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The Journal of Immunology, 1999, 162: 6880-6892.
Copyright © 1999 by The American Association of Immunologists

Human Breast Carcinoma Patients Develop Clonable Oncofetal Antigen-Specific Effector and Regulatory T Lymphocytes1

James W. Rohrer2,*, Adel L. Barsoum*, Donna L. Dyess{dagger}, J. Alann Tucker{ddagger} and Joseph H. Coggin, Jr.*

* Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688; {dagger} Department of Surgery, University of South Alabama College of Medicine, Mobile, AL 36693; and {ddagger} Department of Pathology, University of South Alabama College of Medicine, Mobile, AL 36617


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Oncofetal Ag (OFA) is a 44-kDa glycoprotein expressed during early to mid-gestation fetal development and re-expressed as a surface Ag by tumor cells soon after transformation. The Ag is detectable on all types of human and rodent tumors tested, but is undetectable on normal cells. In experimental animals it is autoimmunogenic and induces potentially protective T cell responses both after experimental immunization and during tumor development subsequent to carcinogenic insult. To determine whether this tumor-associated Ag is also immunogenic for human T lymphocytes, breast carcinoma patients’ peripheral blood mononuclear leucocytes were stimulated in vitro with autologous tumor cells in the presence of IL-2, {gamma}-IFN, and IL-6 for 2 wk. The tumor-reactive cells were then restimulated and cloned by limiting dilution, and the clones were analyzed. We established 24, 19, 11, and 16 tumor-reactive clones from the four respective patients. Of those, 4, 6, 4, and 7, respectively, proliferated specifically to purified OFA. Both CD4 and CD8 OFA-specific clones were established, which responded equally well to purified OFA or 32- to 44-kDa immature laminin receptor protein. All were CD3+, TCR-{alpha}ß+. All CD4 clones secreted {gamma}-IFN, but neither secreted IL-4 nor IL-10. Both IFN-{gamma}-secreting cytotoxic CD8 clones and IL-10-secreting inhibitory CD8 clones were established. Thus, during human cancer development, the same types of OFA-specific effector and regulatory T cells are induced as during murine T lymphomagenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To generate effective immunity to a human or rodent cancer cell, the tumor cells must express antigenic determinants, called tumor-associated Ags (TAA),3 which can be processed by APC and presented on their MHC proteins. Peripheral Th1 cells and CTL must be able to recognize and become activated by those APC MHC protein-presented TAA peptides. Such activated T lymphocytes must then reach the tumor site and be able to kill the tumor cells displaying the immunogenic peptide associated with self-MHC proteins (1). If human tumor cells lack TAA that activate this process, there will be no cell-mediated tumor response save the NK cell response (2).

Several investigators have suggested that spontaneous tumors in humans and experimental animals do not express tumor-associated transplantation Ags (TATA), but that repeatedly passaged tumors, which appear to have such Ags, are due to residual heterozygosity between the tumor cells and the host even when carried in isogeneic animals inbred for years and hundreds of generations (3, 4, 5). However, these authors did not take into account the clonal selection of spontaneous neoplasms over weeks and months in mice and years in humans that may result in the immunoselection of ineffectively immunizing but "modestly" antigenic tumors in older individuals. Indeed Van Waes et al. (6) showed that very aggressive tumors that lack Ags recognized by CTL still express Ags capable of inducing Th cells and that those Th cells can activate cell-mediated immunity against variants of the tumor that still express the CTL-recognized Ags. Besides the lack of immunogenic TATA, tumor cells can avoid immune destruction in several ways (7, 8). Down-regulation of MHC class I proteins makes recognition by the TCR of CD8+ T lymphocytes impossible. Also, the release of soluble TAA by tumor cells can inhibit T cell homing to the tumor site. This process would involve such Ags being picked up by APC away from the tumor bed and thus would allow T cell recognition of the Ag away from the actual tumor site, which may misdirect potentially protective lymphocytes to sites other than where the tumor cells are located. Indeed, oncofetal Ag (OFA) has been shown to be shed by OFA+ cells and is detected in bodily fluids (9). Tumor cell induction of potent anti-TAA Ab responses may result in indirect inhibition of potentially protective anti-TAA cell-mediated immunity because of the release of antiinflammatory cytokines by activated Th2 lymphocytes (10, 11). Indeed, the presence of IL-10 and IL-4 during in vivo exposure to various Ags inhibits the development of CTL and Th1 cells specific for determinants on those Ags (12, 13). The tumor cells themselves can even augment the Th2 cell dominance by secreting TGF-ß, resulting in immune cell overproduction of IL-10 (14) and, thus, inhibit the induction of cell-mediated immunity. If the recognition of MHC protein-presented TATA peptide by TCR occurs in the absence of the B7 costimulator binding to T cell CD28, anergy can be induced (15). Some, but not all, tumors actively evade immune elimination by secreting inhibitory cytokines and activating inhibitory regulatory elements of the immune system (16, 17, 18, 19, 20, 21).

While much of the tumor immunology conjecture has focused on individual tumor-specific transplantation Ag (TSTA)-specific immunity in rodents (22, 23, 24), Leffel and Coggin (25) reported that either of two 3-methylcholanthrene (MCA)-induced BALB/c fibrosarcomas, which had been reported not to share TSTA determinants (26), could induce protective immunity to both. This was confirmed by Hellstrom et al. (23). In fact, a survey of many primary MCA-induced sarcomas of mice had both individual, unshared TSTAs and cross-protective OFA (27). This cross-protection was shown to be due to the expression of 44-kDa and 200-kDa proteins present on early to mid-gestation fetal cells and all tumor cells tested, but not on term fetal or normal neonatal or adult tissues (28); thus termed oncofetal Ag (i.e., OFA). This Ag is present on all human tumors tested (28, 29). This protein can induce cross-reactive protective tumor immunity in vivo and cross-reactive CTL in vitro (30).

Rohrer et al. (31) showed that during development of T cell lymphomas subsequent to x-irradiation of RFM mice, T lymphocytes that were specific for OFA even in irradiated mice that showed no presence of tumor, but had experienced OFA+ preleukemic thymocytes shortly after x-irradiation (32), were induced. The clonable, OFA-specific T cells from the irradiated mice were of several types. IFN-{gamma}-secreting CD4+ Th1 cells and CD8+ CTL cells were detected, but also noncytotoxic CD8+ T cells that secreted IL-10 (33). The last population was able to inhibit both IFN-{gamma} secretion and cytotoxic activity by the effector T cells. Indeed, culturing the IL-10-secreting T cells with neutralizing anti-IL-10 allowed those clones to demonstrate OFA-specific cytotoxic activity (33). Thus, in mice, OFA is both an early marker of transformation (32) and also an autoimmunogen for T cells capable of anti-tumor immunity. However, inhibitory T cells are also induced during tumor development (31, 33).

While OFA is present on all human tumor cells tested (28, 29), it is of interest to determine whether effector and inhibitory T lymphocytes that are specific for OFA are activated during tumorigenesis in humans. In this paper, we report that CD4+ Th1 cells, CD8+ CTL, and IL-10-secreting, noncytotoxic CD8+ T cells that proliferated specifically in response to purified OFA were clonable from all four breast carcinoma patients selected as they became available for biopsy. While the number of tumor-reactive clones able to be established and the profile of the OFA-specific clones varied among the patients, all had all three subclasses of T cell clones.

Interestingly, we also found that recombinant immature laminin receptor protein (iLRP) (34) stimulated the OFA-specific clones’ proliferation in a dose-response identical with that shown using purified OFA. This exactly matched similar experimental results (J.W.R. et al., manuscript in preparation) using iLRP to stimulate murine 44-kDa OFA-specific T cell clones established from survivors of x-irradiation-induced lymphomagenesis (31). This strengthens the identification of OFA as an iLRP, which has been suggested by peptide and cloned gene sequences and mAb reactivity (A.L.B. et al., manuscript in preparation). Thus, it appears that OFA is detectable on human tumor cells and also induces the same type of T lymphocyte immune responses during tumor development in humans as it does in experimental animals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients

The four women were 30–45 years old and had invasive ductal carcinoma of the breast, and tumor tissue and initial blood was taken at the time of mastectomy. Subsequent blood samples were obtained every 2 wk by venipuncture for a further 8 wk.

PBMC purification

PBMC were purified from the breast carcinoma patients’ blood using a modification of the method of Boyum (35). Briefly, the heparinized blood was diluted 1:2 in sterile RPMI 1640 medium, and 4-ml aliquots were layered over 3-ml aliquots of Ficoll-Paque Plus (Pharmacia Biotech, Piscataway, NJ) in 15 ml tissue culture-treated sterile polystyrene centrifuge tubes (Sarstedt, Newton, NC) and then centrifuged for 35 min at 400 x g at 20°C. Following removal of the upper plasma by pipetting, the lymphocyte layer at the interface was removed from each tube, pooled and diluted 1:3 in RPMI 1640 medium, mixed gently to resuspend the cells, and centrifuged at 100 x g for 10 min at 20°C. After removing the supernatant, the pellet was resuspended in 10 ml medium and centrifuged at 100 x g for 10 min at 20°C. The pellet was then resuspended in sterile RPMI 1640 medium and counted for viability using trypan blue dye exclusion.

Preparation of autologous human breast carcinoma cell

Briefly, breast tumor tissue in excess of that needed for pathologic diagnosis was obtained from the surgical Pathology Laboratory at the University of South Alabama-Knollwood Hospital. The tissue was minced into very small pieces, which were then passed through sieves of gradually reduced mesh by applying gentle pressure with the piston of a disposable plastic syringe, and the cells were washed through the sieve in sterile RPMI 1640 medium containing 10% FCS, 100 U/ml penicillin, and 100/µg/ml streptomycin. The cell suspension was then passed through a nylon mesh, centrifuged at 2000 rpm for 10 min, and the sedimented cells were used for T cell stimulation. Such cell preparations were 65–75% viable. Tumor cells not used for in vitro stimulation of T cells at that time were cryopreserved in sterile RPMI 1640 with 10% (v/v) DMSF and 50% (v/v) FCS in a freezer at -70°C. When needed for restimulation, tumor cells were thawed rapidly, diluted in an excess of sterile RPMI 1640 plus 10% (v/v) FCS, and washed twice.

