Spleens Of Tumor-bearing Mice Research Articles

Altered CD3 chain and cytokine gene expression in tumor infiltrating T lymphocytes during the development of mesothelioma.

The mechanisms whereby tumors escape immunosurveillance remain poorly understood. De-activation or deviation of T lymphocyte responses may occur following exposure to tumor-associated or -derived signals. In the present study it is demonstrated that during development of syngeneic malignant mesothelioma in mice, the relative CD3 delta, CD3 gamma and CD3 zeta mRNA levels expressed by tumor infiltrating lymphocytes (TIL) decrease, while CD3 epsilon mRNA levels remain relatively constant. Expression of IFN gamma mRNA by TIL decreased during tumor development, while IL-2 mRNA levels showed slight increases. IL-3 mRNA was not detected at any time during tumor development and IL-4 transcripts were detected in the later stages of tumor development. In the spleens of tumor-bearing mice, IL-2 transcripts were detected throughout the time course from days 1 to 22(24), while IFN gamma mRNA was only detected at early times from days 0-13. Previous work demonstrated a role for tumor cell-derived TGF beta in the immunobiology of mesothelioma. Here it is shown that the suppression of CD3-subunit expression by TIL was ameliorated in tumors where TGF beta -expression was reduced by inducible TGF beta-specific antisense-RNA, thus, suggesting that lymphocytes may become de-activated upon infiltration of the tumor micro-environment.

Dendritic Cells in Antitumor Immune Responses: II. Dendritic Cells Grown from Bone Marrow Precursors, but Not Mature DC from Tumor-Bearing Mice, Are Effective Antigen Carriers in the Therapy of Established Tumors

Antitumor CTL responses were studied in a model tumor bearing a mutant human p53 gene. We found ineffective induction of antitumor CTL in mice bearing these tumors associated with measurable defects in the function of dendritic cells (DC) from these animals. In this study we investigate the mechanism of this defect in mature DC and find that functional DC can be generated by growth from the bone marrow of tumor-bearing animals. Tumor cell supernatants did not affect the function of mature DC obtained from the spleen of tumor-bearing animals, but significantly suppressed the ability to generate functional DC from the bone marrow of control micein vitro.This suggests that tumor cells may release factors which block early stages of DC maturation from precursors. DC generated from the bone marrow of tumor-bearing mice showed normal potential to stimulate allogeneic T cells, to stimulate anti-mutant p53 peptide-specific cytotoxic T cells, and to induce anti-p53 CTL responsesin vivoin control mice. Repeated immunization with peptide-pulsed DC generated from the bone marrow of control mice (every 4–5 days) blocked progression of established tumors. Immunization of mice with peptide-pulsed DC obtained from the spleen of tumor-bearing mice (4 weeks after tumor injection) did not affect the tumor growth, whereas immunization with peptide-pulsed DC generated from bone marrow of tumor-bearing mice resulted in significantly prolonged survival and delayed tumor growth. Tumor progression was associated with change of the balance Th1/Th2 cells in favor of the Th2-like cytokine profile, while effective immunization was associated with a shift to the Th1 phenotype. Thus, frequent immunization of mice with mutant p53 peptide-pulsed DC generated from stem cells of tumor-bearing hosts can induce effective antitumor CTL responses associated with production of Th1 cells and lead to significant antitumor effects.

Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines.

