Local Adoptive Transfer Assay Research Articles

Ocular immune privilege and CD1d-reactive natural killer T cells.

Immune privilege in the eye is, in part, associated with the development of an antigen-specific systemic tolerance termed anterior chamber-associated immune deviation (ACAID). Natural killer T (NKT) cells express T-cell receptor and natural killer (NK) markers and are classified as innate immune cells partly because they produce cytokines within minutes of signals. The aim of this study was to elucidate the role of murine NKT cells in the induction of T regulatory cells in anterior chamber-associated immune deviation. Anterior chamber-associated immune deviation T regulatory cell generation ability was examined in the following NKT cell-deficient mice: SJL mice, CDld or Jalpha281 knockout (KO) mice on C57BL/6 (B6) background, and NKT cell-depleted mice. To detect T regulatory cells, splenic T cells were harvested 7 days after anterior chamber inoculation of ovalbumin (50 microg/2 microL in Hanks balanced salt solution [HBSS]), mixed with ovalbumin-primed T cells (effector) and ovalbumin-pulsed antigen-presenting cells (stimulator), and then cotransferred into the ear pinnae of a syngeneic naive mouse (local adoptive transfer assay). Ear swelling was measured 24 hours later. Anterior chamber-inoculated B6 mice developed T regulatory cells, but all natural killer T cell-deficient mice did not generate T regulatory cells unless they were reconstituted with natural killer T cells. We also found that the number of splenic natural killer T cells were increased in anterior chamber-inoculated B6 mice and those natural killer T cells produced IL-10. CD1d-reactive natural killer T cells are essential for the induction of T regulatory cells in anterior chamber-associated immune deviation through their IL-10 production and are involved in the maintenance of immune privilege of the eye.

Evidence for multiple CD95-CD95 ligand interactions in anteriorchamber-associated immune deviation induced by soluble protein antigen.

We have investigated whether CD95-CD95 ligand interactions are important in anterior chamber-associated immune deviation (ACAID) induced by soluble protein antigen, and if so, to identify the participating cells on which these molecules are expressed. Peritoneal exudate cells as antigen-presenting cells (APC) obtained from B6.lpr/lpr, B6.gld/gld and C57BL/6 mice were cultured with ovalbumin (OVA) and transforming growth factor-beta2 (TGF-beta2) overnight, then injected intravenously into C57BL/6 or B6.lpr/lpr recipients. Some B6.lpr/lpr mice were reconstituted with naive T cells from wild-type C57BL/6 donors. In other experiments, B6. lpr/lpr and B6.gld/gld mice received an anterior chamber injection of OVA followed 7 days later by subcutaneous immunization with OVA plus adjuvant. Delayed hypersensitivity (DH) was assessed with an ear swelling assay. T cells activated in vitro with OVA-pulsed, TGF-beta-treated APC were tested in vivo for their capacity to suppress DH expression in a local adoptive transfer assay. The results indicate that when ACAID was induced by in-vitro generated ACAID-inducing cells, the APC expressed CD95L, and recipient T cells expressed CD95. The capacity of in vitro generated regulatory T cells to suppress DH expression to OVA in vivo was not governed by CD95-CD95L interactions. When OVA was injected into the anterior chamber of naive mice, CD95 expression was required for ACAID induction, although ACAID was readily induced in CD95L-deficient mice. We conclude that CD95-CD95L interactions are required in ACAID for the initial stage of APC presentation of eye-derived antigens to T cells, and that CD95-CD95L interactions participate at one or more additional step in the process by which ACAID is induced by soluble protein antigens.

