Control of B cell proliferation: inhibition of responses to B cell mitogens induced by plasma cell tumors

A multitude of factors has been described that positively and negatively regulate B cell proliferation. A model system for the study of negative control of B cell function is provided by mice bearing plasmacytomas (PC-mice). In PC-mice, the primary immune response, as measured by development of antibody-forming cells (AFC), is severely suppressed. The present report specifically identifies a block in B cell proliferation as the apparent cause of this reduction in AFC production. Thus, the proliferative response of B cells from the spleens of PC-mice (PC-spleens) was significantly impaired when stimulated with four different B cell mitogens (lipopolysaccharide, Salmonella typhimurium mitogen, anti-mu conjugated to Sepharose, and 8-mercaptoguanosine). Nevertheless, the mitogen-responsiveness of these B cells was recovered when they were segregated by various methods from macrophages. These data suggest that the proliferative ability of the B cells in PC-spleens is inherently normal. In concordance with this conclusion, it was shown that suppressor cells from PC-spleens can block the proliferation of normal B cells derived from nontumor-bearing mice. This inhibition does not require direct cell contact and is mediated via soluble factors. The relevance of these results to previous studies of PC-induced immunosuppression and to the control of normal B cell proliferation is discussed.     

Control of B cell proliferation: arrest of B cells in late G1 underlies immunosuppression induced by plasma cell tumors

Immunosuppression in mice bearing plasma cell tumors (PC-mice) provides a model system for the study of negative B cell regulation. Our previous studies demonstrated that B cell proliferation is suppressed in these mice by a cascade of interactions involving macrophages and soluble factors. The present report pinpoints the G1 phase of the cell cycle as the stage of B cell proliferation inhibited in PC-mice. Modulation of surface immunoglobulin (sIg) with anti-mu, an early membrane activation event, occurred normally on B cells from the spleens of PC-mice. However, examination of the size profile and the expression of sIgD and sIgM on B cells from the spleens of PC-mice showed an accumulation of large-sized, low intensity sIgD+ cells, suggesting a block in B cell activation in the late G1 phase of the cell cycle. This was confirmed by experiments in vitro that demonstrated that although LPS-stimulated B cells from the spleens of PC-mice enlarged to a size characteristic of G1 phase, most did not additionally enlarge into S phase even after 3 days of culture, nor did they incorporate significant amounts of [3H]thymidine. Additional confirmation of a block in late G1 was obtained by using analysis of [3H]thymidine incorporation, cell size, and cell cycle after normal cells were cultured in supernatants from cloned PC lines containing the factor(s) that initiates the cascade of events leading to suppression of B cell proliferation. The relevance of these findings to PC-induced immunosuppression and to the regulation of normal B cell proliferation during the G1 phase of the cell cycle is discussed.     

Immune dysfunction in mice with plasmacytomas. I. Evidence that transforming growth factor-beta contributes to the altered expression of activation receptors on host B lymphocytes.

Plasmacytoma-bearing mice (PC-mice) develop a polyclonal B cell immunodeficiency syndrome characterized by marked impairment of: a) primary antibody responses and b) proliferative responses to B cell mitogens. The present investigations used two-color flow cytometry to examine B lymphocytes from the spleens and lymph nodes of PC-mice and found decreased surface membrane expression of surface IgM (sIgM), transferrin receptors (TfR) and IgE FcR (CD23), increased expression of class II MHC, but normal expression of B220, Mel-14, Fc gamma RII, and Fc mu R. These changes were not related to the H chain class or the amount of Ig produced by the plasmacytoma. When cultured with IL-4, B lymphocytes from PC-mice increased their expression of sIgM and class II MHC, but not of CD23. Several findings implicate transforming growth factor-beta 1 (TGF-beta 1) in the mechanism that modulates receptor expression on B lymphocytes in PC-mice: a) ascites fluid from PC-mice contains large quantities of TGF-beta 1; b) supernatants of cultured spleen cells from PC mice contain up to eightfold more TGF-beta than is found with normal spleen cells; c) cloned plasmacytoma cells produce TGF-beta in vitro; and d) the abnormal phenotype of B cells from PC-mice, i.e., decreased CD23, sIgM, and TfR, and increased class II MHC, is induced on normal B cells cultured in the presence of TGF-beta 1. Because sIgM, TfR, class II MHC, and CD23 are molecules that play fundamental roles in the activation of normal B cells, their modulation by TGF-beta 1: a) identifies molecular mechanisms that could account for some of the known immunosuppressive properties of TGF-beta 1 and b) implicates TGF-beta in the pathogenesis of the polyclonal B cell immunodeficiency that is characteristic of plasma cell tumors.     

