Clonal analysis of expression of tumor-associated transplantation antigens and of metastatic capacity

Summary ESb, a spontaneous high metastatic variant of the chemically induced T lymphoma Eb, was found previously to express a tumor-associated transplantation antigen (TATA) that was different from that of the parental line. Syngeneic tumor-specific cytolytic T lymphocytes (CTL) were able to recognize the different TATAs of Eb and ESb in vitro and could therefore be used for routine typing. The object of this study was to investigate tumor antigen expression on a clonal level and to compare the in vitro data with the in vivo behavior of the same cell lines.Our CTL typing analysis of cloned tumor lines revealed that the two populations, Eb and ESb, are distinct and relatively homogeneous with regard to their TATA expression. Furthermore, all ESb clones formed rosettes with antibody-coated erythrocytes, while none of the parental type Eb clones showed this characteristic. The sensitivity to tumor-specific CTL lysis varied with time of tumor cell culture in vitro in a clone-dependent manner.Variability was also noted in vivo in tumor growth and metastatic spread. Of over 50 ESb clones tested, the majority were highly metastatic while a minority were significantly lower in metastatic capacity. High and low metastatic ESb clones could not be distinguished by their expression of TATAs and of Fc receptors. There was also a considerable individual variability in the hosts, although they were genetically identical. This variability was most probably due to differences in the immune status of the animals.     

Characterization of cellular and extracellular plasma membrane vesicles from a low metastatic lymphoma (Eb) and its high metastatic variant (ESb): inhibitory capacity in cell-cell interaction systems

Spontaneously shed extracellular plasma membrane vesicles (ECM) of a highly metastatic murine tumor line (ESb) were compared with plasma membrane vesicles (PM) of the same cells prepared by the nitrogen cavitation method and with ECM and PM preparations of the related low metastatic tumor line Eb. From a previous biochemical analysis it was concluded that the exfoliation of ECM vesicles, which is very pronounced in metastatic ESb cells, is not a random process. This conclusion is further corroborated by the present functional analysis. Compared to ESb PM, ESb-derived ECM were selectively enriched for Fc receptors and depleted in glycoproteins with affinity for hepatocytes. Tumor-derived ECM carried the same tumor antigen as the corresponding tumor line and showed in comparison to PM material an increased inhibitor capacity in a T-cell-mediated tumor-specific cytotoxicity test.     

Fc receptors on mouse effector cells mediating natural cytotoxicity against tumor cells.

Mouse effector cells mediating natural cytotoxicity against tumor cells have been previously thought to be lymphocytes that lack any detectable cell surface markers. The present study presents evidence for receptors for the Fc portion of IgG on these cells. By adsorption of cytotoxic spleen cells on monolayers of sheep erythrocytes (E) plus IgG antibodies to sheep erythrocytes (EA), 50 to 96% of the total cytotoxic reactivity could be removed. Parallel adsorption of cells on E monolayers or on EA monolayers coated with protein A, to block the Fc portion of IgG, resulted in little or no depletion of cytotoxic activity. The presence of Fc receptors on the NK cells was confirmed by combining EA rosette formation with velocity sedimentation at unit gravity. Peak cytotoxicity occurred at the same sedimentation velocity as the peak of Fc-positive cells. After EA rosette formation, there was a shift to a higher sedimentation velocity in the Fc-positive cells and in the natural cytotoxic activity. The increase in sedimentation velocity of NK activity that was observed in these experiments indicated that most of the cells had only bound a small number (three or four) of antibody-coated erythrocytes. Together, these data indicate that cells with Fc receptors account for most of the total lytic activity of normal mouse spleen cells.     

Isotype Specificity and Heterogeneity of Fcγ-Receptors on Guinea Pig Splenic B and T Lymphocytes

The isotype specificity of Fc-receptors for IgG (Fcγ-Rs) on normal guinea pig splenic B and T cells was determined by a rosette assay using sheep erythrocytes sensitized with either homologous IgG1 or IgG2 anti-sheep erythrocyte antibody [EA(IgG1) or EA(IgG2)]. Approximately 70% of the lymphocytes in the highly purified B-cell fraction could form rosettes with EA(IgG2), and 55% of the cells with EA(IgG1). Inhibition experiments with soluble complexes of IgG1 or IgG2 antibody with ovalbumin demonstrated that approximately 20% of the EA(IgG2) rosette-forming B cells bore the Fcγ-R monospecific for IgG2, whereas 80% of the cells had two distinct Fcγ-Rs simultaneously; one monospecific for IgG2 and the other bispecific for IgG1 and IgG2. The existence of a B cell bearing the Fcγ-R monospecific for IgG1 was not definitively demonstrated in the B-cell fraction. In the T cell-enriched fraction, approximately 40% of the cells could form rosettes with EA(IgG2). The EA(IgG1)rosette-forming cells, however, comprised only 6% of the total cells, indicating that most of the EA(IgG2) rosette-forming T cells bear essentially the Fcγ-R monspecific for IgG2 alone. The results obtained revealed that guinea pig splenic lymphocytes bear two distinct Fcγ-Rs, which are not equally distributed on the B- and T-cell populations and also on their respective subsets.     

