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.     

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

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

Role of protein synthesis in the activation of cytotoxic mouse macrophages by lymphokines

The role of protein synthesis during the activation of macrophages (M phi) by lymphokines (LK) was studied. Peritoneal murine macrophages elicited by proteose-peptone (pM phi) were activated with LK (supernatants from normal mouse spleen cells pulsed with concanavalin A) and tested for cytotoxicity in an 18 hr assay against 111In-labeled L5178Y lymphoma target cells. Reversible (cycloheximide and puromycin) or poorly reversible (emetine and pactamycin) inhibitors of protein synthesis were added during activation, and their effects on pM phi-mediated cytotoxicity and pM phi protein synthesis were measured. Minimal concentrations of inhibitors, reducing the rate of protein synthesis by more than 90% without toxic effects on macrophages, were chosen. Exposure of pM phi to LK for 2 to 18 hr in the presence of reversible inhibitors of protein synthesis did not affect the induction of cytolytic activity, indicating that protein synthesis was not required during the activation period. In contrast, activation of macrophages for 2 hr in the presence of poorly reversible inhibitors of protein synthesis resulted in a considerable reduction of cytolytic activity. The impairment of cytotoxic activity was also evident when pM phi were treated with such drugs during the first 2 hr of an 18 hr exposure to LK or when LK-activated macrophages were treated for 2 hr with the drugs before the addition of the targets. These results demonstrate that active protein synthesis is not required during the exposure of pM phi to LK, but that new proteins have to be synthesized to allow the expression of the cytotoxic activity in LK-activated pM phi.     

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.     

Shifts in macrophage (M phi) surface phenotypes during tumor growth: association of Mac-2+ and Mac-3+ M phi with immunosuppressive activity

Rat anti-mouse monoclonal antibodies (mAb), anti-Mac-1, -2, and -3, directed against macrophage (M phi) glycoprotein surface antigens, were used to demonstrate a tumor-induced shift in peritoneal M phi subpopulations. This study of the tumor-induced shift was approached in two steps. First, to show that separate phenotypic M phi subpopulations existed and second, to show that a shift in these populations was involved in immunosuppression of the host during tumor growth. Endogenous peroxidase activity was examined among normal and tumor-bearing host (TBH) M phi. A significant increase in the number of peroxidase-positive M phi occurred during tumor growth. Indirect immunofluorescence showed a decrease in Mac-2+ cells and an increase in Mac-3+ cells in TBH M phi populations. When the mAb, anti-Mac-1,-2, and -3 were used in the presence of complement (C), they were cytotoxic for M phi and showed differential depletion of normal and TBH M phi. Peroxidase-positive TBH M phi were susceptible to C-mediated lysis by anti-Mac-1 and -3 but not by anti-Mac-2, whereas no direct relationship was observed among normal host M phi. To demonstrate differences between normal and TBH M phi subpopulations, soluble inhibitory factors were examined from mAb plus C-modified M phi populations. Anti-Mac plus C-treated normal and TBH M phi produced supernatants with different regulatory capabilities as assessed in the mixed-lymphocyte reaction (MLR). Anti-Mac-2 plus C treatment significantly reduced the ability of TBH M phi to produce a soluble suppressor(s) but did not alter normal host M phi-derived suppressor production. In contrast, anti-Mac-1 and -3 plus C treatment of normal host M phi significantly reduced suppressor production. In the TBH, however, anti-Mac-1 plus C had no effect, while anti-Mac-3 plus C had only a limited reduction as compared to the normal host. Determination of levels of prostaglandin E2 (PGE2) in M phi supernatants showed that normal host Mac-1+ M phi were involved in down regulation of PGE2 production. This control was missing in the TBH M phi. Mac-2+ M phi were the apparent producers of PGE2 which accounts for the factor-mediated MLR suppression attributed to TBH Mac-2+ M phi. Collectively, these data suggest that tumor-induced aberrations in immunoregulation can in part be attributed to differences in anti-Mac mAb-defined M phi subpopulations.     

