Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

H-2 I alloantigens and recall of memory cytotoxic responses

AQR mice were immunized with H-2K and H-2 I encoded alloantigens presented by (Ax6R)F₁ splenocytes. Spleen cells from these alloimmune mice were subsequently restimulated in vitro with B10.A lymphocytes and/or B10.T(6R) lymphocytes, thus presenting them with the immunizing H-2K and H-2 I alloantigens independently. When stimulated with B10.A lymphocytes, alloimmune lymphocytes develop significant cytotoxicity against the immunizing H-2K target antigens. When stimulated with a similar number of B10.T(6R) spleen cells, alloimmune lymphocytes undergo a prominant proliferative response, but develop little, if any, cytotoxicity against the immunizing H-2 K target antigens. The most efficient restimulation of cytotoxicity occurs when the alloimmune spleen cells are simultaneously restimulated by B10.A and B10.T(6R) lymphocytes. Stimulation with the immunizing H-2 I alloantigens alone is not sufficient for regeneration of detectable cytotoxic responses from alloimmune spleen populations. Stimulation with the immunizing H-2K alloantigens alone appears to be both necessary and sufficient to stimulate alloimmune cytotoxic responses. Although the immunizing H-2 I alloantigens are apparently not required to generate alloimmune cytotoxic responses, they markedly potentiate the cytotoxic responses induced by the immunizing H-2K alloantigens.     

Depletion of macrophages expressing I-J antigen results in efficient generation of alloreactive cytotoxic T lymphocytes

The role of suppressor macrophages (S-M phi) produced during generation of cytotoxic T lymphocytes (CTL) stimulated with allogeneic lymphocytes was investigated. Splenic CTL from C3H/He mice (H-2k) were generated by in vivo immunization and subsequent in vitro stimulation by splenic lymphocytes from C57B1/6 mice (H-2b) in mixed lymphocyte reaction (MLR). In addition to in vitro standard 51Cr release assay, the CTL activity was mainly measured in vivo using the Winn assay against EL-4 thymoma cells in B6C3F1 mice (H-2b/k). In mice injected with CTL plus EL-4 cells survival rate was 20% compared with no survival of mice treated with normal spleen cells plus EL-4 cells. The antitumor activity of the CTL was significantly increased when immunized mice were treated with a 5 mg/kg ip dose of indomethacin at the time of immunization (80% survival). Macrophages were depleted from spleen cells of immunized mice by plastic adherence or carbonyl-iron treatment, replaced with an equivalent number of M phi from normal mice, and then introduced into a 5-day MLR. When the antitumor activity of the cells isolated from this MLR was measured in the Winn assay, 90-100% survival in EL-4-bearing mice was observed. In contrast, none of the mice inoculated with EL-4 alone and 20% of the mice that received CTL obtained after alloimmunization followed by MLR in addition to EL-4 survived. These results of CTL activity were confirmed by in vitro cytotoxicity tests. When the M phi isolated from spleens of immunized mice were analyzed for I-Jk antigen expression, a 2.5-fold increase was detected, compared with splenic M phi obtained from normal C3H/He mice. In contrast, Ia and I-Ak antigen expression was equivalent in M phi isolated from normal or immunized C3H/He mice. When immune spleen cells were treated with anti-I-Jk antiserum followed by complement and then, subjected to the MLR, the antitumor activity of CTL was significantly enhanced (80% survival). However, treatment of these cells with anti-I-Ak antiserum and complement did not alter CTL activity. These data suggest that the increase of S-M phi expressing I-Jk+ antigen to be induced during alloimmunization results in suppression of allospecific CTL-generation in MLR.     

