help button home button Am J Pathol R & D Systems
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Le Poole, I. C.
Right arrow Articles by Nickoloff, B. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Le Poole, I. C.
Right arrow Articles by Nickoloff, B. J.
(American Journal of Pathology. 2002;160:521-528.)
© 2002 American Society for Investigative Pathology

Regular Articles

Interferon-{gamma} Reduces Melanosomal Antigen Expression and Recognition of Melanoma Cells by Cytotoxic T Cells

I. Caroline Le Poole*, Adam I. Riker*, M. Eugenia Quevedo*, Lawrence S. Stennett*, Ena Wang{dagger}, Francesco M. Marincola{dagger}, W. Martin Kast{ddagger}, June K. Robinson* and Brian J. Nickoloff*

From the Department of Pathology,*
Cardinal BernardinCancer Center, Skin Oncology Research Program, and the CancerImmunology Program,{ddagger}
Loyola University MedicalCenter, Maywood, Illinois; and the SurgeryBranch,{dagger}
National Institutes of Health,Bethesda Maryland


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
In malignant melanoma, tumor-infiltrating lymphocytes are frequently reactive with melanosomal antigens. Achieving complete remissions by peptide therapy is frequently hampered by metastases evading immune recognition. The tumor microenvironment seems to favor reduced expression of target antigens by melanoma cells. Among candidate factors, interferon-{gamma} (IFN-{gamma}) (102 to 103 U/ml) suppressed expression of antigens MART-1, TRP-1, and gp100 by M14 melanoma cells as shown by immunohistology and fluorescence-activated cell sorting analysis, reducing MART-1 expression by >65%. Northern blot analysis revealed that reduced expression was regulated at the transcriptional level, demonstrating a 79% reduction in MART-1 transcript abundance after 32 hours of IFN-{gamma} treatment. To evaluate consequences of IFN-{gamma} exposure for immune recognition, MART-1-responsive T cells were reacted with pretreated HLA-matched melanoma cells. Cytotoxicity was reduced up to 78% by IFN-{gamma} pretreatment, and was restored by addition of MART-1 peptide AAGIGILTV for 2 hours. Examination of melanoma lesions by quantitative reverse transcriptase-polymerase chain reaction revealed up to 188-fold more abundant IFN-{gamma} transcripts when compared to control skin. Laser capture microdissection and immunohistology localized most IFN-{gamma}-producing T cells to the tumor stroma. Reduced MART-1 expression was frequently observed in adjacent tumor cells. Consequently, IFN-{gamma} may enhance inflammatory responses yet hamper effective recognition of melanoma cells.

Most T cells infiltrating malignant melanoma tumors are reactive with differentiation antigens, expressed by malignant as well as normal melanocytes.1 Such antigens include gp100, MART-1, tyrosinase, TRP-2, and TRP-1.2 By comparison, fewer T cells respond to progression antigens expressed by malignantly transformed cells of diverse lineages such as MAGE-1 and MAGE-3.3,4

Knowledge of target epitopes has provided opportunities for their clinical application to patients with metastatic melanoma. Patients can be immunized with antigenic peptides either directly or after previous incubation with autologous dendritic cells, frequently in combination with expression of immunostimulatory cytokines, ie, interleukin-2, IFN-{gamma}, or granulocyte/macrophage colony-stimulating factor.5-8 Unfortunately, tumor cells develop immune escape mechanisms such as down-regulation of major histocompatibility complex (MHC) molecules at the cell surface, reduced activity of the transporter associated with antigen presentation, and modulation of the proteasome complex.9-11 Dedifferentiation associated with reduced target antigen expression can also lead to immune escape of tumor cells.12

The incentive for dedifferentiation among melanoma cells remains poorly understood to date. The microenvironment may contribute to dedifferentiation by providing circumstances that favor loss of expression of one or more melanosomal differentiation markers.13,14 In this regard, tumor cells at extracutaneous sites encounter different extracellular matrix molecules, cytokines, and growth factors compared to epidermal melanocytes.

