but not of normal mice, mediated suppression of development of CTL in vivo. The active cells within the tolerized PEC have been identified as T cells and macrophages (M phi). Furthermore, PEC from mice tolerized to Mlsd suppressed generation of CTL directed toward TNP-self targets in vitro. T cells and M phi separated from PEC of Mlsd-tolerized mice achieved suppression best in culture when present together. In addition, Lyt-1+ splenic cells from tolerized but not normal mice cooperated to down-regulate CTL generation in vitro with peritoneal M phi from either tolerized or normal mice. Supernatants of 24- to 72-h cultures of PEC from tolerized mice were suppressive of CTL generation when incorporated at 40 to 50% of culture volume. Supernatants of T cells from tolerized PEC or spleen were suppressive in culture only when M phi from normal mice were also present. To achieve suppression dialyzed supernatants of M phi from tolerized mice could replace the M phi.(ABSTRACT TRUNCATED AT 400 WORDS)