Pretreatment of P815 mastocytoma cells with inhibitors of protein synthesis reduces their susceptibility to lysis by cytotoxic thymus-derived lymphocytes

Protein synthesis inhibitors like cycloheximide, emetine, and puromycin diminish the ability of P815, a mastocytoma of DBA/2 mice, to react with anti-H-2^d cytotoxic thymus-derived lymphocytes (CTLs). Compared to untreated P815, tumor cells incubated with the protein synthesis inhibitors exhibited a reduced sensitivity to lysis and a reduced ability to inhibit lysis of untreated P815 cells. Consistent with this reduced reactivity of cycloheximide-treated P815 cells with CTLs was the inability of anti-H-2^d CTLs to form T cell-target cell conjugates with treated P815 cells. As evaluated by the binding of an anti-H-2^d serum, treated P815 cells expressed the same amount of H-2 membrane antigen as untreated cells. However, treated cells were still lysed by CTLs in the presence of the agglutinator, concanavalin A (Con A).     

Anti-tumor effects of an antiserum raised in syngeneic mice to a tumor-specific T suppressor factor

Summary DBA/2 mice inoculated with either cells from the syngeneic P815 tumor or tumor cell membrane extracts develop T suppressor cells which suppress the in vitro generation of cytotoxic T lymphocytes with specificity for the tumor. A soluble suppressor factor with similar properties can be isolated from suppressor cell-enriched populations. It can be highly purified by appropriate immunoadsorption. Antisera to this suppressor factor raised in either DBA/2 or C57BL/6 mice can specifically absorb out suppressor factor and eliminate suppressor cells in the presence of complement. The in vivo effects of these antisera were tested for their ability to modulate the growth of P815 tumors in DBA/2 mice. It was found that the antiserum raised in syngeneic (DBA/2) but not allogeneic (C57BL/6) mice was able to significantly slow the rate of tumor growth and to prolong survival in treated mice. The antiserum was effective in this way only if it was administered early in the course of tumor growth. It was shown that this effect was not attributable to the presence in the serum of antibodies directed to antigens present on P815 cells, and it therefore appears to be due to interference with the function of T suppressor cells arising early in the immune response to the tumor cells.     

The Lyt phenotype of cells involved in the cytotoxic response to syngeneic tumor and of tumor-specific suppressor cells

Suppressor cells, which specifically suppress the in vitro response of syngeneic spleen cells to the DBA/2 mastocytoma, P815, were identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells were determined. DBA/2 allogeneic killer cells were identified as Lyt-¹²⁺, whereas the syngeneic effector cells were found to be predominantly Lyt-²⁺. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-¹⁺ cell is essential in this suppression.     

Alloantigen-induced cytotoxicity against syngeneic tumor cells: analysis at the clonal level

It has been shown that peritoneal exudate cells (PEC) from BALB/c mice immunized with minor histocompatibility antigens presented by DBA/2 or B10.D2 spleen cells are capable of lysing syngeneic YC8 tumor cells in a 4-hr 51Cr-release assay. In this study, we employed limiting dilution analysis to determine the frequency of CTL precursors (CTL-P) reactive against both the specific DBA/2 (or P815) target and the syngeneic tumor YC8. The mean frequency of anti-DBA/2 CTL-P in PEC from BALB/c mice immunized with DBA/2 was 1/302. Between one-third and one-fifth of limiting dilution microcultures that exhibited lytic activity against DBA/2 lymphoblasts (or P815) were also able to lyse YC8. No lysis of YC8 was observed in the absence of a parallel lysis on DBA/2 lymphoblasts or P815 target cells. T cell clones, derived by micromanipulation from microcultures selected for cytotoxic activity against YC8 and/or P815, maintained either the specific anti-allogeneic or the doubly reactive ( antiallogeneic plus anti-syngeneic tumor) phenotype. Fourteen clones (six specific and eight doubly reactive) were tested for cytotoxic activity on a panel of target cells with different haplotypes. All showed H-2-restricted specificity for minor histocompatibility antigens shared by DBA/2 and B10.D2. The restriction element for some of the clones mapped in the K region of the H-2 complex, whereas for other clones the restriction element mapped in the D region; both K- and D-restricted clones were able to lyse YC8. When the clones that exhibited lysis on YC8 were tested on two other BALB/c tumor targets, LSTRA, a Moloney virus induced lymphoma, and RL male-1, a radiation induced lymphoma, two of seven were found to lyse all three syngeneic tumor targets equally well, but not syngeneic BALB/c blasts. These clones were functionally categorized as conventional CTL because they were unable to proliferate when cultured with antigen in the absence of exogenous lymphokines, and were unable to produce lymphokine with IL 2 activity when stimulated by the appropriate splenocytes. When tested in vivo in a Winn assay, a strong anti-tumor activity against YC8 was exerted by the anti-DBA/2 clones DY4 -3 and DY16 -3. These clones lysed both YC8 and the immunizing target cells in vitro. No in vivo effect in neutralizing YC8 tumor growth was observed with clone D2-1, a clone that lysed DBA/2 targets but not YC8 in vitro.     