Antigen presentation by host dendritic cells (DC) is critical for the initiation of adaptive immune responses. We have previously demonstrated in immunogenic murine tumor models that bone marrow (BM)-derived DC pulsed ex vivo with synthetic tumor-associated peptides, naturally expressed by tumor cells, serve as effective antitumor vaccines, protecting animals against an otherwise lethal tumor challenge (Mayordomo, J.I., T. Zorina, W.J. Storkus, C. Celluzzi, L.D. Falo, C.J. Melief, T. Ildstad, W.M. Kast, A.B. DeLeo, and M.T. Lotze. 1995. Nature Med. 1:1297-1302). However, T cell-defined epitopes have not been identified for most human cancers. To explore the utility of this approach in the treatment of tumors expressing as yet uncharacterized epitopes, syngeneic granulocyte/macrophage colony-stimulating factor-stimulated and BM-derived DC, pulsed with unfractionated acid-eluted tumor peptides (Storkus, W.J., H.J. Zeh III, R.D. Salter, and M.T. Lotze. 1993. J. Immunother. 14:94-103) were used to treat mice bearing spontaneous, established tumors. The adoptive transfer of 5 x 10(5) tumor peptide-pulsed DC dramatically suppressed the growth of weakly immunogenic tumors in day 4 to day 8 established MCA205 (H-2b) and TS/A (H-2d) tumor models, when applied in three biweekly intravenous injections. Using the immunogenic C3 (H-2b) tumor model in B6 mice, tumor peptide-pulsed DC therapy resulted in the erradication of established d14 tumors and long-term survival in 100% of treated animals. The DC-driven antitumor immune response was primarily cell mediated since the transfer of spleen cells, but not sera, from immunized mice efficiently protected sublethally irradiated naive mice against a subsequent tumor challenge. Furthermore, depletion of either CD4+ or CD8+ T cells from tumor-bearing mice before therapy totally suppressed the therapeutic efficacy of DC pulsed with tumor-derived peptides. Costimulation of the host cell-mediated antitumor immunity was critical since inoculation of the chimeric fusion protein CTLA4-Ig virtually abrogated the therapeutic effects of peptide-pulsed DC in vivo. The analysis of the cytokine pattern in the draining lymph nodes and spleens of tumor-bearing mice immunized with DC pulsed with tumor-eluted peptides revealed a marked upregulation of interleukin (IL) 4 and interferon (IFN) gamma production, as compared with mice immunized with DC alone or DC pulsed with irrelevant peptides. DC-induced antitumor effects were completely blocked by coadministration of neutralizing monoclonal antibody directed against T helper cell 1-associated cytokines (such as IL-12, tumor necrosis factor alpha, IFN-gamma), and eventually, but not initially, blocked by anti-mIL-4 mAb. Based on these results, we believe that DC pulsed with acid-eluted peptides derived from autologous tumors represents a novel approach to the treatment of established, weakly immunogenic tumors, and serves as a basis for designing clinical trials in cancer patients.

Prevention of Tumor Formation in a Mouse Model of Burkitt’s Lymphoma by 6 Weeks of Treatment with Anti-c-myc DNA Phosphorothioate

Transgenic mice bearing a murine immunoglobulin enhancer/c-myc fusion transgene (Emu-myc) provide a useful model for Burkitt's lymphoma. Groups of 12 Emu-myc mice were treated prophylactically for 6 weeks after weaning with anti-c-myc DNA phosphorothioate (20 mg/kg/day), scrambled control DNA, or saline, delivered by micro-osmotic pumps. Half of the mice treated with saline or scrambled control DNA displayed palpable tumors by 8-9 weeks after birth, and 95% of them did so by 16 weeks, but 75% of the mice treated with antisense DNA were still free of tumors at the age of 26 weeks. Antisense therapy ablated MYC antigen in the spleens of tumor-bearing mice. Plasma physiological parameters indicated no acute toxicity. Long-term tumor resistance after anti-c-myc DNA therapy implies induction of a host response. Prophylactic anti-c-myc DNA therapy might prevent lymphoma in asymptomatic individuals displaying c-myc translocations.

Study of natural lipocortin I. A potent mediator for macrophage-mediated immunosuppression in tumor-bearing mice.