Studies of antibody affinity in plasmacytoma-bearing mice: Evidence for a maturational defect of B lymphocytes

The antibody response of plasmacytoma-bearing mice (PC-mice) is severely reduced. In order to understand the nature of the effect of the tumor on the cells making antibody, quantitative and qualitative studies of the humoral response of PC-mice were undertaken. In these studies, the affinity of the antibody produced by tumor-bearing and normal mice was compared to determine whether the small amount of antibody produced by PC-mice is the product of a normal or an altered population of B cells. Antibody to TNP-Ficoll made by PC-mice 3 days after immunization was less heterogeneous and of an affinity lower than that of antibody made by normal mice. However, at 7 days, the antibody made by PC- and normal mice did not differ significantly. These data suggest that, prior to antigenic stimulation, the B cells of PC-mice are relatively immature, reflecting a possible retardation in the generation and turnover of B lymphocytes. The process of antigen-driven selection of high-affinity antibody-producing cells, however, appears to function normally in PC-mice. These studies, then, reveal a qualitative as well as quantitative defect in the primary humoral response of PC-mice which may reflect an abnormality in the development and differentiation of B cells in these mice.     

A partial characterization of suppressor cells in the spleens of mice conditioned with fractionated total lymphoid irradiation (TLI)

Total lymphoid irradiation (TLI) is a highly effective modality for inducing immunosuppression and transplantation tolerance. The cellular basis for this immunosuppression is not clear, although T cells have been implicated. To study further the effect of TLI on the immune system, we have examined the B cells and suppressor cells in the spleens from TLI-conditioned mice. Our results indicate that after TLI, the spleen is rapidly repopulated with many large, immature cells. The probable source of these cells is the shielded bone marrow (BM). The B cells from TLI-conditioned mice are transiently immature and hyporesponsive in vitro to a T-independent antigen. Spleen cells from TLI-conditioned mice nonspecifically suppress the in vitro T-independent anti-TNP response of normal B cells. The suppressor cells lack both B and T cell markers and adhere to Sephadex G-10. The suppressor cells in spleens from TLI-treated mice bear a number of similarities to those present in normal BM. When normal BM cells were analyzed by indirect immunofluorescence for the presence of the Mac-1 antigen, two populations of suppressor cells could be identified: one was Mac-1+ and the other was Mac-1-. These data are consistent with the possibility that a subpopulation of the suppressor cells found in normal BM and in the spleens from TLI-conditioned mice are immature cells of the monocytic/granulocytic lineage.     

Immunologic deficiency during experimental Chagas' disease (Trypanosoma cruzi infection): role of adherent, nonspecific esterase-positive splenic cells

The involvement of adherent splenic cells in the production of deficient lymphocyte responses during the acute phase of experimental Chagas' disease was investigated. When cultured together, purified adherent splenocytes from mice acutely infected with Trypanosoma cruzi caused a significant reduction in the responses of normal mouse spleen cells to T and B cell-specific mitogens. Similar observations were made when infected mouse adherent splenocytes were co-cultured with normal mouse nonadherent cells. Exchange of adherent cells in infected mouse spleen cells suspensions for adherent cells from uninfected mice resulted in increased responses to stimulation with the T and B cell mitogens tested. Treatment of infected mouse cell suspensions with indomethacin improved the responsiveness of these cells to the mitogens. These results support the concept that the immunosuppression that is characteristic of experimental acute Chagas' disease is at least in part mediated by an adherent cell population and is dependent on a prostaglandin-mediated mechanism.     

