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[Cancer Research 62, 2611-2617, May 1, 2002]
© 2002 American Association for Cancer Research

Immunology

Human CD4+ Effector T Cells Mediate Indirect Interleukin-12- and Interferon-{gamma}-dependent Suppression of Autologous HLA-negative Lung Tumor Xenografts in Severe Combined Immunodeficient Mice1

Nejat K. Egilmez, Stephen D. Hess, Fang-An Chen, Hiroshi Takita, Thomas F. Conway and Richard B. Bankert2

Department of Microbiology, State University of New York at Buffalo, Buffalo, New York 14214 [N. K. E., S. D. H., T. F. C., R. B. B.]; Department of Otolaryngology, Division of Head and Neck Surgery, New York Medical Center, New York, New York 10016 [F-A. C.]; and Buffalo Thoracic Surgical Associates, Buffalo, New York 14209 [H. T.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A human/severe combined immunodeficient mouse chimeric model was used to demonstrate that peripheral blood leukocytes (PBLs) from a patientwith lung cancer completely suppress the growth of an autologous tumorin a PBL dose-dependent fashion repeatedly and over a 4-year period. Suppression of the patient’s tumor required CD4+ T cells, CD56+ natural killer cells, and CD14+ monocytes/macrophages, but was completely independent of CD8+ T cells. The CD4+ effector cells promoted tumor killing indirectly because direct tumor recognition and killing are precluded by the absence of MHC class I and II molecules on the tumor cells. Tumor suppression was found to require both human interleukin-12 (IL-12) and IFN-{gamma}, which were produced and released by the patient’s monocytes and T cells, respectively. These results establish that human CD4+ T cells present in the peripheral blood of a patient with lung cancer are able to orchestrate cytokine-dependent killing of an autologous MHC-negative tumor indirectly and without codependence on CD8+ T cells. We conclude that human tumor suppression is achieved in vivo even in the absence of MHC molecules on tumor cells. This tumor suppression is mediated indirectly by cytokines produced by the patient’s PBLs that ultimately initiate tumor killing via several, presently incompletely defined mechanisms.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MHC class I molecules on the surface of tumor cells are necessary for CD8+ T-cell recognition and direct killing of tumor cells by CTLs (1, 2, 3) . The complete loss of MHC class I antigens by a tumor therefore renders these cells resistant to direct tumor-specific CTL killing (2, 3, 4, 5, 6, 7) . This has led investigators to speculate that altered HLA class I expression, regardless of mechanism, contributes significantly to a tumor’s ability to avoid immune recognition and destruction. However, the loss of HLA class I antigen expression may have a positive influence on effector cell recognition, particularly by NK3 cells. The increased NK susceptibility of tumor cells with MHC class I antigen loss has been demonstrated in several studies (8, 9, 10) . However, some tumor cell lines that are totally lacking MHC class I antigens are resistant to NK cell-mediated lysis in vitro (10 , 11) , and HLA class I antigen-negative tumor cells are able to grow and metastasize in patients. Thus, the functional significance of HLA class I antigen loss by tumors remains open to question, and the role of lymphocytes in the immune surveillance and control of human tumors in vivo is still unclear.

Because most nonhematopoietic tumors do not express MHC class II molecules, which are required for CD4+ T-cell direct recognition, the potential role of CD4+ T cells in controlling solid tumor growth has been rather narrowly defined and is less well understood. The requirement for CD4+ T cells in antitumor responses has been attributed to providing help during priming to achieve full activation and effector function of tumor-specific CD8+ CTLs. CD4+ T cells have been shown to be required for CD8+ T cells to (a) establish long-term radio-resistant, specific memory immunity against tumors (12) ; (b) sustain cytotoxic T-cell responses during chronic viral infection (13) ; and (c) maintain proliferation and viability of adoptively transferred CD8+ T cells in patients (14) .

Several studies, however, have suggested a much broader role for CD4+ T cells in mediating antitumor effector functions that are independent of CD8+ T cells. It has been shown in mouse tumor models that adoptively transferred CD4+ T cells can eliminate cancer cells in vivo in the absence of CD8+ T cells (15, 16, 17, 18) . In several studies, the adoptive transfer of CD4+ T cells (in the absence of CD8+ T cells) led to the complete elimination in vivo of tumor cells that do not express MHC class II antigens (15, 16, 17) . In tumor vaccination studies, CD4+ T cells have been shown to play a direct role in the effector phase of tumor rejection as well as the induction phase of tumor immunity (19) . It has also been shown that vaccination against tumor cells lacking MHC class I expression resulted in tumor rejection that was dependent on CD4+ T cells and NK cells, but not CD8+ T cells (20) . A central role for CD4+ T cells in the antitumor immune response was established by the demonstration that cytokines produced by both TH1 and TH2 cells after tumor cell vaccination activated eosinophils as well as macrophages to mediate tumor killing (21) and by the demonstration that CD4+ T cells can eliminate MHC class II-negative tumor cells in vivo by the indirect effect of IFN-{gamma} release (22) . All of these studies were performed in mouse tumor models, and to date, there has been no convincing evidence for CD4+ effector T cells playing a significant role in tumor rejection in humans.