Cell lines

The anti-OFA IgM-producing hybridoma 115 (28) was carried as an ascites tumor from which ascites fluid was collected, and mAb 115 was purified as described previously (30).

Monoclonal Abs

Monoclonal mouse anti-human CD4 IgG1 from hybridoma 34930.111 was obtained from R&D Systems (Minneapolis, MN). Monoclonal mouse anti-human CD8 IgG1 from hybridoma RPA-T8, monoclonal mouse anti-human CD3 IgG1 from hybridoma UCHT1, and monoclonal mouse anti-human TCR-{alpha}ß IgM from hybridoma V 5T-TCR.01 were obtained from PharMingen (San Diego, CA). Monoclonal mouse anti-HLA-DP, DQ, DR class II protein IgG2a from hybridoma TU39, and monoclonal mouse anti-TNP IgG2a from hybridoma G155–178 were obtained from PharMingen. Monoclonal mouse anti-HLA-A, B, C class I protein IgG2a from hybridoma W6/32.HL was obtained from Sigma (St. Louis, MO).

T cell clone production

PBMC, isolated as described above from breast carcinoma patient heparinized blood, were cultured with irradiated autologous breast carcinoma cells in sterile RPMI 1640 medium containing 2 mM L-glutamine, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, and 10% FCS (complete RPMI 1640). The cultures contained 100 U/ml of recombinant human IL-2, 10 U/ml of recombinant human IFN-{gamma}, and 10 U/ml of recombinant human IL-6 (R&D Systems). We used IL-2 as a growth factor for T cells, IFN-{gamma} to inhibit outgrowth of Th2 cells for Ab production (36), and IL-6 to promote outgrowth and function of CTL cells (37). After 2 wk of culture, the reactive cells (that is, T cells that proliferated during the initial culture of breast carcinoma patient peripheral blood mononuclear leucocytes (PBML) with irradiated autologous breast carcinoma cells in the presence of the cytokine-supplemented RPMI 1640 medium) were restimulated with irradiated autologous tumor cells plus irradiated autologous PBMC in complete RPMI 1640 medium containing IL-2, IL-6, and IFN-{gamma} as described above and cloned by limiting dilution (1 tumor-reactive T cell/5 wells) using essentially the same technique previously published (31) except that the tumor stimulus was the autologous breast carcinoma cells and APC were from the irradiated (autologous) PBMC. After the growth of the clones had stabilized, only recombinant human IL-2 was added (no IL-6 or IFN-{gamma}) at subsequent restimulations. These clones had to be restimulated with autologous tumor cells in the presence of cytokines and autologous PBMC every 2 wk to maintain viability and proliferation. During cultures set up to determine which cytokines were produced by the clones, the clones were restimulated in the presence of irradiated autologous tumor cells and irradiated PBMC that had been depleted of T lymphocytes by negative selection on anti-CD3 mAb-coated petri plates using the method of Wysocki and Sato (38) except that anti-CD3 Ab was used and was added to the plates on the day of the cell separation.

Determination of T cell clone specificity by proliferation in response to Ag

At the time of the 2-wk restimulation of the clones to maintain their proliferation, the cloned cells were harvested, washed in complete RPMI 1640, and a viability count was performed. A portion of the cells was saved to be used in the proliferation assay. The proliferation assay was performed with 10,000 viable cloned cells/well plus irradiated autologous PBMC plus various doses of purified 44-kDa OFA protein from a murine thymic lymphoma cell line or purified 44-kDa protein from normal murine thymus or recombinant iLRP on nitrocellulose (NC) particles or an equivalent amount of unconjugated NC particles in 96-well plates. After 24 h, 10 µl of 5-bromodeoxyuridine (BUdR) was added to each well to a final concentration of 10 µM BUdR/well. Then, the cells were incubated for another 24 h. Proliferation was assayed using the Biotrak BUdR incorporation assay (Amersham, Arlington Heights, IL). At the end of that incubation, the plates were centrifuged at 300 x g for 10 min, and the labeling medium was removed. The cells were dried for 1 h at 60°C. The cells were then fixed with an ethanol fixative for 30 min at room temperature, the fixative was removed, and the wells were coated with blocking buffer (1% protein in 50 mM Tris-HCl, 150 mM NaCl, pH 7.4) and incubated for 30 min at room temperature. The blocking buffer was removed, 100 µl of 1:100 diluted peroxidase-labeled anti-BUdR was added to each well, and the plates were incubated for 90 min at room temperature. The Ab solution was then removed, and the wells were washed three times with 300 µl/well of wash buffer. Then, 200 µl of 3,3',5,5'-tetramethylbenzidine in 15% (v/v) DMSO was added to each well, and the plate was covered and incubated for 5–30 min at room temperature. When the required color density was reached, the reaction was stopped by adding 25 µl of 1 M sulfuric acid to each well, and the plate was read on a microELISA reader at 450 nm.

Determination of the requirement for MHC recognition by T cell clones for the proliferative response to OFA

At the time of the 2-wk restimulation of the clones with irradiated autologous tumor cells to maintain the cloned cells’ proliferation, a portion of the cells from the clones that had proliferated specifically to purified OFA:NC particles (OFA-specific clones) were harvested, washed in complete RPMI 1640, and their viability was determined. The cells were then used in a proliferation assay as described above with the exception that mAbs specific for monomorphic determinants on HLA class I or class II proteins or an isotype control mAb was present during the assay. Also, the irradiated autologous PBML, which served as APCs, were incubated for 30 min at 4°C with anti-HLA or control IgG2a before being added to the assay plate wells. In setting up these assays, a standard order of addition of materials was used: first, irradiated autologous PBML in complete RPMI 1640 medium containing 2 µg/ml of anti-HLA or isotype control mAb plus various doses of purified OFA-conjugated NC particles or the same amount of bare NC particles; and second, the OFA-specific cloned T cells to be assayed. This was a slight modification of the method of Pawelec et al. (39). Each well of the 96-well plate contained 10,000 cloned T cells and 500,000 irradiated autologous PBML. The medium contained 100 U/ml of recombinant human IL-2. These cultures were incubated at 37°C in a 95% air/5% CO2 humidified atmosphere for 24 h and were then pulsed to a final concentration of 10 µM BUdR for an additional 24 h. Proliferation was assessed by quantitating BUdR incorporation using the Biotrak ELISA BUdR incorporation assay (Amersham, Arlington Heights, IL) as described above.

Determination of T cell clone surface Ag phenotype by mAb and complement depletion

One week after Ag restimulation, part of each T cell clone culture was harvested, washed three times by centrifugation in complete RPMI 1640 medium, and a viability count was performed. The cells were diluted to 1 x 106 viable cells/ml, and their surface Ag phenotype was determined by cytotoxicity with mAbs plus facilitating anti-serum plus complement, as previously described (31). The counted cells were pelleted and resuspended in 1 ml of anti-CD4, anti-CD8, anti-CD3, anti-{alpha}ß, or anti-{gamma}{delta} TCR Ab diluted optimally in complete RPMI 1640 medium. For all Abs used, the optimal dilution was 1:15. The control Ab used was normal mouse IgG. After Ab and complement treatment, cells were pelleted by centrifugation, washed three times in complete RPMI 1640 medium, and resuspended in 1 ml complete RPMI 1640. A viability count was performed by trypan blue dye exclusion. The percentage of cells specifically killed or lysed by the experimental Ab and complement treatment was calculated by knowing the number of total and viable cells in each tube at the beginning and comparing the nonspecific killing effect of the control Ab plus facilitating antiserum plus complement with the killing by the experimental Abs plus facilitating Ab plus complement.

ELISA of IFN-{gamma}, IL-4, and IL-10 production by T cell clones

Quantikine assay kits for IFN-{gamma}, IL-4, and IL-10 (from R&D Systems) were used. These kits use HRP-labeled anti-cytokine Ab to detect cytokine that is captured on the anti-cytokine-coated plates. These assays add 3,3',5,5'-tetramethylbenzidine as the substrate, and the color reaction is stopped with 2 N sulfuric acid and color read at 450 nm. The IFN-{gamma} standard curve was linear between 5 pg/ml and 500 pg/ml, and the minimum amount detectable in this assay was 3 pg/ml. The IL-4 standard curve was linear between 8 pg/ml and 2000 pg/ml, with the minimum amount detectable being 4.1 pg/ml. The IL-10 standard curve was linear between 5 pg/ml and 500 pg/ml. The minimum amount of IL-10 detectable was 3 pg/ml.

Determination of T cell clones’ cytotoxic T cell activity against autologous breast carcinoma target cells

Cytotoxicity assays were performed using the CytoTox96 nonradioactive cytotoxicity assay kit produced by Promega (Fisher Scientific, Norcross, GA). The assay quantitatively measures lactate dehydrogenase, a stable cytosolic enzyme that is released upon cell lysis. Released lactate dehydrogenase in culture supernatants was measured with a 30-min coupled enzymatic assay resulting in the conversion of a tetrazolium salt to a red formazan product (40). The amount of color formed is proportional to the number of lysed cells. Color was quantitated using a Titertek Multiskan MC ELISA reader (Fisher Scientific), which measured absorbance at 492 nm. The setup of the assay was the same as previously described for testing mouse clone cytotoxicity against mouse tumors (31), except that the medium used was RPMI 1640 and autologous breast carcinoma cells were used as targets. The percent specific cytotoxicity was calculated using the formula: % cyctotoxicity = [(experimental - effector spontaneous) - target spontaneous]/(target maximum - target spontaneous). There is much less of a spontaneous release of lactate dehydrogenase in this assay than of 51Cr in a traditional 51Cr release cytotoxicity assay, and therefore higher specific cytotoxicity percentages are achieved.