Rat Adenocarcinoma 13762 Expresses Tumor Rejection Antigens but Tumor-Bearing Animals Exhibit Tumor-Specific Immunesuppression

Rat adenocarcinoma 13762 was adapted to continuous growth in culture and used in a variety of experiments to investigate the immune response to inoculation of animals with replication-defective tumor cells. The results demonstrate that 13762 cells express tumor-specific tumor rejection antigens that elicit protective immunity to tumorigenic challenge. By several criteria there is no apparent humoral component of the anti-tumor immunity; however, antitumor immunity is characterized by nylon-wool nonadherent spleen T cells. Anti-tumor T cells demonstrate tumoricidal activity in local adoptive transfer assays and are not found in spleens of naive animals or animals immunized against either nontumorigenic Rat 1 cells or a syngeneic fibrosarcoma. Despite the expression of tumor rejection antigens 13762 tumor, and the demonstrable ability of injection of irradiated tumor to induce anti-tumor immunity, tumors elicited in unimmunized syngeneic animals grow progressively. The reasons for growth of antigenic tumor are unknown but are shown not to be due to defective antigen expression in 13762 tumor since, in addition to being able to elicit T cell immune response in immunized animals, 13762 tumor expresses MHC Class I molecules and can be a target for allogeneic T cell recognition in vitro. These data suggest that in tumor-bearing animals an effective anti-tumor immune response is either not initiated or down-regulated. Since animals bearing 13762 tumors can be immunized against an unrelated syngeneic sarcoma, can produce humoral responses to several protein antigens, and can produce delayed type hypersensitivity response against dinitrofluorobenzene, the immune response to 13762-induced tumors appears specifically suppressed. In support of this contention, 13762 cells express

Impaired cell-mediated immunity in mice bearing healthy orthotopic corneal allografts.

A very high proportion of orthotopically grafted, histoincompatible corneas are accepted in healthy condition for prolonged intervals (> 8 weeks) by naive untreated BALB/c mice. Because these grafts form the anterior surface of the anterior chamber of the eye and because alloantigens placed in the anterior chamber elicit a deviant, stereotypic, systemic immune response known as anterior chamber-associated immune deviation, we have examined the state of alloantigen-specific cell-mediated immunity in BALB/c mice with accepted corneal allografts, as well as mice that had rejected their corneal grafts. Attempts to induce alloantigen-specific delayed hypersensitivity (DH) by s.c. injections of lymphoid cells, genetically identical to the grafts, resulted in positive responses only in "rejector" mice; "acceptor" mice failed to develop or display DH. Spleens cells from acceptors and rejectors were then assayed for the ability to suppress local adoptive transfer of alloantigen-specific DH. It was found that transfer reactions failed to develop if spleen cells from acceptor mice were added to the inoculum, whereas spleen cells from rejected mice had no similar inhibitory effects. The suppression mediated by acceptor spleen cells in local adoptive transfer assays was determined to be specific for the alloantigens expressed on the long-standing corneal allografts. We conclude that, when orthotopic corneal allografts are accepted indefinitely by adult mice, graft success correlates with the induction of alloantigenically specific anterior chamber-associated immune deviation. It is proposed that suppression of alloantigen-specific DH, which is characteristic of mice with accepted grafts, plays a critical role in the success of grafted corneas.

Immunomodulation by adriamycin - effect on the production of cytotoxic and inflammatory antitumor immune-responses.

We have recently demonstrated in a mouse renal adenocarcinoma tumor system that the antitumor effect of adriamycin (ADM) in combination with interleukin-2 (IL2) is superior to treatment with either ADM or IL2 alone. Based on this observation we postulated that modulation of host's immune competence by ADM and/or IL2 may be partly responsible for the antitumor effects of the combination treatment. Indeed, pretreatment with ADM was found to potentiate the production of both cytotoxic and non-cytotoxic immune responses. A single dose of ADM significantly increased the number of nucleated cells in the spleen in a time related fashion. A small but selective increase in CD4+ cells without an apparent change in CD8+ subset of T cells was observed following treatment with ADM. ADM potentiated augmentation of NK activity by IL2, but had no effect on the production of lymphokine activated killer (LAK) cells by IL2. In contrast, treatment with a combination of ADM and IL2 resulted in increased LAK activity and the frequency of LAK-precursors in the spleen. ADM also enhanced the development of tumor specific inflammatory delayed hypersensitivity (DH) response. Mice expressing tumor specific DH were resistant to rechallenge with viable tumor cells and their spleen cells inhibited the tumor growth in a local adoptive transfer assay. Thus, antitumor effects of combined ADM/IL2 treatment may in part involve augmented production of cytotoxic and T cell-mediated inflammatory antitumor immune responses.