The role of host lymphocytes and host macrophages in antitumor reactions after injection of sensitized lymphocytes and tumor target cells into naive mice

Summary DBA/2 mice were immunized i.p. against syngeneic SL2 lymphosarcoma cells. At various days after the last immunization peritoneal and spleen lymphocytes were collected. The lymphocyte suspensions were enriched for T-cells by nylon wool filtration.The peritoneal T-cells from immunized mice (a) expressed direct specific antitumor cytotoxicity in vitro, (b) induced macrophage cytotoxicity in vitro, and (c) exerted tumor neutralization measured in a Winn-type assay. Spleen T-cells from these immunized mice (a) expressed no direct specific antitumor cytotoxicity in vitro, (b) only induced moderate macrophage cytotoxicity in vitro, but (c) exerted tumor neutralization in a Winn assay.For effective tumor neutralization in vivo effector target cell ratios of 1000:1 were required. When the effector/target ratio of 1000:1 was maintained but the absolute numbers of effector and target cells were lowered from 10⁶ to 10⁵ lymphocytes and 10³ to 10² target cells respectively, no tumor neutralization was obtained.The major effect of the sensitized-transferred T-lymphocytes seemed to be the induction of cytotoxic macrophages in the (naive) recipient mice, as the peritoneal macrophages collected from the recipient mice 7 days after i.p. injection of a mixture of sensitized T-cells and tumor cells were cytotoxic. Purified peritoneal T-lymphocytes collected from these recipient mice were able to induce macrophage cytotoxicity in vitro but expressed no cytotoxic T-cell activity.In conclusion, our results show that in the tumor system used, tumor neutralization after transfer of sensitized lymphocytes is not dependent on the presence of cytotoxic T-lymphocytes. Lymphocytes with the strongest potency to render macrophages cytotoxic (in vitro and in vivo) also induce the best tumor neutralization in vivo, suggesting an important role for host macrophages as antitumor effector cells.     

Studies on the induction and expression of T cell-mediated immunity. IV. Non-overlapping populations of alloimmune cytotoxic lymphocytes with specificity for tumor-associated antigens and transplantation antigens.

Two non-overlapping populations of alloimmune cytotoxic T cells with specificity for tumor-associated antigens (TAA) and for histocompatibility antigens (H-2) were characterized by two independent methods. The heterogeneity of cytotoxic cells was demonstrated in spleen cells derived from BALB/c (H-2d) mice sensitized to EL-4 (H-2b) tumor and from C57BL/6 (H-2b) mice sensitized to G-35 (H-2d) tumor cells. Adsorption of immune lymphocytes on monolayers prepared with cells bearing the sensitizing H-2 antigens abrogated the in vitro cell-mediated cytotoxicity (CMC) directed against 51Cr-labeled normal target cells (spleen cells or ConA-activated spleen blasts), whereas significant cytolytic activity to the corresponding 51Cr-tumor cells was still retained. Likewise, in competitive inhibition assays, CMC to 51 Cr-tumor target cells was only partially inhibited by unlabeled normal cells, whereas CMC to 51Cr-normal target cells was completely abrogated. These results suggested that alloimmune cytotoxic lymphocytes are heterogeneous and can be subdivided into two independent populations of restricted specificity. Several experiments suggested that the effector cell population directed against TAA can no longer elicit a graft-vs-host (GVH) reaction in vivo. This was demonstrated by adoptive transfer into lethally-irradiated allogeneic recipients of cytotoxic or primed spleen cells fractionated on host target cell monolayers. Furthermore, these results demonstrated that both effector cells and memory cells possess high affinity binding receptors to corresponding H-2 antigens. The potential use of fractionated immune lymphocytes sensitized to tumor allografts in adoptive immunotherapy is discussed.     

Contrasting effects of alloantiserum and xenoantiserum on cell-mediated lysis of tumor cells

The effect of anti-EL-4 serum on antibody-dependent cytotoxicity (ADCC) and cell-mediated cytotoxicity (CMC) was studied in allogeneic and xenogeneic systems. Inbred strains of BALB/c mice and Lewis rats were immunized with EL-4 tumor cells. Using microcytotoxic assays of ⁵¹Cr release from labeled EL-4 cells, complement-dependent cytolysis, ADCC, and CMC were determined. Complement-dependent cytolysis was observed in both systems. Although ADCC was demonstrated in both systems, the kinetics of cytolysis were different. Xenoantisera and alloantisera had opposite effects on CMC. Incubation of EL-4 target cells with BALB/c anti-EL-4 serum resulted in inhibition of CMC by immune BALB/c spleen cells. In contrast, treatment of EL-4 target cells with Lewis anti-EL-4 serum potentiated the CMC of immune Lewis spleen cells. It is thought that differences in the strength of response, antibody characteristics, and effector cells may determine the degree of inhibition or potentiation observed in these systems.     