The existence of antibody-like activities against the weakly immunogenic minor histocompatibility antigens

BALB/c mice develop cytotoxic lymphocytes as well as produce specific antibodies against the minor histocompatibility antigens when injected with DBA/2 P815 cells. P815 cells grown in BALB/c mice have IgG antibodies on their surface as demonstrated by the binding of ¹²⁵I-labeled goat anti-mouse IgG and by complement-dependent cytotoxicity. Serum from BALB/c mice hyperimmunized with P815 cells blocked lymphocyte-mediated cytotoxicity by BALB/c immune peritoneal exudate cells. This blocking activity was removed by absorbing hyperimmune serum with DBA/2 spleen cells or P815 cells. This result suggests that specific antibodies were generated against the minor histocompatibility differences between BALB/c and DBA/2 mice. The experimental procedures described may be very useful in demonstrating minute quantities of antibody against minor histocompatibility antigens on tumor cells.     

The preparation of xenoantiserum specific for a murine mastocytoma cell line

Summary A method is described whereby a xenoantiserum directed toward membrane components of DBA/2 murine mastocytoma P815 cells was raised. This antiserum was found to be specific for tumor cell extracts and had no reactivity with comparable extracts of normal cells when tested by complement fixation. The antiserum was capable of killing P815 cells in the presence of guinea-pig complement but had no reactivity with L1210 leukemia cells or a variety of normal DBA/2 cell preparations. When mixed with varying numbers of tumor cells and injected IP to either DBA/2 or B6D2 Fl mice, the antiserum demonstrated a protective effect by either prolonging survival time or, when low numbers of cells were injected, apparently facilitating complete removal from the body of tumor cells. When administered following resection of SC grown tumors in B6D2 Fl mice, the antiserum prevented tumor recurrence in 50% of treated mice in comparison to animals treated with normal rabbit serum, in which recurrence occurred in all test animals.     

Helper cells active in the generation of cytotoxicity to a syngeneic tumor

We have examined a population of thymic lymphocytes that augment the generation in vitro of specific cytotoxic T cells against P815.X2, a syngeneic tumor in DBA/2 mice. These helper cells are evident in the thymus of DBA/2 mice 5 to 7 days after subcutaneous injection of live P815 cells. They are Thy-1 positive, resistant to gamma radiation, antigen specific, and appear to exert their influence most effectively when added at the beginning of the culture.     

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.     

Failure of specific adoptive immunotherapy owing to survival and outgrowth of variant cells

Summary Adoptive immunotherapy, the transfer of spleen cells from immunized mice to mice with a small tumor, was usually curative for mice with the P815 mastocytoma provided that steps were taken to prevent the generation of tumor-induced suppressor cells in the recipient animal. However, failure of adoptive immunotherapy of the P815 tumor, resulting in regrowth of either the primary intradermal or a metastatic tumor, was observed in 10 out of 112 animals receiving graded doses of 7.5×10⁷ to 3.0×10⁸ immune spleen cells. Examination of the ten tumors in mice that failed to respond to therapy revealed that seven of them were significantly less susceptible than the original P815 tumor to rejection in vivo by transferred anti-P815-specific effector cells. In addition, nine of the ten therapy-failure tumors were also less susceptible than the original P815 tumor to lysis in vitro by P815-specific, but not DBA/2-specific, cytotoxic T lymphocytes. Sensitivity to lysis by tumor-specific cytotoxic T cells was not, however, strongly correlated with sensitivity to rejection in vivo by P815-specific effector spleen cells. Neither in vivo sensitivity to rejection, nor sensitivity to cytotoxic T cells, was correlated with alterations in class I major histocompatibility complex antigen expression. These results suggest that the survival and outgrowth of variant tumor cells was frequently the cause of failure of specific adoptive immunotherapy of the P815 tumor, and that selection for cells with a reduced sensitivity to killing by cytotoxic T cells was only one mechanism that might lead to an immunotherapeutic failure.This work was supported by a grant from RJR Nabisco Inc., a grant from the J. M. Foundation, and by USPHS grant CA-40597 awarded by the National Cancer Institute     