Active Specific Chemoimmunotherapy of Lymph‐node Metastasis from a Poorly Immunogenic Murine Fibrosarcoma

The fibrosarcoma MCA‐SP, which was recently induced with methylcholanthrene (MCA) in C3H/ HeJ mice, displays poor immunogenicity in in vivo prophylaxis. A cell variant MCA‐SPN1, which bears a tumor‐specific transplantation antigen (TSTA) cross‐reactive with the parental line MCA‐SP, was selected because of its proclivity for axillary lymph‐node metastases. Although these lymph‐node metastases were resistant to sinecomitant (post‐excisional) immunity, they were susceptible to combined active and passive specific Chemoimmunotherapy, using tumor‐specific, 1‐butanol‐extracted, preparative isoelectric focusing‐purified, TSTA (1 fig weekly sc injections), cyclophosphamide (CY, a single intraperitoneal 20 mg/kg dose), and adoptive transfer of immune splenic T lymphocytes, which had been re‐stimulated in vitro with extracted TSTA and interleukin‐2. This triple regimen both reduced the incidence of spontaneous lymph‐node metastases, and prolonged the survival of tumor‐bearing, as well as tumor‐resected hosts. The results from local adoptive transfer assay using T‐lymphocyte snbpopulations of spleen and lymph nodes in these treated hosts suggested that Lyt 2+ cytotoxic T‐lymphocytes (CTL) mediated in vivo tumor‐neutralization. Thus TSTA/CY/CTL therapy activates tumoricidal host responses effective against the poorly immunogenic MCA‐SP tumor and its lymph‐node metastases.

Characterization of variant and parental-cross-protective immunity to immunogenic variants of a murine fibrosarcoma using the local adoptive transfer assay.

The purpose of this study was to characterize the lymphocyte populations responsible for rejection of immunogenic (Imm+) tumor variants, and the cross-protective immunity engendered by Imm+ variants against the weakly immunogenic parental tumor. Immunogenic clones of the weakly immunogenic methylcholanthrene-induced fibrosarcoma MCA-F have been generated using 1-methyl-3-nitro-1-nitrosoguanidine, 5-aza-2'-deoxycytidine, or ultraviolet radiation (UV-B; 280-320 nm). These clones grow progressively in immunosuppressed adult-thymectomized irradiated mice, but are rejected by immunocompetent syngeneic hosts. The parental MCA-F tumor grows progressively in both groups. Mice that have rejected a challenge of 1 x 10(5) Imm+ cells show an anamnestic immune response against both the Imm+ clone and the parental MCA-F tumor. Using the local adoptive transfer assay and depletion of T-cell subsets with antibody plus complement, we show that immunity induced by the Imm+ variants against the parent MCA-F was mediated by the Thy1.2+, L3T4a+ population without an apparent contribution by Lyt2.1+ cells. Although antivariant immunity was also dependent upon Thy1.2+ cells, depletion of either the L3T4a+ or the Lyt2.1+ cells failed to abolish immunity against the variant. A role for Lyt2.1+ T lymphocytes in antivariant immunity, but not antiparent immunity, was supported by the results of cytotoxic T lymphocyte (CTL) assays. Following immunization with high numbers (1 x 10(5) to 5 x 10(5) of viable Imm+ cells, antivariant, but not antiparent CTL activity was detected in mixed lymphocyte tumor cell cultures. Immunization with lower numbers (3 x 10(4] of viable Imm+ or with high numbers of mitomycin-C-treated Imm+ engenders only antivariant immunity without parental cross-protection. Under these conditions lymphocytes mediating immunity against the variant in the local adoptive transfer assay were exclusively of the Thy1.2+, L3T4a+ phenotype, with no contribution from the Lyt2.1+ cells. Identical results were obtained for Imm+ clones of MCA-F induced by methylnitronitrosoguanidine, 5-azadeoxycytidine, and UV-B, suggesting that the nature of the antitumor immunity engendered by Imm+ is not significantly affected by the agent used. Furthermore, these results demonstrate that the cross-reactivity and cellular effectors of antitumor immunity in this system are influenced by the immunizing dose of Imm+ cells: the predominant effectors of both antivariant and parental-cross-reactive immunity were of the CD4+ T cell subclass, with a CD8+ cytotoxic population contributing to antivariant immunity only after high-dose immunization.

