CD8+ cell activation to a major mastocytoma rejection antigen,P815AB: requirement for tum− or helper peptides in priming for skin test reactivity to a P815AB-related peptide

Delayed-type hypersensitivity (DTH) responses, mediated by CD8⁺ cells and detected by skin test assay, occur in sensitized mice in response to challenge with class I-restricted antigenic peptides of mutagenized (tum^?) P815 mastocytoma cells. In contrast, a nonapeptide related to a tumor rejection antigen, P815AB, failed in this study to elicit DTH after sensitization of mice with irradiated tumor cells or adoptive transfer of P815AB-pulsed dendritic cells. Unresponsiveness, however, could be overcome by immunization with tumor cells co-expressing P815AB and tum^? antigens. When used for cell pulsing in vitro, a mixture of P815AB and tum^? peptides was also highly effective in inducing anti-P815AB reactivity, as was the combined use of P815AB and class II-restricted peptides of tetanus toxin or Plasmodium berghei circumsporozoite protein. While the effector phase of the CD8⁺ cell-mediated DTH to P815AB was unaffected by the ablation of CD4⁺ cells, the same treatment, or neutralization of IFN-γ, negated the induction of reactivity if it occurred at the time of sensitization. Thus, defective activation of CD4⁺ cells may contribute to the poor immunogenicity of P815AB. Besides providing an insight into the mechanisms of anti-tumor protection induced by tum^? cells, these data offer useful information for the design of vaccination strategies against identified tumor antigens.     

Immunogenic variants obtained by mutagenesis of mouse mastocytoma P815. III. Clonal analysis of the syngeneic cytolytic T lymphocyte response

The cytolytic T lymphocyte (CTL) response of syngeneic mice to antigenic variants obtained by mutagenesis of mastocytoma P815 was analyzed at the clonal level. Estimates of the frequency of CTL precursor cells in spleens from mice immunized with P 815 variants ranged from lo⁴ to 2 X 10^?3. This frequency could be increased approximately 100 times by stimulating immune spleen cells in mass culture for 6-8 days with the immunizing variant. Specificity analysis of a large number of individual CTL clones demonstrated the existence of two distinct populations of CTL. Some CTL clones lysed exclusively the immunizing variant, while others lysed equally well all P815 targets, but not syngeneic tumor L1210. These results provide direct evidence for the existence of new, individual specificities on P815 variants in addition to a common antigen already present on the original P815 cells. This confirms that a variety of new antigens can be induced by mutagenesis on the same background cell. Stable, highly active CTL clones specific for either individual variant antigens or common P815 antigens could be maintained in long-term culture.     

The use of P815: a warning

It is generally known that many malignancies are accompanied by forms of impairment of the immune system of the host. Also P815 mastocytoma cells can exert suppressive effects on the immune response reaction in vivo and in vitro. However, we have found a product released by P815 cells, which is able to activate macrophages non-specifically; macrophages normally not cytotoxic against tumor cells become cytotoxic against various tumor cell lines upon incubation with the P815 product. Nevertheless the same product which induces macrophage activation in vitro, seems to cause enhancement of tumor growth in vivo. We feel, if the activity of a tumor product depends so strongly on the experimental conditions used, one should be very careful in interpreting the results of experiments in which P815 cells are used as target cells.     

小鼠肥大细胞瘤P815模型的建立与初步应用

目的 建立小鼠肥大细胞瘤Ｐ８１５种植瘤模型，并对其体内诱发特异性ＣＴＬ应答机理进行初步的研究。方法 经有限稀释法筛选Ｐ８１５单克隆瘤系，用ＲＴ－ＰＣＲ及产物ＤＮＡ测序鉴定肿瘤抗原Ｐ１Ａ基因的表达；在此基础上，用活瘤苗免疫同系小鼠，经标准＾５１Ｃｒ释放试验检测在体特异性ＣＴＬ的活性。结果 筛选出了一个Ｐ８１５单克隆成瘤系Ｐ８１－Ｆ３，并鉴定该瘤存在Ｐ１Ａ基因的表达，用活瘤疫苗免疫６只步鼠，在３只体内     

Effector cells of low-dose IL-2 immunotherapy in tumor bearing mice: tumor cell killing by CD8+ cytotoxic T lymphocytes and macrophages.

