Antibody-mediated immunosuppression of a cytotoxic cell response not involving a simple antigen-masking mechanism.

An in vitro cytotoxic cell response against alloantigens was inhibited by antibody directed against the alloantigens and also by anti-trinitrophenyl antibody provided that the allogeneic stimulator cells were modified with trinitrophenyl. Inhibition of an anti-allogeneic cytotoxic cell response against trinitrophenyl-coupled allogeneic stimulator cells by anti-trinitrophenyl antibody is dependent upon the intact Fc portion of inhibitory antibody. The magnitude of the difference between intact and F(ab')2 anti-trinitrophenyl antibody in immunosuppressive activity suggests that the Fc portion emits negative signals rather than simply adding to a steric hindrance effect.     

Properties of H-2L locus products in allogeneic and H-2 restricted, trinitrophenyl-specific cytotoxic responses.

The role of the recently defined L antigen (a second D region product) in allogeneic and TNP-specific syngeneic primary CML responses has been investigated. The lysis by anti-L specific cytotoxic effector cells was not inhibited when the target cells were pretreated with an antiserum directed against K and D, whereas an antiserum against L completely abrogated this response. Therefore, H-2L products are recognized on the target cell independently of H-2K and H-2D locus products. Both A.SW cells as well as B10 cells were found to respond to Ld alloantigens, in addition to Dd alloantigens when stimulated by cells differing only in the D region. The results of cold target blocking and antiserum inhibition experiments failed to detect cytotoxic cells with specificity of L antigens in association with TNP, under conditions in which TNP-specific effectors to K and D antigens were demonstrable. These findings suggest that there is a more limited involvement of H-2L locus products than the H-2K or H-2D locus products in the induction and specificity of these responses.     

Inhibition of antiskin allograft immunity induced by infusions with photoinactivated effector T lymphocytes (PET cells)

Induction of tolerance for skin allotransplantation requires selective suppression of the host response to foreign histocompatibility antigens. This report describes a new approach which employs pre-treatment with 8-methoxypsoralen (8-MOP) and ultraviolet A light (UVA) to render the effector cells of graft rejection immunogenic for the syngeneic recipient. Eight days after BALB/c mice received CBA/j skin grafts, their splenocytes were treated with 100 ng/ml 8-MOP and 1 J/cm2 UVA prior to reinfusion into naive BALB/c recipients. Recipient mice were tested for tolerance to alloantigens in mixed leukocyte culture (MLC), cytotoxicity (CTL), delayed-type hypersensitivity assays (DTH), and challenge with a fresh CBA/j graft. Splenocytes from BALB/c recipients of photoinactivated splenocytes containing the effector cells of CBA/j alloantigen rejection proliferated poorly in MLC and generated lower cytotoxic T-cell responses to CBA/j alloantigens in comparison with sensitized and naive controls and suppressed the MLC and CTL response to alloantigen from sensitized and naive BALB/c mice. In vivo, the DTH response was specifically suppressed to the relevant alloantigen in comparison with controls. BALB/c mice treated in this fashion retained a CBA/j skin graft for up to 42 days post-transplantation without visual evidence of rejection. These results showed that reinfusion of photoinactivated effector cells resulted in an immunosuppressive host response which specifically inhibited in vitro and in vivo responses that correlate with allograft rejection and permitted prolonged retention of histoincompatible skin grafts.     

Specific inhibition of cytotoxic memory cells produced against UV-induced tumors in UV-irradiated mice.

Cytotoxic responses of UV-irradiated mice against syngeneic UV-induced tumors were measured by using a 51Cr-release assay to determine if UV treatment induced a specific reduction of cytotoxic activity. The in vivo and in vitro primary responses against syngeneic tumors and allogeneic cells were unaffected, as was the "memory" response (in vivo stimulation, in vitro restimulation) against alloantigens. In contrast, the memory response of UV-treated mice against syngeneic, UV-induced tumors was consistently and significantly depressed. The cytotoxicity generated by tumor cell stimulation in vivo or in vitro was tumor-specific and T cell-dependent. Since the primary response against syngeneic UV-induced tumors produces apparently normal amounts of tumor-specific cytotoxic activity, UV-treated mice may not reject transplanted syngeneic tumors because of too few T effector memory cells. These results imply that, at least in this system, tumor rejection depends mostly on the secondary responses against tumor antigens and that at least one carcinogen can, indirectly, specifically regulate immune responses.     

