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.     

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.     

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.     

Characterization of a soluble factor that specifically suppresses the in vitro generation of cells cytotoxic for syngeneic tumor cells in mice.

A tumor-specific soluble factor found in extracts of thymocytes from mice bearing small P815 tumors has been demonstrated. This factor is capable of significantly suppressing the in vitro generation of syngeneic cells cytotoxic for P815 targets if it is added to culture vessels within the first 30 hr of culture. The suppressive factor eluted from Sephadex G-100 after hemoglobin and was estimated to have a m.w. in the range of 40 to 60,000. Preparative isoelectric focusing of thymic extracts established that the suppressive material has an isoelectric point in the range of pH 4.6 to 4.9. The suppressive activity of extracts could be removed by passage of the material through immunoadsorbent columns prepared from membrane proteins of P815 cells but not by analogous columns prepared by using L1210 membrane proteins. The suppressive material was not removed by its passage through immunoadsorbent columns containing anti-mouse immunoglobulin.     

Characterization of suppressor cells in mice bearing syngeneic mastocytoma.

Suppressor cells from syngeneic P815 mastocytoma-bearing DBA/2 mice that inhibit in vitro generation of specific anti-tumor cytotoxicity were characterized. Suppressive activity was almost completely eliminated by treating suppressive spleen cells with anti-theta serum and complement. Treatment with anti-mouse lg serum and complement or with carbonyl iron did not affect their suppressive activity. When suppressive thymocytes from P815 tumor-bearing DBA/2 mice were tested for their capacity to inhibit the generation of cytotoxicity against L1210 cells, a leukemia line in DBa/2 mice, they did not affect the activity, indicating that the supressor cells in the thymocytes of P815 tumor-bearing mice are specific to the tumor. When Ficoll-Hypaque density cell separation was carried out with cytotoxic spleen cells and suppressive spleen cells from 815 tumor-bearing mice, the dense fraction was enriched for kiler cells whereas the suppressive activitty was mainly recovered in the light fraction. Therefore, killer cells and suppressor cells in P815 tumor-bearing mice are thought to be distinct populations.     

Induction of suppressor cells by a tumor-derived suppressor factor

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.     

The blocking activity of antisera raised to either tumor antigens or alloantigens in cytotoxicity assays using syngeneic or allogeneic killer cells on the P815 murine mastocytoma target

Methods have been published whereby a tumor-specific antigen associated with membranes of the P815 mastocytoma of DBA/2J mice was purified. Antiserum, raised in rabbits, to this material demonstrated specificity for P815 as opposed to other cells or materials of DBA/2J origin when tested by either complement-mediated target cell lysis or the enzyme-linked immunosorbent assay ELISA. This antiserum was tested for its ability to block killing by in vitro raised syngeneic lymphocytes cytotoxic for P815. It was found that this antiserum as well as antiserum raised in rabbits to normal DBA/2J membrane components and anti-H-2^d antiserum (raised in congenic mice) were all able to block killing when ⁵¹Cr-labeled P815 targets were pretreated with these antisera. On the other hand, only the anti-DBA/2 serum and the anti-H-2^d serum were capable of slightly blocking syngeneic killing of L1210 cells. Similarly, C57B1/6 cytotoxic lymphocytes raised against DBA/2 cells were blocked by pretreatment of ⁵¹Cr-labeled P815 targets with the rabbit anti-DBA/2 serum and the anti-H-2^d serum but not by the anti-P815 serum. The implications of these observations are discussed.     