Murine IgA binding factors produced by Fc alpha R(+) T cells: role of Fc gamma R(+) cells for the induction of Fc alpha R and formation of IgA-binding factor in Con A-activated cells

The relationship between the production of a T cell factor having affinity for IgA (IgA-binding factor(s); IgA BF) and the expression of Fc receptors specific for IgA (Fc alpha R) was studied by using murine spleen cells activated with concanavalin A (Con A blasts). Fc alpha R was detected by the cytophilic binding of anti-TNP murine IgA myeloma protein (MOPC 315 IgA) to Con A blasts as determined by an indirect rosette method with trinitrophenylated sheep red blood cells (TNP-SRBC). After 18 hr preculture with IgA, Fc alpha R was expressed on 15 to 20% of Con A blasts, which released IgA BF suppressing the in vitro IgA synthesis of the spleen cells stimulated with pokeweed mitogen (PWM). Without preculture with IgA, there was neither induction of Fc alpha R nor the production of IgA BF from Con A blasts. Fc alpha R was not induced on Con A blasts by IgA if Fc gamma R(+) cells were depleted from the blasts by rosetting with SRBC sensitized with rabbit IgG antibody (EA gamma). Even after preculture with IgA, the suppressive IgA BF was undetectable in the culture supernatant of Con A blasts depleted of the Fc gamma R(+) cell population. By using a double rosette method with EA gamma and trinitrophenylated quail red blood cells, Fc alpha R proved to be co-expressed on Fc gamma R(+) precursor T cells in the Con A blasts. The results suggested that both Fc gamma R and Fc alpha R could be co-expressed on Con A blasts, as is the case with T2D4 Fc gamma R(+), Fc alpha R(+) T hybridoma cells, which are known to produce IgG-binding factor(s) (IgG BF) and IgA BF. The relationship between Fc gamma R and Fc alpha R on a single cell was studied by using monoclonal anti-Fc gamma R antibody (2. 4G2 ). The reactivity of 2. 4G2 antibody with T cell Fc gamma R was proved by the inhibition of EA gamma rosette formation by Con A blasts or T2D4 cells. The addition of 2. 4G2 monoclonal antibody, however, did not affect the induction of Fc alpha R on Con A blasts by IgA. Furthermore, the binding of IgA to Fc alpha R already expressed on L5178Y T lymphoma cell line cells was not inhibited by the monoclonal antibody. The results confirmed that Fc alpha R are distinct from Fc gamma R co-expressed on the same Con A blasts, and that the expression of Fc alpha R on Fc gamma R(+) T cells and their production of suppressive IgA BF may be induced by the binding of IgA to Fc alpha R.     

New antigens presented on tumor cells can cause immune rejection without influencing the frequency of tumor-specific cytolytic T cells

The specific immune response against syngeneic tumors by T cells is dependent on the existence of tumor-associated transplantation antigens (TATA). In the case of the chemically induced DBA/2-derived lymphoma Eb and its highly metastatic variant ESb the immunogenicity of these antigens is not sufficient to prevent tumor growth. Therefore we tested in two systems the influence of additional antigens as possible helper determinants for the generation of tumor-specific immune responses. In the Eb tumor system additional antigens were induced by mutagenization. The frequency of cytotoxic T lymphocytes (CTL) in response to mutagenized Eb cells was higher than that in response to untreated Eb cells. Fine specificity analysis revealed there there was no increase in the CTL response against the original TATA, but an activation of additional CTL clones responding to mutagen-induced antigens. In the ESb tumor system we tested the effect of additional recognition of minor histocompatibility antigens on the frequency of TATA-specific CTL. Transplantation of ESb tumor cells into B10.D2 mice, which are H-2-identical but differ in minor antigens, results in strong tumor rejection responses. In a limiting dilution mixed-leukocyte-tumor microculture system it was found that the minor antigens are recognized at the clonal level as independent antigens. The overall frequency of anti-tumor CTL in ESb-immunized B10.D2 mice was about 1/3000. Among these, the frequency of TATA-specific CTL was 1/16,709 and thus not significantly different from that of syngeneic DBA/2 mice. Thus neither minor antigens nor mutagen-induced antigens acted in the Eb/ESb tumor system as helper determinants and did not increase the frequency of tumor-specific CTLs.     