Determination of the ability of anti-IL-10 to convert noncytotoxic CD8, OFA-specific T cell clones to cytotoxic clones

To determine whether the clones from patient MP and EP that were secreting IL-10 were being inhibited by it (IL-10), the cells were harvested 1 day before the 2-wk restimulation culture and cultured with 10 µg/ml mouse monoclonal anti-human IL-10 IgG1 (from hybridoma mAb 217; R&D Systems) or normal mouse IgG for 24 h as described previously (31). The cells were then harvested, washed three times with complete RPMI 1640 medium, and viability counts were performed. The cells were then added to a 4-h cytotoxicity assay against autologous and allogeneic breast carcinoma cells as described above and previously (31), except that anti-IL-10 or control IgG was added to a final concentration of 10 µg/ml.

ELISA for OFA/iLRP on breast carcinoma cells

Flat-bottom 96-well plates (Nunc-Immunoplate I; Vanguard International, Neptune, NJ) were coated with riLRP (300 ng/100 µl/well) and postcoated with 1% BSA in PBS, pH 7.2. A direct binding curve for the anti-OFA mAb 115 was first generated by incubating 100 µl of a serial dilution of the Ab in 0.5% BSA in PBS at 37°C for 1 h with the riLRP. The plate was washed four times for 5 min each with PBS-T solution (PBS containing 0.5% Tween-20). The plate was further incubated with a biotinylated goat anti-mouse µ-chain specific Ab at 1:5000 dilution in 0.5% BSA in PBS for 1 h. The plate was washed again as described previously and 100 µl of an AB reagent (avidin:biotinylated HRP; Vector Laboratories, Burlingame, CA; one drop of each in 10 ml PBS-T) were added to each well of the microplate and incubated for 30 min at room temp. The plate was washed as described previously. Finally, 100 µl of the substrate solution (2,2'-azino-di-(3-ethylbenz-thiazoline sulfonate in 0.1 citrate puffer, pH 4) was added to each well. After an incubation period of 30 min, the colored product was measured spectrophotometrically at 410 nm in a microELISA reader. The tests were done in triplicate.

For assaying for OFA/riLRP on breast carcinoma cells, a competitive ELISA was performed using a dilution of the mAb 115 that was below the saturation point (about 70% of the plateau level) to obtain maximal sensitivity to inhibition. A total of 5 x 104 breast carcinoma cells from each patient were incubated with 0.5 ml of the Ab dilution overnight at 10°C. The cells were sedimented by centrifugation, 100 µl of the supernatant was applied to each of three wells on a microplate coated with riLRP, and the ELISA reaction was continued as described above. Although we were unable to obtain sufficient normal breast tissue cells from the patients, all assays were run with OFA+ MCA1315 fibrosarcoma cells as a positive control and normal BALB/c mouse spleen cells (OFA-) as a negative control. The percent inhibition was calculated from the formula: [1 - (preabsorption Ab OD410 - background OD410)/(postabsorption Ab OD410 - background OD410)] x 100. Experimental values are presented as the mean ± SEM of the number of individual assays. This assay has been very reproducible since its development in our laboratory in 1985 (28). As previously published (28), only 2–9% absorption of the 115 IgM anti-OFA mAb is seen with normal human tissues, while human tumors of various types absorb from 22–89% of the 115 IgM anti-OFA activity. Thus, ~10 times as much Ab is reproducibly absorbed by cancer cells as is by normal human tissue.

Recombinant iLRP

The cDNA-encoding iLRP was cloned from a 7-day gestation embryonic library prepared from Swiss/Webster mouse. The coding region was cloned into an expression vector under control of the Taq promoter, and the protein was expressed in Escherichia coli. Inclusion bodies were isolated and solubilized in 6 M guanidine hydrochloride in 20 mM Tris, pH 8.0, 0.1 M NaCl, 2 mM EDTA, and 0.02% azide. The solubilized protein was added to six volumes of 20 mM Tris, pH 8.0, 1 M guanidine HCl, 2 mM reduced glutathione, and 0.2 mM oxidized glutathione, was renatured for 18 h at 4°C, and then was dialyzed against 20 mM Tris, pH 8.0, 0.1 M NaCl, and 0.04% azide.

Preparation and solubilization of 5T plasma membranes

Membrane fractions from 5T lymphoma cells grown in culture or normal thymus cells were prepared using the method of Standring and Williams (41). Protease inhibitors aprotinin (100 KIU/ml), PMSF (1 µM), leupeptin (15 µM), N-{alpha}-p-tosyl-L-lysine chloromethylketone (50 µM), and soybean trypsin inhibitor (5 µg/ml) were used to minimize membrane protein degradation.

Preparation of Ag-bearing NC particles

OFA was isolated from 5T lymphoma membrane extract by immunoaffinity chromatography on a 115 mAb-Sepharose column, as previously described (28). Eluted 5T membrane material or the normal thymus membrane preparation was mixed with the sample buffer (v/v) and subjected to 10–20% gradient SDS-PAGE according to the method of Laemmli (42). Separated proteins were transferred to NC (43), made visible by staining with Ponceau S (44), and a NC strip carrying the 44-kDa bands was cut. Each NC strip was processed to obtain Ag-bearing particles using the method of Abou-Zeid et al. (45).

Statistics

Where multiple experiments were performed on each tissue, the data was analyzed for significant differences using Student’s t test. The data from experiments in which dose-response curves were generated were analyzed using ANOVA. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The breast carcinoma patients’ tumors expressed OFA

To be sure that any lack of OFA-reactive T cell clones did not just reflect the lack of sensitization during tumor development, tumor tissue taken from each patient was assayed for its ability to absorb anti-OFA mAb 115 reactivity to riLRP in an ELISA absorption assay (28). Fig. 1Go shows that all patients’ tumors that were tested absorbed 57%–78% of the anti-OFA/iLRP activity. Patient JR’s tumor cells had been used up in the other assays and therefore could not be tested. Thus, the breast carcinomas from the three patients that were tested were expressing OFA. We were unable to obtain sufficient normal breast tissue from the patients to use as direct negative controls. However, all assays were performed not only with the human breast carcinoma tissue, but also BALB/c mouse fibrosarcoma cells as positive controls and BALB/c mouse spleen cells as OFA-negative normal tissue controls. This assay has been very reproducible over the past 13 years, and normal human tissue always absorbs at least one-tenth as much Ab as does human cancer tissue (28).



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  		FIGURE 1. The expression of OFA by breast carcinoma patients’ autologous tumor cells. The patients’ breast carcinoma cells were tested for their ability to absorb monoclonal anti-OFA IgM 115 before addition of the Ab to an indirect ELISA using recombinant OFA/iLRP-conjugated plates. The data shown represent the mean ± SEM inhibition of maximal reaction by absorption with the tumor cells in three repeat assays.

 
Human breast carcinoma patients’ peripheral blood contains clonable CD4+ and CD8+ tumor-reactive T lymphocytes

The culture of PBML with irradiated autologous breast carcinoma cells in the presence of recombinant human IL-2, IFN-{gamma}, and IL-6 followed by restimulation with irradiated autologous tumor cells and limited dilution cloning allowed the establishment of 24, 19, 11, and 16 tumor-reactive T cell clones from the four patients, respectively (Fig. 2Go). The term "tumor reactive" is an operational definition in that these T cells grew from cultures of breast carcinoma patient’s PBML cultured with irradiated autologous breast carcinoma cells in the presence of the cytokines mentioned above and were restimulated by autologous breast carcinoma cells in the presence of irradiated autologous PBML and cytokines during cloning. Thus, presumably, the T cells that proliferated did so because of a recognition of some epitope on the autologous tumor cells, because subsequent culture with only irradiated autologous PBML and cytokines did not stimulate proliferation or cytokine production by the clones (Fig. 3Go and data not shown; see also Figs. 7Go and 8Go). All of the clones express CD3 and {alpha}ß TCR. More than 87% of the cloned cells were killed with monoclonal anti-CD3 plus facilitating anti-mouse IgG Ab plus complement or monoclonal anti-human TCR-{alpha}ß IgM plus complement. Isotype control Abs plus facilitating Ab plus complement killed <2.2% of the cells. Anti-{gamma}{delta} TCR IgM plus complement similarly killed <3% of the cells. Of the CD3+, TCR-{alpha}ß+ clones, 36.8 (±2.4)% were CD4+, CD8- T cell clones and 63.2 (±2.4)% were CD4-, CD8+ T cell clones. However, analysis of uncloned, CD3+ cells (T lymphocytes) purified from the peripheral blood of the tumor patients showed 64.9 ± 4.8% CD4+ cells and 35.1 ± 4.8% CD8+ cells (data not shown). Thus, while the tumor-reactive T cells had an inverted frequency of CD4:CD8 T cells (0.58), the uncloned peripheral blood T cells showed a normal 1.85 CD4:CD8 ratio. This may reflect some artifact of the cloning culture or may reflect a skewing toward CD8 T cells in the tumor-reactive portion of the total T cell population during breast carcinoma development. A similar CD8 predominance was also seen in clones derived from irradiation-induced T lymphoma developing RFM mice (31).



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  		FIGURE 2. The surface Ag phenotype of the tumor-reactive T cells cloned from the four breast carcinoma patients. Monoclonal anti-CD4-, anti-CD8-, anti-CD3-, anti-TCR-{alpha}ß-, and anti-{gamma}{delta} TCR Ab plus facilitating Ab plus low-toxicity rabbit complement (for use with human cells)-mediated killing of breast carcinoma patient T cell clones was analyzed.