To address the question of the functional significance of HLA loss of tumors with respect to immune surveillance and to investigate the role of human CD4+ T cells in tumor rejection, we studied a patient’s antitumor response to a MHC class I- and class II-negative tumor resected from that patient. This was achieved by establishing a tumor cell line from the patient’s (MHC-negative) primary lung tumor biopsy tissue and by monitoring the ability of the patient’s PBLs to suppress the growth of tumor xenografts after coengraftment of the autologous tumor and PBLs into SCID mice (23) . In the following studies, we establish that CD4+ T cells (but not CD8+ T cells) are required for the arrest of tumor growth. We also show that human IL-12 and human IFN-{gamma} are essential for the elimination of the tumor cells and that the antitumor effect of the cytokines appears to be mediated indirectly via multiple effector mechanisms that involve monocytes/macrophages and NK cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice.
C.B.17 scid/scid mice were obtained from our breeding colony. All mice were maintained in microisolation cages (Lab Products Inc., Maywood, NJ) under pathogen-free conditions in the animal maintenance facilities of Roswell Park Cancer Institute. Male or female mice 8–12 weeks of age were used in the experiments.

Tumor Cells.
Human non-small cell lung carcinoma cell line RPCI-9530 was established in our laboratory from a patient’s resected primary tumor. Cells were maintained in DMEM-nutrient mixture F-12 (Life Technologies, Inc., Grand Island, NY), supplemented with 10% heat-inactivated FCS and were incubated at 37°C in 5% CO2. Cells were allowed to grow to ~80% confluence and were harvested with 0.25% trypsin and 1 mM EDTA in DMEM.

Case Summary of the Patient.
The patient (no. 9530) was diagnosed with a 3.7-cm undifferentiated adenocarcinoma of the left upper lobe of the lung, stage IIB. The patient’s preoperative functions were extremely poor and indicative of advanced chronic obstructive pulmonary disease. In view of this condition, the lung lesion was removed by local resection with a narrow margin around the tumor instead of a lobectomy, which would be the standard approach for a patient without chronic obstructive pulmonary disease. Despite this less-than-optimal surgical therapy, the patient is still alive with no evidence of tumor for 4 years.

Human Peripheral Blood Cells.
HuPBLs were obtained by leukapheresis of peripheral blood donated by the lung cancer patient at various times after the resection of the primary lung carcinoma RPCI-9530. RBCs were lysed with freshly prepared 0.83% NH4Cl solution, and the HuPBLs were washed twice and resuspended at a concentration of 1 x 107 cells/ml in DMEM.

Genotyping of Patient Tumor and PBLs.
DNA samples obtained from the original tumor biopsy tissue, the cell line RPCI-9530, and patient PBLs were genotyped by a PCR reverse dot-blot assay using the AmpliType PM PCR Amplification and Typing kit as recommended by the manufacturer (Perkin-Elmer, Foster City, CA).

Lymphocyte Subset Depletions.
After the RBCs were lysed, CD4+, CD8+, CD14+, or CD56+ cells were depleted either by first pulsing with the appropriate monoclonal antibody followed by incubation with magnetic beads coated with goat antimouse IgG (Advanced Magnetics, Cambridge, MA) as described previously (23) or by direct binding to antimouse CD4, CD8, CD14, or CD56 MACS microbead columns (Miltenyi Biotec, Auburn, CA) as recommended by the supplier. Briefly, cells were resuspended in cold MACS buffer (0.5% BSA-2 mM EDTA in PBS) at a concentration of 1 x 107 cells/80 µl and mixed with 20 µl of microbeads specific for the population to be depleted in the same buffer. The mixture was incubated at 4°C for 15 min, diluted with 10 ml of cold MACS buffer, and centrifuged at 500 x g for 5 min at 4°C. The pellet was resuspended in MACS buffer (1 x 108 cells/0.5 ml) and passed through the column. The flow-through was collected and kept on ice. The column was washed three times with 0.5 ml of MACS buffer, and the flow-through was collected in the same tube. The cells were washed once with DMEM, and an aliquot was counted to determine yield. The efficacy of depletion was confirmed by FACS analysis of depleted cell populations, and >95% depletion was found for all subsets of depleted cells.