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.     

"Nonspecific" immunoenhancing T cells in tumor-bearing mice include anti-idiotypic subsets

Splenocytes from DBA/2 mice inoculated 3 wk earlier with syngeneic P815 mastocytoma tumor cells produce increased numbers of antibody plaque-forming cells (PFC) when stimulated with either sheep red blood cells (SRBC) or phosphorylcholine (PC) on Streptococcus pneumoniae R36a in vitro. The nature of this nonspecific hyperreactivity was investigated in mixed cultures of purified splenic T and B cells. The addition of T cells from P815 tumor-bearing mice (TP815) into the cultures of normal B cells produced a significant enhancement of the PFC response to both SRBC and PC, when compared with the effect of normal T cells added to control cultures. The idiotypic profile of the enhanced anti-PC response was studied by a PFC-inhibition assay with monoclonal antibodies against two distinct idiotopic determinants (Id) of the T15 family. Normal B cells produced greater than 90% of T15 Id-positive (Id+) PFC. Addition of normal T cells diminished the proportion of T15 Id+ PFC to approximately 60%, whereas the rest of PFC were Id-. Addition of the immunoenhancing TP815 cells into the normal B cells cultures elevated the number of both T15 Id+ and Id- PFC responses, proportionally. However, when TP815 cells were first incubated on T15 protein-coated dishes and the non-adherent fraction was added to B cell cultures, the anti-PC PFC response remained enhanced but consisted of predominently T15 Id- PFC. These observations suggest that the early stage of P815 tumor growth activates various populations of specific helper/amplifier T cells including subsets with anti-idiotypic activity and that the generalized increase of antibody response to various antigens in tumor-bearing mice may be regarded as a polyclonal activation of specific T cells.     

Macrophage activation for tumor cytotoxicity: Control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines

Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.     

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     

Suppressor deletion therapy: selective elimination of T suppressor cells in vivo using a hematoporphyrin conjugated monoclonal antibody permits animals to reject syngeneic tumor cells

Summary A MAb (B16G) which recognizes a constant epitope on TsC and their soluble factors in DBA/2 mice has been described previously. In this study, we show that when this MAb is covalently linked to the photoactivable molecule Hp, and injected i.v. into P815 tumor-bearing mice which were subsequently exposed to light, tumors undergo permanent regression in 10%–40% of these mice (depending on the individual experiment). All control animals died within an average of 22–24 days after tumor cell injection. It is suggested that tumor regression is attributable to immune mechanisms facilitated by the elimination of a population of TsC. When splenocytes of B16G-Hptreated mice were assayed in vitro for the generation of CTL active against P815 tumor cells, it was found that 24 h after treatment, a significant increase in killer cell activity was noted but that this effect was gone by 48h. We also show that B16G-Hp conjugates are capable in vitro of specifically killing cells of a TsC hybridoma, A10 (which has been shown previously to secrete a T suppressor factor reactive with P815 cell surface antigens). This conjugate had no cytotoxic effect on P815 cells under conditions in which A10 cells were killed.Abbreviations CTL cytotoxic T lymphocytes - C-MAb control monoclonal antibody - EDCI 1-ethyl-3-(3-diemthylaminopropyl) carbodiimide - Hp hematoporphyrin - MAb monoclonal antibody - PBS phosphate-buffered saline - RMIg rabbit antimouse Ig - TsC T suppressor cell - TsF T suppressor factor - FCS fetal calf serum - DME Dulbecco's modified Eagle's medium     

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.     