 


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  		FIGURE 3. Proliferative response of the patients’ CD4+ T cell clones to 75 ng/well of purified OFA bound to NC particles. A total of 10,000 viable cloned T cells taken 2 wk after their latest restimulation with irradiated autologous breast carcinoma cells were incubated with 5 x 105 irradiated autologous PMBC plus 75 ng/well of purified RFM mouse 5T lymphoma 44-kDa OFA conjugated to NC particles (solid bars), 75 ng/well of purified normal thymus 44-kDa protein (p44) conjugated to NC particles (hatched bars), or an equivalent amount of bare NC particles (open bars) for 24 h, pulsed for an additional 24 h with BUdR, and then assayed for BUdR incorporation using monoclonal anti-BUdR Ab on the cells after fixation in an ELISA.

 


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  		FIGURE 7. Secretion of IFN-{gamma}, IL-4, and IL-10 by breast carcinoma patients’ OFA-reactive, CD4+ T cell clones upon Ag stimulation. CD4+ clones taken 2 wk after their most recent restimulation with irradiated autologous tumor cells were cultured for 48 h with irradiated, T cell-depleted autologous PBMC with or without irradiated autologous tumor cells in complete RPMI 1640 medium containing 100 U/ml of recombinant human IL-2. Culture supernatants from those cultures were then harvested and assayed for human IFN-{gamma}, human IL-4, and human IL-10 by quantitative ELISA. Data are shown as pg/ml of cytokine secreted by each clone. A, Cytokine secretion subsequent to culture with irradiated autologous T cell-depleted PBML plus irradiated autologous breast carcinoma cells. B, Cytokine secretion subsequent to culture with irradiated autologous T cell-depleted PBML in the absence of autologous breast carcinoma cells.

 


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  		FIGURE 8. Secretion of IFN-{gamma}, IL-4, and IL-10 by breast carcinoma patients’ OFA-reactive, CD8+ T cell clones upon Ag stimulation. CD8+ clones taken 2 wk after their most recent restimulation with irradiated autologous tumor cells were cultured for 48 h with irradiated, T cell-depleted autologous PBMC with or without irradiated autologous tumor cells in complete RPMI 1640 medium containing 100 U/ml of recombinant human IL-2. Culture supernatants from those cultures were then harvested and assayed for human IFN-{gamma}, human IL-4, and human IL-10 by quantitative ELISA. Data are shown as pg/ml of cytokine secreted by each clone. A, Cytokine secretion subsequent to culture with irradiated autologous T cell-depleted PBML plus irradiated autologous breast carcinoma cells. B, Cytokine secretion subsequent to culture with irradiated autologous T cell-depleted PBML in the absence of autologous breast carcinoma cells.

 
Both CD4+, TCR-{alpha}ß+ and CD8+, TCR-{alpha}ß+ T cell clones that are OFA-reactive are established from breast carcinoma patient peripheral blood

Fig. 3Go shows that from 20% to 50% (mean, 32.1 ± 7.6%) of the CD4+ tumor-reactive clones from the four breast carcinoma patients proliferated specifically to 75 ng/well of purified 44-kDa OFA protein:NC particles. While the OFA was purified from the 5T mouse thymic lymphoma, the proliferation was specific in that there was no incorporation of BUdR over background levels when the clones were cultured in the presence of 75 ng/well of purified normal murine thymus 44-kDa protein (not OFA):NC particles or unconjugated NC particles. The stimulation index for OFA-reactive CD4 clones to purified 44-kDa OFA:NC particles compared with the response to bare NC particles was from 34.1 to 65.4 (mean ± SEM, 49 ± 4.9). Fig. 4Go similarly shows that from 14.3% to 42.9% (mean ± SEM, 32 ± 7.4%) of the CD8+ tumor-reactive clones from the four breast carcinoma patients proliferated specifically to 75 ng/well of purified 44-kDa OFA protein:NC particles, but no clones responded above background levels to normal murine thymus p44:NC particles. The stimulation index for the OFA-reactive CD8 clones to purified 44-kDa OFA:NC particles compared with bare NC particles was from 9.3 to 68.8 (mean ± SEM, 28.9 ± 6.2). The culture of the clones with normal thymus p44:NC particles induced no more proliferation than with bare NC particles (Figs. 3Go and 4Go). Thus, ~30% of the breast carcinoma patients’ tumor-reactive T cell clones specifically proliferate to OFA presented by irradiated autologous PBML. However, ~70% of both CD4 (18/26) and CD8 (31/44) clones, which proliferate in response to autologous tumor cells, do not proliferate when exposed to purified OFA:NC particles in the presence of autologous PBML (Figs. 3Go and 4Go), and thus, must be recognizing some non-OFA epitope on the autologous tumor cells. There also appear to be two populations of CD8 anti-OFA T cells in all patients observed in that some proliferate vigorously to 75 ng/well of OFA while others proliferate approximately only one-seventh as much to the same dose of OFA:NC particles. This was also previously seen in irradiated RFM mice, which had survived T lymphoma development (31).



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  		FIGURE 4. Proliferative response of the patients’ CD8+ T cell clones to 75 ng/well of purified OFA bound to NC particles. A total of 10,000 viable cloned T cells taken 2 wk after their latest restimulation with irradiated autologous breast carcinoma cells were incubated with 5 x 105 irradiated autologous PBMC plus 75 ng/well of purified RFM mouse 5T lymphoma 44-kDa OFA conjugated to NC particles (solid bars), 75 ng/well of purified normal thymus 44-kDa protein (p44) conjugated to NC particles (hatched bars), or an equivalent amount of bare NC particles (open bars) for 24 h, pulsed for an additional 24 h with BUdR, and then assayed for BUdR incorporation using monoclonal anti-BUdR Ab on the cells after fixation in an ELISA.

 
The proliferation of OFA-reactive clones to purified OFA is inhibited in the presence of monoclonal anti-HLA Abs

Fig. 5Go shows that all of the CD4 T cell clones that proliferate in response to 75 ng/well of purified OFA-conjugated NC particles in the presence of autologous irradiated PBML and IL-2 are inhibited 85.6–94.6% (mean ± SEM, 90.1 ± 1.1%) if the assay is done in the presence of anti-HLA class II monomorphic monoclonal IgG2a TU39 compared with when the same amount of monoclonal isotype control anti-TNP IgG2a is included in the proliferation assay. Similarly, all of the CD8, OFA-reactive T cell clones’ proliferation to purified OFA-conjugated NC particles was inhibited 84.2–92.9% (mean ± SEM, 89.4 ± 0.9) when monoclonal anti-HLA class I monomorphic Ab W6/32.HL was present during the assay compared with when monoclonal isotype control anti-TNP IgG2a is present (Fig. 6Go). Thus, it appears that both specific recognition of OFA plus MHC of the appropriate class is required for optimal stimulation of T cell clone proliferation.



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  		FIGURE 5. Proliferation of OFA-specific, CD4+ T cell clones to OFA is inhibited by the presence of monoclonal anti-HLA-DP, DQ, and DR monomorphic Ab in the proliferation assay culture. A total of 10,000 viable cloned T cells taken 2 wk after their latest restimulation with irradiated autologous breast carcinoma cells were incubated with 5 x 105 irradiated autologous PBMC plus 75 ng/well of purified RFM mouse 5T lymphoma 44-kDa OFA conjugated to NC particles in the presence of monoclonal anti-HLA class II IgG2a (2 µg/ml) (cross-hatched bars), monoclonal anti-TNP IgG2a control Ab (2 µg/ml) (solid bars), or an equivalent amount of bare NC particles in the presence of anti-TNP IgG2a control Ab (2 µg/ml) (open bars) for 24 h, pulsed for an additional 24 h with BUdR, and then assayed for BUdR incorporation using monoclonal anti-BUdR Ab on the cells after fixation in an ELISA.

 


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  		FIGURE 6. Proliferation of OFA-specific, CD8+ T cell clones to OFA is inhibited by the presence of monoclonal anti-HLA-A, B, and C monomorphic Ab in the proliferation assay culture. A total of 10,000 viable cloned T cells taken 2 wk after their latest restimulation with irradiated autologous breast carcinoma cells were incubated with 5 x 105 irradiated autologous PBMC plus 75 ng/well of purified RFM mouse 5T lymphoma 44-kDa OFA conjugated to NC particles in the presence of monoclonal anti-HLA class I IgG2a (2 µg/ml) (cross-hatched bars), or monoclonal anti-TNP IgG2a control Ab (2 µg/ml) (solid bars), or an equivalent amount of bare NC particles in the presence of anti-TNP IgG2a control Ab (2 µg/ml) (open bars) for 24 h, pulsed for an additional 24 h with BUdR, and then assayed for BUdR incorporation using monoclonal anti-BUdR Ab on the cells after fixation in an ELISA.

 
Cytokine profiles of CD4+ OFA-specific clones from breast carcinoma patients

Supernatants taken from 48-h restimulation cultures of OFA-specific CD4 T cell clones in the presence of recombinant human IL-2 and irradiated T cell-depleted autologous PBMC with or without irradiated autologous breast carcinoma cells were assayed for IFN-{gamma}, IL-4, and IL-10 by ELISA. None of the CD4 T cell clones from any of the patients secreted amounts of IL-4 or IL-10 above the level of sensitivity, while all secreted >2000 pg/ml of IFN-{gamma} (Fig. 7GoA). That these clones express the cytokine profile of Th1 cells is to be expected because they were initially cultured, cloned, and expanded in the presence of IFN-{gamma}, which inhibits the growth of Th2 CD4+ T cells (36). That the IFN-{gamma} was the product of the T cell clones that were being restimulated and was induced by OFA is suggested by the fact that cultures containing the CD4 clones plus irradiated autologous T cell-depleted PBML and IL-2, but lacking the irradiated autologous tumor cells, did not have any IFN-{gamma} detectable above the level of sensitivity of the assay (Fig. 7GoB).

