Trypanosoma cruzi: Responses by cells from infected mice to alloantigens

In unidirectional mixed lymphocyte cultures containing (as responders, stimulators, or regulators) spleen cells from mice infected with Trypanosoma cruzi, alloantigen responses were less than in cultures containing normal spleen cells only. Depletion of plastic adherent cells from infected spleen cells (stimulators or regulators) reversed their inhibitory effect on normal spleen cells (responders); removal of adherent responder cells and/or B lymphocytes did not alter the low alloantigen responses of normal spleen cells (stimulated by infected spleen cells) or infected spleen cells (stimulated by normal spleen cells). Infected spleen cells were effective in regulating mixed lymphocyte cultures only when added at the initiation of the culture. Serum from infected mice suppressed mixed lymphocyte cultures containing responder spleen cells syngeneic to the serum donor if added up to 24 hr after initiation of cultures, whereas the “suppressor serum” had to be present at the initiation of cultures when responder cells were allogeneic to the serum donor. Cultures of infected spleen cells (whole or macrophage enriched) produced a factor which was suppressive when added to mixed lymphocyte cultures containing syngeneic responder cells at initiation. It is proposed that the serum suppressor substance regulates cell-mediated immune responses directly by suppressing the response-potential of cells and indirectly by triggering the release of a factor from adherent splenic cells which induces a hyporesponsive state in T lymphocytes.     

A partial characterization of suppressor cells in rat fetal liver cells

Rat fetal liver cells (FLC) obtained at 18–20 days gestation suppressed mixed lymphocyte reactions(MLR) of adult lymph node cells. The suppression was not strain specific: both syngeneic and allogeneic FLC were capable of suppressing the MLR. The same suppressor activity was observed with fetal spleen cells but not with fetal thymus cells. Removal of phagocytic cells from FLC failed to inhibit the suppressor activity. The suppressor cells were separated into two different types by BSA density gradient: one is radiosensitive, the other radioresistant. A stronger suppressor activity was observed in radiosensitive cells. The suppressor cells belonged to the fraction agglutinated with peanut agglutinin. The data suggest that the suppressor cells in rat FLC may be a proliferating blastoid-type cell rather than mature lymphocytes or mature macrophages.     

Splenic suppressor cells and cell-mediated cytotoxicity in murine schistosomiasis.

An impairment of the capacity to generate alloantigen-specific cytotoxic T lymphocytes (CTL) was observed in mixed lymphocyte cultures (MLC) established with spleen cells from mice infected with Schistosoma mansoni. This impairment, which was observed as early as the eighth week of infection, could be abrogated by the fractionation of spleen cell suspensions by the carbonyl iron/magnet method prior to the establishment of MLC. Cocultivation of normal spleen cells with increasing numbers of splenocytes from S. mansoni-infected syngeneic mice resulted in a dosage-dependent suppression of CTL generation. This "infectious suppression" was not sensitive to antiserum against mouse thymic lymphocyte antigen (MTLA). The present studies suggest the role of a macrophage rather than a T cell as the suppressor cell in this model of cell-mediated immunity in schistosome-infected mice.     

Fractionation of lymphocyte subpopulations which regulate mixed lymphocyte reactions.

Four days after injection of allogeneic lymphocytes BALB/c splenic T cells suppress proliferation of syngeneic cells in mixed lymphocyte reactions (MLR). Conversely, lymph node cells from the same mice amplify MLR responses. To further characterize these functional subpopulations, alloantigen-primed lymphocyte suspensions from both organs were fractionated by velocity sedimentation at unit-gravity. After fractionation MLR suppressor cells from spleens localized exclusively in rapidlly sedimenting fractions of large cells. MLR suppressor activity of cells from these fractions, as well as that of unfractionated spleen cell suspensions, was abolished by treatment with anti-Thy-1.2 serum and complement. Spleen cell fractions of similar sedimentation velocity also secreted a soluble MLR suppressor into culture supernatants. Although inhibitory of MLR, spleen cells of rapid sedimentation velocity did not suppress responses to T cell mitogens. In marked contrast with the effects of spleen cells, large 4-day-alloantigen-primed lymph node cells had no suppressive activity in MLR. MLR amplifier cells of uncertain derivation were found in fractions of medium sedimentation velocity from both spleens and lymph nodes. Fractionation of alloantigen-primed lymph node cell suspensions did reveal, however, a subpopulation of small cells with MLR suppressor acitivty which was unaffected by treatment with anti-Thy-1 serum and complement. The data thus indicate that large alloantigen-activated lymphocytes are not intrinsically suppressive nor are cells which suppress MLR necessarily large. We consequently conclude that regulation of MLR responses by alloantigen-primed lymphocytes involves a complex interaction between distinct functional subpopulations of cells which are separable both by physical and biologic properties.     