We previously reported a possible role of lipocortin I secreted from Mac-1+, -2+ macrophages in immunosuppression in tumor-bearing mice. In this study, we purified natural lipocortin I from the spleens of tumor-bearing mice and compared its immunosuppressive activity with recombinant lipocortin I produced by Escherichia coli. The culture supernatants of splenic macrophages from tumor-bearing mice suppressed the mitogenic responses in splenic lymphocytes. The culture supernatants contained higher levels of lipocortin I, but not PGE2, which was considered as a major immunosuppressive factor. Anti-lipocortin I antiserum neutralized these inhibitory activities. Natural lipocortin I purified from the spleens of tumor-bearing mice showed a potent suppressive activity, which was > or = 200 times higher than that of recombinant mouse lipocortin I at protein level. Alkaline phosphatase treatment of natural lipocortin I decreased immunosuppressive activity to the level of recombinant lipocortin I. N-glycosidase F treatment failed to decrease the immunosuppressive activities. These results suggest that the protein modification of lipocortin I, possibly phosphorylation but not glycosylation, is critical for the potent immunosuppressive activity in tumor-bearing mice.

Role of colony-stimulating factor-1 in macrophage activation in tumor-bearing mice.

We previously reported a dramatically increased number of macrophages in tumor-bearing mice. In this study, we investigated the involvement of CSF in that phenomenon. CSF-1 responding cells as macrophages precursors increased significantly in number in the spleens of tumor-bearing mice as compared with those in normal mice. Splenic cells and sera from the tumor-bearing mice respectively expressed CSF-1 in mRNA and serum protein levels, but failed to express the other CSF (granulocyte-macrophage-CSF or IL-3). Nonadherent splenic mononuclear cells (< 0.5% macrophages) from normal mice proliferated and differentiated into mature macrophages in culture within 7 days with recombinant mouse CSF-1 (rCSF-1). Both macrophages harvested from tumor-bearing mice and those activated in vitro with rCSF-1 expressed mostly Mac-1, -2 (and -3) Ag, showed yeast phagocytosis, produced IL-1 but not IL-2 or IL-3, and displayed potent cytotoxicity against NK cell resistant Meth-A tumor cells. These macrophages also expressed lipocortin I mRNA and secreted lipocortin I protein, and suppressed mitogenic responses of splenic lymphocytes. rCSF-1-activated macrophages derived from nonadherent splenic cells expressed both CSF-1 and CSF-1 receptor (c-fms) mRNA. Administration of rCSF-1 into normal mice induced hemopoietic and immunologic alternations similar to those observed in tumor-bearing mice. These results suggest that CSF-1 is involved in the dramatic increase of macrophages in tumor-bearing mice, possibly through an autocrine or paracrine loop.

Antitumor effect of a Coliolus preparation, PSK: induction of macrophage chemotactic factor (MCF) in spleens of tumor bearing mice.

The effect of administration of PSK (Polysaccharide Kureha), a Coliolus preparation, in Meth-A solid tumors was analyzed in BALB/c mice. Spleen cells prepared from normal, non-treated Meth-A bearing, PSK-treated normal and PSK-treated tumor bearing mice were examined for induction of macrophage chemotatic factor (MCF). Only spleen cells from the latter mice produced MCF after 48 hrs of cultivation in the presence of Meth-A cells or concanavalin A (Con A). MCF-producing cells were indicated to be Lyt-1 positive, L3T4 positive and Lyt-2 negative cells in the negative elimination assay. There were no differences in the production of other cytokines including interleukin-2, interferon and tumor necrosing factor, spleen cells obtained other different groups of mice. The antitumor effect of either crude or purified MCF (molecular weight 100,000) was examined by daily consecutive intratumoral injections into Meth-A tumor tissues, and a significant inhibitory effect was detected.

Modulation of the immune response to tumors by a novel synthetic compound, (4R)-3-benzoyl-N-[(1R)-1-phenylethyl]-4-thiazolidinecarboxamide (RS-0481).