Differing time courses of spleen leukocyte MIF synthesis and cytotoxicity during rejection of a murine lymphoma allograft

In vitro spleen leukocyte MIF synthesis and direct cytotoxicity were studied during growth and rejection of the EL-4 murine lymphoma in allogeneic tumor-bearing BALB/c mice to compare the time courses of these events. Rejection of EL-4 tumors occurred between 6 and 8 flays after intraperitoneal inoculation. Spleen leukocyte cytotoxicity measured by isotope release from chromium-51 labeled EL-4 target cells was first detected on day 6 and was maximal between days 11 and 18. In contrast, spleen leukocyte MIF synthesis stimulated by intact EL-4 cells was sometimes observed on day 11 and was maximal between 18 and 25 days after tumor challenge. These results show that maximal spleen cytotoxicity and MIF synthesis occur after completion of, rather than during ip tumor rejection and, in addition, that these two in vitro lymphocyte responses follow independent, significantly different time courses (P < 0.05). This asynchrony of MIF synthesis and cytotoxicity suggests that these in vitro correlates are mediated by distinct lymphocyte subpopulations.     

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.     

Induction of high-grade anti-tumor immunity by use of a recombinant H-2Kb/avian erythroblastosis virus erbB gene transfectant

Summary The recombinant H-2K^b-erbB gene, encoding for a part of the H-2 class I antigen and the kinase domain of the V-erbB peptide, was successfully introduced into murine mastocytoma P815 variant P1.HTR cells, which resulted in low but significant cell-surface expression of the hybrid gene product. When the chimeric gene transfectant was inoculated into the CDF₁ mice, it soon grew but regressed thereafter. The tumorigenicity of this transfectant was lower than the H-2K^b gene transfectant that expressed the H-2K^b antigen at a comparable level. These CDF₁ mice that had received the chimeric gene transfectant obtained a high-grade anti-tumor immunity against the challenge of a high dose of parental tumor. Corresponding to these observations, anti-tumor cytotoxic T lymphocytes, which lyse parental P1.HTR cells but not syngeneic L1210 or NS-1 tumor cells, were developed in the peritoneal cavity of mice that had been inoculated with the transfectant and parental tumor. Definite antibody activity binding to parental P1.HTR tumor cells was also demonstrated in the sera of these mice, precipitating 40-kDa, 74-kDa and 98-kDa molecules from the surface of the radiolabeled P1-HTR tumor cells. The results suggested that the chimeric H-2-erbB gene transfectant efficiently triggers both cellular and humoral anti-tumor immune responses.     

Interaction of peritoneal exudate lymphocytes with histocompatibility antigens.

The effect of allo-immunization on the response of mouse peritoneal exudate lymphocytes (PELs) in mixed lymphocyte culture (MLC) and cell mediated cytotoxicity was described. In characterizing these responses, the results were compared with data obtained in similar experiments with splenic lymphocytes (SpLs). While unimmunized BALB/c SpLs (H-2^d) showed strong reactivity in the one way MLC against irradiated C57BL/6 spleen cells, unimmunized PELs gave a barely detectable response. Subcutaneous (sc) but not intravenous (iv) allo-immunization resulted in a transient but marked increase in MLC reactivity by PELs. Immunization by either route resulted in an augmented MLC by SpLs. Further, sc, but not iv, allo-immunization resulted in the transient appearance of cellular cytotoxic lymphocytes in PELs. It was concluded that PELs were unique among secondary lymphoid populations in that they contained very few histocompatibility antigen reactive cells in the absence of immunization; and as such represented a population committed solely to antigens which the animal had recently experienced.     