Studies on the specificity of in vitro-induced lymphocytotoxicity to methylcholanthrene-induced fibrosarcoma cell lines

Cytotoxic effector lymphocytes were induced by in vitro immunization of lymph node and spleen cells from CS7B16(H2^b) and Balb/c(H2^d) mice to syngeneic or allogeneic methylcholanthrene-induced fibrosarcoma (MCAF) cell lines. The T cell-dependent cytotoxicity was specific to target cell lines to which the lymphocytes were immunized in vitro. Normal fibroblasts as stimulator cells did not induce lymphocytotoxicity to syngeneic MCAF cells or to normal syngeneic fibroblasts. The results indicate that the in vitro-immunized lymphocytes recognize individual specific tumor-associated antigens of the MCAF cells. In experiments in which the lymphocytes were immunized in vitro to allogeneic MCAF cells, cytotoxic reactions to alloantigens, but not to tumor-associated antigens, were detected. Incubation with phytohemagglutinin (PHA) during the sensitization period modified the specificity of the cell-mediated lysis of MCAF cells: Allogeneic as well as syngeneic target cells were destroyed by these effector cells. PHA induced a nonspecific cytotoxic effect which increased the specific lysis of target cells. The cytotoxicity of the in vitro-immunized lymphocytes was inhibited by incubation with membrane protein preparations from the syngeneic MCAF cell lines. In contrast to the specificity of the cytotoxic effect to the different syngeneic cell lines, the membrane extract of one individual syngeneic MCAF cell line was able to inhibit the lymphocytotoxicity to all other syngeneic cell lines. Membrane protein preparations from allogeneic MCAF cells or from normal syngeneic fibroblasts were not inhibitory. The in vitro-immunized cytotoxic lymphocytes did not impair the tumor growth in vivo as could be demonstrated by passive transfer of the lymphocytes in a Winn assay.     

Transfer of cell-mediated hyperacute rejection reaction: The role of active sensitization

The transfer of lymph node cells draining graft sites of allogeneic murine skin results in adoptive immunization of syngeneic recipients, as per hyperacute rejection of allogeneic test skin grafts. The transfer of spleen cells from mice sensitized by i.p. injection of allogeneic cells does not have this result unless the cells undergo a secondary in vitro sensitization. The resultant hyperacute rejection is due in part to adoptive immunization via the spleen cells primed during the in vivo sensitization and rendered transfer effective by the in vitro exposure; in part it is due to active sensitization by carried-over antigen from the in vitro exposure. It follows that the transfer effect of spleen cells sensitized in vivo and in vitro is only in part abrogated by exposure to α-Thy. 1 serum and complement.     

Regulation of cellular immune response against autologous human melanoma. I. Evidence for cell-mediated suppression of in vitro cytotoxic immune response

The potential existence of down-regulation of cytotoxic immune response against an autologous human melanoma line was investigated as a possible explanation for cytotoxic unresponsiveness against the autologous melanoma cells. The melanoma cell line, PJ-M, was established and lymph node resident lymphocytes (LNL) were isolated from a lymph node which was partially infiltrated with the melanoma cells. Autologous peripheral blood lymphocytes (PBL) were sensitized in in vitro co-culture (IVC) against radiated PJ-M cells in the presence or absence of PJ-M-sensitized LNL and enriched suppressor (OKT8+) or inducer (OKT4+) LNL populations, and were assayed for cytotoxicity in a 4-hr 51Cr-release microcytotoxicity assay. Significant cytotoxic response against PJ-M could be generated in the PBL, but not in the LNL. The addition of sensitized, unfractionated LNL, OKT8+, or OKT4+ LNL populations abrogated cytotoxic response in the PBL against PJ-M. The suppression of cytotoxic response was induced selectively against the PJ-M targets, because IVC of PBL in the presence of the sensitized LNL did not affect the generation of polyclonal cytotoxic alloreactivities, nor did they abrogate the generation of cytotoxic response against allogeneic targets in IVC against the corresponding allogeneic targets. These results suggest the possibility that cytotoxic immune response against the autologous melanoma cells might have been suppressed by the individual's own immunoregulatory circuit.     