Flow Cytometry.
Tumor cells or HuPBLs were resuspended in PBS containing 2% FCS and stained with one of the following phycoerythrin-labeled antibodies (Becton Dickinson, San Jose, CA): anti-MHCI class, anti-MHCII class, anti-Fas, anti-CD80, anti-CD86, anti-intercellular adhesion molecule-1, and anti-epidermal growth factor receptor in the case of tumor cells, or anti-CD4, anti-CD8, or anti-CD56 in the case of HuPBLs as described previously (23) . Aliquots from each sample (at least 5000 cells) were analyzed using a FACScan cell sorter (Becton Dickinson).

Tumor Cell-HuPBL Engraftment into SCID Mice.
One day before tumor inoculation, SCID mice received i.p. injections containing monoclonal antibody TM-ß1 (specific for the murine IL-2R ß subunit) to deplete murine NK cells (24) . Freshly harvested RPCI-9530 tumor cells were mixed with HuPBLs at the indicated ratio and were inoculated s.c. (1 x 106 RPCI-9530 cells and the indicated number of HuPBLs in 0.2 ml DMEM/mouse) into the ventral caudal midline area of SCID mice. Tumor growth was monitored once a week for 8–12 weeks or until the tumor reached a diameter of 1.5 cm. Tumor volume was determined using the formula: a2 + b/2, where a is the shortest and b is the longest perpendicular dimensions of the tumor.

Effect of IFN-{gamma} on the Proliferation of RPCI-9530 Cells in Vitro and in Vivo.
The effect of rhIFN-{gamma} on the proliferation of RPCI-9530 in vitro was assessed using a MTT assay (25) . Briefly, RPCI-9530 tumor cells were cultured overnight in a 96-well plate (1 x 104 cells/well) in 100 µl of complete medium (DMEM/F-12 + 10% FCS). After overnight culture, 100 µl of complete medium containing rhIFN-{gamma} were added to each well at the following concentrations: 10, 100, and 1000 units/ml. Complete medium without any rhIFN-{gamma} was used as a negative control, whereas medium containing 2.0 mg/ml G418 was used as a positive control for inhibition of cell growth. One group of cells cultured with 1000 units/ml rhIFN-{gamma} also received 20 µl of 1:4 diluted antihuman IFN-{gamma} ascites (hybridoma B133.3.1, a generous gift from Dr. G. Trinchieri, Dardilly, France). After the addition of rhIFN-{gamma}, cells were cultured at 37°C in 5% CO2 for 4 days without any change in medium. After 4 days of in vitro culture, a 4-h MTT assay was performed to determine the relative concentration of viable cells in each well. Medium (100 µl) was removed from each well, and 20 ml of 0.5% MTT solution (made fresh in PBS) were added. Cells were incubated at 37°C for 4 h. A solution of 0.04 M HCl in isopropanol (200 µl/well) was used to solubilize MTT formazan. The absorbance of each well was measured at 540/660 nm with a dual wavelength microplate reader. Each concentration of IFN-{gamma} was tested in 12 replicate wells.

To assess the in vivo effects of IFN-{gamma} on tumor cell growth, SCID mice were depleted of murine NK cells as described previously and received s.c. inoculations of 1 x 106 RPCI-9530 tumor cells in 200 µl of DMEM with or without 1000 units of rhIFN-{gamma}. Subsequently, mice received daily i.p. injections of rhIFN-{gamma} in DMEM (500 units/200 µl) or DMEM only (control) for 10 consecutive days. Tumor measurements were obtained weekly, and tumor volumes were calculated as described.

In Vivo Neutralization of IL-12, IFN-{gamma}, and iNOS.
To neutralize human IL-12 in vivo, NK-depleted SCID mice received injections of the monoclonal antibody BH6 (26) at a final concentration of 50 µg/200 µl in PBS. Human IFN-{gamma} was neutralized by injecting 200 µl of antihuman IFN-{gamma} ascites (hybridoma B133.3.1) diluted 1:4 in PBS into the mice. iNOS activity was inhibited by injection of 0.2 mg/200 µl of L-NAME in PBS. Control mice received injections of the inactive isoform, D-NAME (0.2 mg/200 µl) in PBS or PBS only. All injections were given i.p. for 10 consecutive days, with the first injection given ~6 h before s.c. inoculation of tumor + PBLs.