Murine CD8+ cytotoxic T lymphocytes lyse Toxoplasma gondii-infected cells

Studies were performed to determine whether CTL against Toxoplasma gondii-infected cells could be induced in a murine model of T. gondii infection in which CD8+ T lymphocytes have been shown to play a major role in resistance against this parasite. In 51Cr release assays, nylon wool nonadherent spleen cells from BALB/c (H-2d) mice immunized with the temperature-sensitive (ts-4) mutant strain of T. gondii were cytotoxic for T. gondii-infected P815 (H-2d) mastocytoma cells but not for uninfected cells. This cytotoxic activity was remarkably increased after in vitro stimulation with T. gondii-infected syngeneic spleen cells. The effector cells were shown to be CD8+ T lymphocytes, because the cytotoxicity was significantly inhibited by depletion of CD8+ T lymphocytes but not by depletion of CD4+ T lymphocytes. This cytotoxic activity was genetically restricted. Spleen cells from T. gondii-immune BALB/c mice were not cytotoxic for T. gondii-infected EL4 (H-2b) thymoma cells, whereas spleen cells from T. gondii-immune C57B1/6 (H-2b) mice were cytotoxic for T. gondii-infected EL4 cells but not for T. gondii-infected P815 cells. The cytolytic activity of CD8+ T lymphocytes against T. gondii-infected cells might be a mechanism whereby these cells confer resistance against T. gondii.     

Continuously proliferating T killer cells specific for H-2b targets: selection and characterization.

BALB/c (H-2d) thymus-derived lymphocytes sensitized to C57BL/6 (H-2b) alloantigens have been propagated in vitro for over 9 months. These T lymphocytes are specifically cytotoxic to H-2b target cells but are stimulated to proliferate by both H-2b and H-2k spleen cells. This indicates that for these selected cells the antigen requirements for cell proliferation are different from those for cell-mediated cytotoxicity. If not continuously stimulated with allogeneic spleen cells, the cytotoxic cultures fail to divide and rapidly lose their cytotoxic activity. Allogeneic erythrocytes do not stimulate cell proliferation in "quiescent" cell cultures and allogeneic tumor cells do so only in the presence of spleen cells. However, "quiescent" cell cultures display cytotoxicity in the presence of phytohemagglutinin A as do cell cultures which have lost their cytotoxic activity although they proliferate upon allogeneic stimulation. The significance of these findings is discussed.     

Enhancement by interferon of natural killer cell activity in mice.

Injection of mice with several interferon inducers, Newcastle Disease virus, polyinosinic-polycytidylic acid and tilorone resulted in an increase in spleen cell cytotoxicity for ⁵¹chromium-labeled mouse YAC tumor target cells in 4-hr in vitro assays. This increase in spleen cell cytotoxicity was abrogated by injection of mice with potent anti-mouse interferon globulin. Inoculation of mice with mouse interferon (but not human leucocyte or mock interferon preparations) also resulted in a marked enhancement of spleen cell cytotoxicity. The extent of enhancement of spleen cell cytotoxicity was directly proportional to the amount of interferon injected and a significant increase was observed after inoculation of as little as 10³ to 10⁴ units of interferon. An effect could be detected as soon as 1 hr after injection of interferon. The increase of spleen cell cytotoxicity after inoculation of an interferon inducer was not due to a localization and accumulation of cytotoxic cells in the spleen but reflected a general increase in cytotoxic cell activity in various lymphoid tissues (except the thymus). The splenic cytotoxic cells from interferon or interferon-inducer-injected mice had the characteristics of natural killer (NK) cells since (i) interferon enhanced spleen cell cytotoxicity in athymic (nu/nu) nude mice, (ii) classical spleen cell fractionation procedures by nylon wool columns, anti-Thy 1.2 serum plus complement, anti-Ig columns, and depletion of FcR+ rosette-forming cells, failed to remove the effector cells generated in vivo or in vitro. Therefore like NK cells, interferon-induced cytotoxic cells lack the surface markers of mature T and B lymphocytes, are not adherent, and are devoid of avid Fc receptors. Furthermore like NK cells, the spleen cells from interferon-treated mice lysed various target cells (known for their sensitivity to NK cells) without H-2 or species restriction. Incubation in vitro of normal spleen cells with interferon also resulted in an increase in cytotoxicity for YAC tumor cells. We conclude that interferon acts directly on NK cells and enhances the inherent cytotoxic activity of these cells.     