The presence and cytotoxicity of tumor infiltrating cells is described in mice during effective immunotherapy with interleukin 2 (IL-2). DBA/2 mice were inoculated i.p. with 2 x 10(4) tumor cells on day 0 and treated with daily i.p. injections with 20,000 units IL-2 on days 10-14. Mice bearing a large syngeneic i.p. tumor burden (SL2 lymphoma, P815 mastocytoma, L5178Y lymphoma, or L1210 lymphoma) could be cured by i.p. immunotherapy with these low doses of IL-2. In the peritoneal cavity of these mice an infiltrate of mononuclear cells was present. Similar numbers of lymphocytes (10(6)-10(7)) and macrophages (+/- 10(7)) were present in control tumor bearing mice and IL-2 treated tumor bearing mice. The ratio of CD4+/CD8+ T lymphocytes in the peritoneal cavity of mice rejecting the SL2 tumor was smaller than 0.5, whereas this ratio is about 2 in naive mice. In the spleens of IL-2 treated tumor bearing mice only a minor decrease of CD4+/CD8+ ratio was observed from 2.1-2.4 to 0.9-1.9. T cells isolated from the peritoneal cavity of mice inoculated with SL2 tumor cells and treated with IL-2, were highly cytotoxic to SL2 cells: at E:T ratio 2:1 the cytotoxicity index was 37 +/- 3. This cytotoxicity was specific and mediated by CD8+ T lymphocytes. Macrophages that were present in the peritoneal cavity of mice treated with IL-2 were also highly cytotoxic. The C.I. of these cells was 63-76% at E:T ratio 1:1. Cytotoxic macrophages were also present in untreated tumor bearing mice. The i.p. injections of IL-2 (20,000 units/day) caused a four-fold increase in the local NK-activity in the peritoneal cavity in naive mice. These IL-2 injections did not generate LAK-activity in vivo. Specificity of the in vivo tumor rejection was tested by injection SL 2 i.p. on day 0 and P815 i.p. on day 10, or vice versa, followed by IL-2 treatment. Only the tumor cells that were injected on day 0 were rejected. These in vivo experiments point to specific tumor rejection. In conclusion, both cytotoxic macrophages and CTL's are present in a sufficient number and with sufficient cytotoxicity to explain the killing of tumor cells in the peritoneal cavity. The CTL-activity seems of decisive importance for tumor rejection as this is induced by IL-2.     

"Natural" killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype.

In the spleens of young, adult mice there exist naturally occurring killer lymphocytes with specificity for mouse Moloney leukemia cells. The lytic activity was directed against syngeneic or allogeneic Moloney leukemia cells to a similar extent, but was primarily expressed when tested against in vitro grown leukemia cells. Two leukemias of non-Moloney origin were resistant and so was the mastocytoma line P815. Although killer activity varied between different strains of mice, the specificity of lysis was the same as indicated by competition experiments using unlabeled Moloney or other tumor cells as inhibitors in the cytotoxic assays. Capacity to compete and sensitivy to lysis by the killer cells were found to be highly positively correlated. Analysis of the kinetics of the cytotoxic assay revealed a rapid induction of lysis within one to four hours, arguing against any conventional in vitro induction of immune response. No evidence was found of soluble factors playing any role in the cytolytic assay.     

IL-12对小鼠肥大细胞瘤基因疫苗的免疫学作用

目的 研究小鼠肥大细胞瘤P815基因疫苗和鼠IL-12对该疫苗的免疫学作用。方法 将小鼠肥大细胞瘤P815特异抗原基因P1A克隆到真核表达质粒pCI-neo中；用P815细胞对DBA/2小鼠右腹侧皮下注射，构建P815小鼠肿瘤模型；以重组基因疫苗单独或与鼠IL-12真核表达质粒一起肌肉注射，观察肿瘤的消长，特异细胞毒T淋巴细胞激活和抗全的生成情况。结果 重组基因疫苗在体外有很好的表达，注射后CTL的杀伤效率为40%，IL-12共注射的CTI，杀伤效率达到60%，免疫后，30%小鼠的肿瘤出现消退；同IL-12共注射则有50%的小鼠的肿瘤出现消退，2种情况下都不能检测到任何特异抗体的产生。结论 重组P1A肿瘤疫苗能有效激活机体的肿瘤特异免疫应答；基因疫苗对小鼠P815肿瘤的治疗作用主要归因于细胞免疫；IL-12有增强这种免疫应答的作用。     