The antimetastatic function of concomitant antitumor immunity. II. Evidence that the generation of Ly-1+2+ effector T cells temporarily causes the destruction of already disseminated tumor cells

Progressive growth of the P815 mastocytoma in an immunocompetent host evokes the generation of an antitumor immune response that can be measured in terms of the production of cytolytic Ly-1+2+ T cells in the draining lymph node and spleen. This immunity, designated concomitant immunity, is present on day 6 of tumor growth, peaks on day 9, and decays progressively thereafter. It fails to develop in mice made T cell deficient by thymectomy and lethal whole-body gamma-radiation, and reconstituted with syngeneic bone marrow cells (TXB mice). Employment of a mouse survival assay, capable of enumerating metastatic P815 cells in cell suspensions, showed that the P815 tumor metastasizes to the draining lymph node and spleen at the same rate in normal and TXB mice for the first 6 days of growth of an intradermal P815 tumor. By day 6 of tumor growth there were approximately 10(3) P815 cells in the draining lymph node in both types of mice. However, during the generation of concomitant immunity between days 6 and 9, the number of metastatic P815 cells in the draining lymph nodes and spleens of normal tumor-bearing mice declined by nearly 90%. After day 12, however, the number of tumor cells in the nodes and spleens increased concordantly with the decay of concomitant immunity. These findings, together with the demonstration that T cell-deficient mice failed to restrain the number of metastatic P815 cells in the draining lymph node and spleen, suggest that concomitant immunity is an important defense mechanism against the development of systemic disease. Additional evidence consistent with this interpretation was provided by studies which showed that adoptive immunization with spleen cells from concomitant immune donors significantly prolonged the median survival time of TXB tumor-bearing mice by destroying a substantial proportion of P815 tumor cells already seeded in the draining lymph node. Adoptive immunization also delayed the appearance of metastatic tumor cells in the spleen.     

Failure of infiltrating precursor cytotoxic T cells to acquire direct cytotoxic function in immunologically privileged sites

Minor H incompatible P815 tumor cells inoculated into the anterior chamber (AC) of the eyes of BALB/c mice grow progressively, revealing this to be an immunologically privileged site. By contrast, a similar inoculation of tumor cells is rapidly rejected from nonprivileged ocular sites (subconjunctiva). Mice with progressively growing AC-tumors and those that reject their ocular subconjunctiva tumors both have expanded clones of tumor-specific cytotoxic precursor cells (pTc) in their spleens and cervical lymph nodes. In an effort to determine why the expanded pool of primed pTc is unable to effect rejection of AC intraocular tumors, we have examined the susceptibility of the tumor cells growing within the immunologically privileged AC to lysis by cytotoxic T cells and the cytotoxic function of tumor-infiltrating lymphocytes. P815 tumor cells extracted from intraocular tumors and P815 cells maintained in routine tissue culture are equally susceptible to lysis when exposed in vitro to fully differentiated, DBA/2-specific cytotoxic T cells. Thus, progressively growing tumor cells within the AC are not insensitive to immune-mediated lysis by cytotoxic T cells. We have been able to harvest significant numbers of DBA/2-specific pTc from these same intraocular tumors. When the tumor-infiltrating lymphocytes are driven in vitro with exogenous IL-2, they acquire the capacity to lyse specifically P815 tumor cells. However, no evidence of fully cytotoxic, tumor-specific T cells was found among lymphocytes harvested from intraocular tumors, i.e., when the harvested cells were tested immediately for cytolytic activity. Inasmuch as we have reported that directly cytotoxic T cells are present during tumor rejection at nonimmunologically privileged ocular sites, such as the subconjunctival space, we conclude that progressive growth of P815 tumor cells within the anterior chamber is due in part to a failure of infiltrating pTc to differentiate in situ into fully functional cytotoxic effector cells.     

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).     

Lysis of murine macrophages by lymphokine-activated killer-like cells induced by BCG in vitro.