Generation of suppressor lymphocytes during sensitization in culture against a syngeneic tumor: affinity chromatography on insolubilized histamine

We investigated the effect of depletion of histamine-binding lymphoid cells on immunological properties of lymphocytes sensitized in culture against tumor cells. C57BL/6 spleen cells that were sensitized in vitro on monolayers of the syngeneic Lewis lung carcinoma (3LL) became cytotoxic to the tumor cells in vitro after 3 to 5 days of sensitization. Sensitized cells harvested after 4 days of sensitization occasionally enhanced tumor growth in vivo. Fractionation of the sensitized lymphocytes over insolubilized histamine-rabbit serum albumin-Sepharose (HRS) columns decreased or abolished the enhancing activity in vivo and specifically increased the in vitro cytotoxic activity of the depleted lymphocytes. A similar increase in the cytotoxic activity of HRS-fractionated cells was observed in an allogeneic combination of C57BL spleen cells sensitized against C3H fibroblasts. The effect of HRS chromatography on the in vitro cytotoxic activity increased with prolonged incubation of the depleted effector cells with the target cells.     

Allogeneic restriction of the delayed inflammatory reaction in the rat.

Delayed-type hypersensitivity (DTH) to Listeria monocytogenes was measured in rats that were recipients of syngeneic, semisyngeneic, and allogeneic immune thoracic duct lymphocytes (TDL). DTH could be transferred only to recipients that shared at least one haplotype with the TDL donors. The restriction was expressed in an inability of sensitized lymphoblasts to localize efficiently at antigen injection sites in the pinna of the ear and peritoneal cavity. Failure of allogeneic lymphoblasts to extravasate in more than trace numbers into Listeria-antigen-induced exudates was reflected in an absence of other lymphocyte-mediated expressions of DTH. Thus, lymphocyte-dependent MCA was not detected in Listeria-antigen-induced peritoneal exudates borne by recipients of allogeneic immune TDL and blood monocytes were not recruited in increased numbers into such exudates as they were in exudates borne by syngeneic rats. But allogeneic restriction of the delayed inflammatory response to Listeria antigen was overcome, at least in part, when antigen-presenting macrophages of the same MHC type as the immune TDL donors were implanted in the peritoneal cavity. The results encourage the belief that the observed failure of immune TDL to transfer DTH to allogeneic recipients is related to the inability of sensitized donor T cells to recognize antigen displayed by allogeneic macrophages.     

Studies of thymopoietin pentapeptide (TP5) on experimental tumors: I. TP5 relieves immunosuppression in tumor-bearing mice

The allogeneic and syngeneic immune responses of tumor-bearing mice (C57BL/6 mice bearing 3LL and DBA mice bearing P815) were evaluated by the cytotoxic lymphocyte precursor unit (CLP-U) and MLC. In general, tumor-bearing mice showed slightly enhanced immune responses 4 days after tumor inoculation. This enhanced immune response rapidly declined and about 7–10 days after tumor inoculation, both allogeneic and syngeneic responses were markedly lower than normal. Mice treated with TP5, starting 2 weeks before tumor inoculation, retained normal or enhanced allogeneic and syngeneic responses up to 3 weeks after tumor inoculation. When this tumor-induced suppressive effect was studied in cell transfer experiments, spleen cells from tumor-bearing mice enhanced the growth of tumors in syngeneic recipients whereas spleen cells from TP5-treated mice inhibited the growth of tumors in syngeneic recipients. Moreover, the spleen cells from TP5-treated mice also showed enhanced cytotoxic activity against tumor cells in vitro. These findings suggest that the tumors, after a transient stimulatory phase, induced immune suppressive mechanisms in the hosts' immune defenses. Treatment with TP5 prevented the development of these immune suppressive effects and spleen cells from TP5-treated tumor-bearing mice inhibited tumor growth in freshly tumor-inoculated recipients.     