Cytokine profiles of CD8+ OFA-specific clones from breast carcinoma patients

Supernatants taken after 48-h restimulation cultures of OFA-specific CD8 T cell clones in the presence of recombinant IL-2 and irradiated T cell-depleted autologous PBML with or without irradiated autologous breast carcinoma cells were assayed as described above for IFN-{gamma}, IL-4, and IL-10. None of the CD8 T cell clones secreted detectable amounts of IL-4. However, two nonoverlapping populations of CD8 clones were shown by their IL-10 and IFN-{gamma} secretion profiles (Fig. 8GoA). Patients JR and EP had 50% of their CD8 OFA-specific clones secreting IFN-{gamma} and 50% secreting IL-10. Patient SL had three of four CD8 clones secreting IL-10 and patient MP had two of three CD8 clones secreting IL-10. The clones not secreting IL-10 secreted IFN-{gamma}. Approximately equivalent amounts of cytokine were secreted no matter which cytokine was secreted in that they all secreted between 400 and 1000 pg/ml of either IL-10 or IFN-{gamma}. Thus, the established CD8 clones express the cytokine profiles we previously described for murine cytotoxic T cells (IFN-{gamma}) and for noncytotoxic, inhibitory T cells (IL-10) established from long-term survivors of irradiation-induced lymphomagenesis (31). That the IFN-{gamma} and IL-10 were the products of the T cell clones that were being restimulated and were induced by OFA is suggested by the fact that cultures containing the CD8 clones plus the irradiated autologous T cell-depleted PBML and IL-2, but lacking the irradiated autologous tumor cells, did not have any IFN-{gamma} or IL-10 detectable over the level of the sensitivity of the assays (Fig. 8GoB).

The CD8 OFA-specific clones kill only autlologous breast carcinoma cells and that cytotoxicity is inhibited by IL-10 secretion

Because we had previously found that IL-10-secreting clones were CTL cells, but could only be shown as such in the presence of neutralizing anti-IL-10 Ab (33) and because autologous tumor cells were limited in number, we combined analysis of the cytotoxic activity of the CD8+ OFA-specific T cell clones from patients MP and EP with analysis of the specificity of the cytotoxicity and the IL-10 inhibitory activity on that cytotoxicity. Thus, the CD8 clones were cultured with autologous or allogeneic breast carcinoma cells at an E:T ratio of 50:1 for 48 h with or without monoclonal anti-human IL-10 or mouse IgG as an isotype Ab control. Fig. 9GoA shows that only one of three CD8 MP clones and two of four CD8 EP clones were cytotoxic to autologous tumor cells in the presence of isotype control Ab. No clones were cytotoxic to nonautologous tumor cells. However, in the presence of anti-IL-10 (Fig. 9GoB), all CD8 clones were cytotoxic to their autologous tumor cells, but were still not cytotoxic to allogeneic breast carcinoma cells. The clones that required anti-IL-10 to be able to be cytotoxic were less cytotoxic than were those three clones that could kill the tumor cells in the absence of anti-IL-10. Those clones that were cytotoxic without any Ab addition being required were also the clones that were secreting IFN-{gamma} (clones M4, E4, and E5). These were also clones that proliferated more vigorously to OFA (stimulation index of 62.3, 68.8, and 40.8, respectively). Thus, the CD8+ OFA-specific clones that secrete IL-10 are inhibited from acting as effector cells by the IL-10 they are secreting and, presumably, may act as inhibitors of neighboring effector cells in vivo due to their secretion of IL-10.



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  		FIGURE 9. All CD8+, OFA-reactive clones from patients MP and EP are cytotoxic to their autologous tumor cells, but the IL-10-secreting clones become cytotoxic only in the presence of neutralizing anti-IL-10 Ab. Cytotoxic activity against autologous and allogeneic breast carcinoma cells at a 50:1 E:T ratio using OFA-reactive CD8 T cell clones from breast carcinoma patients MP and EP in the presence of 10 µg/ml of normal mouse IgG (A) or monoclonal mouse anti-human IL-10 IgG1 (B).

 
Because both patient’s CD8 T cell clones kill the autologous tumor cells significantly better (p = 0.01) than the other patient’s tumor cells and both tumors express OFA (Fig. 1Go), the cytotoxicity may be restricted by MHC and thus the clones may be traditional CTL cells. This is true for both the IFN-{gamma}-secreting CTL clones (Fig. 9GoA) and for the IL-10-secreting CD8 clones whose cytotoxicity is apparent only when they are assayed in the presence of neutralizing anti-IL-10 Ab (Fig. 7GoB). While we do not have the patients’ MHC types to directly demonstrate MHC restriction, the fact that the clones are CD3+, CD8+, TCR-{alpha}ß+, and are almost completely inhibited from proliferating to purified OFA:NC particles presented by autologous irradiated PBML by the presence of monoclonal anti-HLA class I Ab (Fig. 6Go) is suggestive that the requirement for the tumor targets to be autologous to the effector cells to get cytotoxicity is due to MHC restriction of the cytotoxic T cell clones.

Human OFA-specific T cell clones recognize iLRP as well as OFA

Fig. 10Go shows the proliferative response of the CD4 clones (A) of patient JR and the CD8 clones (B) of patient JR to various doses of purified 44-kDa OFA, normal thymus 44-kDa protein (p44), and recombinant iLRP. While there was no proliferative response to normal thymus 44-kDa protein (not OFA) by any of the clones, all of the clones responded by proliferation to both purified 44-kDa thymic lymphoma-derived OFA and iLRP. In fact, while three of the four clones were optimally responding to OFA at a dose of 75 ng/well and the other clone could respond to no less than 75 ng/well, the dose-response curve of each to OFA was identical to the dose-response curve to iLRP. Thus, the human T cell clones induced during breast carcinoma development that recognized OFA also recognized, in a quantitatively identical manner, iLRP. The data shown are only those for patient JR’s OFA-specific T cell clones, but they are representative of the other three patients’ clones as well (data not shown).



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  		FIGURE 10. Proliferation dose response of patient JR’s OFA-reactive CD4+ or CD8+ T cell clones to purified 5T thymic lymphoma 44-kDa OFA, purified normal thymus 44-kDa protein, or iLRP conjugated to NC particles as measured by ELISA determination of BUdR incorporation. Responses to OFA (closed circle and closed square), control normal thymus 44-kDa protein (open square and open diamond), or iLRP (closed and open triangles) of OFA-reactive CD4 and CD8 clones taken 2 wk after their latest restimulation with irradiated autologous tumor cells are shown. T cell clones were cultured for 48 h with irradiated autologous PBML and various doses of purified 44-kDa OFA, recombinant iLRP, or purified normal thymus p44 in the presence of 100 U/ml of recombinant human IL-2. A, Response of CD4 clones. B, Response of CD8 clones.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Many studies have shown that the injection of irradiated tumor cells into normal animals or the excision of an already developed tumor induces cell-mediated immunity that protects the animal against subsequent challenge with tumor cells (23, 25, 46, 47). It has been demonstrated by several laboratories that while anti-tumor immunity exists in a tumor-bearing animal, which can be adoptively transferred with lymphocytes, the established tumor is not rejected (25, 48). This has been termed concomitant immunity (48, 49). It has been shown that the established tumor can evade immune destruction by a number of methods (8, 19, 20, 50). While in mice, much attention has been paid to individual TSTA (22, 23, 24), an OFA has been found to be present on all tumor cells tested (28, 29), can cross-protect against tumor challenge in vivo, and can induce CTL in vitro (30). Although there is a suggestion that spontaneous tumors in man actually lack tumor Ags (4), a number of human tumor-type specific Ags have been found to be able to induce CTL (51, 52, 53, 54) and have been used in immunotherapy regimens (55, 56). OFA is also present on human tumors (28, 29). We previously showed that in mice that survived fractionated irradiation-induced lymphomagenesis without any apparent tumors developing, there were clonable memory Th1 cells and CTL cells that recognized 44-kDa OFA, which suggested that while transformation had occurred subsequent to irradiation, the survivor mice had killed the tumor cells before they could present clinically (31). However, those mice did not have enhanced protection against subsequent tumor challenge, which may have been due to the presence of CD8 cells that secreted IL-10 and the IL-10 inhibited CTL cytotoxic activity (31, 33). The data presented herein suggest that similar events may occur during the development of breast carcinomas in humans and show that just as in experimental animals OFA is an autoimmunogen for T lymphocytes that have potential protective effector activity, but also leads to induction of T lymphocytes that secrete IL-10 that inhibits cytotoxic activity (Fig. 9Go).

While for some tumors, the cytotoxic T cells appear to kill both autologous and allogeneic tumor cells in man (57, 58), it is clear from Fig. 9Go, A and B that both the outright cytotoxic CD8 T cells and the CTL whose cytotoxicity is blocked by their IL-10 secretion are both able to kill autologous, but not allogeneic breast carcinoma cells even though OFA is expressed by both targets (Fig. 1Go). Some TCR-{alpha}ß+ T cells express NK1.1 (59) and appear to be restricted by CD1 instead of MHC (60). Those NK T cells can be important in anti-tumor cytotoxicity (61, 62) and in IL-12-induced immunity (63). Therefore, it is important that our OFA-reactive CD8 clones are restricted, at least, to autologous tumor cells (which is probably due to MHC restriction) and thus are probably classical cytotoxic T cells. Our ability to block our clones from proliferating to purified OFA in the presence of irradiated PBML APC with monoclonal anti-HLA Abs further suggests that these TCR-{alpha}ß+ clones are recognizing tumor-associated OFA in an MHC-restricted manner and thus are traditional Th1 cells and CTL. Because our clones react to iLRP and iLRP has been shown to induce {gamma}{delta} TCR+ T cells (64), our work shows that typical TCR-{alpha}ß+ Th1 cells and CTL reactive to OFA and iLRP also are induced during breast carcinoma development in humans.