The lymphokine-activated killer (LAK)-like activity was found to be induced in mouse splenocytes cultured together with Bacillus Calmette-Guérin (BCG). The killer cells induced by BCG were capable of killing both NK-sensitive (YAC-1, P388D1) and NK-resistant (P815) tumor cells. As an important finding, they also lysed syngeneic macrophages (M phi). The anti-M phi killer activity appeared on day 2, and reached a peak on day 5 of culture. Phenotype analysis of the killer cells by depletion techniques using monoclonal antibody (mAb) and complement indicated that the majority of these anti-M phi killer cells were Thy-1+ and asialo GM1+. This M phi cytolysis could be inhibited by the addition of cold M phi, YAC-1 tumor cells, and P815 tumor cells, suggesting that the same population of the effector cells recognize M phi and tumor cells. The addition of anti-MHC class I, anti-MHC class II, anti-L3T4, or anti-Ly-2 mAb directly to assay cultures did not affect anti-M phi cytolysis, suggesting that the MHC molecules are not involved in the cytolysis of M phi by the BCG-induced killer cells. The addition of anti-LFA-1 mAb partially inhibited the cytotoxicity, suggesting importance of the contact between targets and effectors in the cytolysis. Our present data suggest that activation of murine lymphocytes with BCG induces LAK-like cells capable of killing a wide variety of tumor cells as well as M phi and this anti-M phi cytolysis is mediated by nonspecific killer cells.     

Therapeutic effectiveness of the immunity elicited by P815 tumor cells engineered to express the B7-2 costimulatory molecule

 It is well accepted that inoculation of B7-1-transfected tumor cells into normal mice leads to tumor rejection and subsequent resistance to challenge. However, the effectiveness of B7-2-transfected tumor cells in eliciting protective antitumor immunity is less clear. Here we show that B7-2-transfected P815 tumor cells (B7-2⁺) are as effective as B7-1-transfected P815 tumor cells (B7-1⁺) in eliciting protective immunity in normal DBA/2 mice. In addition, B7-2⁺ cells were found to be at least as effective as B7-1⁺ cells in retarding tumor progression when admixed with parental P815 tumor cells prior to inoculation into normal mice. Moreover, the B7-2⁺ cells and the B7-1⁺ cells were equivalent in their ability to retard tumor growth when administered peritumorally into mice bearing established (approx. 3 mm in diameter) parental P815 tumors. Finally, P815 tumor cells infected with a recombinant replication-defective adenovirus encoding the murine B7-2 gene were effective in retarding the growth of established parental P815 tumors. Thus, B7-1 and B7-2 are comparable in terms of their ability to stimulate the generation of tumor-eradicating immunity in normal mice as well as in mice bearing established parental tumors. Moreover, adenovirus vectors can be used to generate B7-2-expressing tumor cells effective in the immunotherapy of established parental tumors. Received: 10 January 1996 / Accepted: 23 February 1996     

The antimetastatic function of concomitant antitumor immunity. I. Host Ly-1+2+ effector T cells prevent the enumeration of metastatic tumor cells in a biological assay

A mouse survival assay was evaluated for its suitability to enumerate metastatic P815 tumor cells in the draining lymph node and spleen of a B6D2 F1 (H-2b X H-2d) host bearing a primary intradermal P815 tumor. The mouse survival assay is based on the linear relationship between the log10 number of P815 tumor cells (H-2d) injected i.p. into mice and their mean survival time. It was found that the assay is capable of quantifying as few as 10 tumor cells in lymph node and spleen, but only if cell suspensions of these organs are treated with anti-H-2b serum and complement, in order to selectively destroy H-2bd host cells. This was necessary because host cells from the lymph node and spleen of a tumor-bearing host possessed antitumor functions, in that they were capable of destroying the H-2d P815 tumor cells when admixed with the tumor cells and injected i.p. into 800-rad irradiated test recipients. The kinetics of acquisition and loss of host cells with antitumor function and the Ly phenotype of these host cells suggest that they are the same cells that give the tumor-bearing host the capacity to express concomitant immunity against a tumor implant.     