Cyclophosphamide and abrogation of tumor-induced suppressor T cell activity

Summary Previously we have demonstrated that the in vitro generation of P815-specific anti-tumor cytotoxic T lymphocytes (CTL) was suppressed by splenic suppressor T cells from late tumor-bearing hosts (TBH). Suppression is not caused by in vitro growth of P815 from splenic metastases, since suppression was also seen with spleen cells from late TBH mice bearing a hypoxanthine/aminopterin/thymidine-sensitive subline (PHS-5) of P815 in the presence of HAT. Cyclophosphamide has been shown to inhibit theinduction of suppressor cells selectively in a number of immune responses, but evidence that it can inhibit active tumor-induced suppressor T cells is limited. We have found that suppressor T cells already induced by P815 in syngeneic late TBH are sensitive to low doses of cyclophosphamide (50 mg/kg) given 1 day before spleen harvest, but the in vitro CTL response of late TBH spleen cells could not be restored by pretreating the mice with cyclophosphamide, even when exogenous interleukin-2 was added to the cultures. Although 50 mg/kg cyclophosphamide did not inhibit the CTL response of spleen cells from mice immunized with P815 +Corynebacterium parvum, the same dose of cyclophosphamide eliminated the CTL response of spleen cells from early TBH. Interleukin-2 (IL-2) did not overcome this effect of cyclophosphamide, suggesting a direct effect on CTL. Ultra-low-dose cyclophosphamide (10 mg/kg) did not adversely effect early TBH CTL but was still able to eliminate suppressor T cell activity from late TBH. Nevertheless, late TBH CTL remained unresponsive after pretreatment of mice with ultra-low-dose cyclophosphamide, even when exogenous IL-2 was added in vitro. CTL precursor frequency analyses demonstrated that cyclophosphamide pretreatment had little or no effect on the numbers of CTL precursors from early TBH. Late TBH CTL precursor cells were not detectable in these studies, with or without suppressor T cell inhibition by cyclophosphamide pretreatment. Thus, it appears that most CTL precursor cells may be lost or irretrievably inactivated in the spleens of late TBH mice.This work was supported by grants CA42443, CA48075 and T32-CA09210 from the National Cancer Institute, Department of Health and Human Services, and an American Cancer Society Clinical Oncology Career Development Award (H. D. Bear)     

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.     

Immunization of DBA/2 mice with a T cell hybridoma-derived TsF increases immune resistance to the syngeneic tumors P815 and L1210

The ability of a tumor-specific T suppressor factor (TsF) isolated from a T cell hybridoma, A10, to act as an immunogen in DBA/2 mice was investigated. The TsF was affinity purified from ascites over an immunoadsorbent column containing a monoclonal antibody (B16G) that has specificity for the TsF molecule, or over columns containing membrane extracts of the P815 mastocytoma (the tumor for which A10 is specific). The specificity control was BW5147 (the fusion partner for A10) membrane extracts treated in the same way as A10. DBA/2 mice were immunized with the affinity-purified material or PBS and were subsequently challenged with either the P815 tumor or the L1210 DBA/2 thymoma. When mice were immunized with material affinity purified over B16G, eluted material from both A10 ascites and BW5147 membrane extracts enhanced resistance to both P815 and L1210 challenge, indicating that B16G was binding immunogenic material derived from both preparations, which exerted a tumor-protective effect. However, when a P815 affinity column was used, protective material was eluted only from A10 ascites, and this bestowed resistance to both P815 and L1210. When irradiated whole cells were used as immunogens, only A10 cells stimulated anti-tumor immunity, and this appeared to be directed specifically to the P815 tumor. The implications of these findings in terms of the potential for immune modulation with anti-suppressor therapy, and the specificity of the B16G monoclonal, are discussed. The demonstration of B16G binding material (TsF) in the membranes (but not the ascites) of the BW5147 line is also of significance to investigators using BW5147 fused suppressor hybridomas.     

Augmentation of killer activity of murine spleen cells against allogeneic and syngeneic tumor cells by inoculation of alloantigen in vivo