RS-0481, (4R)-3-benzoyl-N-[(1R)-phenylethyl]-4-thiazolidinecarboxamide, is a compound that can re-establish the function of certain lymphoid cell populations impaired by the presence of a growing tumor in an animal. The compound markedly augmented the tumor-specific cytotoxic T lymphocytes, TDTH (delayed-type hypersensitivity T cells), and the nonspecific lymphokine-activated-killer-cell-like cell responses. It also enhanced the tumor-inhibitory effect of macrophages in tumor-bearing mice, but not in normal mice, indicating that it enhances the antitumor immune responses. Lymphocytes from RS-0481-treated tumor-bearing mice released significantly higher amounts of macrophage-activating factor(s) (MAF) and interleukin-2(IL-2)-like factors in culture compared with lymphocytes from untreated animals. Also, sera from treated tumor bearers showed elevated colony-stimulating factor (CSF) activity. Although the compound did not influence the factor-producing activity in mice without tumor, it enhanced the responsiveness of their bone marrow cells, T cells, and macrophages to CSF, IL-2, and MAF. It seems therefore possible that the compound enhances the responsiveness of immunocompetent cells to cytokines, resulting in a marked augmentation of antitumor T cell responses in tumor-bearing mice. Consistently it inhibited the development of lymph node metastasis of transplanted X5563 plasmacytoma, and we showed that T cells play a decisive role in this inhibition. The compound also counteracted the development of suppressor T cell activity in the spleen of tumor-bearing mice.

Evaluation of recombinant human tumor necrosis factor by cell electrophoresis. II. In vivo effect of recombinant human tumor necrosis factor on cell populations and natural killer‐activity in spleen of tumor‐bearing mice

AbstractThe effect of recombinant human tumor necrosis factor (rH‐TNF) on the composition and natural killer (NK)‐activity of lymphocytes in the spleen of normal and tumor‐bearing CBA mice was investigated. The characterization of cell composition was based on the analytical separation of splenic subpopulations differing in electrophoretic mobility. In untreated tunor mice, an increase of the electrophoretically intermediate population was found at the final stage of tumor growth. Treatment with TNF depressed this intermediate cell population in the spleen of tumorbearing mice, the electrophoretic distribution of splenocytes approaching the normal pattern. Also in normal mice TNF treatment affected the electrophoretic pattern. Already the first injection of TNF induced an increase of the intermediate cell population in the spleen. Tumor‐bearing mice showed a decreased NK‐activity at the final stage. In normal but not in tumor‐bearing mice repeated injections of TNF seemed to depress the NK‐activity. Cell electrophoresis represents a useful method for evaluating the effect of TNF on the cell populations in the spleen of mice.

Antitumor cells found in tumor-bearing mice given ubenimex.

Antitumor effector cells in spleens of tumor-bearing mice given ubenimex were investigated. The administration of ubenimex, starting 7 days after the tumor inoculation, was effective in inhibiting growth of IMC carcinoma. Spleen cells taken from these mice showed a marked suppression of the tumor growth by the WINN assay. The antitumor activity of spleen cells was reduced by treatment to remove T cells or NK cells, whereas spleen cell preparations enriched for T cells showed the strongest antitumor activity. Moreover, NK activity against YAC-1 cells was found in the spleen. These results indicate that the administration of ubenimex to IMC carcinoma-bearing mice generates cytotoxic T cells and NK cells in the spleen. The antitumor effect of ubenimex was not observed in X-ray-irradiated and in anti-asialo GM1 serum-treated mice.

T-cell recruitment from the thymus to the spleen in tumor-bearing mice. II. Functional characteristics of recruited T-cells.