Effects of the removal of adherent and phagocytic cells on the spleen cell lymphoproliferative response of tumor-bearing mice

Summary Cell-mediated immunity was investigated in two BALB/c mouse tumor systems using the lymphoblastogenesis test with phytohemagglutinin as the mitogen. This lymphoproliferative response was quantitated using the Stimulation Index (SI). There was little evidence for suppressor cell activity in cell mixing experiments in which spleen cells from #51 cell-injected mice were mixed with spleen cells from normal mice. Following macrophage removal by Sephadex G-10 columns and carbonyl iron ingestion, there were no significant changes in the SI values for spleen cells from the #51 cell-injected mice. In contrast, spleen cells from mice injected with H238 cells, a herpes virus-transformed cell line, had a significantly lower SI value than that of normal mice. Suppressor cell activity was demonstrated in cell mixing experiments in which spleen cells from H238 cell-injected mice were mixed with normal spleen cells. Removal of adherent cells from spleen cells from H238 cell-injected mice by Sephadex G-10 columns restored the SI value to that of normal mice. An increased SI value was also seen after removal of phagocytic cells by carbonyl iron. These results suggested that cells with the functional properties of macrophages played an important part in the immunosuppression observed in the H238 tumor system. Comparison of the two macrophage depletion methods suggested that another cell population was also involved in the suppressive effect. Results of immunofluorescent techniques with anti-Lyt-1 and anti-Lyt-2 monoclonal antibodies show these cells to be Ly 1^–, Ly 2,3⁺ phenotypes of T-lymphocytes.     

Trypanosoma cruzi-induced suppressor substance III. Activation of suppressor cells

Serum from mice infected with Trypanosoma cruzi (SSS) is known to interact with normal spleen cells to induce an immunosuppressed condition and activate splenic suppressor cells. The induction of immunosuppression by SSS was shown to be independent of, and precede the activation of, suppressor cells. Suppressor-cell activation, however, was demonstrable only after the induction of immunosuppression. Furthermore, mice that were given two aliquots of SSS at different intervals of time, exhibited suppression of humoral responses of similar duration and magnitude, regardless of the SSS transfer regimen, whereas both the length and degree of suppressor-cell activity was critically dependent on the interval of time between SSS transfers. SSS interacted with spleen cells via a trypsin-sensitive membrane site which was regenerable within a 4- to 5-hr period, yet the suppressive effects of SSS on spleen cells following interaction was resistant to treatment with trypsin. The interaction between SSS and spleen cells during brief adsorption protocols leads to immunosuppression only because extensive washing of SSS-treated spleen cells did not reverse the immunosuppression process, but did prevent the development of detectable suppressor cells. The phenomenon of suppressor-cell activation was further distinguished from immunosuppression in that supernates from culture of spleen cells derived from SSS-treated mice or T. cruzi-infected contained a factor that activated suppressor cells, but did not directly induce a state of suppression in the responding cell population.     

Plasmodium berghei: Reduction of the mouse's specific lymphoproliferative response in relation to corticosterone and pregnancy

Spleen cells from mice immune to Plasmodium berghei exhibited a significantly increased in vitro proliferative response to parasitized reticulocytes compared to spleen cells from normal mice. The specific response to malaria antigen was decreased in spleen cells from pregnant immune mice in contrast to the nonspecific response to the mitogen phytohemagglutinin. Addition of mouse serum to spleen cell cultures of immune mice depressed both the phytohemagglutinin and the specific proliferative response, whereas serum of pregnant mice exerted an even stronger inhibition than serum of nonpregnant mice. Charcoal adsorption of mouse sera for the elimination of steroid hormones removed the serum dependent immunosuppression from normal as well as pregnant serum. Corticosterone added to the spleen cell cultures depressed also the proliferative response. These findings demonstrate that the response to malaria antigen is decreased in immune mice during pregnancy. The possible effect of serum corticosterone on the depression of the immune response is discussed.     