Induction of suppressor cells in autologous mixed lymphocyte culture (AMLC) in humans

T cells stimulated for 6-7 days in autologous mixed lymphocyte culture (AMLC) showed suppressive effects when added to fresh mixed cultures where autologous lymphocytes (A) were stimulated by Mitomycin C-treated allogeneic lymphocytes (Xm), in a ratio of A:Xm:AMLC-activated cells of 1:1:0.5. Both cytotoxic and proliferative activities in second cultures, as assayed after 6 days of incubation, were significantly inhibited (percentage suppression of cytotoxic activity observed in 17 experiments was 75.3 +/- 22.4; percentage suppression of proliferation was 60.6 +/- 18.2). Suppressor cells (SC) generated in AMLC were Mitomycin C sensitive and nonspecific in their action; not only A/Xm but also X/Am and X/Ym cultures were suppressed to the same extent. AMLC-Activated cells showed a considerable degree of proliferation in response to alloantigens but failed to express any cytotoxic activity against autologous or allogeneic phytohemagglutinin blasts. Thus, the inhibitory effect observed in this system is not due to cytotoxic elimination of responding or stimulating cells in the second culture but rather reflects a true regulatory (suppressive) mechanism.     

Morphological and functional characteristics of cells infiltrating and destroying tumor multicellular spheroids in vivo.

EMT6 mammary sarcoma cells were grown in vitro as multicellular spheroids to model for the heterogeneity of microenvironments and structural changes which develop in many tumors, including micrometastases. Spheroids of 700-900 micron diameter were implanted into and recovered at different times from the peritoneal cavities of sensitized or nonsensitized allogeneic and syngeneic mice. The colony forming efficiency of spheroid tumor cells recovered at 24 and 48 h from sensitized allogeneic mice was markedly decreased as compared with those from nonsensitized allogeneic or syngeneic animals. These recovered spheroids were extensively infiltrated by both lymphocytes and macrophages, which ultrastructurally had very close membrane associations with tumor cells. Host cells recovered from spheroids exhibited cytotoxic activity in an in vitro 51Cr release assay. Thus, multicellular spheroids in vivo provide a unique experimental model to study the functional capacity of host cells within a spheroical tumor. Although lacking the stroma and the vasculature of in vivo solid tumors, this model does have many similarities to in vivo tumors and is thus suitable for studying the tumor cell-host cell interactions within the tumor microenvironment. In addition, the system offers the potential for quantitative study of the effects of treatment modalities on tumor cell-host cell interactions.     

Generation of cytotoxic lymphocytes to syngeneic tumor by using co-stimulator (Interleukin 2)

Cytotoxic lymphocytes (CL) highly active against the syngeneic mastocytoma, P815, were generated from spleen cells of DBA mice cultured with co-stimulator (Interleukin 2) and P815. More CL activity was generated from spleen cells of P815 tumor-bearing mice than from spleen cells of normal mice. Thymus cells from tumor-bearing mice, however, did not produce increased CL activity. Most of the CL were Thy 1 and Ly 1 positive. The optimal culture conditions and kinetics were similar to those for the generation of allogeneic cytotoxic T lymphocytes. The cytotoxic activity against syngeneic P815 was similar in magnitude to the response of DBA spleen cells to allogeneic tumor lines and to the response of allogeneic CBA spleen cells to P815. Although CL generated from tumor-bearing mice did not lyse normal DBA cells, they did lyse, to a much lesser degree, a number of tumor cell lines other than the sensitizing P815. This nonspecific lysis was not H2 restricted nor was it restricted to tumors of lymphoid origin. Generation of nonspecific cytolytic activity was antigen independent, occurring in the presence of co-stimulator alone.     

Recruitment of effector lymphocytes by initiator lymphocytes. Recruited lymphocytes are immunospecific and include graft-versus-host-reactive lymphocytes.