Characterization of effector lymphocytes associated with immunity to murine sarcoma virus (MSV) induced tumors. I. Physical properties of cytolytic T lymphocytes generated in vitro and of their immediate progenitors.

Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.     

The differentiation of suppressor cell populations as revealed by studies of the effects of mitogens on the mixed lymphocyte reaction and on the generation of cytotoxic lymphocytes.

The regulation by concanavalin A (Con A) and bacterial lipoloysaccharide (LPS) of the mixed lymphocyte reaction (MLR) and of the generation of cytotoxic lymphocytes (CL) was studied in congenic resistant mice using cortisone resistant thymocytes as the responding cells. LPS enhances the generation of CL selectively when suboptimal numbers of allogeneic cells are present in mixed lymphocyte cultures and also results in the augmentation of the MLR. Mitogenic concentrations of Con A on the other hand suppress the generation of CL regardless of alloantigen dose. The mechanism of suppression cannot be ascribed to the presence of suppressor T cells, since the addition to the cultures of syngeneic cortisone resistant thymocytes activated by Con A does not change the immune response. However, prospective suppressor cells that can be activated by Con A are located in secondary lymphoid organs such as spleen and lymph node. Suppressor activity by those cells is abolished by anti θ plus complement. Con A activated spleen cells suppress the MLR, whereas Con A activated thymocytes amplify the proliferation of responding cells.     

In vivo generation of suppressor T cells by thymosin in congenitally athymic nude mice

Treatment of nude mice with thymic factors such as thymosin has been mostly ineffective in generating effector T cells. This study examined the effects of treating nude mice with thymosin fraction 5 on the induction of cells that could participate in and/or regulate cytotoxic T lymphocyte (CTL) generation by normal spleen cells in vitro. Splenic lymphocytes from BALB/c nude mice injected with thymosin fraction 5 every other day for 2 wk were tested for their ability to generate CTL in vitro. Two days after the last subcutaneous injection of thymosin, nude spleens were removed, mixed with normal BALB/c spleen cells, and placed into a mixed lymphocyte tumor culture (MLTC) against allogeneic RBL 5 tumor cells. After a 5-day incubation, cultures were tested for the presence of CTL in a 4-hr 51Cr-release assay. Spleen cells from thymosin-treated nude mice did not generate CTL but suppressed the ability of normal spleen cells to generate CTL in vitro. Characterization of the thymosin-induced nude mouse suppressor cells showed them to be Thy 1 positive, nonadherent, cyclophosphamide-sensitive T cells. These data demonstrate that some T cell maturation occurs in vivo under thymosin influence. However, the activity of these cells is initially limited to a regulatory function. These studies suggest that maturation of functional suppressor T cells occurs before CTL. Further immunologic manipulation appears to be necessary in order to induce CTL effector cells in nude mice.     

Syngeneic mixed lymphocyte reaction in mice: strain distribution, kinetics, participating cells, and absence in NZB mice.

Co-culture of mouse spleen nonadherent (T-enriched cells with mitomycin C-treated unfractionated syngeneic spleen cells resulted in increased DNA synthesis in the responding T cells. The kinetics of this syngeneic mixed lymphocyte reaction (SMLR) showed that peak DNA synthesis occurred on day 5 of culture compared to day 4 for conventional mixed lymphocyte reaction (MLR). Anti-T cell antiserum plus complement treatment of the responding cell population abolished the reaction, and similar treatment of the stimulator population enhanced SMLR. These studies indicate that SMLR represents the response of T cells to non-T cells. Studies on the generation of cytotoxic T lymphocytes (CTL) in parallel cultures of T cells activated by syngeneic or allogeneic spleen cells showed no cytotoxicity of SMLR-activated cells for either PHA- or LPS-induced blasts but did show a good CTL response of allo-activated cells to both targets. Studies on the strain distribution of SMLR revealed that NZB mice manifested poor or no stimulation in SMLR whereas all other strains tested exhibited strong SMLR. This defect in NZB mice may be pathogenetically related to the autoimmune disease that develops in these mice.     