Specificity of the T cells that mediate and suppress adoptive immunotherapy of established tumors

This study was designed to investigate the specificity of the T cells that express and suppress antitumor immunity in a model of adoptive immunization against established tumors. Results obtained with the P815 mastocytoma, L5178Y lymphoma, and P388 lymphoma showed, in agreement with previous findings from this laboratory, that an intravenous infusion of splenic T cells from immunized mice can cause the regression of a tumor growing in T-cell-deficient mice. So far as specificity of adoptive immunity is concerned, reciprocal passive transfer experiments with these three tumors revealed that T cells from donor mice immunized against the P815 tumor are not capable of causing regression of the P388 tumor or L5178Y tumor, even if both the P815 and L5178Y tumors are growing in the same host. Similarly splenic T cells from mice immunized against the P388 tumor or L5178Y tumor had no effect on growth of the P815 tumor. Suppression of adoptive immunity was also specific, in that passively transferred suppressor T cells from mice bearing a progressive P815 tumor were capable of suppressing adoptive T-cell-mediated regression of the P815 tumor, but not the P388 tumor growing in T-cell-deficient recipients. Reciprocally, P388 suppressor spleen cells from mice bearing a progressive P388 tumor prevented adoptive T-cell-mediated regression of the P388 tumor, but not the P815 tumor. The results indicate, therefore, that the T cells from immunized mice that mediate adoptive antitumor immunity and the T cells from tumor-bearing mice that suppress the expression of this immunity are specific for the tumor that evokes their generation.     

Use of a skin test assay to determine tumor-specific CD8+ T cell reactivity

We have observed delayed-type hypersensitivity (DTH) reactions in immunized mice challenged subcutaneously with class I-binding peptides related to rejection antigens recognized by cytotoxic T lymphocytes on mutagenized (tum^?) variants of mastocytoma P815. As observed by skin test in virally infected mice challenged with viral peptides, the intrafootpad injection of tum^? peptides resulted in a dose-dependent DTH that peaked at approximately 24 h. The response was mediated by CD8⁺ cells and could be induced by previous vaccination of mice with live tumor cells, intrasplenic deposition of the eliciting peptide, or adoptive transfer with peptide-pulsed syngeneic dendritic cells. These sensitization procedures resulted in an immunologically specific footpad reaction detectable for up to 2–6 months after priming. The evaluation by DTH in cancer patients of long-lived CD8⁺ anti-tumor T cell responses following local challenge with tumor-specific peptides may be of great interest in human immunotherapy trials involving immunization against identified tumor antigens.     

Experimental gene therapy of cancer using tumor cells engineered to secrete interleukin-13

Cytokines locally delivered to the site of a tumor boost both specific and nonspecific host anti-tumor defenses. Interleukin (IL)-13 is a recently described cytokine produced by mouse type 2 helper T lymphocytes. The aim of this study was to evaluate the inhibition of tumor growth induced by IL-13 delivered locally within or around transplanted tumor cells in mice. We observed that local administration of IL-13 at the site of transplanted tumor cells in vivo had potent inhibitory effects on growth of both immunogenic (P815 mastocytoma, H-2^d) or nonimmunogenic (3LL lung carcinoma, H-2^b) tumor cells. Mice injected with transfected P815 cells secreting large amounts of IL-13 rejected the P815 tumor and developed systemic specific anti-tumor immunity leading to long-lasting specific anti-tumor protection. Less efficient anti-tumoral effects were obtained with the nonimmunogenic 3LL tumor model when local administration of IL-13 was achieved by co-inoculating xenogeneic chinese hamster ovary (CHO) IL-13 cells. Several local injections of CHO IL-13 cells were needed to obtain rejection of 3LL tumors and no induction of long-lasting anti-3LL memory was obtained. Several studies were performed to elucidate the IL-13 anti-tumoral effects. Experiments with nude mice indicated that IL-13 can also stimulate nonspecific anti-tumor defenses. The histological examination of P815 IL-13 cells undergoing rejection showed monocytic cells and neutrophils infiltrating the tumor. Studies indicated that IL-13 administered in vitro did not directly stimulate the cytotoxicity of peritoneal macrophages and natural killer cells. However, experiments with Boyden chemotaxis chambers indicated that IL-13 was chemotactic for macrophages. Finally, preliminary experiments in vitro suggest that IL-13 improved antigenic presentation of P815 membranes. Thus, anti-tumor effects of IL-13 in vivo most probably result from pleiotropic effects including recruitment of nonspecific cells and improved stimulation of immune-specific anti-tumor effectors.     