Characterization of IL-2-dependent cytotoxic T-cell clones. III. Inhibition of killing activity by monosaccharides

The effects of monosaccharides on the cytotoxic activity of cytotoxic T lymphocytes (CTL) and three cloned long-term cytotoxic T-lymphocyte lines (CTLL) are compared. Uncultured CTL and clones CTLL-A2 and CTLL-A11 were derived from the peritoneal cavity of C57BL/6 mice immunized against the H-2Dd determinants on the BALB/c sarcoma Meth A. Clone CTLL-R5 was derived from spleen of (BALB/c X C57BL)F1 mice immunized against a unique determinant on the BALB/c radiation-induced leukemia RL male 1. The cell-surface phenotype of the clones is Lyt-1+,2+,3+. Cytotoxic activity of CTLL-A2 and CTLL-R5 as determined by a 4-hr 51Cr-release assay was inhibited over 50% by 1 mM 2-deoxy-D-glucose. CTLL-A11 and the uncultured cytotoxic T cells were more resistant to inhibition by 2DG (40% at 20 mM). Surprisingly, it was found that the addition of D-mannose, D-galactose, D-glucose, L-fucose, alpha-methyl-D-mannose, and N-acetyl-D-glucosamine also inhibited, in a dose-related manner, the cytotoxicity of CTLL-A2 and CTLL-A11. CTLL-R5 showed a more restricted inhibition pattern: only D-mannose and D-galactose were inhibitory. The mechanism of inhibition remains to be clarified.     

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.     

Mechanisms of leukemogenesis. I. Generation of autoreactive lymphocytes in response to a murine leukemia virus.

Examined in this paper is the capacity of 334C murine leukemia virus (MuLV) to stimulate the generation of virus-specific cytotoxic effector cells in mice of the C57BL/6 strain that are relatively resistant to Friend, Moloney, and Rauscher (FMR) MuLV-induced leukemia, and in BALB/c mice that are relatively susceptible to leukemia induced by FMR MuLV. Generation of cytotoxicity requires in vivo administration of the virus followed by in vitro culture of lymphoid cells from virus-injected animals. Lymphoid cells from MuLV-resistant C57BL/6 donors develop high levels of specific cytotoxicity after secondary in vitro stimulation with syngeneic MuLV-induced tumor cells. Cells derived from these same donors, cultured in the absence of MuLV-induced tumor cells, fail to exhibit cytotoxicity. Secondary in vitro stimulation of lymphocytes from MuLV-susceptible BALB/c animals results not only in generation of cytotoxic reactivity against syngeneic MuLV-induced tumor cells but also induces apparently autoreactive effector cells capable of lysing other H-2d tumor cells as well as normal peritoneal cells bearing H-2d antigens. Moreover, generation of cytotoxicity by BALB/c lymphocytes occurs whether or not MuLV-induced tumor cells are included in the secondary culture system.     

Investigation on the effect of peptides mixture from tumor cells inducing anti-tumor specific immune response

The peptides mixture was prepared from tumor cells by freezingthawing cells, precipitation by heating, followed by acidification of the solution. The activation and proliferation of mouse splenocytes by HSP70-peptide complex, formed by the binding of HSP70 and peptides in vitro, were observed, so was the specific cytotoxicity of the proliferative lymphocytes to tumor cells. The phenotypes of the proliferative lymphocytes were analyzed by a flow cytometer. BALB/c mice inoculated with H22 hepatocarcinoma cells in peritoneal cavity or hind thigh were immunized by injection with HSP70-peptides complex to observe the inhibitory effect of the immunization on tumor and lifetime of tumor-bearing mice. On the other hand, blood samples were collected from the immunized mice to check the functions of liver and kidney. The results showed that the peptides mixture from tumor cells contained tumor-specific antigen peptides which could be presented by HSP70 to activate lymphocytes in vitro, the proliferative lymphocytes were T cells which were specifically cytotoxic to tumor cells, the in vivo growth of both ascitic and solid carcinoma could be suppressed by immunization with HSP70-peptides and the lifetime of tumor-bearing mice was prolonged, the in vivo immunization with HSP70-H22-peptides had no impact on the function of mouse liver and kidney, suggesting that there was no occurrence of autoimmunity in vivo after immunization.     