The effect of prostaglandin E2 (PGE2) on characteristics of splenic T-cells in tumor-bearing mice (TBM) was studied from the viewpoint of PGE2-mediated cell-recruitment system from the thymus to the spleen. The splenic T-cells of TBM enhanced tumor growth. In the TBM, the total number of thymocytes decreased, and the number of splenic T-cells concurrently increased. In adult thymectomized tumor bearers, these tumor growth-enhancing T-cells were absent in the spleen, and the number of splenic T-cells remained unchanged. Such suppressor T-cells in the spleens of TBM may relate to recruitment of immature T-cells from the thymus. The levels of PGE2 in the plasma were depressed in the TBM. Replenishment by exogenous PGE2 prevented both an increase in splenic T-cell population and an augmented generation of T-cells that had an enhancing effect on tumor growth. Our findings show that a low level of PGE2 induced the T-cell recruitment from the thymus to the spleen and that such recruited cells led to an acceleration of tumor growth.

T cell recruitment from the thymus to the spleen in tumor-bearing mice. I. Analysis of recruited cells by surface markers.

After inoculation of tumor cells (methylcholanthrene-induced sarcoma), the number of Thy 1+ cells and PNA (peanut agglutinin) binding cells, which were shown to be different subpopulations were increased in the spleen of thymus-intact mice, in contrast this increase was not observed in adult thymectomized mice. In experiments performed concurrently with splenic cell analysis, we found that the plasma PGE2 levels declined in parallel with the tumor growth. Prevention of such a decline of plasma PGE2 level by replenishment with exogenous PGE2 inhibited the splenic cell increase in tumor bearers. In the tumor-bearing mice, cell traffic systems from the thymus to the periphery was ascertained by injecting fluorescein diacetate (FDA) into the thymus and observing fluorescein positive cells in the periphery. We suggest that increased recruitment of thymic cells to the periphery may be mediated by PGE2 in the presence of a tumor.

Kinetic Analysis of the Inhibition of Natural Killer (NK) Activity by Multiple Populations of Tumor-Activated Suppressor Cells

Previous studies have shown that the progressive growth of the M-1 fibrosarcoma in DBA/ 2J mice is associated with the activation of suppressor cells which inhibit both mitogeninduced proliferative responses and antibody synthesis. In this study, we have analyzed the effect of tumor growth on NK cell activity. Mice in the advanced stages of tumor growth did have a significant depression in NK activity, and this depression could not be overcome by the injection of the interferon inducer, polyinosinic-polycytidylic acid (Poly I:C). The decline in NK activity was associated with the presence in the spleens of suppressor cells capable of inhibiting the NK activity of spleen cells from Poly I:C-treated syngeneic mice. In order to characterize the suppressor cells, we used a combination of negative selection procedures and kinetic analysis. These studies demonstrated that the spleens of tumor-bearing mice contained two distinct populations of suppressor cells which were not evident in normal mice. One population was non-adherent to nylon wool, Thy-1 -, non-phagocytic, did not bind target cells, and had a non-competitive mechanism of suppression. The second population was adherent, Thy-1 -, phagocytic, and had a competitive mechanism of suppression. In addition, the spleens of both normal and tumor-bearing mice contained an adherent, non-competitive suppressor cell population which was enriched following negative selection procedures removing T cells or phagocytic cells.

Immunological effects of mouse ceruloplasmin.

Mouse ceruloplasmin, the level of which in serum increases on administration of antitumor polysaccharides, was shown to have enhancing effects on the antibody production of mouse spleen cells toward sheep red blood cells and restorative effects on the suppressed mixed lymphocyte reaction of spleen cells of tumor-bearing mice. The proliferation of interleukin-2 dependent cytotoxic T cells and the suppressed concanavalin A response of the tumor bearing mice spleen cells were also enhanced by this serum glycoprotein, weakly but reproducibly. Furthermore, this serum glycoprotein was found to enhance glucose consumption and interleukin-1 production and cytotoxic activities of mouse peritoneal macrophages. These results suggest that mouse ceruloplasmin plays some roles in the natural resistance surveillance against tumors.

A granulocytosis-inducing tumor inhibits the production of B lymphocytes in murine bone marrow.