Potent Systemic Antitumor Immunity Induced by Vaccination with Chemotactic-Prostate Tumor Associated Antigen Gene-Modified Tumor Cell and Blockade of B7-H1

We previously reported that several DNA fragments from human prostate-specific membrane antigen (hPSM), mouse prostatic acid phosphatase (mPAP), and human prostate-specific antigen (hPSA) genes were selected and fused to create a novel hPSM-mPAP-hPSA fusion gene (named 3P gene), and human secondary lymphoid tissue chemokine (SLC), 3P, and human IgG Fc genes were inserted into pcDNA3.1 to construct a DNA vaccine, designated pSLC-3P-Fc. In this report, to establish a more efficient treatment for immunotherapy against prostate cancer, the construct was transfected into B16F10 to generate gene-modified tumor cell vaccine (named B16F10-SLC-3P-Fc). In poorly immunogenic B16F10 mouse melanoma model, the immunization with B16F10-SLC-3P-Fc resulted in a strong antitumor response and 50% of tumor-bearing mice achieved long-term survival (>120 days). In vivo depletion of lymphocytes indicated that CD8⁺ T cells were involved in the direct tumor killing, whereas CD4⁺ T lymphocytes were required for the induction of CD8⁺ CTL response in B16F10-SLC-3P-Fc-immunized mice. Splenocytes from B16F10-SLC-3P-Fc-immunized mice specifically recognized and lysed PSM, PAP, PSA, and 3P expressing tumor cells. The combined therapy of B16F10-SLC-3P-Fc plus anti-B7-H1 MAbs further enhanced the immune response. Rechallenge experiment showed that a persistent memory response was successfully induced by the combined therapy. These observations suggest pSLC-3P-Fc-modified tumor cells could serve as a vaccine against prostate cancer, and the therapy combined with anti-B7-H1 MAbs further enhanced the antitumor immune response.     

Paralysis of B7 co-stimulation through the effect of viral IL-10 on T cells as a mechanism of local tolerance induction

The Epstein-Barr virus (EBV) encodes an open reading frame with significant homology to the cellular IL-10 gene. This viral IL-10 (vIL-10) might enable EBV to evade antiviral T cells. We employed transfectants of a murine tumor cell line (P815) to investigate whether vIL-10 interferes with the first (antigenic) or second (co-stimulatory) signal of T cell activation. Untransfected P815 cells caused tumors in syngeneic DBA/2 mice after s.c. inoculation. In contrast, transfectants that provided either a strong antigenic stimulus (P815-K^b cells) or a strong co-stimulatory signal (P815-B7 cells) were rejected. Injection of double-transfected P815 cells expressing K^b and secreting high levels of vIL-10 (P815-K^b -vIL-10) did not result in tumor growth. We then investigated whether vIL-10 could paralyse co-stimulation by B7 under the same conditions. Therefore P815-B7 cells were mixed with vIL-10-secreting P815-K^b cells and co-injected into DBA/2 animals. Most of these mice developed a tumor. Explanted tumor cells expressed the B7 molecule but not the K^b antigen. These observations in vivo were mirrored by experiments in vitro: vIL-10 could induce T cell tolerance towards P815-B7 cells but not P815-K^b cells. Taken together our results suggest that vIL-10 acts directly on T cells to inhibit co-stimulatory signals mediated via B7 receptors such as CD28 or CTLA-4.     