We have shown previously that initiator T lymphocytes (ITL), sensitized in vitro against fibroblast antigens, recruit effector T cells in vivo. After injection into hind footpads of syngeneic recipients, sensitized ITL migrated to the draining popliteal lymph nodes (PLN) and activated a trapping mechanism by which circulating lymphocytes were recruited in the PLN. This paper reports experiments designed to test the immunospecificity of these recruited T lymphocytes (RTL). We found that immunospecific RTL were depleted from other lymphoid organs during recruitment in the PLN. However, immunospecific ITL were not depleted from spleens during PLN recruitment. Thus ITL and RTL are functionally distinguishable. We show that specific GVH reactive lymphocytes were also lost from spleens and distal lymph nodes during trapping of RTL in the PLN. Thus, the trapping phase of the recruitment response is immunospecific, as are the sensitization and effector phases. The trapped RTL are antigen-specific, and include the pool of GVH-reactive-lymphocytes committed to the same alloantigen. Thus, it appears that GVH-reactive cells respond to syngeneic ITL sensitized against allogeneic fibroblasts.     

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.     

In vitro sensitization of human lymphoid cells to antigens on cultured melanoma cells: II. Sensitization against melanoma-associated antigens

Peripheral blood lymphoid cells from patients with malignant melanoma can be sensitized on allogeneic or autochthonous melanoma monolayers. Peak cytotoxicity occurred after 5 days of sensitization. Sensitization appeared to be directed against melanoma-associated antigens, as judged by the pattern of cytotoxic reactivity. Sensitized cells were cytotoxic against autochthonous or allogeneic melanoma cells, but not against autochthonous fibroblasts or allogeneic tumor cells of different histologic types. Sensitization of responder lymphoid cells from melanoma patients on allogeneic melanoma cells usually resulted in more pronounced cytotoxicity against autochthonous melanoma target cells than did sensitization on autochthonous melanoma monolayers. These results indicate that cell cultures of human malignant melanoma contain tumor-associated antigens which can sensitize human peripheral blood lymphoid cells in vitro. These results also support the concept that there are cross-reactive tumor-associated antigens in human malignant melanomas.     

Normal T-cell receptors for alloantigens

To study the diversity of normal mouse T lymphocytes capable of mediating allograft immunity, we modified a cell culture system so that both induction of sensitization and target cell damage could be studied in vitro. Mouse lymph node lymphocytes were sensitized in vitro against allogeneic fibroblasts. The sensitized lymphocytes produced immunospecific cytotoxic effects against target fibroblasts in vitro. We found that T lymphocytes were directly involved in both sensitization and cytotoxicity.We used this allograft system to separate nonsensitized mouse lymphocytes on the basis of their ability to bind to allogeneic fibroblasts. Adhering lymphocytes were found to be enriched in effector cells following sensitization. The nonadhering lymphocytes showed a decreased ability to undergo sensitization against fibroblasts that were syngeneic to the ones used for adsorption. However, they were able to become sensitized against unrelated fibroblasts of another H-2 phenotype.These findings indicate that specific receptors for histocompatibility antigens pre-exist on diverse populations of normal mouse T lymphocytes.     

Cyclosporin A suspends transplantation reactions in the marine sponge Microciona prolifera

Sponges are the simplest extant animals but nevertheless possess self-nonself recognition that rivals the specificity of the vertebrate MHC. We have used dissociated cell assays and grafting techniques to study tissue acceptance and rejection in the marine sponge Microciona prolifera. Our data show that allogeneic, but not isogeneic, cell contacts trigger cell death and an increased expression of cell adhesion and apoptosis markers in cells that accumulate in graft interfaces. Experiments investigating the possible existence of immune memory in sponges indicate that faster second set reactions are nonspecific. Among the different cellular types, gray cells have been proposed to be the sponge immunocytes. Fluorescence confocal microscopy results from intact live grafts show the migration of autofluorescent gray cells toward graft contact zones and the inhibition of gray cell movements in the presence of nontoxic concentrations of cyclosporin A. These results suggest that cell motility is an important factor involved in sponge self/nonself recognition. Communication between gray cells in grafted tissues does not require cell contact and is carried by an extracellular diffusible marker. The finding that a commonly used immunosuppressor in human transplantation such as cyclosporin A blocks tissue rejection in marine sponges indicates that the cellular mechanisms for regulating this process in vertebrates might have appeared at the very start of metazoan evolution.     

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.     

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

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