Mice bearing a transplantable CE mammary carcinoma have been shown to have greatly augmented rates of neutrophil production coupled with a marked diminution of bone marrow lymphocytes. The objective of the present study was to test whether the loss of lymphocytes, and especially of B cells, from the bone marrow and spleen of tumor-bearing animals was due to a reduced rate of cell production and if so, at what level this response was regulated. A modified 3H-TdR pulse and chase analysis was used to assess the rates of production of small lymphocytes and B cells (stained for c mu and s mu) at weekly intervals after CE tumor transplantation. 3H-TdR was infused continuously for 24 hr, and radioautographs were prepared of bone marrow and spleen cells 0, 24, and 48 hr after termination of the infusion. Pre-B cells (c mu+s mu-) essentially disappeared from the femoral bone marrow by the end of 1 wk of tumor growth, followed by a great reduction in the number of c mu+s mu+ cells in the marrow and s mu + cells in the spleen. Although pre-B cells appeared in the peripheral marrow (caudal vertebrae, metatarsal bones) and spleen of tumor-bearing mice, these cells could not compensate for the continued decrease in the numbers of more mature B cells. In normal mice, during the 48-hr chase period, newly formed, 3H-TdR-labeled, small lymphocytes and s mu+ cells continued to emerge from the prelabeled precursor compartment at a steady rate, but after 1 wk of tumor growth, the number of small lymphocytes and s mu+ cells emerging from the precursor compartment fell steadily during the 48-hr chase period. During the second and third weeks of tumor growth, a steady state appears to have been reached in B cell production, which was at a level approximately 10 times below that of normal. Because pre-B cells are normally maintained by a less mature precursor population (2), the initial disappearance of c mu+s mu- cells suggests that the CE mammary carcinoma exerts its modulatory influence on primary B cell production by inhibiting or eliminating the cells that eventually feed into the pre-B compartment. The nature of the regulatory factors apparently secreted by the tumor and the more precise identity of the target cells are under investigation.

Importance of the concomitant presence of palpable MOPC-315 tumor in stimulation of splenocytes by C-type MOPC-315 virus in Vitro

BALB/c mice inoculated with MOPC-315 tumor cells developed an antiviral response against C-type particles extracted from subcutaneous tumors of plasmacytoma-bearing mice as shown by in vitro stimulation of spleen cells from tumor-bearing mice by virus-containing preparations. Induction of blastogenic response by virus-containing preparations was found to occur in unfractionated spleen cell populations, the glass-wool non-adherent fraction (depleted of macrophages and tumor cells) and the nylon-wool non-adherent (T-enriched) fraction of spleen cells. The antiviral response was no more detectable in spleens of tumor-bearing mice cured by melphalan. Cured mice developed a strong antitumor immune response as shown by their resistance to challenge with a tumorigenic dose of MOPC-315 tumor cells. However, challenge with tumor cells of cured, resistant mice did not induce reappearance of antiviral response.

Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

The spleens of mice bearing large M-1 fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleens of tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1 +, Lyt 1 +2 + and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody response to T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressor cell assays. These data suggest that cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressor cells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors.

Alterations in biosynthesis and homeostatis of cholesterol and in lipoprotein patterns in mice bearing a transplanted lymphoid tumor

The membrane fluidity of murine lymphoid GRSL tumor cells has been shown to depend on their site of growth in the host. Tumor cells located in ascites, in contrast to those in the enlarged spleen, show a very high plasma membrane fluidity, mainly due to a decreased level of cellular (membrane) cholesterol. Yet, the rate of cholesterol biosynthesis in the tumor cells as estimated by the activity of HMG-CoA reductase and the incorporation of [ 14C]acetate into cholesterol was extremely high when compared to various lymphoid cells in normal control mice. Also the rate of biosynthesis and the cholesterol content in liver and spleen of tumor-bearing mice were substantially higher than in the organs of control mice. Blood plasma cholesterol, however, was decreased in tumor-bearing mice as a result of altered lipoprotein patterns. Outgrowth of the tumor was accompanied by a strong reduction in plasma high-density lipoproteins. Low-density lipoproteins became transiently increased, but eventually all lipoproteins, and consequently the plasma cholesterol content decreased to very low levels, especially so in the ascites plasma. The low transfer of [ 14C]cholesteryl ester-labeled lipoproteins between blood and ascites plasma after either intravenous or intraperitoneal injection suggested a hampered flow between the two compartments. Also apparent differences in cholesteryl ester fatty acid composition between lipoproteins of the blood and ascites plasma indicated the lack of a rapid equilibration between the two compartments. The results suggest that the limited availability of lipoproteins as an additional source of cholesterol to the rapidly growing ascites cells promotes their high membrane fluidity.