Prolongation of survival of mice bearing the Eb and ESb lymphoma by treatment with interferon inducers alone or in combination with Corynebacterium parvum

Summary The objective of this study was to evaluate if pretreatment with Corynebacterium parvum (C. parvum) augments the effects of interferon (IFN) inducers on survival of DBA/2 mice transplanted with two syngeneic lymphoma variants, the low metastatic Eb and the high metastatic ESb tumor. The involvement of IFN in the treatment effects was investigated. As inducers of IFN-/ Newcastle disease virus (NDV), polyinosinic-polycytidylic acid (polyI:polyC), and 10-carboxymethyl-9-acridanone (CMA) were injected i. p. at the site of tumor transplantation. The Eb tumor was found to be sensitive to the antiproliferative action of IFN-/ in vitro. In vivo single injections of each of the inducers retarded growth of the Eb tumor. In C. parvum-pretreated mice the effects of the inducers on survival were markedly increased. There was a correlation between prolonged survival and local IFN levels in response to polyI:polyC or CMA but not upon NDV. Injections of each of the inducers increased cytotoxicity of peritoneal exudate cells against the Eb tumor cells in vitro especially when mice were pretreated with C. parvum. Although other mechanisms cannot be excluded IFN-mediated activation of host defence and also direct antiproliferative effects of endogenously produced IFN seem to be involved in the antitumor effects by these IFN inducers in the Eb model. In the ESb tumor model irrespective of additional pretreatment with C. parvum survival was only slightly prolonged by the treatments and endogenous IFN induction did not result in any real benefit for the animals. When compared with Eb cells the ESb cells were less sensitive to the antiproliferative action of IFN-/ in vitro and less sensitive to in vitro cytotoxicity by the host cells. Although other mechanisms may additionally be active in vivo the different susceptibility of the Eb and ESb tumor cells to the direct and indirect actions of IFN seems to contribute to the different responsiveness of these tumor cell lines to the treatments with IFN inducers.     

Effects of anti-beta-2 microglobulin antibodies on human lymphocytes.

Anti-β2 microglobulin antisera prepared in rabbits immunized with β2m purified from the urine of a single patient were cytotoxic for human T and B lymphocytes of all donors tested; lymphocytotoxicity could be fully inhibited by all human sera tested, not by serum from other animal species. Anti-β2 microglobulin antibodies and their F(ab′)2 fragments had little effect on E and EAC rosette formation, suggesting that β2m is not closely associated with receptors for sheep erythrocytes on T lymphocytes or receptors for C3 on B cells. Anti-β2m IgG and F(ab′)2 fragments inhibited EA rosette formation though the latter did not impair lysis of antibody-coated xenogeneic erythrocytes by lymphocytes bearing receptors for the Fc portion of IgG. Some of the antisera had a mild mitogenic effect, all of them inhibited mitogen and antigen-induced lymphocyte proliferation at high concentrations whereas they potentiated these responses at low concentrations. In mixed lymphocyte cultures pretreatment of responding cells markedly depressed the response whereas coating of stimulating cells with β2m antibodies had little or no effect.     

Evidence for clustering of H-2K, H-2D, and the Fc receptor on the membranes of B cells.

Alloantisera to H-2K, H-2D, and Ia antigens markedly inhibited the binding of EA but not FITC-IgG by the B cell Fc receptor. EA rosette formation approached normal levels when masked H-2 but not Ia specificities were allowed to cap on the membranes of B cells. beta2-mu coated SRBC were bound by the Fc receptor, and high concentrations of soluble beta2-mu were found to moderately inhibit EA rosette formation while lower concentrations enhanced binding. The data support the concept of Fc/Ia identity, and they suggest that H-2K, H-2D, and the Fc receptor may be closely grouped on the membranes of B cells. Further, these observations suggest that the beta2-microglobulin associated with H-2 could serve to link T cells with the Fc receptor of B cells during the inductive phase of antibody synthesis.     