ELISA to Quantify Human IFN-{gamma}.
Human IFN-{gamma} levels in the sera of the mice were determined by an ELISA developed in our laboratory as described previously (23) . Briefly, 96-well microtiter plates were coated with a mouse antihuman IFN-{gamma} monoclonal antibody (M-700A; Endogen, Cambridge, MA) overnight at 4°C. Standards and unknowns were added to the well and incubated with biotin-labeled antihuman IFN-{gamma} monoclonal antibody M-701 (Endogen). Bound antibody was detected by the addition of avidin-horseradish peroxidase conjugate and the substrate o-phenylenediamine (Sigma Chemical Co., St. Louis, MO).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment and Characterization of Human Non-Small Cell Lung Tumor Cell Line RPCI-9530.
Small (1 mm3) pieces of biopsy tissue obtained from a patient (patient 9530) diagnosed with a poorly differentiated carcinoma of the lung were first implanted s.c. into SCID mice. Tumors were allowed to engraft and grow to approximately 1 cm3. The tumor xenografts were removed, and a single-cell suspension was placed into culture as described in "Materials and Methods." The cell line derived from this tissue was identified as RPCI-9530.

DNA obtained from this cell line, the corresponding patient biopsy tissue, and the patient’s PBLs was genotyped with a PCR reverse-dot blot assay using the AmpliType PM PCR Amplification and Typing Kit (Perkin-Elmer). The results of this analysis established that the cell line was of human origin and was indistinguishable from the original tumor biopsy tissue.

FACS analysis of immunofluorescently stained RPCI-9530 tumor cells established that the cells were positive for Fas, intercellular adhesion molecule-1, and epidermal growth factor receptor, but expressed undetectable levels of MHC class I or class II antigens on their surface. This was the first human lung tumor of 21 established in our laboratory that expressed neither MHC class I or II antigens. Western blot analysis of the RPCI-9530 tumor revealed the presence of low levels of intracytoplasmic MHC class I {alpha}-chain, but no detectable ß2-microglobulin. No message for ß2-microglobulin was detected by gene array analysis of cDNA obtained from tumor xenografts.

Patient’s PBLs Suppress Growth of RPCI-9530 Tumor Xenografts in a Cell Dose-dependent Fashion.
PBLs were obtained by leukapheresis of patient 9530. The RPCI-9530 tumor cells (1 x 106/mouse) were injected s.c. into SCID mice either alone or with increasing numbers of the patient’s PBLs, resulting in PBL:tumor cell ratios of 2.5:1, 5:1, 10:1, and 20:1. Mice in all groups were monitored weekly for tumor growth. The results presented in Fig. 1Citation show that the patient’s PBLs suppressed tumor growth in a cell dose-dependent fashion. At ratios of 2.5:1 and 5:1, tumors grew progressively, as did tumors in mice that received tumor cells only. In mice inoculated with 1 x 107 PBLs (10:1) there was a delay in tumor growth, but ultimately tumors grew progressively in all mice. In mice that received 2 x 107 PBLs (20:1), tumor growth was completely suppressed in three of five mice. Complete arrest of tumor growth was observed in all mice that received 4 x 107 PBLs (40:1; data not shown.) These results are representative of four separate experiments over a period of 4 years. The first experiment with the patient’s PBLs was conducted 6 months after the tumor was excised.



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  		Fig. 1. Patient’s PBLs suppress the growth of autologous tumors in a dose-dependent manner in the SCID/Winn model. RPCI-9530 cells (1 x 106) were mixed with increasing doses of PBLs obtained from patient 9530 and engrafted s.c. into SCID mice as described in "Materials and Methods." Tumor growth was monitored for a period for 8 weeks by weekly tumor measurements (see "Materials and Methods"). Each group consisted of five mice. Bars, SD. Complete tumor suppression was observed only at a ratio of 20 PBLs to 1 tumor cell in three of five mice.