Effect of melphalan administration on the activity of natural killer and natural cytotoxic spleen cells of normal and tumor-bearing mice

The effect of melphalan ( l-PAM; l-phenylalanine mustard) administration on natural killer (NK) and natural cytotoxic (NC) activities of spleen cells from normal noninoculated BALB/c mice and mice bearing large MOPC-315 plasmacytoma tumors was investigated. Injection of l-PAM (7.5 or 15.0 mg/kg) had no effect on NK and NC activities of spleen cells from normal mice whereas stimulation by PHA was markedly suppressed. NK activity was markedly suppressed in spleens of tumor-bearing mice whereas NC activity was not affected. Therapy with l-PAM of tumor-bearing mice led to transient recovery of NK activity which was possibly related to the effect of l-PAM on macrophage suppressive activity on NK cells. It is concluded that NK and NC cell activities and the response to PHA stimulation are selectively affected by l-PAM administration.

Influence of Mammary Tumor Progression on Phenotype and Function of Spleen and In Situ Lymphocytes in Mice&lt;xref ref-type="fn" rid="FN2"&gt;2&lt;/xref&gt;

Peripheral immunity to an immunogenic chemically induced mouse mammary adenocarcinoma has been demonstrated in the syngeneic host, i.e., BALB/c mice, by several in vivo and in vitro cell-mediated immune assays. Analysis of the responsiveness of the immune cells within the in situ population may prove to be more indicative of the actual interactions between host and tumors. A centrifugal elutriation procedure was used to isolate tumor-infiltrating lymphocytes. A fluorescence-activated cell sorter revealed that most in situ cells were of the T-cell lineage as determined by the absence of surface immunoglobulin (sIg) and the presence of the Thy 1.2 antigen on their surfaces, while in mice with large tumors there were 27.4% Thy 1.2+ and 47.9% sIg+ cells Further studies using fluorescein-conjugated monoclonal anti-Lyt 1 or anti-Lyt 2 antibodies revealed a decrease in the levels of Lyt 1+ cells and an increase in the percentage of Lyt 2+ lymphocytes in the spleens of mice bearing large tumors. Within the Thy 1.2+ population infiltrating mammary tumors, the proportion of lymphocytes presenting the Lyt 1 marker was decreased in comparison to that of spleens of normal mice (0.58 vs. 0.83). At the same time the relative proportions of Lyt 2+ cells within the Thy 1.2+ population was increased in the in situ population (0.50) compared to those of spleens of normal mice (0.28). Examination of the functional abilities of the in situ lymphocytes (ISL) revealed that ISL derived from small tumors responded to the T-cell mitogens phytohemagglutinin (PHA) and concanavalin A (Con A), although at lower levels than those of the splenocytes of the animals from which they were obtained. Responses to PHA were lost rapidly in ISL from large tumors, whereas Con A responses were more durable. In contrast to the spleen cells of tumor bearers, ISL failed to respond to tumor-associated antigens (TAA) at any stage of the disease. The changes in the subsets of T-cells in the spleens of tumor-bearing mice and in ISL may provide an explanation for the decreased activation of ISL by mitogens and for the lack of responsiveness to TAA observed in the splenocytes of mice with large tumors and in the ISL isolated from the mammary adenocarcinomas.