Marker profile, enzyme activity, and function of a human myelomonocytic leukemia cell line.

Morphological and functional characteristics of a permanent human leukemia cell line (DD) that possesses myelomonocytic features were investigated. The cells bear a second type Fc gamma receptor and form rosettes with sheep erythrocytes sensitized with rabbit IgG (EA). However, the surface-bound EA is not internalized. The cell line lacks the surface markers CD2, CD19, CD14, HLA-DR, Fc gamma receptor I, Fc gamma receptor III, and CR3. alpha 1-Antitrypsin, lysozyme, Factor XIII a subunit of blood coagulation, and acid phosphatase reactions were negative. A terminal differentiation of the DD cell line was observed when the expression of CD14, CR3, Fc gamma receptor I, and Fc gamma receptor III was induced. The DD cells induced with 12-O-tetradecanoylphorbol-13-acetate or Escherichia coli lipopolysaccharide can internalize EA via Fc gamma receptor II and complement-coated yeast in the function of the inducers. The phagocytic ability appears to be parallel with the appearance of enzymes which participate in phagocytosis.     

Transfer of long-lasting tumor immunity by immune T cells from MHC congenic mice: Migration,survival and tumor-protectivity of cytotoxic donor cells

Immunocompetent B10.D2 (H-2^d) mice are able to reject the highly malignant lymphoma ESb of DBA/2 (H-2^d) origin very effectively. Seven days after intravenous injection of the ESb tumor cells, B10.D2 mice developed a strong tumor-rejection response which was associated with the generation of anti-tumor T cells in their spleens with direct cytotoxic activity. Most of the cytotoxic potential was directed against the minor histocompatibility differences as demonstrated by the lysis of unrelated DBA/2 derived Eb tumor cells and normal DBA/2 but no B10.D2 derived ConA lymphoblasts. A previously performed clonal analysis, however, revealed a minority population of CTL clones which specifically recognized the ESb specific transplantation antigen (ESb-TATA). When transferred systemically into DBA/2 mice, the B10.D2 anti-ESb immune spleen cells could delay the outgrowth of s.c. transplanted ESb tumor cells. When the ESb tumor cells were experimentally distributed in a s.c. implanted sponge-matrix, the i.v. injected B10.D2 immune cells could confer complete protective immunity against the metastatic tumor, provided the recipients were pre-treated with 5 Gy to allow a better take of the allogeneic cells. The distribution of intravenously injected B 10D2 donor spleen cells was assessed in the recipients up to 50 days by cytotoxicity testing and assaying for the expression of the ₂ microglobulin allelic form b ( ₂m^b). These tests revealed a high propensity of donor cells to populate the spleen and lymph nodes of the DBA/2 recipients. Again this was particularly marked in sublethally irradiated mice where a long-lasting lymphoid chimerism was established.     

An appraisal of Fc receptors on human peripheral blood B and L lymphocytes.

Human circulating lymphocytes with easily detectable surface immunoglobulin have been divided into two populations, B cells and L cells. This second population lacks membrane-incorporated Ig, but has a receptor for membrane-labile cytophilic IgG. In this study purified B and L lymphocytes were examined for Fc receptors that bind aggregated IgG and IgG complexed to erythrocytes. Purified lymphocyte populations were prepared by nylon columns and by negative selection with rosetting techniques. L lymphocytes bound aggregated guinea pig and human IgG, and formed rosettes with human erythrocytes sensitized with Ripley IgG (EA). Treatment of L lymphocytes with trypsin had no effect on the receptors for IgG. B lymphocytes did not bind EA and attachment of aggregated IgG was variable; up to one-third of these cells fixed aggregated human IgG to the cell membrane. Trypsin treatment abolished binding of Agg-IgG to B cells in sharp contrast to its effect on L cells. Furthermore, double-label immunofluorescence studies showed that cells with both membrane-incorporated Ig and receptors for aggregated guinea pig IgG were rare. These studies indicate that human peripheral blood B lymphocytes lack a high affinity, trypsin-resistant Fc receptor that is present on L lymphocytes.     

Immunobiology of the human placenta. I. IgGFc receptors in trophoblastic villi.