 
Patient’s CD4+ T Cells and NK Cells, but not CD8+ T Cells, Are Required for Rejection of Tumor Xenografts.
To determine which cells in the patient’s PBLs were required for the suppression of RPCI-9530 tumor xenografts, PBLs were depleted of CD4+ or CD8+ T cells or CD56+ NK cells before engraftment of the tumor cells into SCID mice. As illustrated in Table 1Citation , removal of the CD4+ T cells abrogated the tumor suppression: 9 of 10 mice given CD4-depleted PBLs (in a 20:1 ratio of PBLs to tumor cells) developed progressively growing tumors by 8 weeks. In addition to CD4+ T cells, NK cells in the PBLs also contributed to the tumor suppression: 8 of 10 mice receiving PBLs depleted of CD56+ cells developed tumors 8 weeks after engraftment of the tumor and PBLs. There was no evidence of coparticipation of CD8+ T cells: tumors developed in only 2 of 10 mice given CD8-depleted PBLs in a PBL:tumor ratio of 20:1 (the same as in the control group, in which tumors developed in 2 of 10 mice given nondepleted PBLs). All mice inoculated with tumor cells only (i.e., no PBL) developed tumors within 8 weeks (data not shown). These results establish that the patient’s CD4+ T cells and CD56+ NK cells, but not CD8+ T cells, were required for the suppression of the MHC-negative tumor xenografts. Because the tumor was MHC class II negative and, therefore, direct recognition and killing of tumors by CD4+ T cells was not possible, the most likely mechanism of CD4+ T-cell-mediated tumor killing would be cytokines released by the PBLs. This could result in either a direct suppressive effect of the cytokine on the tumor or an indirect effect of the cytokine on accessory cells such as NK cells or macrophages, which are then activated to kill the tumor. To investigate these possibilities, we monitored the sera of experimental and control mice for the presence of human cytokines and assessed the effect of cytokine-specific function-blocking antibodies on tumor suppression.


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  		Table 1 Rejection of RPCI-9530 tumor xenograft is dependent on the patient’s CD4+, but not CD8+ T cells

 
Rejection of RPCI-9530 Tumor Xenografts by the Patient’s PBLs Is Dependent On Human IL-12 and IFN-{gamma}.
SCID mice inoculated s.c. with the patient’s PBLs and tumor cells were shown to have elevated levels of human IFN-{gamma} in their sera 1 week after engraftment of the human cells. No human IFN-{gamma} was detected in control mice inoculated with tumors only. The production of human IFN-{gamma} depended on CD4+ T cells and CD14+ monocytes and macrophages because mice that received tumor cells and PBLs depleted of these cells did not develop detectable levels of IFN-{gamma} in their sera (Fig. 2)Citation . The production of human IFN-{gamma} was independent of both CD8+ T cells and CD56+ NK cells. To address the possible functional significance of IFN-{gamma} with respect to the patient’s PBL-mediated tumor suppression, SCID mice inoculated with PBLs and the patient’s RPCI-9530 tumor cells were treated with function-blocking antihuman IFN-{gamma}-specific antibodies. The results of these experiments are presented in Fig. 3Citation . All mice in these experiments were inoculated s.c. with the patient’s PBLs and tumor cells at a ratio of 20 PBLs to 1 tumor cell. Antihuman IFN-{gamma} antibodies completely abrogated the tumor-suppressive activity of the PBLs. All mice (five of five) treated with anti-IFN-{gamma} antibodies developed rapidly progressing tumors, whereas only two of five control mice showed any evidence of tumor growth by 10 weeks after engraftment, and these tumors were significantly smaller than the ones observed in the anti-IFN-{gamma}-treated mice.



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  		Fig. 2. Effect of PBL subset depletion on serum levels of human IFN-{gamma}. PBLs were obtained from patient 9530 by leukapheresis and depleted of the various PBL subsets in vitro, using antibody-coated magnetic beads (see "Materials and Methods"). After depletion, PBLs (2 x 107) were mixed with RPCI-9530 tumor cells (1 x 106) and injected s.c. into NK-depleted SCID mice. On day 5 postinoculation, mice were bled, and sera from individual mice were assayed for the presence of human IFN-{gamma} by ELISA. Data are presented as an average of five mice/group (bars, SD). Human IFN-{gamma} was not detected in any of the mice inoculated with CD4+-depleted or CD14+-depleted PBLs. The differences between groups with no IFN-{gamma} (CD4+ and CD14+ depletion) and groups with detectable IFN-{gamma} (no depletion, CD8+ depletion, and CD56+ depletion) were statistically significant (P < 0.05, unpaired Student’s t test). No significant difference existed among the three groups with detectable levels of serum IFN-{gamma} (P > 0.4, unpaired Student’s t test).

 


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  		Fig. 3. Effect of human IL-12 and human IFN-{gamma} neutralization on tumor growth in the SCID/Winn model. PBLs and tumor cells from patient 9530 were mixed at a ratio of 20:1 and inoculated s.c. into NK-depleted SCID mice (n = 5 for the PBS only and anti-IFN-{gamma} groups; n = 4 for the anti-IL-12 group). Mice received daily i.p. injections of neutralizing antibodies to human IL-12 or human IFN-{gamma}, whereas control mice received injections of an isotype control antibody only (see "Materials and Methods" for doses and schedules). Tumor growth was assessed by weekly measurements, and the average tumor volumes from all mice in each group were plotted (bars, SD).