After C57BL/6 (B6) mice were inoculated with BALB/c spleen cells via tail vein, kinetics of cytotoxic activities in the B6 mice against sensitizing alloantigens (H-2d) and against syngeneic antigens were investigated using, as target cells, P815 mastocytoma cells (H-2d) and B16 melanoma cells (H-2b). Cytotoxic activity against P815 in the B6 spleen cells reached a peak 3 days after alloantigen inoculation, decreased drastically on day 5 and rose again thereafter. The profile of anti-B16 cytotoxic activity was similar to that of anti-P815 activity. The cytotoxic activity against P815 was inhibited partially by cold B16, but that against B16 was not inhibited by cold P815. Surface phenotype of cytotoxic cells against P815 was Lyt2+, Thy1+, Asialo GM1+ and that of cytotoxic cells against B16 was Lyt2-, Thy1+/-, and Asialo GM1+. The results indicate that inoculation of B6 mice with allogeneic BALB/c spleen cells induce two types of cytotoxic cells; one is similar to lymphokine-activated killer (LAK) cells and the other is activated natural killer cells.     

Antigen and receptor-driven regulatory mechanisms. VI. Demonstration of cross-reactive idiotypic determinants on azobenzenearsonate-specific antigen-binding suppressor T cells producing soluble suppressor factor(s)

The first detectable suppressor T cell (Ts) arising after i.v. administration of azobenzenearsonate- (ABA) conjugated syngeneic spleen cells to A/J mice has been studied for its receptor specificity and ability to produce soluble suppressor factor(s). This cell, termed Ts1, has a specific receptor for the eliciting antigen ABA, as demonstrated by selective binding to ABA protein- but not TNP protein-coated plastic dishes. The activity of ABA-Ts1 can be abrogated by treatment with anti-idiotypic antibodies made against anti-ABA antibodies of A/J mice (anti-CRI), indicating that these ABA-binding cells possess a surface receptor structure sharing idiotypic determinants with antibodies of the same specificity. Finally, soluble extracts from, antigen-adherent ABA-Ts1, but not nonadherent cells from the same spleen cell population, possess suppressive activity when assayed directly for afferent suppression or tested for their ability to trigger a second population of Ts (Ts2) in naive recipients. These findings demonstrate a close concordance between a T cell surface receptor, soluble T suppressor factors, and B cell derived antibody, all capable of direct recognition of the eliciting ABA antigen.     

Characterization of an anti-idiotypic T cell hybridoma involved in the regulation of the immune response to the P815 mastocytoma

We report the isolation and characterization of a T cell hybridoma (A29) which secretes a factor that exhibits anti-idiotypic and immune-modulating characteristics. The A29 cell line is thought to represent the hybrid analog of the Ts2 suppressor cell population in the cascade regulating the immune response to the P815 tumor in DBA/2 mice. The putative TsF2 molecule is reactive with the monoclonal antibody B16G, shown previously by us to bind a public specificity of T suppressor factors (TsF). A29 TsF also exhibits specific binding to a TsF1 secreted by another T cell hybridoma, A10, which shows specificity for antigen from the P815 tumor (this has been described previously). A29 itself does not exhibit binding to P815 antigens. Affinity-purified material from A29 appears to share characteristics with A10 molecules in that the predominant material has an apparent m.w. of 70,000. Studies with calcium flux of A29 cells showed that they respond significantly and specifically on exposure to A10 TsF stimulus. We showed further that affinity-purified A29 TsF molecules can specifically suppress the in vitro generation of syngeneic CTL to the P815 tumor, and that panning of DBA/2 splenocytes over A29-TsF-coated plates renders cell populations capable of generating a higher in vitro CTL response to P815 than appropriately treated controls.     

Characterization of the suppressor cell(s) responsible for anterior chamber-associated immune deviation (ACAID) induced in BALB/c mice by P815 cells