Qualitative and quantitative studies were performed on IgGFc receptors in the trophoblastic villi of human placentae ranging in gestational age from less than 4 weeks to full term. IgGFc receptors were detected on cells of the syncytiotrophoblast (ST) using two different assay systems; EA rosette formation and direct immunofluorescence with deaggregated human IgG. The ST IgGFc receptors had high affinity for native IgG molecules (deaggregated IgG) as well as affinity for antigen-antibody complexes (EA). The receptors for deaggregated IgG were present on a majority of ST cells in first and second trimester trophoblast, but were significantly less frequent on ST cells in older placentae. Similar receptors also were detected on cells lining some fetal vessels in the trophoblastic villi. Not only were the IgGFc receptors expressed on ST cells, but in vivo bound IgG was detected in association with the ST and the two patterns of IgG binding were essentially identical. In contrast to the qualitative nature of the receptors on ST cells, cells in the stromal (central) region of the trophoblastic villi expressed IgGFc receptors that had high affinity for EA but failed to bind the deaggregated IgG except at a high concentration. The results are discussed with respect to the possible role of the IgGFc receptors in the specific transfer of IgG from maternal to fetal circulations.     

The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of theH-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera againstK-, I-, andD-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab′)₂ fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against Ia antigens failed to inhibit bone marrow RFCs, although antibody against H-2K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in theK andD regions as well as theI region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.     

Maturation-related changes in the expression of Fc gamma RII and Fc gamma RIII on mouse mast cells derived in vitro and in vivo.

The expression and function of Fc gamma RII and Fc gamma RIII on three mouse mast cell populations that differ in maturity as assessed by secretory granule constituents were analyzed by cellular and immunochemical approaches. As quantified by flow cytometric analysis of the binding of the rat 2.4G2 anti-Fc gamma RII/III mAb, mouse serosal mast cells (SMC) purified from the peritoneal cavity expressed more receptors per cell than did mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC), which are progenitors of SMC. Coculture of BMMC with mouse 3T3 fibroblasts for 2 wk, which alters the secretory granule composition toward that of SMC, also increased receptor epitope expression to a level equivalent to that of SMC. As assessed by rosette assays with mouse mAb to SRBC, all three mast cell populations bound IgG1, IgG2a, and IgG2b, essentially all binding was inhibited by 2.4G2 antibody, and greater quantities of the antibody were required to block immune adherence by cocultured mast cells and SMC as compared with BMMC. Immunoprecipitation and SDS-PAGE analysis of Fc gamma RII and Fc gamma RIII from BMMC, cocultured mast cells, and SMC that were surface radiolabeled with Na125I revealed predominant native forms of 62, 57, and 56 kDa, respectively, and an additional surface form of 43 kDa in SMC. Removal of N-linked carbohydrate from immunoprecipitates demonstrated that BMMC expressed peptide cores of 38 kDa (Fc gamma RII-1 gene product) and 31 kDa (Fc gamma RII-2 gene product), and barely detectable amounts of a 28-kDa (Fc gamma RIII gene product) core. The expression of all three was increased by coculture with 3T3 fibroblasts, consistent with the increased expression of their common epitope by cytofluorographic analysis. SMC expressed primarily the Fc gamma RII-1 and some Fc gamma RIII gene product. Thus, the three populations of mast cells express different amounts and ratios of the Fc gamma RII and Fc gamma RIII gene products, and maturation of BMMC during coculture with fibroblasts in vitro and in the peritoneal cavity in vivo augments cell-surface expression of the receptors and immune adherence function.     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.     

Fc receptor heterogeneity: immunofluorescent studies of B, T, and "third population" lymphocytes in human blood with rabbit IgG b4/anti-b4 complexes.

IgG Fc receptors on human peripheral blood lymphocytes (PBL) were characterized by immunofluorescence studies with defined rabbit IgG b4 allotype/anti-allotype complexes. Three discrete types of Fc receptor-bearing cells, totaling approximately 33% of PBL, were identified. Fc receptors of the three types differed in their sensitivity to trypsin and in either absolute or localized density (topography) as determined by variable requirements for anti-IgC cross-linking in order to visualize bound complexes microscopically. The question of additional heterogeneity related to differences in individual Fc receptor affinity for complexed IgG was not approached in this study. Ten to 15% of PBL had pronase-sensitive, trypsin-resistant Fc receptors readily detected by direct immunofluorescence by using large fluorescein-conjugated complexes prepared near equivalence. Double label and lymphocyte fractionation experiments established this population to be largely distinct from suface IgM+ B cells and T cells, and identical to EA Ripley rosette-forming cells. Approximately 50% of surface IgM+ B cells and approximately 10% of T cells had lower density Fc receptors identified by indirect immunofluorescence with small complexes prepared in antigen excess or by cross-linking fluorescein-conjugated complexes with anti-rabbit IgG anti-serum. An additional approximately 15% peripheral T and B cells had very low density Fc receptors detectable by complexing the IgG on the cell surface by sequential incubations of cells with b4 IgG and anti-b4. Fc receptors on B and T cells were sensitive to both pronase and trypsin digestion. The heterogeneity of IgG Fc receptors on different lymphocyte subpopulations as defined by these these experiments may be of relevance for further analysis of normal and abnormal immune function.     