Involvement of lymphocyte mediators in the rejection of a murine tumor allograft

Macrophage migration inhibition by peritoneal leukocytes was studied in BALB/c mice bearing intraperitoneal allogeneic EL-4 lymphomas to explore the role of this immune effector function in allograft rejection. The nonadherent peritoneal leukocyte population harvested between 8 and 10 days after allograft inoculation inhibited migration of nonimmune murine macrophages as demonstrated by both direct and indirect migration assays using the agarose droplet method. This host response also contained large numbers of adherent macrophages which others have shown to be cytotoxic to EL-4 target cells. These findings provide direct evidence for lymphokine activity in allograft rejection and suggest that lymphocyte mediators may attract and activate the cytotoxic macrophages observed in this response.     

Augmentation of syngeneic tumor-specific immunity by semiallogeneic cell hybrids

Hybrid cell lines were established from fusions between lipopolysaccharide- (LPS) stimulated C57BL/6J spleen cells and MPC-11 tumor cells (45.6TG1.7, abbreviated M45), and were tested for their ability to immunize semiallogeneic mice against a parental tumor challenge. These hybrids were tumorigenic in syngeneic (BALB/c X C57BL/6J) F1 (CB6F1) mice but did not grow in semiallogeneic (BALB/c X A/J) F1 (CAF1) mice. All hybrids express both parental major histocompatibility antigens (H-2b and H-2d) as detected by indirect immunofluorescence and by their ability to function as either stimulators or targets for allogeneic cytotoxic lymphocytes (CTL). M45 tumor-associated antigens (TAA) were expressed on the hybrid surface as shown by their ability to act as either stimulators or targets for syngeneic CTL specific for M45 TAA. Immunization of semiallogeneic CAF1 mice with the hybrids i.p. followed by a challenge with M45 tumor cells resulted in extended survival when compared to untreated mice or animals immunized i.p. with M45 tumor cells. This immunity was specific and was not due to an allogeneic effect; immunization with an unrelated H-2bd tumor, 70Z/3, or H-2bd B6D2F1 spleen cells or with semiallogeneic spleen cells plus M45 did not protect mice from M45 challenge. Interestingly, prophylactic priming with semiallogeneic hybrid tumor cells or parental myeloma cells led to M45-specific CTL and "help" for an in vitro CTL response; however, the degree of CTL priming by hybrid tumors was not augmented when compared to the level of CTL achieved with parental tumor alone. Hence, stimulation of CTL activity per se by hybrid tumor cells cannot explain the protective effect of hybrid tumor immunization. These studies nevertheless confirm that semiallogeneic hybrids, which we show express TAA and alloantigens, can be used to immunize mice against a lethal syngeneic myeloma tumor challenge.     

Participation of the humoral immune system in the myeloma-specific transplantation resistance

Studies are reported that examine the participation of humoral and cellular responses in the myeloma-specific transplantation resistance induced by preimmunization of BALB/c mice with purified myeloma protein M315. A plasmacytoma spleen colony-forming assay was used to provide a quantitative estimate of the tumor immunity in recipients of unfractionated spleen cells or serum from mice immunized four to seven times with M315. The primary findings were: 1) that the tumor immunity can be transferred by immune spleen cells provided that a boost of M315 is given to adoptive hosts, and 2) that passively transferred serum containing anti-M315 idiotype antibody (a-Id315) can also inhibit tumor growth. Adoptive transfer was successful in the presence of minute amounts of a-Id315, whereas passive transfer required relatively large amounts of activity. The passive transfer experiments involved an extended injection schedule and thus could not discriminate between direct effects of antibody or idiotype-specific humoral substances on tumor cells or inductive effects on the cellular immune system. Experiments examining the properties of the immune elements involved in the transplantation immunity demonstrated that, once established, they are resistant to acute radiation (to 800 R) and cortisone damage.