Spontaneous adherence and rosette formation of lymphocytes to Leydig cells: an in vitro technique

A spontaneous rosette phenomenon occurs in vitro when suspensions of mouse testicular cells are mixed with suspensions of lymphocytes. The core cell of these rosettes are the Leydig cells, and the coronas are formed by lymphocytes. No other testicular cell, either seminal or Sertoli, participates in lymphocyte binding.Autogeneic, syngeneic, allogeneic, and xenogeneic lymphocytes from thymus, spleen, and peripheral blood similarly react with mouse Leydig cells. Lymphocytes from spleens of athymic nude mice and from chicken bursa also give Leydig rosettes. It is suggested that mouse Leydig cells carry membrane receptors spontaneously recognized by lymphocytes.     

Enhanced lymphocyte-mediated killing of tumour cells after tumour irradiation in vivo.

The effect of local X-irradiation of a syngeneic carcinoma of fibrosarcoma growing in the leg of W/Fu rats on the ability of host spleen lymphocytes to kill syngeneic and allogeneic tumour cells in vitro was examined. Lymphocytotoxicity was found to be enhanced one day after local X-irradiation (4000 R) when compared with unirradiated tumour-bearing rats (p = 0.05 to 0.01). This enhancing effect of local irradiation was not observed when the lymphocytes were tested 1 week or later after X-irradiation, nor when normal legs of non-tumour bearing rats were irradiated. Mechanisms which might possibly explain these results are a reduction in release of tumour-specific antigen which can act as an inhibitor of cell-mediated cytotoxicity, or depletion of suppressor cells in the lymphocyte population. These findings may be relavant to clinical studies of cellular immunity in patients undergoing radiotherapy of malignant tumours.     

Stimulation and suppression of cellular immunity in mice during liver regeneration

Augmentation of proliferative activity of spleen cells from (CBA X C57BL/6)F1 mice was demonstrated in allogeneic mixed lymphocyte reaction on day 3 after partial hepatectomy, with the decrease of proliferative response observed on day 10 after operation. The decrease was due to the activity of suppressor cells in the spleen of hybrid mice. The immune response of mice in graft-versus-host reaction as well as cytotoxicity of natural killer cells and cytotoxic T lymphocytes were also reduced on days 10-11 after partial hepatectomy.     

Transplantation of cultured thymic fragments. VI. H-2 recombinant donors

Athymic (nude) mice were transplanted with cultured thymic fragments from syngeneic, allogeneic, and partially allogeneic (recombinant) mice. Lymphocyte proliferation and cytotoxicity in vitro were measured to assess immunologic reconstitution. Transplanted nude mice were immunocompetent whether donor and recipient were disparate for class I, class II, or both H-2 gene types. Furthermore, allotolerance for thymic H-2 class I antigens was achieved independently of class II antigen allotolerance. Class I antigen tolerance was not broken during lymphocyte responses to unrelated alloantigens, ruling out insufficient help as the tolerance mechanism. Splenocytes, isolated from nude mice transplanted with fully allogeneic or syngeneic thymic fragments and stimulated in vitro with trinitrophenyl-modified cells, displayed H-2-restricted, hapten-specific cytotoxicity. Cytotoxic cells from allotolerant mice were restricted to either host or thymic H-2 antigens, depending on the stimulating cell haplotype. Response levels for thymic and host trinitrophenyl-modified cells were comparable. We have shown that allogeneic thymic epithelium transplanted into adult nude mice can induce allotolerance to class I and II H-2 antigens equally, and permits T lymphocyte interaction with cells bearing thymic donor or host H-2 antigens. Our results are consistent with a model wherein T lymphocyte self-receptors retain their genomic repertoire but can be selectively mutated or expanded by appropriate H-2 antigen presentation by the thymus.     

Effects of sodium periodate modification of lymphocytes on the sensitization and lytic phases of T cell-mediated lympholysis.