Prostaglandin synthesis by lymphoid tissue of mice experiencing a graft-versus-host reaction: relationship to immunosuppression

Studies were carried out on the induction of PGE synthesis during the GVH reaction and its role in GVH-induced immunosuppression. The results demonstrated that spleen, lymph node cells and, to a much lesser degree, thymus cells obtained from adult C57BL/6 × AF₁ mice treated with 50–75 × 10⁶ C57BL/6 lymphoid cells were stimulated to produce PGE during the course of the GVH reaction. The spleen and lymph node PGE production peaked at Day 9 post-GVH induction (30- and 15-fold higher than normal, respectively). Thereafter, it declined to near normal levels by Days 25–30 post-GVH induction. Passage of GVH spleen cells through a rayon column removed macrophages but not mitogen-responsive T and B cells and also removed nearly all of the PGE-producing cells, except during the later course of the GVH reaction. Removal of PGE-producing cells from GVH-immunosuppressed spleen cells significantly reconstituted the mitogen response to PHA and LPS. Treatment of mice experiencing a GVH reaction with indomethacin delayed the onset of suppression of the plaque-forming cell response to sheep erythrocytes. These results suggest that early GVH-induced immunosuppression which may represent an amplified normal regulatory mechanism is mediated by increased macrophage production of PGE which suppresses both B- and T-cell functions, whereas at later stages other immunosuppressive mechanisms become operational.     

Graft-versus-host induced immunosuppression: depressed T cell helper function in vitro.

The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.     

Factors influencing hematopoietic spleen colony formation in irradiated mice. IV. The effect of erythropoietic stimuli

A variety of erythropoietic stimuli influenced the number of endogenous spleen colonies in irradiated mice and the number of transplantable colony forming cells in the spleen and marrow of unirradiated mice. Bleeding was the most effective stimulus. Bleeding before irradiation resulted in a 30-fold increase in endogenous spleen colonies and in increases in spleen weight, spleen iron and iododeoxyuridine uptake and volume of packed red cells ten days after irradiation. Bleeding unirradiated mice produced a 10-fold increase in the number of transplantable colony forming cells in the spleen and a slight decrease in the total number in the humerus. Bleeding before irradiation resulted in a significant reduction in 30-day post irradiation deaths, an effect abolished by splenectomy. Plasma from bled mice induced an increase in endogenous colonies when injected before irradiation into normal mice. Injection of erythropoietin, testosterone or testosterone plus cobalt induced effects which were, in general, qualitatively similar to those of bleeding, although they were less effective quantitatively. Except for a slight effect induced by ten injections of erythropoietin, post-irradiation stimulation in normal mice proved ineffective. Erythropoietin increased colony numbers and spleen iron uptake when given after irradiation to hypertransfused mice. The results of these studies do not support the concept that the colony forming cell and the erythropoietin sensitive cell are separate entities.     

Enhanced interferon-alpha/beta (IFN-alpha/beta) and defective IFN-gamma production in chronic graft versus host disease: a potential mechanism for immunosuppression

Immunosuppression is a well-characterized consequence of chronic graft-versus-host disease (GVHD). We have previously shown that interferon (IFN) is produced in high levels during acute GVHD. Our objective in this study was to determine if IFN, as a cytokine with known immunosuppressive qualities, could be detected in mice experiencing chronic GVHD-induced immunosuppression. Two different experimental models were used to induce chronic GVHD. The first model involved the injection of parental strain spleen cells into adult F1 hybrids (AJ----B6AF1), while the second model utilized GVHD induced across minor histocompatibility barriers (B10.D2----BALB/c). Results indicated that significant levels of serum IFN-alpha/beta are present in mice undergoing chronic GVHD. Spleen cells from chronic GVHD mice were also shown to produce significant levels of IFN-alpha/beta upon in vitro culture in medium only. This IFN-alpha/beta production was greatly increased when GVHD spleen cells were cultured with either concanavalin A (Con A) or IL-2. In contrast, IFN-gamma production was undetectable in these Con A- or IL-2-containing cultures. Additionally, these same spleen cells which produced high levels of IFN-alpha/beta were immunosuppressed as measured by mitogen-induced cell proliferation. These results suggest that IFN-gamma production is defective in GVHD spleen cells, and that the presence of high IFN-alpha/beta production by GVHD mice may contribute to the immunosuppression associated with chronic GVHD.     