All patients’ tumors in this study expressed OFA, which confirms previous data showing OFA to be present (28, 29) and suggesting that it should serve as a TAA in humans as it does in mice (30, 31, 32, 33). Also, all patients had peripheral blood T lymphocytes that reacted very vigorously to irradiated autologous tumor cells in the presence of exogenous IL-2, IFN-{gamma}, and IL-6. Unlike the mouse spleen cells taken either from tumor-immune animals or from tumor-surviving animals, the human breast cancer patients’ T lymphocytes proliferated enough from the beginning of culture with autologous tumor cells that the culture had to be split within 2–3 days after initiation. After about 12 days, the cultures began to show some cell death, which is normal. Subsequent to restimulation with irradiated autologous tumor cells in the presence of the same cytokines as above, limited-dilution cloning resulted in from 11 to 24 tumor-reactive clones. None of the clones subsequently responded by proliferation or cytokine production to culture with irradiated autologous PBML and the cytokines described above unless irradiated autologous tumor cells or purified OFA:NC particles were present. Thus, they were operationally defined as tumor reactive and 16.7–43.8% of those clones were reactive to the tumor due to specific recognition of 44-kDa OFA/iLRP in the context of autologous MHC expression by the tumor cells.

During breast carcinoma development, the CD4:CD8 ratio appeared to be inverted in the tumor-reactive and OFA-reactive clones compared with uncloned peripheral blood T lymphocytes from the same patients. While this may represent an artifact of our cloning procedure, a similar inversion was previously seen in the tumor-reactive clones from lymphomagenesis survivor mice (31). Because OFA is present on early to mid-gestation fetuses in experimental animals and humans, it is of interest that a similar CD8:CD4 inversion occurs during pregnancy in humans concurrent with a reduction in the potency of immunity (65). That this may actually reflect a change in the phenotype of circulating tumor-reactive T cells in vivo during tumor growth is suggested by the observation of a similar inversion during IL-2 treatment of lymphoma- and mastocytoma-bearing mice (66). In that case, an increase in therapeutic, anti-tumor CD8 CTL cells occurred at the site of the tumor. Tumor-infiltrating T lymphocytes in human renal cell carcinoma also had a lower CD4:CD8 ratio than is normal (67).

The survival of established tumors in hosts in which concomitant immunity can be demonstrated through adoptive transfer or partial tumor excision and rechallenge with the same tumor at a different site has often been explained by selection for poorly immunogenic tumor cells subsequent to induction of immunity (8) or by tumor-induced immune suppression (16, 21). It has been demonstrated that IL-10 is secreted by tumor cells (19). Also, tumor cell-secreted TGF-ß can induce IL-10 secretion and promote Th2 skewing of anti-tumor immune responses (14). Tumor-infiltrating lymphocytes in human renal cell carcinoma have been shown to have a high frequency of IL-10-secreting cells (68, 69) and inhibited in vitro cytotoxicity to autologous tumor cells (70). We have previously shown that the irradiation-induced lymphomagenesis survivor mice CD8 T cell clones that were noncytotoxic and did not secrete IFN-{gamma} secreted IL-10, which could inhibit cytotoxic T cell activity in vitro and may have explained the enhancement of tumor development in those survivor mice in vivo (31, 71). We find that the same populations of T lymphocytes appear to be clonable from breast carcinoma patients in this report. While there are too few patients from which to draw a conclusion, it is of interest to note that patients SL and MP, who had 75% and 67% of their CD8 clones secreting IL-10, respectively, both had to re-enter the hospital for a second mastectomy operation during the course of this study. This may suggest that the profile of T cells reactive to OFA may be predictive of the therapeutic outcome. Considerably more patients will have to be observed before any conclusion can be made about this possibility.

We (A.L.B. et al., manuscript in preparation) have shown that the sequence of five full-length OFA cDNA clones from MCA1315 fibrosarcoma cells were identical. Also, sequence alignments revealed 99.4% identity between the nucleotide sequence of OFA and the published nucleotide sequence for murine iLRP (72). The predicted amino acid sequence of OFA and that deduced from the murine iLRP gene were 99.7% identical with a phenylalanine in OFA at amino acid position 18, where leucine is recorded in the published murine iLRP sequence (72). Also, we found that monoclonal anti-iLRP Abs bind OFA and that monoclonal anti-OFA Abs are inhibited from binding OFA by iLRP. We show herein that the CD4 and CD8 human OFA-reactive T cell clones show the same dose response to iLRP as to purified OFA. Murine anti-OFA T cell clones also respond to iLRP (J.W.R. et al., manuscript in preparation). Thus, it appears that OFA may be iLRP. Besides the sequence and immunological cross-reactivity, functional and temporal similarities also are found. OFA is detectable on cells exposed to carcinogenic insult before they are histologically visible as transformed cells (32). This protein is also expressed in early to mid-gestation during fetal development, but not expressed in term fetus, normal neonatal, or adult tissues (28). The 32- to 44-kDa iLRP is expressed during the same time frame in fetal development (73), is conserved like OFA between many species (34), and is overexpressed in cancer cells and correlates with their metastatic potential (74).

iLRP is 32–44 kDa, but precursor LRP appears to dimerize to form one component of the high-affinity mature 67-kDa LRP (75). However, this dimer is combined noncovalently with a galactoside-binding protein, galectin-3, to form the mature high-affinity LRP (76). During tumor progression, galectin-3 is down-regulated (77), while the 32- to 44-kDa monomeric form is over-expressed as the tumor becomes more aggressive (78, 79, 80, 81). Because we have found that iLRP (OFA)-reactive effector and inhibitor T lymphocytes are induced during the development of breast carcinomas, it is of interest that the expression of iLRP on breast carcinomas (78, 79), carcinoma of the colon, (80) and in uterine adenocarcinoma (81) appears to be associated with poor prognoses for the patients. Thus, the frequency of IL-10-secreting CD8+ anti-iLRP T cells may also be predictive for the success of therapy of such tumors. Indeed, in renal cell carcinomas, CTL that have infiltrated the tumor bed are dysfunctional (70, 82) and IL-10 is often present in the tumor-bed microenvironment (83), with part of it being produced by the anti-tumor T lymphocytes that infiltrate the tumor site (68, 69). Thus, we see in breast carcinomas a tumor Ag (iLRP), which is important for tumor cell invasiveness, induces potentially protective T lymphocytes, but also induces other T lymphocytes to secrete a cytokine (IL-10) capable of inhibiting the effector T cells.


   Acknowledgments
 
We thank Dr. J. P. Houchins at R&D Systems for cloning and expressing the iLRP gene and supplying us with purified recombinant iLRP.


   Footnotes
 
1 This work was supported by R&D Systems, Minneapolis, MN. Back

2 Address correspondence and reprint requests to Dr. James W. Rohrer, Department of Microbiology and Immunology, LMB Building, University of South Alabama College of Medicine, Mobile, AL 36688-0002. E-mail address: Back

3 Abbreviations used in this paper: TAA, tumor-associated Ag; OFA, oncofetal Ag; TATA, tumor-associated transplantation Ag; TSTA, tumor-specific transplantation Ag; NC, nitrocellulose; BUdR, 5-bromodeoxyuridine; iLRP, 32- to 44-kDa immature laminin receptor protein; PBML, peripheral blood mononuclear leucocytes; MCA, 3-methylcholanthrene; RFM, RFM/UnCr strain mice; TNP, 2,4,6-trinitrophenyl. Back