 
Because IL-12 is known to have significant antitumor activity and is a potent inducer of IFN-{gamma}, it was also of interest to determine whether human IL-12 was present in the sera and whether function-blocking antibodies specific for IL-12 had any effect on the PBL-mediated tumor suppression. Although no human IL-12 was detected in the sera, all mice (four of four) inoculated with PBLs and tumor cells at a 20:1 ratio and treated with antihuman IL-12 function-blocking antibodies developed rapidly progressing tumors (Fig. 3)Citation . In the anti-IL-12 antibody-treated mice, serum levels of IFN-{gamma} were significantly reduced compared with the control mice inoculated with PBLs and tumor cells (data not shown).

We conclude from these results that human IFN-{gamma} produced by CD4 + T cells is required for PBL-dependent tumor suppression and that endogenous IL-12 (most likely produced by monocytes/macrophages within the PBL inoculum) plays a role in this response by augmenting IFN-{gamma} production by CD4+ T cells.

Effect of IFN-{gamma} on MHC Class I and II Expression in RPCI-9530 Tumor Cells.
Because IFN-{gamma} is known to be a potent stimulator of MHC class II expression and the IFN-{gamma} was shown to be required for tumor suppression, one possible explanation for the CD4+ T-cell-dependent tumor suppression was that IFN-{gamma} up-regulates MHC class II antigen on the tumor, leading to direct recognition and killing of the tumor by CD4+ T cells. This possibility was addressed by determining the level of MHC class II antigens on RPCI-9530 cells in the presence or absence of recombinant human IFN-{gamma}. RPCI-9530 tumor cells did not express detectable levels of MHC class II antigens, and there was very little or no induced expression of HLA-DR, -DP, or -DQ after incubation of the cells for 46 h with IFN-{gamma} (data not shown). A positive control human lung tumor cell line, RPCI-9055, which expresses MHC class I and II antigens constitutively, revealed an up-regulation of class II expression after a 46-h incubation with IFN-{gamma}. We conclude that the effect of IFN-{gamma} on the suppression of RPCI-9530 tumor/PBL xenografts is not attributable to an up-regulation of MHC class II antigens on the tumor to make it directly recognizable by CD4+ effector T cells.

In the Absence of Patient’s PBLs, IFN-{gamma} Fails to Suppress or Inhibit the Growth of RPCI-9530 Tumor Xenografts in SCID Mice.
IFN-{gamma} could be acting directly on tumor cells to suppress their proliferation. To address this possibility, mice were inoculated with tumors (in the absence of PBLs) and treated with rhIFN-{gamma}. A total of 1 x 106 RPCI-9530 tumor cells were injected s.c. into SCID mice with or without human IFN-{gamma}. In the absence of human PBLs, no significant difference was observed in tumor growth between the control and cytokine-treated tumor-bearing mice. Cultivation of the RPCI-9530 tumor cells in vitro with IFN-{gamma} was shown to have only a very modest effect on cell proliferation. Therefore, although IFN-{gamma} at a very high dose has a slight direct effect on tumor growth in vitro, it is insufficient by itself (i.e., in the absence of the patient’s PBLs) for the complete elimination of tumor xenografts in vivo. Another possibility is that IFN-{gamma} is mediating its antitumor effect indirectly by activating accessory cells such as monocytes and macrophages. To address this possibility, we undertook additional cell depletion studies.

CD14+ Monocytes and Macrophages Are Required for the Suppression of RPCI-9530 Tumor Xenografts.
Macrophages and monocytes were present within the PBL/tumor xenografts and were the most likely source of the endogenous IL-12 shown above to be involved in tumor suppression. These cells also had the potential dual capacity of presenting antigen to T cells and of killing tumor cells. With respect to the latter, it is well established that IFN-{gamma} activates macrophages and that the resultant tumoricidal macrophages are able to recognize and kill tumor cells through various mechanisms. To determine whether monocytes/macrophages were required for the rejection of tumor xenografts, CD14+ cells were depleted from the patient’s PBLs before engraftment into SCID mice with RPCI-9530 tumor cells at a PBL:tumor ratio of 20:1. In this experiment, CD4+ and CD8+ T cells were also eliminated individually from PBLs before injection into SCID mice. The results are summarized in Table 2Citation . Once again, the depletion of CD8+ T cells had no effect on tumor growth, whereas tumors grew in all but one mouse that received CD4+ T-cell-depleted PBLs (Table 2)Citation . A complete loss of tumor suppression was observed after the depletion of CD14+ cells. These results, which were repeated in three independent experiments, establish that in addition to CD4+ T cells and NK cells, macrophages/monocytes are required for the complete suppression of tumor xenografts.