Recruitment and activation of tumor-specific immune T cells in situ. CD8+ cells predominate the secondary response in sponge matrices and exert both delayed-type hypersensitivity-like and cytotoxic T lymphocyte activity

This study analyzes the involvement of CD4+ and CD8+ T cells in a secondary cellular immune response to the highly metastatic murine lymphoma ESb in situ. This tumor line expresses tumor-associated transplantation Ag which can induce protective immunity in vivo and specific CTL in vitro. In tumor-immune mice the injection of a tumor vaccine (x-irradiated ESb tumor cells) into s.c. implanted vascularized sponges resulted in the generation of a specific secondary immune response characterized by massive leukocyte recruitment and generation of strong CTL activity at the restimulation site. During the antitumor immune response the CD4+:CD8+ T cell ratio decreased significantly and specifically in the restimulated sponges. Depletion of CD8+ but not CD4+ T cells from the tumor immune mice before restimulation significantly reduced the delayed-type hypersensitivity-like response and totally blocked the generation of tumor-specific CTL activity in situ. Only a minority of the CD8+ immune T cells which predominated the secondary response in situ expressed IL-2R and lymph node homing receptors as detected by the mAb MEL-14.     

Suppression of adult B cell differentiation in pokeweed mitogen-stimulated cultures by Fc(IgG) receptor-negative T cells from cord blood.

Unfractionated T lymphocytes from cord blood suppressed adult B cell differentiation into immunoglobulin-producing cells in pokeweed mitogen-stimulated co-culture system. Cord blood T cells were fractionated into T cells bearing Fc receptors for IgG (Tgamma cells) and T cells lacking Fc receptors for IgG(Tnon-gamma cells) by rosette formation with ox erythrocytes coated by the IgG fraction of rabbit antisera followed by Ficoll-Hypaque gradient sedimentation. T gamma cells from cord blood, even though isolated after the interaction with immune complexes, showed no suppressor activity on adult B cell differentiation, whereas Tnon-gamma cells exerted strong suppression to a similar extent to that by unfractionated cord T cells. The suppressor activity on B cell differentiation by Tnon-gamma cell as well as by unfractioned T cells from cord blood was completely abrogated by irradiation with 2000 rads. These results indicated that, contrary to suppressor function found in adult T cells, the suppressor activity in cord T cells might be exerted by a T cell subset lacking Fc receptors for IgG(Tnon-gamma cells).     

Enhancement of anti-tumor CD8 immunity by IgG1-mediated targeting of Fc receptors

Dendritic cells (DCs) function as professional antigen presenting cells and are critical for linking innate immune responses to the induction of adaptive immunity. Many current cancer DC vaccine strategies rely on differentiating DCs, feeding them tumor antigens ex vivo, and infusing them into patients. Importantly, this strategy relies on prior knowledge of suitable “tumor-specific” antigens to prime an effective anti-tumor response. DCs express a variety of receptors specific for the Fc region of immunoglobulins, and antigen uptake via Fc receptors is highly efficient and facilitates antigen presentation to T cells. Therefore, we hypothesized that expression of the mouse IgG1 Fc region on the surface of tumors would enhance tumor cell uptake by DCs and other myeloid cells and promote the induction of anti-tumor T cell responses. To test this, we engineered a murine lymphoma cell line expressing surface IgG1 Fc and discovered that such tumor cells were taken up rapidly by DCs, leading to enhanced cross-presentation of tumor-derived antigen to CD8+ T cells. IgG1-Fc tumors failed to grow in vivo and prophylactic vaccination of mice with IgG1-Fc tumors resulted in rejection of unmanipulated tumor cells. Furthermore, IgG1-Fc tumor cells were able to slow the growth of an unmanipulated primary tumor when used as a therapeutic tumor vaccine. Our data demonstrate that engagement of Fc receptors by tumors expressing the Fc region of IgG1 is a viable strategy to induce efficient and protective anti-tumor CD8+ T cell responses without prior knowledge of tumor-specific antigens.