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo

Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose-based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA-specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA-specific CD4(+)/CD8(+) T cell and antibody responses. MDO also targeted lymph node DC to cross-present OVA, leading to OTI CD8(+) T cell proliferation. Moreover, upon challenge with B16-OVA tumor cells, tumors in mice pre-immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA-immunized mice. This mannose-based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.     

Specific and nonspecific antitumor immunity. III. Specific T lymphocyte-mediated cytolysis of P815 mastocytoma and SL2 lymphoma by draining lymph node cells from syngeneic tumor-bearing DBA/2J mice.

Tumor-specific cytolytic activity, as measured by the 51Cr release assay, has been demonstrated in the draining lymph node cells from DBA/2 mice bearing the syngeneic P815 mastocytoma or SL2 lymphoma. This lytic activity is mediated by cytolytic T lymphocytes (CTL), since cytotoxicity is eliminated by treatment of the effector cells with anti-Thy 1.2 (theta) serum plus complement but is enhanced or unaffected by anti-Thy 1.2 serum alone, antimouse immunoglobulin plus complement, normal or aggregated mouse immunoglobulin, or removal of adherent cells. The time course of the CTL response has been analyzed and is similar for both P815 and SL2, with a peak around Days 10 to 12 after tumor grafting. Detectable CTL activity then wanes despite continued antigenic stimulation from the growing tumor. The ability of the immunotherapeutic agent Corynebacterium parvum to augment such specific CTL responses is documented as one antitumor pathway by which this agent may act.     

Regulation of Plasminogen-Activator in Mastocytoma Cells by Lymphokines

Culture supernatants from mitogen-stimulated splenocytes were found to stimulate protease production in P815Y mastocytoma cells. Such supernatants increased cell-associated plasminogen activator levels in a dose-dependent fashion, and under serum-free conditions. In contrast to peritoneal exudate cells, tumor-cell plasminogen activator was not enhanced by the mitogen ConA alone. The tumor cell line P815Y may, thus, be used as a homogenous cell source for the quantitation of lymphocyte factors which activate or inhibit plasminogen activator activity.     

Regulation of T-cell function in lung tissue by pulmonary alveolar macrophages.

We have examined by limit dilution analysis the frequency of several types of DBA/2-specific precursor cells found in the draining lymph nodes of BALB/c mice following anterior chamber or subconjunctival inoculations of P815 tumour cells. Assays for precursors of cytotoxic T cells (pTc) and T-helper cells [interleukin-2 (IL-2)- and IL-4-producing cells] were conducted periodically during a 6-month interval after injection of tumour cells. The results indicate that nodes of both sets of recipients contained primed P815-specific CD8+ pTc that were detectable within 2 weeks of tumour implantation, and persisted throughout the 6-month observation period. Early after tumour inoculation, but not thereafter, these CD8+ cells also secreted Il-2. By contrast, only lymph nodes from mice that received P815 cells into the subconjunctival space contained CD4+ cells that secreted both IL-2 and IL-4; eventually, IL-4-secreting cells formed the vast majority of P815-specific CD4+ cells in these mice. Lymph nodes of mice that received P815 cells in the anterior chamber contained CD4+ T cells that were clonally expanded, and secreted IL-2, but not IL-4. These IL-2-secreting cells proved to be short-lived and were not present 6 months after inoculation. It is proposed that the IL-2- and IL-4-secreting T cells found in lymph nodes of subconjunctival tumour recipients are in vivo homologues of Th0 cells, that these cells can mediate delayed hypersensitivity responses, and that they are the forerunners of, or are themselves, memory T cells. These data indicate that the failure of mice that receive P815 tumour cells in the anterior chamber to display antigen-specific delayed hypersensitivity results from an inability to convert antigen-activated, IL-2-only-secreting CD4+ T cells (pTh) into Th0 cells. These findings also imply that mice with anterior chamber-associated immune deviation (ACAID) fail to develop memory CD4+ T cells.     