Amniotic Fluid Enhances Allogeneic Cytotoxic T Cell Responses,Whereas It Suppresses Mitogen-Stimulated Lymphocyte Proliferation

Mouse amniotic fluid has been shown to suppress T lymphocyte proliferation and suggested to be important in regulating immunity during pregnancy. In allogeneic pregnancy, cytotoxic T cells in pregnant lymphocytes against paternal transplantation antigen are impaired. We examined the effect of amniotic fluid to the alloreactive CTL responses. Although the amniotic fluid suppressed Con A or LPS stimulated lymphocyte proliferation as previously reported, the amniotic fluid taken from syngeneic C57BL/6 pregnant mice or allogeneic C57BL/6 × BALB/c pregnant mice enhanced the anti-H-2^d or anti-H-2^k CTL responses dose-dependently. We speculate that amniotic fluid contains not only immunosuppressive factors but also immunoenhancing factors which upregulate the allogeneic CTL responses.     

Activation of specific suppressor cells with heat-treated allogeneic tumor cells

The ability of heat-treated allogeneic cells to induce suppressor cells was examined. The tumor cell lines EL-4 (H-2^b) and P815-X2 (H-2^d), were heated to 56 °C for 10 min and injected intravenously into mice of the DBA/2J (H-2^d) and C57BL/6J (H-2^b) strains, respectively. After 4 days, the splenocytes of the treated mice were mixed with normal spleen cells and cultured for 5 days with allogeneic tumor cells. The cytotoxic T-cell response was reduced in cultures of these cell mixtures. An allogeneic difference was required to induce suppression because the syngeneic combination did not induce suppressor cell activity. Furthermore, the induction of cytotoxic T cells to the C118 cell line (H-2^k) was not suppressed by this procedure, which suggests that the suppression was haplotype specific. These suppressor cells were sensitive to anti-Thy 1.2 and complement, cortisone, and cyclophosphamide, but insensitive to irradiation. These are characteristics similar to suppressor cells activated by intact cells. Heat treatment abrogated the tumor cell's ability to induce a proliferative and a primary, but not a secondary, cytotoxic T-cell response. The heat-treated cells also lost their ability to function as cold target inhibitor cells, but retained the same quantity of serologically detected antigens as the intact cells. These results suggest that the serologically detected antigens are responsible for the activation of the suppressor cells of the cytotoxic T-cell response.     

Cell-mediated cytotoxicity against syngeneic tumors

Spleen cells from DBA/2 mice bearing the DBA/2 P815X mastocytoma for approximately 2 weeks can be stimulated in vitro by mastocytoma cells to generate cytotoxicity measured as ⁵¹Cr release from mastocytoma cells in a 4-hr assay. These cytotoxic cells will not kill allogeneic cell lines but will kill a series of first transplant generation syngeneic tumors. T cells are involved in that treatment of the responding or the cytotoxic cell populations with either anti-T or anti-theta antibody + complement will abrogate all cytotoxicity. Anti-Ly 2.1 antibody + complement treatment of either responder cells (prior to the in vitro culture with irradiated tumor cells) or effector cells after culture markedly decreases cytotoxicity whereas treatment with anti-Ly 1.1 was more effective prior to culture compared to its effect on cytotoxic cells per se. These T cells are in the small lymphocyte class and occur either singly or in aggregates. Suppression of antisyngeneic tumor cytotoxicity by antibody inhibits preferentially the expression of cytotoxicity in the aggregate fractions.     