Specific chemoimmunotherapy in tumor-bearing mice with extracted antigen, cyclophosphamide, and intrasplenic administration of interleukin-2.

The effect of daily intrasplenic (I-SP) injection of interleukin-2 (IL-2) in conjunction with specific chemoimmunotherapy with extracted tumor antigens and cyclophosphamide was assessed with the use of methylcholanthrene (MCA)-induced fibrosarcoma in C3H/HeJ mice. Injections of 80 U of human IL-2 were delivered transcutaneously into the spleen (I-SP), which had been relocated to the subcutis with its blood supply intact. Six daily I-SP injections into mice bearing MCA-F tumors activated immune spleen cells (SPC), as evidenced by specific neutralization of the MCA-F, but not the antigenically different MCA-D tumor, in local adoptive transfer assays. The immune cell phenotype was Thy 1.2+ Lyt 2+, based upon abrogation of tumor neutralization after depletion with monoclonal antibodies and complement. In a second series of experiments, primary hosts bearing established MCA-F tumors underwent therapy with 1 microgram 1-butanol extracted, isoelectrophoretically purified TSTA injected subcutaneously, a single intraperitoneal (IP) dose of 20 mg/kg cyclophosphamide (CY), and/or either IP or I-SP IL-2 injection. Triple chemoimmunotherapy with I-SP, but not IP, IL-2 retarded tumor outgrowth more effectively than single or double treatment protocols. Furthermore, in a third series of investigations, the triple therapy group showed both decreased numbers and incidence of spontaneous metastases from a subcutaneous implant of clone 9-4, a highly metastatic variant of MCA-F. Indeed, 35% of hosts that received triple therapy, but none of the animals treated with other regimens, were free of lung metastasis (P less than 0.02). Thus, tumor resistance engendered by chemoimmunotherapy with TSTA and CY is potentiated in vivo by I-SP administration of IL-2.

Mechanism of the A/Ph.MC.S1 tumor graft rejection in syngeneic mice.

A considerable number of normal mice injected with a low dose of A/Ph.MC.S1 syngeneic methylcholanthrene-induced tumor cells showed regression of the tumor after a transient period of growth. The regressor mice eliminated a challenge inoculum in an accelerated fashion. Splenic lymphocytes from such regressor mice inhibited the growth of the same tumor in the local adoptive transfer assay. This capacity required the presence of thymus-derived cells. The regressor animals developed a delayed-type hypersensitivity response against the tumor In vivo and their lymph-node cells produced macrophage migration inhibitory factor in the presence of tumor cells in vitro. The growth of this tumor was facilitated by treating the primary recipients with carrageenan, and the strong tumor-inhibitory capacity of regressors was also depressed by this agent. Early macrophage infiltration of the tumor in regressor mice was demonstrable histochemically, preceding the inhibition of tumor growth. A lymphokine-producing T cell-mediated, macrophage-dependent delayed-type hypersensitivity-like mechanism is proposed to be the dominant mechanism of the resistance in this tumor-host model.