Anterior chamber-associated immune deviation (ACAID) is a complex set of immune responses induced by the inoculation of antigens into the anterior chamber of the eye. Histocompatibility antigens, tumor-specific antigens, reactive haptens, and viral antigens have been shown to induce this phenomenon, which comprises the following specific host responses: high titer humoral antibodies, primed cytotoxic T cells, but specifically, impaired skin graft rejection and delayed-type hypersensitivity (DTH). Using the model system of ACAID induced by inoculation of P815 mastocytoma cells into the anterior chambers of H-2-compatible, but minor H-incompatible, BALB/c mice, we demonstrate that the impaired capacity of these animals to develop and express DTH is due to the activation of suppressor T cells. Generation of these cells requires an intact spleen, is not inhibited by cyclophosphamide pretreatment, and is abrogated by systemic treatment of the host with anti-I-J monoclonal antibodies. This splenic suppressor cell(s) can transfer suppression of DTH adoptively to naive syngeneic mice. One suppressor cell is Thy-1.2, Lyt-2.2, and I-Jd positive. A minority of these cells (or a second population of suppressor cells) also expresses the L3T4 surface marker. Suppression is exerted on the efferent limb of DTH expression, although afferent suppression is not excluded. P815-induced ACAID suppressor cells resemble similar cells induced by haptenated spleen cells inoculated into the anterior chamber of the eye. We propose that induction of these suppressor cells, whose target of action is selective for T DTH cells, but not for other types of T cells, is responsible for the phenomenon of immune privilege in the anterior chamber of the eye.     

Mechanism of loss of natural killer activity in P815 ascites tumor bearing DBA/2 mice

P815 tumor cells (10(7] were administered intraperitoneally to DBA/2 mice. As the ascites tumor grew in the syngeneic host, a decline leading to a total loss of host spleen natural killer (NK) activity could be demonstrated. Removal of T and B cells or macrophages from the tumor-bearing (TB) mouse spleen cells did not raise the level of NK activity. Spleen cells from TB mice did not inhibit the NK activity of normal spleen cells. Comparable target (YAC cells) binding capacity could be demonstrated in spleen cells derived from normal or TB mice, but interferon failed to significantly stimulate the NK activity of TB mouse spleen cells. In adoptive transfer experiments, transfer of spleen or bone marrow cells from TB mice resulted in the development of significant levels of spleen NK activity in lethally X-irradiated recipient DBA/2 mice. These results indicate that the impairment of NK cell differentiation pathway rather than active suppression at the level of effector cells may be the mechanism of loss of NK activity in P815 TB DBA/2 mice.     

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     

Altered immune response patterns in murine syngeneic pregnancy: presence of natural null suppressor cells in maternal spleen identifiable by monoclonal antibodies

Expression of certain autologous lymphocyte-activating antigenic determinants on the developing embryo is known to provide a stimulus for maternal anti-fetal autoproliferative responses. If left unregulated these responses could exert negative influences on the reproductive process by converting to autoaggressive forms of immune reactivity. In normal circumstances, immunological reactions of this nature are therefore likely to be under the control of pregnancy-associated immunoregulatory elements found within the maternal/fetal environment. In the present investigation we describe a naturally occurring splenic inhibitory cell type devoid of conventional T, B, and macrophage surface markers associated with syngeneic murine pregnancy that is capable of exerting potent immunosuppressive effects on an in vitro expression of fetal/newborn T cell autoreactivity, namely the autologous mixed lymphocyte reaction (AMLR). Maternal spleen cells inhibitory for AMLR were found to be highly resistant to cytotoxic pretreatment with a panel of conventional antisera directed against T cell-specific antigenic determinants. The non-T nature of the natural splenic suppressor cell was further indicated by experiments showing that purified spleen T cells had no inhibitory activity. Pregnancy spleen cell populations that were effectively depleted of macrophages retained full ability to inhibit AMLR. Maternal suppressor activity could be localized to the spleen cell population bearing receptors for the B cell-specific lectin, soybean agglutinin (SBA). A panel of monoclonal antibodies prepared against enriched populations of suppressor cells was screened and selected for specific reactivity using an ELISA against glutaraldehyde-fixed SBA+ spleen cell subpopulations from pregnant versus virgin animals. Several of the monoclonals developed against suppressor-enriched spleen cell populations from isopregnant as well as allopregnant animals were effective in reducing or eliminating suppressor cell activity following cytotoxic pretreatment in the presence of complement. The novel set of anti-suppressor monoclonal antibodies described here should prove useful in furthering the isolation and characterization of pregnancy-associated suppressor cells and in determining their relationship to natural suppressor cell populations described in other systems.     