Changes in spleen histology in response to antigenic stimulation in the snapping turtle,Chelydra serpentine

Proliferative and migratory changes of lymphoid cells in the spleen were observed in turtles stimulated with KLH and maintained at 30°C. Small foci of pyroninophilic lymphoblasts were first seen in the white pulp at about day 5. Progressive enlargement of these centers continued and peaked by days 8-12. By days 15-20 the white pulp returned to a normal (unimmunized) state, while the number of pyroninophilic cells, primarily plasma cells, increased markedly in the red pulp. At days 22-25, the number of plasma cells returned to normal levels and the spleen appeared normal for the remainder of the 60 day observation period. These events suggest that at 30°C,-the turtle is capable of a strong and prompt proliferative response in the white pulp sheaths, followed by migration and differentiation of lymphoblasts into plasma cells, n the red pulp., Observations of pyroninophilic cells in sinuses, venules and veins of the spleen and a concomitant depletion of cells in red pulp, further suggest a migration from the spleen to other sites. Following a second antigenic challenge, at day 60, no significant histological changes were observed at 30°C. Nor were any changes observed following primary or secondary antigenic challenge, in animals maintained at 10°C. These findings are discussed with regard to immunological memory and low temperature immunosuppression in ectothermic vertebrates.     

Suppression of in vitro antibody responses by Listeria primed spleen cells.

Spleen cells from mice primed with virulent Listeria monocytogenes do not develop an anti-SRBC plaque forming cell response to SRBC in culture. Furthermore, when Listeria primed spleen cells are co-cultured with normal spleen cells and SRBC, the anti-SRBC response of the normal cells is suppressed. Listeria primed spleen cells from T cell depleted donors are equally effective at immunosuppression. The immunosuppressive effect does not appear to be due to the presence of the bacterium or its products per se in the cultures. Furthermore, the effect cannot be transferred across a 0.45 μm pore membrane. Kinetic studies show that the immunosuppressive effect develops by 2 days post-Listeria inoculation and peaks by Day 6. Low doses of Listeria are not immunosuppressive and produce some enhancement effect. From these results, it is suggested that a population of non-T cell dependent cells develop in Listeria primed hosts that nonspecifically suppress the response of B cells to an unrelated antigen in culture.     

The Peripheral Myeloid Expansion Driven by Murine Cancer Progression Is Reversed by Radiation Therapy of the Tumor

Expansion of myeloid-lineage leukocytes in tumor-bearing mice has been proposed as a cause of systemic immunosuppression. We demonstrate that radiation therapy of tumors leads to a decline in myeloid cell numbers in the blood and a decrease in spleen size. The frequency of myeloid cells does not decline to the level seen in tumor-free mice: we demonstrate that metastatic disease can prevent myeloid cell numbers from returning to baseline, and that tumor recurrence from residual disease correlates with re-expansion of myeloid lineage cells. Radiation therapy results in increased proliferation of T cells in the spleen and while T cell responses to foreign antigens are not altered by tumor burden or myeloid cell expansion, responses to tumor-associated antigens are increased after radiation therapy. These data demonstrate that myeloid cell numbers are directly linked to primary tumor burden, that this population contracts following radiation therapy, and that radiation therapy may open a therapeutic window for immunotherapy of residual disease.     

Stimulation of lymphoid cells from normal and immune mice by syngeneic BALB/c plasma cell tumors.

Lymphoid cells from normal and immunized BALB/c mice could be stimulated in vitro by syngeneic PCT contrasted with an absence of response to a number of other tumors. Maximal responses of normal cells to PCT were found to occur 5 days after the initiation of the cultures at an optimal responding:stimulation cell ratio of 1:2. MLTI activity of normal cells could not be blocked or enhanced by PCT myeloma protein products indicating that MLTI reactivity was directed against non-idiotypec cell surface determinants. Lymphoid cells from immunized mice demonstrated increased MLTI responses to cells of the immunizing tumor but not to other PCT, indicating that the post-immunization MLTI responses were primarily to individual rather than shared tumor cell surface antigens. Activity of both normal and immunized spleen cells was found to involve thymus-derived lymphocytes. The persistence of residual MLTI activity after treatment with anti-theta serum and complement, however, implicated participation of non-theta antigen-bearing cells in MLTI reactivity. From these data, we conclude that lymphoid cells from un-immunized mice are capable of T cell-dependent reactivity to syngeneic PCT-associated antigens and that elevations in these reactivities after immunization may reflect specific cellular immune responses.     