Modulation of macrophage activation and resistance by suppressor T lymphocytes in chronic murine Schistosoma mansoni infection

Activated macrophages (M phi) appear responsible for at least part of the concomitant resistance in mice infected with Schistosoma mansoni. We found that as murine S. mansoni progressed from acute (8 to 12 wk of infection) to chronic (16 to 24 wk) stages, acquired resistance decreased (57% resistance to challenge with cercariae at 8 wk vs 28% by 24 wk, p less than 0.05), as did macrophage activation (21% +/- 2 killing of schistosomula by 8 wk M phi vs 8% +/- 2 for 24 wk M phi, p less than 0.01). T cells from the spleens of 8 wk-infected mice were capable of activating M phi from naive animals when stimulated with worm antigens (24% +/- 2 killing vs 8% +/- 2 induced by normal T cells, p less than 0.01); T cells obtained from 24 wk-infected mice did not activate M phi (13% +/- 2 killing). Furthermore, T cells from 24 wk-infected animals suppressed activation of M phi by 8 wk T cells. The addition of 10(5) 24 wk T cells to 3 X 10(5) antigen-stimulated 8 wk T cells reduced subsequent M phi killing from 27% +/- 4 to 13% +/- 2 (p less than 0.05). Week 24 T cells (3 X 10(5] reduced this additionally to 9% +/- 1 (p less than 0.01), a value no greater than that of unstimulated M phi. The subpopulation of T cells responsible for suppression of M phi activation was Lyt-2+1- nonadherent T cells. Cyclophosphamide treatment of chronically infected mice resulted in enhanced resistance (41%), M phi activation (18% +/- 1 killing), and T cell activation of naive M phi (10% +/- 1 killing). Thus, during chronic S. mansoni infection, resistance to reinfection wanes in parallel to and perhaps because of development of suppressor T cells that interfere with T-dependent M phi activation.     

Interleukin-2-activated murine cell lines with macrophage- and B-lymphoblast-lytic activity.

Interleukin-2 (IL-2)-activated murine killer cell lines with macrophage- and B-lymphoblastic-lytic activity were established, and their target specificity, surface markers, recognition-related structures, and requirements for optimal cell growth were characterized. Sustained growth of IL-2-activated lymphocytes was supported by the combination of IL-2 and IL-4-enriched T cell conditioned medium (CM), but was not supported by IL-2 alone or the combination of IL-2 and IL-3-containing CM in the presence of macrophages (M phi). The established line required continuous contact with M phi to maintain anti-M phi cytolytic activity. Flow cytometric analysis showed that the original line isolated by the first cloning was Thyl+, CD4-, and weakly CD8+, FcR+. The majority of these cells were CD3+ and TCR-V beta 8+. From this line, the CD3+, TCR-V beta 8+ and CD3-, TCR-V beta 8- clones were isolated by subcloning. The former clone showed Thyl+, CD3+, CD4-, CD8-, TCR-V beta 8+, FcR(+)-phenotype, and the latter clone showed Thyl+, CD3-, CD4-, CD8-, TCR-V beta 8-, FcR- phenotype. The original line and subclones showed a similar target specificity and killed resident or thioglycollate (TG)-induced peritoneal M phi and B-lymphoblasts, but did not kill T-lymphoblasts. Allogeneic M phi, M phi-like cell line P388D1, and B cell hybridoma were sensitive, whereas fresh lymphocytes, T cell lymphoma BW5147, natural killer (NK)-sensitive YAC-1, and NK-resistant P815 tumor cells were resistant to lysis by these cytotoxic lines. The addition of anti-H-2 heteroserum, anti-MHC class 1, anti-MHC class II, anti-CD3, or anti-TCR-V beta 8 monoclonal antibody (mAb) to assay cultures did not inhibit the anti-M phi cytolysis by these killer cells. In addition, the CD3- TCR-V beta 8- clone killed M phi and B lymphoblasts better than the CD3+, TCR-V beta 8+ clone. These results suggest that cytotoxic lines established in this study do not use the T cell receptor (TCR) molecules to recognize target cells and the MHC molecules are not involved in recognition. Anti-LFA-1 mAb partially inhibited anti-M phi-lysis, suggesting that the cell contact between targets and effectors is important in cytolysis. Our present data suggest that the culture condition containing IL-2, IL-4, and M phi may support the continuous growth of non-MHC-restricted killer cells with relative target specificity against M phi and B-lymphoblasts.     

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.