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  		Table 2 CD14+ monocytes/macrophages are critical to tumor suppression that is dependent on CD4+ T cells and CD56+ NK cells in the HuPBL/tumor SCID mouse xenograft model

 
We conclude that PBLs from a patient with lung cancer are able to suppress the growth of an autologous MHC class I- and II-negative tumor in vivo. Moreover, this suppression is mediated indirectly by cytokines released from CD4+ T cells and monocytes, which activate accessory cells (including, but not restricted to NK cells, monocytes, and macrophages) that are, in part, responsible for the observed inhibition of the tumor. Other possible mechanisms contributing to the tumor suppression are noted in the "Discussion."


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We showed that CD4+ T cells in the peripheral blood of a patient diagnosed with lung cancer are able to orchestrate the suppression of autologous tumor xenografts. The suppression of the tumor xenografts was indirect because the tumor cells were MHC class I and II negative, and it was dependent on human IFN-{gamma} and IL-12 produced by the coengrafted patient PBLs. The antitumor CD4+ T cells were present in the patient’s peripheral blood at least 6 months after the surgical resection of the tumor and present for up to 4 years after surgery. During this period, the patient showed no clinical evidence of new tumor growth or metastasis. In the SCID mouse model, the CD4+ T-cell-mediated tumor rejection occurred with the coparticipation of CD56+ NK cells and CD14+ monocytes/macrophages, but without any evidence of CD8+ T-cell involvement. All of the data presented here were derived from multiple experiments with tumor and lymphocytes derived from a single patient whose tumor was MHC negative. Similar experiments have now been conducted with another patient whose tumor was MHC class I positive (patient 10319). Data from these studies have once again established that cells in the patients’ PBLs are able to suppress autologous tumor xenografts in a cell dose-dependent fashion and that this response is dependent on human IFN-{gamma} and IL-12 produced by the coengrafted PBLs. However, in addition to NK cells, monocytes, and CD4+ T cells, the patients’ CD8+ T cells were found to contribute to the suppression of the MHC class I-positive tumor (data not shown). Although all of the mechanisms or combinations of events responsible for the tumor suppression observed with both patients remain unresolved, one likely scenario is that IL-12 released by monocytes and macrophages stimulates the production and release of IFN-{gamma} from the T cells. The IFN-{gamma} activates NK cells and macrophages, resulting in generation of tumoricidal cells.

Several other possible mechanisms could be contributing to the IFN-{gamma}-mediated tumor killing. IFN-{gamma} is known to directly suppress tumor growth or to induce the expression of MHC class I and class II antigens, making the tumor more easily recognized by CD8+ or CD4+ T cells (27) . Both of these possibilities have been ruled out here. IFN-{gamma} in the absence of human PBLs had no effect on the growth of the RPCI-9530 tumor xenografts, and had no direct effect on the proliferation of the RPCI-9530 cells in vitro (data not shown). The lack of an IFN-{gamma} effect on class I expression was expected because the tumor was negative for ß2-microglobulin. The possibility that IFN-{gamma} had an effect on class II expression in a small subset of tumors could not be ruled out, but there was no detectable effect on the majority of the tumor cells. Other possible mechanisms contributing to the IFN-{gamma}-dependent tumor killing have been considered. Another possible mechanism is via the IFN-{gamma}-induced production of iNOS by macrophages, leading to the release of NO, which is cytotoxic to both normal and neoplastic cells (27) . The possible role of iNOS in the patient’s PBL-mediated tumor suppression was addressed in our model by use of the iNOS inhibitor L-NAME. The results demonstrated that this inhibitor had no effect on the PBL-mediated suppression of tumor xenografts (Table 3)Citation or on the production of IFN-{gamma} (data not shown).


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  		Table 3 Inhibition of iNOS activity has no effect on PBL-mediated tumor suppression

 
At present, an attempt is being made to identify the molecular events that may contribute to the PBL-mediated tumor suppression by monitoring increases or decreases in the expression of genes encoding selected cytokines, chemokines, integrins, angiogenic factors, costimulatory molecules, and their receptors in tumor xenografts with or without the patient’s PBLs. Analysis of gene expression patterns in tumor xenografts with and without the patient’s PBLs has revealed many significant changes (i.e., both increases and decreases) in gene expression patterns associated with the addition of patient PBLs that suggest events that may contribute to tumor rejection. The changes observed in gene expression are consistent with events that would occur with both a cognitive and innate immune response, including up-regulation of cytokine and chemokine receptors and activation and costimulatory molecules (data not shown). Significant increases in genes encoding two chemokines, IP-10 and MIG, suggest the possibility that angiostasis may contribute to IFN-{gamma}-dependent tumor suppression because others have established that these ELR-negative CXC chemokines inhibit neovascularization (28 , 29) and the overexpression of MIG has been shown to inhibit the growth of human lung tumor xenografts in SCID mice (30) . It is also possible that IFN-{gamma} may have a direct antiangiogenic effect on the xenografts by interacting with endothelial cells that may express IFN-{gamma} receptors (31) . The changes in gene expression patterns are provocative and suggestive of many possible antitumor mechanisms; however, further studies are needed to determine which changes in gene expression also lead to changes in protein expression and then to establish which proteins are contributing to the arrest of the tumor.