Sensitization of mouse splenic lymphocytes in vitro with sodium borohydride, suggesting that the biologic effects of sodium periodate are-treated autologous spleen cells stimulated a one-way mixed lymphocyte reaction and led to the generation of thymus-derived cytotoxic effector cells. These effectors were capable of lysing in 4 hr periodate-treated syngeneic and, to a lesser extent, periodate-treated allogeneic target cells. These results suggest that sensitization by periodate-treated autologous cells could result either from a specific reaction to modified self components or from a nonspecific mitogenic stimulation. Effector cells generated by allogeneic sensitization were detected on periodate-modified targets, irrespective of the H-2 antigens expressed by the targets. The effects of periodate modification on both stimulator and target cells were reversible by sodium periodate are dependent on the formation of a free aldehyde group on cell surface glycoproteins. Pretreatment of stimulator cells with neuroaminidase prevented the effect of periodate treatment, suggesting that the sensitization involves oxidized sialic acid residues. During the 4-hour 51Cr-release assay periodate-treated targets could be used to detect cytotoxic effector cells of any specificity. Fresh spleen cells and lymphocytes cultured for 5 days without antigen or in the presence of lipopolysaccharide did not lyse periodate-treated targets. An increasing level of cytotoxicity was detected on periodate-treated targets when the effector cells were generated, respectively, by stimulation with concanavalin A, by sensitization with periodate-modified autologous cells. Although the lysis of periodate-treated targets is itself nonspecific, effector cell specificity could be determined by selective blocking of the lytic phase with cells syngeneic to the stimulators. These results indicate that a nonspecific interaction can occur between lymphocytes and periodate-treated target cells, but that this interaction leads to lysis only when the lymphocytes were activated to become cytotoxic effectors.     

In Vitro Reconstitution of Anti-Sheep Erythrocyte Antibody Response of T Cell-Depleted Spleen Cells by Allogeneic T Cells or by Factors Derived from Them

Restoration of the impaired antibody response to sheep erythrocytes (SRBC) in cultures of mouse spleen cells, which were deprived of thymus-derived lymphocytes (T cells) by treatment with anti-mouse brain-associated θ (BAθ) antiserum and complement, was studied by adding a small portion of syngeneic or allogeneic normal spleen cells in vitro. Allogeneic spleen cells had a far greater effect than syngeneic spleen cells on the restoration, as far as the normal spleen cells added were able to recognize the alloantigens on the anti-BAθ serum-treated spleen cells (bone marrow-derived lymphocytes). Treatment of the allogeneic spleen cells with mitomycin C did not affect their activity in the restoration of the impaired antibody response. The possibility that the role of T cells in the antibody response to SRBC may be replaced by a nonspecific mediator derived from T cells reacting with allogeneic cells was proven by the finding that supernatant of the mixed allogeneic spleen cell cultures restored the impaired anti-SRBC antibody response of the T cell-depleted spleen cells. The effect of such culture supernatant on the restoration of the antibody response was greatest when it was added to the T cell-depleted spleen cell cultures one day after cultivation with SRBC, suggesting that the effectiveness may result from triggering of the proliferation and differentiation of antibody-forming cell precursors, which have already reacted with the antigen, to antibody-forming cells.     

Suppressive Effect by Cooperation of Splenic and Peritoneal Adherent Cells on Generation of Cytotoxic T Lymphocytes

When spleen cells primed in vivo against allogeneic lymphoid cells were used as responder cells in secondary mixed lymphocyte cultures, a high degree of cytotoxicity was generated even in the absence of splenic adherent cells. However, removal of adherent cells from such primed responder spleen cells reduced the cytotoxicity to some extent. On the other hand, when these responder cells were transferred into the peritoneal cavity of irradiated syngeneic mice together with antigenic cells, unseparated responder cells generated a lower degree of cytotoxicity than did adherent cell-depleted responder cells. In an in vitro system, peritoneal adherent cells also suppressed the generation of cytotoxic T lymphocytes by unseparated responders; however they augmented the cytotoxic T lymphocyte generation by adherent cell-depleted responders. These adherent cell populations with augmenting activity became inhibitory when they coexisted. The mechanism of this inhibitory action remains unclear.     

Macrophage activation of allogeneic lymphocyte proliferation in the guinea pig mixed leukocyte culture.

The role of the macrophage in the guinea pig mixed leukocyte culture was investigated. Macrophages obtained from oil-induced peritoneal exudates, peritoneal wash-out cells, spleen, and alveolar washings were found to be effective stimulators of allogeneic lymph node and splenic lymphocyte DNA synthesis. The stimulatory properties of macrophages proved radioresistant but viability dependent. Unfractionated lymph node cells or adherence column purified lymph node lymphocytes and thymocytes were only minimally active as stimulators, even in the presence of macrophages syngeneic to the responder lymphocytes. Allogeneic fibroblasts, polymorphonuclear leukocytes, L2C leukemia cells, and xenogeneic (murine) macrophages failed to simulate. These data provide evidence that the macrophage is the predominant stimulator of the mixed leukocyte culture in the guinea pig.     