Changes in spleen morphology and lymphoid cell activity during tumor progression

Morphologic alterations in the spleen and dynamic changes in the biologic activity of its cellular components occur concomitant with the neoplastic progression of a murine methylcholanthrene induced fibrosarcoma. Spleen size, weight and cell number increased with tumor growth. Using the local adoptive transfer assay (LATA), spleens from tumor bearing mice in the early and late stages of neoplastic growth were shown to possess non-specific, tumor-facilitating cells which were radioresistant, phagocytic and adherent, presumably macrophages. On the other hand, spleens from mice in the intermediate and late stages of tumorigenesis displayed specific, tumor-neutralizing cells, which were radiosensitive and Thy 1.2 positive, presumably T-cells. Thus, a balance of tumor-facilitating and neutralizing cells may determine neoplastic progression in susceptible, syngeneic hosts.

Specific and non-specific immunologic mechanisms of tumor growth facilitation.

Specific and non-specific mechanisms of tumor growth facilitation were studied using methylcholanthrene (MCA)-induced fibrosarcomas in C3H/HeJ mice. In early and late stages of tumor growth, mice possessed non-specific, tumor-facilitating cells detected by local adoptive transfer assay (LATA). These cells appeared to be macrophages; they were radioresistant (700 rads), phagocytic, and adherent to plastic. Specific tumor facilitation was induced by treatment either with crude 3M KCl extracts, or with an acidic pIEF fraction (pI 3.5). After treatment with this material, animals displayed facilitated outgrowth of only MCA-F, but not the antigenically distinct MCA-D or MCA-C tumors. Thus in addition to non-specific stimuli, which accelerate neoplastic growth, tumors bear tumor-specific transplantation antigens (TSTA), which induce specific facilitation.

The development of in vitro and in vivo anti-tumor cytotoxicity in noncytotoxic, MOPC-315-tumor-bearer, spleen cells ?educated? in vitro with MOPC-315 tumor cells

Spleen cells from mice bearing various sizes of MOPC-315 plasmacytoma tumors were not cytotoxic in the 51Cr release assay or the local adoptive transfer assay. These noncytotoxic spleen cells became cytotoxic, however, upon in vitro cocultivation with MOPC-315 tumor cells. The maximal level of in vitro anti-tumor cytotoxicity (51Cr release) with in vitro “educated” tumor-bearer spleen cells was obtained on the fifth day of the cocultivation and was equal to or lower than the level of cytotoxicity seen with in vitro educated normal spleen cells. On the other hand, the level of in vivo anti-tumor cytotoxicity (Winn assay) achieved with tumor-bearer spleen cells educated in vitro was at least equal to, but usually greater than the level of anti-tumor cytotoxicity obtained with normal spleen cells educated in vitro. Thus, in vitro education can generate anti-tumor cytotoxicity in autochthonous lymphoid cells from tumor-bearing hosts. Such educated histocompatible cells should be useful for immunotherapy regimens that might be applicable to man.

Progression of the immune response to solubilized tumor antigens.

Local adoptive transfer assays (LATA) were used to analyze and compare the progression of the immune response in C3H/HeJ mice to irradiated tumor cell vaccine and to crude 3M KCl solubilized antigens extracted from a syngeneic methylcholanthrene-induced fibrosarcoma. Sequential LATA performed 2, 6, 9, 12 and 15 days after soluble antigen pretreatment of spleen cell donors revealed a sinusoidal evolution of lymphoid cell activity. An initial brief period of potent tumor facilitation (days 6-9) was followed by a phase of tumor neutralization (days 9-12) which decayed by day 15. On the other hand, spleen cells from donors sensitized with irradiated tumor cells exhibited consistent tumor neutralization which was sustained throughout 15 days. Thus the tumor growth facilitation observed only in CSA-treated donors may represent a qualitative difference in the immune state induced by soluble, as opposed to cellular, forms of tumor antigen.