Received for publication March 30, 1998. Accepted for publication March 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Lotze, M. T., O. J. Finn. 1990. Recent advances in cellular immunology: implication for immunity to cancer. Immunol. Today 11:190.[Medline]
  2. Berke, G.. 1991. Lymphocyte-triggered internal target cell disintegration. Immunol. Today 12:396.[Medline]
  3. Scott, O. C. A.. 1991. Tumor transplantation and tumor immunity. Cancer Res. 51:757.[Free Full Text]
  4. Hewitt, H. B., E. R. Blake, A. S. Walder. 1976. A critique of the evidence for active host defence against cancer, based on personal studies of 27 murine tumours of spontaneous origin. Br. J. Cancer 33:241.[Medline]
  5. Embleton, M. J.. 1991. Letter to the Editor. Cancer Res. 51:5433.[Free Full Text]
  6. Van Waes, C., J. L. Urban, J. L. Rothstein, P. L. Ward, H. Schreiber. 1986. Highly malignant tumor variants retain tumor-specific antigens recognized by T helper cells. J. Exp. Med. 164:1547.[Abstract/Free Full Text]
  7. Boon, T.. 1992. Toward a genetic analysis of tumor antigens. Adv. Cancer Res. 58:177.[Medline]
  8. Restifo, N. P., F. Esquivel, A. L. Asher, H. Stotter, R. J. Barth, J. R. Bennink, J. J. Mule, J. W. Yewdell, S. A. Rosenberg. 1991. Defective presentation of endogenous antigens by a murine sarcoma: implications for the failure of an anti-tumor response. J. Immunol. 147:1453.[Abstract]
  9. Barsoum, A., Jr J. Coggin. 1991. Isolation and partial characterization of a soluble oncofetal antigen from murine and human amniotic fluids. Int. J. Cancer 48:248.[Medline]
  10. Paludan, S. R.. 1998. Interleukin-4 and interferon-{gamma}: the quintessence of a mutual antagonistic relationship. Scand. J. Immunol. 48:459.[Medline]
  11. Enk, A. H., V. L. Angeloni, M. C. Udey, S. I. Katz. 1993. Inhibition of Langerhans cell antigen-presenting function by IL-10: a role for IL-10 in induction of tolerance. J. Immunol. 151:2390.[Abstract]
  12. Powrie, F., S. Menon, R. L. Coffman. 1993. Interleukin-4 and interleukin-10 synergize to inhibit cell-mediated immunity in vivo. Eur. J. Immunol. 23:3043.[Medline]
  13. Wang, L., E. Goillot, R. I. Tepper. 1994. IL-10 inhibits alloreactive cytotoxic T lymphocyte generation in vivo. Cell. Immunol. 159:152.[Medline]
  14. Maeda, H., A. Shiraishi. 1996. TGF-ß contributes to the shift toward Th2-type responses through direct and IL-10-mediated pathways in tumor-bearing mice. J. Immunol. 156:73.[Abstract]
  15. Harding, R. A., J. G. McArthur, J. A. Gross, D. H. Raulet, J. P. Allison. 1992. CD28-mediated signaling co-stimulates murine T cells and prevents induction of anergy in T cell clones. Nature 356:607.[Medline]
  16. Vaage, J.. 1971. Concomitant immunity and specific depression of immunity by residual or reinjected tumor tissue. Cancer Res. 31:1655.[Abstract/Free Full Text]
  17. Berg, D., R. G. Lynch. 1991. Immune dysfunction in mice with plasmacytomas. I. Evidence that transforming growth factor ß contributes to the altered expression of activation receptors on host B lymphocytes. J. Immunol. 146:2865.[Abstract]
  18. Smith, D. R., S. L. Kunkel, M. D. Burdick, C. A. Wilke, M. B. Orringer, R. I. Whyte, R. M. Strieter. 1994. Production of interleukin-10 by human bronchogenic carcinoma. Am. J. Pathol. 130:136.[Abstract]
  19. Yu, A., H. Watts, N. Jaffe, R. Parkman. 1977. Concomitant presence of tumor-specific cytotoxic and inhibitor lymphocytes in patients with osteogenic sarcoma. N. Engl. J. Med. 297:121.[Abstract]
  20. Finke, J. H., A. H. Zea, J. Stanley, C. L. Longo, H. Mizoguchi, R. R. Tubbs, R. H. Wiltrout, J. J. O’Shea, S. Kudoh, E. Klein, R. M. Bukowski, A. C. Ochoa. 1993. Loss of T cell receptor {zeta} chain and p56lck in T-cells infiltrating human renal cell carcinoma. Cancer Res. 53:5613.[Abstract/Free Full Text]
  21. Bost, K. L., S. C. Bieglik, B. M. Jaffe. 1995. Lymphokine mRNA expression by transplantable murine B lymphocyte malignancies: tumor-derived IL-10 as a possible mechanism for modulating the anti-tumor response. J. Immunol. 154:718.[Abstract]
  22. Srivastava, P. S., L. J. Old. 1988. Individually distinct transplantation antigens of chemically induced mouse tumors. Immunol. Today 9:78.[Medline]
  23. Hellstrom, K. E., I. Hellstrom, J. P. Brown. 1978. Unique and common tumor-specific transplantation antigens of chemically induced mouse sarcomas. Int. J. Cancer 21:317.[Medline]
  24. De Leo, A. B., H. Shikn, T. Takahashi, M. John, L. J. Old. 1977. Cell surface antigens on chemically induced sarcomas of the mouse. I. Murine leukemia virus-related antigens and alloantigens of cultured fibroblasts and sarcoma cells: description of a unique antigen on BALB/c Meth A sarcoma. J. Exp. Med. 146:720.[Abstract/Free Full Text]
  25. Leffel, M. S., Jr J. H. Coggin. 1977. Common transplantation antigens on 3-MCA-induced sarcomas detected by three methods of assay. Cancer Res. 37:4112.[Abstract/Free Full Text]
  26. Hellstrom, K. E., I. Hellstrom. 1967. Cell bound-immunity to autologous and syngeneic mouse tumors induced by methylcholanthrene and plastic discs. Science 156:981.[Abstract/Free Full Text]
  27. Jr Coggin, J. H.. 1989. Cross-reacting tumor associated transplantation antigen on primary 3-methylcholanthrene-induced BALB/c sarcomas. Mol. Biother. 1:223.[Medline]
  28. Jr Payne, W. J., Jr J. H. Coggin. 1985. Mouse monoclonal antibody to embryonic antigen: development, cross-reactivity with rodent and human tumors, and preliminary polypeptide characterization. J. Natl. Cancer Inst. 75:527.
  29. Gussack, G. S., S. D. Rohrer, R. B. Hester, P. I. Liu, Jr J. H. Coggin. 1988. Human squamous cell carcinoma lines express oncofetal antigen 44 kilodalton polypeptide defined by monoclonal antibody to mouse fetus. Cancer 62:57.
  30. Barsoum, A. L., Jr J. H. Coggin. 1989. Immunogenicity of a soluble, partially purified OFA from murine fibrosarcoma in syngeneic mice. J. Biol. Resp. Modifiers 8:579.
  31. Rohrer, J. W., C. Culpepper, A. L. Barsoum, Jr J. H. Coggin. 1995. Characterization of RFM mouse T lymphocyte anti-oncofetal antigen immunity in apparent tumor-free, long-term survivors of sublethal x-irradiation by limiting dilution T lymphocyte cloning. J. Immunol. 155:2266.
  32. Rohrer, S. D., R. N. Sarli, A. L. Barsoum, R. B. Hester, Jr J. H. Coggin. 1992. Expression of 44 kilodalton oncofetal antigen as a premalignancy marker in x-irradiation-induced murine T cell lymphoma. J. Natl. Cancer Inst. 84:602.[Abstract/Free Full Text]
  33. Rohrer, J. W., Jr J. H. Coggin. 1995. CD8 T cell clones inhibit antitumor T cell function by secreting IL-10. J. Immunol. 155:5719.[Abstract]
  34. Menard, S., V. Castronovo, E. Tagliabue, M. E. Sobel. 1997. New insights into the metastasis-associated 67 kD laminin receptor. J. Cell. Biochem. 67:155.[Medline]
  35. Boyum, A.. 1968. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21, 97:S77.
  36. Gajewski, T. F., F. W. Fitch. 1988. Antiproliferative effect of IFN-{gamma} in immune regulation. I. IFN-{gamma} inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones. J. Immunol. 140:4245.[Abstract]
  37. Rogers, L. A., A. Zlotnik, F. Lee, K. Shortman. 1991. The maintenance of lytic specificity during the development of clones of cytotoxic T lymphocytes from single precursor cells. J. Immunol. Methods 143:241.[Medline]
  38. Wysocki, L. J., V. L. Sato. 1978. Panning for lymphocytes: a method for cell selection. Proc. Natl. Acad. Sci. USA 75:2844.[Abstract/Free Full Text]
  39. Pawelec, G., A. Ziegler, P. Wernet. 1985. Dissection of human allostimulatory determinants with cloned T cells: stimulation inhibition by monoclonal antibodies TU22, 34, 35, 36, 37, 39, 43, and 58 against distinct human MHC class II molecules. Human Immunol. 12:165.[Medline]
  40. Decker, T., M.-L. Lohmann-Mathes. 1988. A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity. J. Immunol. Methods 15:61.
  41. Standring, R., A. F. Williams. 1978. Glycoproteins and antigens of membranes prepared from rat thymocytes after lysis by shearing or with detergent Tween-40. Biochim. Biophys. Acta 508:85.[Medline]
  42. Laemmli, U. K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[Medline]
  43. Towbin, H., T. Staehlin, J. Gordon. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76:4350.[Abstract/Free Full Text]
  44. Harlow, E., L. Lane. 1988. Antibodies: A Laboratory Manual 494. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
  45. Abou-Zeid, C. E., J. Steele Filley, G. A. W. Rook. 1987. A simple new method for using antigens separated by polyacrylamide gel electrophoresis to stimulate lymphocytes in vitro after converting bands cut from Western blots into antigen-bearing particles. J. Immunol. Methods 98:5.[Medline]
  46. Wahl, D. V., W. H. Chapman, I. Hellstrom, K. E. Hellstrom. 1974. Transplantation immunity to individually unique antigens of chemically induced bladder tumors in mice. Int. J. Cancer 14:114.[Medline]