Immunogenetic basis for immunologic privilege in the anterior chamber of the eye

DBA/2 mastocytoma (P815) cells are able to grow when inoculated into the anterior chamber of eyes of histoincompatible mice. Tumor growth is unrestrained in recipient mice differing from the DBA/2 strain at multiple minor H loci, but sharing the H-2 ^d haplotype; progressive tumor growth in these animals involves the entire eye, invades the orbit and kills the hosts by extension into the cranial vault. Alternatively, P815 cells grow initially but are unable to sustain continued growth in the anterior chambers of recipient mice differing from DBA/2 at the H-2 complex. Recipients differing from DBA/2 at either the K or D regions of H-2 develop anti-DBA/2 immunity that destroys the intraocular tumor within 20 days of inoculation. Severe inflammatory reactions in these eyes produce innocent bystander destruction of ocular tissue and produce blindness. Recipients differing from DBA/2 at both K and D regions of H-2 also mount vigorous anti-DBA/2 immunity that destroys the intraocular tumor. In these instances, the nonspecific component of the rejection reaction is minimal: when the tumor cells are destroyed, the eyes are restored to anatomic and functional integrity. These results indicate that immunologic privilege in the anterior chamber of the eye is afforded to allogeneic tissues to varing degrees depending upon allodisparity at H-2 loci encoding class I MHC products. The results further imply that the precision of the alloimmune response may be under the control of these same MHC products.     

Further characterization of thymic suppressor cells and a factor that suppress the generation of cells cytotoxic for a syngeneic tumor in DBA/2 mice.

Antigen-specific suppressor cells and suppressive extracts obtained from the thymuses of DBA/2 mice bearing small syngeneic P815 mastocytomas were compared for their immunogenetic properties and requirements. The assay for specific suppression involved the ability of either cells or extracts to inhibit the primary in vitro cytotoxic response of normal DBA/2 splenocytes to mitomycin-treated P815 cells. It was shown that pretreatment of suooressor cell populations with anti-Iad antiserum plus rabbit complement removed the suppressive activity. Similarly, absorption of the suppressor factor with anti-Iad antiserum removed the suppressive properties of the material. It was found that the suppressor cells, generated in DBA/2 tumor bearers, were capable of specifically suppressing the anti-P815 response of B6D2 F1 radiation chimeras possessing lymphoid cells of the H-2b or H-2t2 haplotype equally as well as they could suppress the response of H-2d-bearing effector cells. This indicates that the suppressor cells are not H-2 restricted with respect to K or D markers on the responder cells in this system.     

Cytotoxic T cell responses to a syngeneic tumour: conditions for primary activation in vitro.

Primary cytotoxic responses of DBA/2 lymph node cells to a syngeneic tumour (the mastocytoma P815) have been generated in vitro. The development of these responses is dependent on the addition of a soluble factor (CSCS) which is produced by concanavalin A-activated spleen cells. The response is mediated by T lymphocytes, can be detected at low effector to target cell ratios and is directed against P815 tumour-associated antigens.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An H-2 antigen variant, referred to as ?4 + 31 clone 1, was selected by its resistance to an anti-H-2D^d antiserum (BALB.G anti-BALB/c.H-2 ^g antiH-2 ^d ). When tested by cell-mediated cytolysis, this variant was found to be sensitive to cytolytic T lymphocytes raised in the same donor-host combination as that used in raising the antiserum. Further CML characterization of this variant, reported here, indicates that the cell line is in fact resistant to anti-H-2D^d killer cells raised in a more restricted immunization, viz. BALB.G anti-BALB/cH-2 ^db ,H-2 ^g anti-H-2 ^db . It is, however, sensitive to cytolytic cells raised in (BALB.B xBALB/c-H-2db) F¹ H-2 ^b /H-2 ^db ) against the BALB/c strain. These results suggest that the variant does not express H-2D^d itself, but probably expresses CML target antigens that are missing in theH-2 ^db mutation. This in vitro-isolated variant might thus be the complementary mutation to the in vivoobtainedH-2 ^db mutation.     

Lymphotoxin and interferon production by rossette-separated human peripheral blood leukocytes.