γ-Irradiation facilitates the expression of adoptive immunity against established tumors by eliminating suppressor T cells

It was found that sublethal (550 rad) whole-body gamma-irradiation of mice bearing established immunogenic tumors enabled tumor-sensitized spleen cells infused intravenously 1 h later to cause complete tumor regression in all mice. In contrast, gamma-irradiation alone caused only a temporary halt in tumor growth, and immune cells gave practically no therapeutic effect at all. This result was obtained with the SA1 sarcoma, Meth A fibrosarcoma, P815 mastocytoma, and P388 lymphoma. Additional experiments with the Meth A fibrosarcoma revealed that the spleen cells from tumor-immune donors that caused tumor regression in gamma-irradiated recipients were T cells, as evidenced by their functional elimination by treatment with anti-Thy-1.2 antibody and complement. It was shown next that adoptive T-cell-mediated regression of tumors in gamma-irradiated recipients was inhibited by an intravenous infusion of spleen cells from donors with established tumors, but not by spleen cells from normal donors. The spleen cells that suppressed the expression of adoptive immunity were functionally eliminated by treatment with anti-Thy-1.2 antibody and complement. Moreover, they were destroyed by exposing the tumor-bearing donors to 500 rad of gamma-radiation 24 h before harvesting their spleen cells. The results are consistent with the interpretation that gamma-radiation facilitates the expression of adoptive T-cell-mediated immunity against established tumors by eliminating a population of tumor-induced suppressor T cells from the tumor-bearing recipient.     

Cellular basis of tolerance to serum albumin in adult mice. I. characterization of T suppressor and T helper cells.

The surface markers and size of suppressor cells were determined in adult (BALB/c x C57BL/Ka)F1 mice which were tolerized with a single injection of deaggregated bovine serum albumin (BSA). Suppressor cells from the spleens of tolerazided donors were assayed in a cell transfer system in which graded numbers of cells were injected into irradiated syngeneic mice along with limiting numbers of T cells primed to BSA and an excess of B cells primed to DNP-BSA. Adoptive hosts were challenged with DNP-BSA in saline, and the anti-DNP response was measured. Suppressor cells were antigen specific as shown by the inhibitory activity of BSA-tolerant spleen cells on the response to DNP-BSA, but not to DNP-BGG. Suppressor cells were eliminated by in vitro treatment with anti-Thy 1.2, anti-Ly-2.2, anti-I-J subregion antisera and C, but not with anti-Ly-1 or anti-I-A subregion antisera. Neither unprimed nor primed helper T cells were detected in the spleen of tolerized donors after in vitro treatment with anti-Ly-2.2 antisera. Both helper and suppressor T cells from the spleens of primed or tolerized donors, respectively, showed a rapid sedimentation velocity (S greater than 3.7 mm/hr).     

Modulation of natural cytotoxicity by alloantibodies. I. Alloantisera enhancement of cytotoxicity of mouse spleen cells toward a human myeloid cell line.

Natural killer activity of spleen cells obtained from different strains of mice against the human myeloid leukemia cell line, K562, and two mouse cell lines P815 and L1210 was measured by using the 4-hr chromium release assay. The level of cytotoxic activity of spleen cells against the K562 target was usually less than 4% lysis. However, treatment of the spleen cells with a specific anti-H-2 antiserum resulted in a dose-dependent augmentation of the degree of lysis of K562 cells. The augmentation of cytotoxic activity could be obtained by pretreatment of the spleen cells with antisera or by directly adding the antisera to the cytotox-incubation medium. Anti-thy-1 and anti-immunoglobulin antisera had no enhancing effect under similar conditions. The specific alloantisera-treated spleen cells did not show any increase in cytotoxicity against P815 and L1210 target cells. Spleen cells responsible for the alloantiserum-mediated augmentation of cytotoxicity against K562 cells appear to be different from T or B cells as indicated by their resistance to anti-thy-1 and complement treatment and lack of adherence to nylon wool columns.