Preferential replication of vaccinia virus in the ovaries is independent of immune regulation through IL-10 and TGF-β

Vaccinia virus (VACV) exhibits a strong tropism for ovarian tissue and can cause ovary pathology and sterility. Why VACV preferentially accumulates in this organ is not known. Here we show that multiple immune cell populations infiltrated the ovaries following VACV infection, including virus-specific CD8 T cells making both IFN-γ and TNF. This was also accompanied by the induction of interleukin (IL)-10 and TGF-β, suggesting that VACV may exploit the ovarian environment for immune evasion via induction of these suppressive cytokines. To test this we used several strategies, including neutralizing these cytokines, and exogenous targeting of the T-cell response, to determine if this inhibited virus replication in the ovaries. We found that the VACV-specific CD8 T-cell immunity and the clearance of virus were not enhanced in the ovaries of infected mice in which IL-10 receptor (IL-10R) was blocked with antagonist antibody. VACV replication was also only moderately affected in the ovaries of infected IL-10 knockout mice. Similarly, blockade of TGF-β with antagonist antibody demonstrated no effect on CD8 T-cell immunity or VACV replication. Lastly, an agonist antibody targeting the tumor necrosis factor receptor superfamily member OX40 (TNFRSF4) enhanced the number of VACV-specific CD8 T cells producing IFN-γ in lymphoid tissue, but had no effect on CD8 T-cell infiltration of the ovaries or on the viral load. Collectively, the results indicate that preferential replication of VACV in the ovaries may not be dependent on immune suppressive mechanisms in this tissue.     

Regulatory Role of CD8 in Major Histocompatibility Complex-Unrestricted Tumoricidal Activity of Mouse T Cells Activated with Anti-CD3 Monoclonal Antibody

Antigen-nonspecific CD8⁺ cytotoxic T cells induced with anti-CD3 monoclonal antibody (mAb) are able to kill tumor cells in a major histocompatibility complex (MHC)-unrestricted fashion. However, the role of CD8 in the MHC-independent tumoricidal activity of anti-CD3-activated killer T (AK-T) cells has not been investigated. Here we show that anti-CD8α mAb inhibits, in a dose-dependent fashion, lysis of P815 and YAC-1 tumor cells by mouse AK-T cells. The inhibition of MHC-unrestricted cytotoxicity by anti-CD8α mAb cannot be attributed to interference with an adhesion-like function of CD8 towards class I MHC molecules on the target cells because anti-CD8α mAb (i) had equal inhibitory effects on the cytolysis of tumor target cells regardless of their relative level of class I MHC molecule expression and (ii) did not interfere with the formation of conjugates between AK-T cells and class I MHC-bearing P815 tumor cells. However, anti-CD8α mAb abrogated AK-T cell granule exocytosis in the presence of P815 tumor cells, indicating a regulatory role for CD8 in the signal transduction events which result in lysis of the tumor target cell. Immunoblot analysis of the post-nuclear fraction of lysates from AK-T cells exposed to P815 tumor cells in the presence of anti-CD8α mAb revealed reduced phosphorylation of tyrosine residues on a protein with an Mr of -62 kDa. Taken together, these data suggest that CD8 is able to affect the tumoricidal activity of MHC-unrestricted AK-T cells independent of class I MHC molecules on the target cell.     

Effect of tumor cells on the generation of cytotoxic T lymphocytes in vitro. I. Accessory cell functions of mouse tumor cells in the generation of cytotoxic T lymphocytes in vitro: replacement of adherent phagocytic cells by tumor cells or 2-mercaptoethanol.

In agreement with previous reports, the primary in vitro response to alloantigens has been shown to be dependent on the presence of macrophages (Mphs). Splenocytes extensively depleted of adherent phagocytic cells did not generate cytotoxic T lymphocytes, and this activity could be completely restored by small numbers of adherent peritoneal cells (accessory cells). Either P388D1 (Mph-like tumor), P388 ("null" tumor) or P815 (mastocytoma) tumor cells, or 2-mercaptoethanol, could completely replace the accessory function normally mediated by accessory cells. These tumor cells did not non-specifically "enhance" the cytotoxic activity generated with normal nondepleted spleen cells. The restored cultures maintained killing specificity to H-2 targets which was mediated by effector T cells as shown by sensitivity to anti-theta and complement. Therefore, Mphs seem not to be the sole cells capable of mediating an accessory function in a primary response to alloantigens in vitro.