When normal (CBA × DBA)F₁ spleen cells are cultured for 4 days in polyacrylamide vessels, clones of cytotoxic lymphocytes (CLs) are generated. The specificity of these apparently spontaneous CL clones has been investigated by assaying cells from individual clones against pairs of different target cells. CL clones were found to discriminate between the two parental strain splenic blasts, between splenic blasts and syngeneic tumour cells, and between two F₁ splenic blasts induced with different mitogens (LPS and PHA). The CL clones generated spontaneously in culture also discriminate between semisyngeneic targets [DBA blasts and (CBA × DBA)F₁ blasts]. Significant cross-reactivity however, was detected when CL clones were assayed against normal P815 targets and TNP-modified P815 targets.     

Spontaneous autocytotoxicity against an unexpected H-2 d haplotype in MRL/lpr (H-2 k) autoimmune disease-prone mice

The MRL/lpr (H-2 ^k) inbred strain, a model for the autoimmune disease systemic lupus erythematosus, differs from the healthy inbred strain MRL +/+ (H-2 ^k) by only 0.1 % of its genome. Southern blot analysis using class I and class II probes confirmed the H-2 ^k genotype of both strains. Among the Ia^k-positive peritoneal cells, cells with an unexpected expression of Ia^d specificities were detected in a radioimmunoassay using several monoclonal antibodies and one conventional antiserum. This was only found in aged (6- to 9-month-old) mice both in the MRL/lpr strain (32 % Ia^d-positive mice) and in the MRL +/+ strain (42% Ia^d-positive mice). Furthermore, 24% of aged MRL/lpr mice exhibited strong spontaneous cytotoxic T lymphocyte (CTL) activities against P815 (H-2 ^d) target cells, and 57% had a weaker but still detectable level of cytotoxicity. In contrast, such a CTL activity has never been found in the MRL +/+ strain. These results suggest that the anti-H-2^d d CTL plays a role in the onset of the autoimmune process in MRL/lpr mice.     

Anti-H-2 antibodies induced by syngeneic immunization

Alloreactive cytotoxic antibodies were induced in BALB/c mice by syngeneic immunization with normal lymphoid cells. Sixteen out of 41 mice produced antibodies with distinct anti-H-2 specificity. Anti-K^k antibodies were present in all positive sera, but the individual sera produced different reactivity patterns when tested on a panel ofH-2 haplotypes. Absorption and immunoprecipitation experiments confirmed the H-2 specificity of the syngeneic sera. We hypothesize that virus-modified H-2^d structures have triggered alloreactive B-cell clones to produce anti-H-2 antibodies.     

Adoptive immunotherapy of a BALB/c lymphoma by syngeneic anti-DBA/2 immune lymphoid cells: Characterization of the effector population and evidence for the role of the host's non-T cells

Summary It has been previously shown that the BALB/c lymphoma YC8 is susceptible to lysis by syngeneic anti-DBA/2 lymphocytes and that YC8-bearing BALB/c mice can be cured by adoptive transfer of such immune effectors. In this study in vivo and in vitro functions of the curative immune lymphocytes have been evaluated together with the role of the host immune system in the mechanism of tumor eradication. It was found that the curative anti-DBA/2 lymphocytes were not directly cytotoxic to YC8 cells although they developed into YC8-lytic cells after in vitro restimulation with YC8. In vivo, the immune lymphocytes were able to mediate a tumor-specific delayed type hypersensitivity reaction against YC8 but had a low tumor-neutralizing activity in the Winn assay. Proliferation of infused BALB/c anti-DBA/2 lymphocytes was necessary for the in vivo therapeutic effect, since irradiation of effector cells or treatment of the donor immune lymphocytes with vinblastine abolished their curative capacity. Immunodepression of the T cell compartment of the prospective tumor-bearing animals by thymectomy plus irradiation or its abrogation in B mice (thymectomized, lethally irradiated, and reconstituted with fetal liver cells) did not interfere with the therapeutic effect of the transferred anti-DBA/2 lymphocytes. Blocking the macrophage functions of the host by carrageenan, however, abolished the therapeutic effect of immune lymphocytes. These data indicate that a radiation-resistant, non-T cell is involved in the tumor eradication induced by anti-DBA/2 lymphocytes. It was also shown that cured mice, tested 90 days after therapy, become resistant to 5×10³ LD₈₀ YC8 cells and that this resistance was due to the presence of memory cells derived from the transferred and not from the host lymphocyte population.