Interactions of bone marrow cells from young and old mice with syngeneic and allogeneic thymic tissue

Age-related changes manifested in MHC-linked recognition of bone marrow (BM) cells by the thymic stroma were studied in vitro model of thymus-BM chimeras. Fetal thymuses (FT) depleted of self-lymphocytes were colonized with BM cells from syngeneic and allogeneic donor mice. When cells from young (3-month-old) or old (24-month-old) donors syngeneic to the stroma were seeded in a mixture with cells of allogeneic young origins (C57BL/6J-Thy1.2 and ARK/J-Thy1.1 seeded onto C57BL/6J FT), the syngeneic cells showed an age-related developmental advantage. Accordingly, cells from the old syngeneic mice manifested a significantly reduced capacity to compete with allogeneic cells when compared with the young syngeneic cells. When allogeneic BM cells from young or old mice were seeded onto the thymic stroma in a mixture with BM cells from young donors syngeneic to that stroma (BALB/c-Thy1.2 mixed with C57BL/Ka-Thy1.1 seeded onto C57BL/6J or C57BL/Ka FT), the Thy1+ cells which developed were mainly of syngeneic origin. The age of the allogeneic cells had no significant effect on the results. However, when old allogeneic cells were mixed with old syngeneic cells, the developmental advantage of the syngeneic cells was not manifested. When seeding of allogeneic cells was followed 1 day later by seeding of syngeneic cells, the syngeneic advantage was eliminated, suggesting that the MHC-linked competition began during the first 24 hr of contact with the thymic tissue. When BM-derived thmocytes grown in FT explants were transferred onto second FT recipient explants of the same genotype as the first ones, the syngeneic advantage was abolished, suggesting either that the thymic microenvironment was modified as a result of colonization or that it induced a change in the BM cells. In this respect, the young allogeneic BM-derived thymocytes showed a significant advantage when compared with the old cells. Thus, the MHC-linked syngeneic preference in the early development of BM cells is also manifested in aging mice, yet at a level that is significantly reduced compared with that seen in the young mice.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Virus-specific T-cell sensitization

Syngeneic, semiallogeneic, or allogeneic spleen lymphocytes were transferred intonu/nu BALB/c mice, which were infected with vaccinia virus. Specific Sensitization of transferred thymus-derived cells was determined in vivo by mean survival time and virus titer in the spleen six days after infection, and in vitro by cell-mediated cytolysis of vaccinia virus-infected syngeneic target cells. Virus-specific Sensitization took place only after transfer of syngeneic or semiallogeneic spleen lymphocytes; allogeneic lymphocytes had no influence on mean survival time or virus titer and showed no virus-specific cytolytic activity in vitro. Infection of mice with vaccinia virus-strain WR, Elstree, DIs, or DIs-infected syngeneic fibroblasts resulted in the generation of virus-specific effector cells, while injection of a high amount of inactivated virus particles caused no Sensitization. These results suggest H-2 homology for production of virus-specific effector cells. Propagation of virus is not necessary, since early surface antigens, combined with syngeneic H-2 antigens, suffice for Sensitization of cytolytic T lymphocytes.Abbreviations used in this paper are as follows CMC cell-mediated cytolysis - CTL cytolytic T lymphocyte - LCM lymphocytic choriomeningitis - MHC major histocompatibility complex - MST mean survival time - T cell thymus-derived cell - TCID₅₀ 50 percent tissue culture infective dose     

Existence of T cells manifesting self-reactivity indistinguishable from alloreactivity

The studies reported here were designed to analyze the phenotypic characteristics of self-reactive T lymphocytes induced in culture by allogeneic effect factor (AEF), as well as the control of their functional activities by the major histocompatibility complex (MHC). Unprimed T cells cultured with AEF in the absence of exogenous stimulating target cells become activated against self-antigens, as evidenced by their ability to manifest two distinct activities. First, such cells could lyse syngeneic target cells. This cytolytic activity was directed against H-2K antigens and was mediated by Lyt-2+ T cells. Second, the AEF-activated T cells could be stimulated in a secondary culture to high levels of proliferative activity by irradiated syngeneic spleen cells. The stimulator cells in this syngeneic mixed lymphocyte reaction (MLR) were found to be Thy-1-negative, Ia-positive splenic adherent cells. Stimulation in the secondary syngeneic MLR was provided by I-region specificities, and the majority of the proliferating cells were Lyt-1+ cells. Finally, AEF-induced T cells were effective in serving as effectors of graft-vs-host reactions in vivo in syngeneic recipients. These results prove that, under appropriate conditions, murine T lymphocytes can display aggressive patterns of self-reactivity that are similar in both quantity and quality to the classical patterns of alloreactivity and may have great significance for our understanding of MHC recognition processes.