Our results and those of others (31) emphasize and support the concept that effective tumor immunity is complex and does not rely exclusively on direct tumor recognition and killing by effector cells. Moreover, the loss or absence of MHC antigens on the tumor does not abrogate the ability of immunocompetent cells to mount an effective antitumor response. CD4+ T cells have been shown to act as antitumor effector cells in a wide variety of MHC class II-negative animal tumor models (32, 33, 34) . In these models, the CD4+ T cells reject tumors without evidence of coparticipation of CD8+ T cells and in the absence of direct interaction of the effector T cells with antigen on the tumor. These and many other animal studies have been performed in vivo, using both cell depletion analyses and adoptive transfer studies, and have revealed that tumor killing occurs indirectly by cytokines that connect both the cognitive and innate immune systems in a complex and as yet poorly defined network (reviewed in Refs. 35 , 36 ). These complex mechanisms are best recognized and defined by in vivo systems. The lytic capacity of CTLs in vitro is often a poor predictor of the antitumor response in vivo (35) . Furthermore, in vitro systems are unable to account for or recognize the complete network of events that may contribute to tumor suppression in vivo. The human/SCID mouse chimeric model used here has made it possible to study the panoply of molecular events that contribute to human immune cell-mediated tumor rejection and to evaluate immunotherapeutic strategies for cancer (37) .

Although the chimeric model described here does not precisely reflect all of the physiological events within a patient, it can provide valuable insights that are not feasible with in vitro systems. However, one major potential limitation of the model is that some of the mechanisms that can inhibit tumors in this model occur in the absence of a tumor microenvironment and before the tumor has had a chance to attach, invade, and vascularize. Whether the mechanisms observed in the SCID model are relevant to established tumors in patients remains to be resolved. One new approach being taken is to engraft nondisrupted pieces of fresh human tumor biopsy tissues to establish xenografts that include a tumor microenvironment consisting of tumor cells, associated inflammatory cells, stromal cells, and an extracellular matrix (37) . Studies using this SCID model have revealed that the patients’ inflammatory cells remain viable and responsive to cytokines for prolonged periods and that these cells can be recruited to attack and destroy the tumor (26) . Changes in gene expression patterns observed in the intact microenvironment of the tumor xenograft after a local and sustained release of human IL-12 are very similar to those that have been observed in the SCID model reported here, i.e., a CD4+ T-cell-mediated IFN-{gamma}-dependent tumor suppression (37) . This newer SCID model is expected to more closely reflect events within a tumor microenvironment in a patient and will be the focus of future studies evaluating cancer immunotherapeutic strategies.


   ACKNOWLEDGMENTS
 
We thank Charlene Romanello and Cheryl Zuber for typing the manuscript. We also thank Sandra Yokota, Robert Parsons, and Jenni Loyal for outstanding technical support, and Dr. Harry Slocum and the Tissue Procurement Staff for their support in securing tumor biopsy tissues. We are also grateful to the entire Department of Laboratory Animal Resources staff at Roswell Park Cancer Institute, who provided us with valuable support in the care and maintenance of the mice used in this study. Finally, we are greatly indebted to patients 9530 and 10319 for providing the PBLs that made this study possible.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by NIH Grants CA96528, CA75235, and CA79879 and Roswell Park Cancer Institute Core Grant CA16056. Back

2 To whom requests for reprints should be addressed, at Department of Microbiology, State University of New York at Buffalo, 138 Farber Hall, 3435 Main Street, Buffalo, NY 14214. Phone: (716) 829-2701; Fax: (716) 829-2169; E-mail: rbankert{at}buffalo.edu Back

3 The abbreviations used are: NK, natural killer; HuPBL, human peripheral blood leukocyte; IL-12, interleukin-12; SCID, severe combined immunodeficient; FACS, fluorescence-activated cell sorting; rhIFN-{gamma}, recombinant human IFN-{gamma}; MTT, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; iNOS, inducible nitric oxide synthase; L-NAME and D-NAME, N-nitro-L-arginine methyl ester and N-nitro-D-arginine methyl ester, respectively. Back

Received 10/29/01. Accepted 2/26/02.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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