  47. Wepsic, H. T., B. Zbar, H. J. Rapp, T. Borsos. 1970. Systemic transfer of tumor immunity: delayed hypersensitivity and suppression of tumor growth. J. Natl. Cancer Inst. 44:955.
  48. North, R. H., D. P. Kirstein. 1977. T cell-mediated concomitant immunity as the expressors of nonspecific immunity to unrelated tumors and bacterial parasites. J. Exp. Med. 145:275.[Abstract/Free Full Text]
  49. Gershon, R. K., R. L. Carter, K. Kondo. 1967. On concomitant immunity in tumor-bearing hamsters. Nature 213:674.[Medline]
  50. Chen, L., S. Ashe, W. A. Brady, I. Hellstrom, K. E. Hellstrom, J. A. Ledbetter, P. McGowan, P. S. Linsley. 1992. Costimulation of anti-tumor immunity by the B7 counterreceptor for the T lymphocyte molecules CD28 and CTLA-4. Cell 71:1093.[Medline]
  51. Traversari, C., P. van der Bruggen, I. F. Luescher, C. Lurquin, P. Chromez, A. Van Pel, E. De Plaen, A. Amar-Costesec, T. Boon. 1992. A nonapeptide encoded by human MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E. J. Exp. Med. 176:1453.[Abstract/Free Full Text]
  52. Gaugler, B., B. Ban den Eynde, P. van der Bruggen, P. Romero, J. J. Gaforio, E. De Plaen, B. Lethe, F. Brasseur, T. Boon. 1993. Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes. J. Exp. Med. 179:921.[Abstract/Free Full Text]
  53. Bernhard, H., J. Karbach, T. Wolfel, P. Busch, S. Storkel, M. Stockle.. 1994. Cellular immune response to human renal cell carcinomas: definition of a common antigen recognized by HLA-A2-restricted cytotoxic T lymphocyte clones. Int. J. Cancer 59:837.[Medline]
  54. Kittlesen, D. J., L. W. Thompson, P. H. Gulden, J. C. A. Skipper, T. A. Colella, J. A. Shabonowitz, D. F. Hunt, V. H. Engelhard, Jr C. L. Slingluff. 1998. Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: implication for tumor vaccine development. J. Immunol. 160:2099.[Abstract/Free Full Text]
  55. Kawakami, Y., S. Eliyahu, C. H. Delgado, P. F. Robbins, K. Sakaguchi, E. Apella, J. R. Yannelli, G. J. Adema, T. Miki, S. A. Rosenberg. 1994. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc. Natl. Acad. Sci. USA 91:6458.[Abstract/Free Full Text]
  56. Rosenberg, S. A., B. S. Packard, P. M. Aebersold, D. Solomon, S. L. Topalian, S. T. Toy, P. Simon, M. T. Lotze, J. C. Yang, C. A. Seipp, C. simpson, C. Carter, S. Bock, D. Schwartzentruber, J. P. Wei, D. E. White. 1988. Use of tumor infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma: preliminary report. N. Eng. J. Med. 319:1676.[Abstract]
  57. Kim, T. Y., A. C. Von Eschenbach, M. D. Filaccio, K. Hayakawa, D. R. Parkinson, C. M. Balch, K. Itoh. 1990. Clonal analysis of lymphocytes from tumor peripheral blood and nontumorous kidney in primary renal cell carcinoma. Cancer Res. 50:5263.[Abstract/Free Full Text]
  58. Mitropoulos, D., S. Kooi, J. Rodriguez-Villanueva, C. D. Platsoucas. 1994. Characterization of fresh (uncultured) tumour-infiltrating lymphocytes (TIL) and TIL-derived T cell lines from patients with renal cell carcinoma. Clin. Exp. Immunol. 97:321.[Medline]
  59. MacDonald, H. R.. 1995. NK1.1+ T cell receptor-{alpha}ß+ cells: new clues to their origin, specificity, and function. J. Exp. Med. 182:633.[Free Full Text]
  60. Bendelac, A., O. Lantz, M. E. Quimby, J. W. Yewdell, J. R. Bennink, R. R. Brutkiewicz. 1995. CD1 recognition by mouse NK1+ T lymphocytes. Science 268:863.[Abstract/Free Full Text]
  61. Ballas, Z. K., W. Rasmussen. 1991. Lymphokine activated killer (LAK) cells. VI. NK1.1+, CD3+ LAK effectors are derived from CD4-, CD8-, NK1.1- precursors. Cell. Immunol. 134:296.[Medline]
  62. Takeda, K., S. Seki, K. Ogasawara, R. Anzai, W. Hashimoto, K. Sugiura, M. Takahashi, M. Satoh, K. Kumagai. 1996. Liver NK1.1+ CD4+ {alpha}ß T cells activated by IL-12 as a major effector in inhibition of experimental tumor metastasis. J. Immunol. 156:3366.[Abstract]
  63. Anzai, R., S. Seki, K. Ogasawara, W. Hashimoto, K. Sugiura, M. Sato, K. Kumagai, K. Takeda. 1996. Interleukin-12 induces cytotoxic NK1+ {alpha}ß T cells in the lungs of euthymic and athymic mice. Immunology 88:82.[Medline]
  64. Ferrarini, M., S. Heltai, S. M. Pupa, S. Menard, M. R. Zocchi. 1996. Killing of laminin receptor-positive human lung cancers by tumor-infiltrating lymphocytes bearing {gamma}{delta}+ T cell receptor. J. Natl. Cancer Inst. 88:436.[Abstract/Free Full Text]
  65. Barnett, M. A., R. P. Learmouth, E. Pihl, E. C. Wood. 1983. T helper lymphocyte depression in early human pregnancy. J. Reprod. Immunol. 5:55.[Medline]
  66. Mass, R. A., P. A. M. Roest, M. J. Becker, I. S. Weimar, H. F. J. Dullens, W. Denotter. 1992. Effector cells of low dose IL-2 immunotherapy in tumor-bearing mice: tumor cell killing by CD8+ cytotoxic T lymphocytes and macrophages. Immunobiol. 186:214.[Medline]
  67. Elasser-Beile, U., U. Wetterauer, W. Schultze-Seemann, H. Gallati, J. S. Monting, S. von Kleist. 1996. Analysis of the immune reactivity of infiltrating and peripheral lymphocytes from patients with renal cell carcinoma by measuring cytokine secretion. Cancer Immunol. Immunother. 42:93.[Medline]
  68. Wang, W., C. Redovan, R. Tubbs, T. Olencki, E. Klein, S. Kudoh, J. Finke, R. M. Bukowski. 1995. Selective cytokine gene expression in renal cell carcinoma tumor cells and tumor-infiltrating lymphocytes. Int. J. Cancer 61:780.[Medline]
  69. Maeurer, M. J., D. M. Martin, C. Castelli, E. Elder, G. Leder, W. J. Storkus, M. T. Lotze. 1995. Host immune response in renal cell cancer: interleukin-4 (IL-4) and IL-10 mRNA are frequently detected in freshly collected tumor-infiltrating lymphocytes. Cancer Immunol. Immunother. 41:111.[Medline]
  70. Van den Hove, L. E., S. W. Van Gool, H. Van Poppel, L. Baert, L. Coorevits, B. Van Damme, J. L. Ceuppens. 1997. Phenotype, cytokine production and cytolytic capacity of fresh (uncultured) tumor-infiltrating T lymphocytes in human renal cell carcinoma. Clin. Exp. Immunol. 109:501.[Medline]
  71. Jr Coggin, J. H., J. W. Rohrer, A. L. Barsoum. 1997. A new immunobiological view of radiation-promoted lymphomagenesis. Int. J. Radiat. Biol. 71:81.[Medline]
  72. Rao, C. N., V. Castronovo, M. C. Schmitt, U. M. Wewer, A. P. Claysmith, L. A. Liotta, M. E. Sobel. 1989. Evidence for a precursor of the high affinity metastasis-associated murine laminin receptor. Biochemistry 28:7476.[Medline]
  73. Van den Brule, F. A., J. Price, M. E. Sobel, R. Lambotte, V. Castronovo. 1994. Inverse expression of two laminin binding proteins, 67LR and galectin-3, correlates with the invasive phenotype of trophoblastic tissue. Biochem. Biophys. Res. Commun. 201:388.[Medline]
  74. Sobel, M. E.. 1993. Differential expression of 67 kD laminin receptor in cancer. Cancer Biol. 4:311.
  75. Landowski, T. H., E. A. Dratz, J. R. Starkey. 1995. Studies of the structure of the metastasis-associated 67 kDa laminin binding protein: fatty acid acylation and evidence supporting dimerization of the 32 kDa gene product to form the mature protein. Biochemistry 34:11276.[Medline]
  76. Castronovo, V.. 1993. Laminin receptors and laminin-binding proteins during tumor invasion and metastasis. Invasion Metastasis 13:1.[Medline]
  77. Castronovo, V., F. A. Van Den Brule, P. Jackers, N. Clausse, F. T. Liu, C. Gillet, M. E. Sobel. 1996. Decreased expression of galectin-3 is associated with progression of human breast cancer. J. Pathol. 179:43.[Medline]
  78. Pellegrini, R., S. Martignone, E. Tagliabue, D. Belotti, R. Bufalino, N. Cascinelli, S. Menard, M. I. Colnaghi. 1995. Prognostic significance of laminin production in relation with its receptor expression in human breast carcinomas. Breast Cancer Res. Treat. 35:195.[Medline]
  79. Martignone, S., S. Menard, B. Bufalino, N. Cascinelli, R. Pellegrini, E. Tagliabue, S. Andreola, F. Rilke, M. I. Colnaghi. 1993. Prognostic significance of the 67 kilodalton laminin receptor expression in human breast carcinomas. J. Natl. Cancer Inst. 85:398.[Abstract/Free Full Text]
  80. Mafune, K., T. S. Ravikumar, J. M. Wong, H. Yow, L. B. Chen, Jr G. D. Steele. 1990. Expression of a Mr 32,000 laminin-binding protein messenger RNA in human colon carcinoma correlates with disease progression. Cancer Res. 50:3888.[Abstract/Free Full Text]
  81. Van den Brule, F. A., C. Cuicu, A. Berchuck, R. C. Bast, M. Deprez, F. T. Liu, D. N. Cooper, C. Pieters, M. E. Sobel, V. Castronovo. 1996. Expression of the 67-kD laminin receptor, galectin-1, and galectin-3 in advanced human uterine adenocarcinoma. Hum. Pathol. 27:1185.[Medline]
  82. Kudoh, S., C. Redovan, P. Rayman, M. Edinger, R. R. Tubbs, A. Novick, J. H. Finke, R. M. Bukowski. 1997. Defective granzyme B gene expression and lytic response in T lymphocytes infiltrating human renal cell carcinoma. J. Immunother. 20:479.
  83. Angevin, E., F. Kremer, C. Gaudin, T. Hercend, F. Triebel. 1997. Analysis of T-cell immune response in renal cell carcinoma: polarization to type 1-like differentiation pattern, clonal T-cell expansion and tumor-specific cytotoxicity. Int. J. Cancer 72:431.[Medline]



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