Mechanisms of tumor-induced immunosuppression: evidence for contact-dependent T cell suppression by monocytes.

BACKGROUND: The progressive growth of tumors in mice is accompanied by down-regulation of specific T cell responses. The factors involved in this suppression are not completely understood. Here, we have developed a model to examine the role of host immune effector cells in the inhibition of T cell function. In this model, progressive growth of a colon carcinoma line, CT26, is accompanied by loss of T cell response to alloantigens in both cytolytic and proliferation assays. MATERIALS AND METHODS: The CT26 tumor was inoculated into BALB/c syngeneic mice. Tumor growth, cytolytic T cell responses, lymphocyte proliferation, and flow cytometric analysis was performed in tumor-bearing animals 7 or 28 days after tumor inoculation. RESULTS: Spleen cells from tumor-bearing mice were found to suppress the proliferative response of spleen cells from normal mice to alloantigens. Examination of the spleen cell population by FACS analysis revealed an increase in the percentage of monocytes as defined by expression of CD11b, the Mac-1 antigen. Removal of the Mac-1-positive cells from the tumor-bearing hosts spleen relieved suppression of the tumor-bearing mouse spleen cell proliferative response to alloantigens, and addition of the Mac-1-positive enriched cells suppressed proliferation of normal T cells in response to alloantigens. Cell contact was required for this inhibition. CONCLUSIONS: Tumor induction of suppressive monocytes plays an important role in the general immunosuppression noted in animals bearing CT26 tumors. Identification of the mechanisms responsible for this effect and reversal of tumor-induced macrophage suppression may facilitate efforts to develop effective immunotherapy for malignancy.     

Experimental cutaneous leishmaniasis. I. Nonspecific immunodepression in BALB/c mice infected with Leishmania tropica

Leishmania tropica in BALB/c mice causes a fatal infection accompanied by the development of multiple metastatic lesions. Spleen cells from these mice were shown to have depressed proliferative responses to concanavalin A (Con A), phytohemagglutinin (PHA), and lipopolysaccharide (LPS). Coinciding with this immunodepression was the development of a cell population capable of suppressing normal spleen cell responses to Con A. This suppressor cell activity was first observed at 6 wk and was present throughout the remainder of the infection. At 12 wk the suppressor cells could be removed by Sephadex G-10 passage or carbonyl iron treatment; however, Sephadex G-10 passage could not reverse the suppression at 18 wk. Indomethacin, a prostaglandin synthetase inhibitor, was found to abrogate the activity of the adherent suppressor cell, suggesting that prostaglandin production may be involved in the immunosuppression seen in these mice. In addition, Sephadex G-10 passage and indomethacin were found to markedly augment spleen cell responses to leishmanial antigen, indicating that the adherent suppressor cell is capable of regulating specific immunologic responses.     

TCR zeta down-regulation under chronic inflammation is mediated by myeloid suppressor cells differentially distributed between various lymphatic organs

T cell AgR zeta chain down-regulation associated with T cell dysfunction has been described in cancer, infectious, and autoimmune diseases. We have previously shown that chronic inflammation is mandatory for the induction of an immunosuppressive environment leading to this phenomenon. To identify the key immunosuppressive components, we used an in vivo mouse model exhibiting chronic inflammation-induced immunosuppression. Herein, we demonstrate that: 1) under chronic inflammation secondary lymphatic organs display various immunological milieus; zeta chain down-regulation and T cell dysfunction are induced in the spleen, peripheral blood, and bone marrow, but not in lymph nodes, correlating with elevated levels of Gr1(+)Mac-1(+) myeloid suppressor cells (MSC); 2) MSC are responsible for the induction of such an immunosuppression under both normal and inflammatory conditions; and 3) normal T cells administered into mice exhibiting an immunosuppressive environment down-regulate their zeta expression. Such an environment is anticipated to limit the success of immunotherapeutic strategies based on vaccination and T cell transfer, which are currently under investigation for immunotherapy of cancer.