To explore a mechanism for immune evasion, the potential of IFN-{gamma} generated by activated tumor-infiltrating lymphocytes to modulate target antigen expression and subsequent immune recognition of melanoma cells was investigated. Consequences of IFN-{gamma} exposure for target antigen expression were tested at the protein and RNA levels in sporadically metastatic M14 cells.15 Functional immune recognition of tumor cells pretreated in the presence or absence of IFN-{gamma} was assessed using cytotoxic MART-1-reactive T cells and HLA-matched melanoma cells.16 Finally, the abundance of IFN-{gamma} in primary and metastatic melanoma lesions was assessed by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), laser capture microscopy, and immunohistology. Such investigations can contribute to a further understanding of the dedifferentiation process, representing a potential immune escape mechanism for malignant melanoma cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cultured Cells

The melanoma cell line M14 was cultured in 10% fetal bovine serum and standard antibiotics (100 IU/ml penicillin, 100 g/ml streptomycin, and 25 mg/ml amphotericin; Life Technologies, Inc., Gaithersburg, MD) in glutamine containing Dulbecco’s modified Eagle’s medium (Life Technologies, Inc.).15 The melanoma cell line 624.38 was cultured in glutamine containing Iscove’s modified Dulbecco’s medium (Mediatech Inc., Herndon, VA) with 10% normal human AB serum (Sigma Chemical Co., St. Louis, MO), 1% standard antibiotics and ciprofloxacin at 10 µg/ml (Bayer Corporation, Kankakee, IL).16 Ciprofloxacin is routinely added to patient-derived cultures to prevent mycoplasma infestation. Similarly maintained melanoma cell cultures F002, F003, and F010 were derived from metastases by fine needle aspiration and used within 20 passages.16 The MART-1-reactive, HLA-A*0201-restricted A42 T-cell line was likewise maintained in medium described for 624.38 in the presence of 1000 IU/ml interleukin-2 (R&D Systems, Minneapolis, MN).17

Normal melanocytes were obtained from skin by proteolytic treatment as described previously and maintained in Medium 154 (Cascade Biologicals, Portland, OR) supplemented with 4% fetal bovine serum and standard antibiotics (Life Technologies, Inc.), 0.6 ng/ml basic fibroblast growth factor, 10 nmol/L endothelin-1, 10 nmol/L {alpha}-melanocyte stimulating hormone ({alpha}-MSH), 1 µg/ml {alpha}-tocopherol, 5 µg/ml insulin, and 1 µg/ml transferrin (Sigma).18

Treatment by Inflammatory/Melanogenic Modulators

M14 cells were treated with 1000 IU/ml interferon-{gamma} (IFN-{gamma}), 100 ng/ml interleukin-13, or 10 µg/ml transforming growth factor-ß (all from R&D Systems) or with 0.6 ng/ml basic fibroblast growth factor, 1 nmol/L endothelin-1, 10 nmol/L {alpha}-MSH, 5 µg/ml insulin (all from Sigma) for 72 hours. Cells were subsequently fixed in acetone for immunohistology or in 2% paraformaldehyde for fluorescence-activated cell sorting (FACS) analysis, or alternatively washed and harvested in Tri-Reagent (Sigma) for RNA isolation.

Immunohistology

Indirect immunostaining was performed on fixed adherent cells or tissue sections by preincubation in 10% normal human serum in phosphate-buffered saline (PBS), followed by incubation in primary antibodies. Biotinylated antibodies to mouse immunoglobulins (Igs) were used as secondary antibodies to MEL5 (anti-TRP-1; Signet Laboratories, Dedham, MA), NKI-Beteb (anti-gp100; Sanbio, Uden, The Netherlands), M2-7 C10 (anti-MART-1; NeoMarkers, Fremont, CA), MEL-1 (anti-GD3; Signet Laboratories), and anti-CD3 (Pan-T cell marker; Becton Dickinson, San Jose, CA) in single-staining procedures, followed by peroxidase-conjugated streptavidin in the tertiary step. Alternatively, in double-staining procedures isotype-specific antibodies labeled with peroxidase or alkaline phosphatase were used in the second step (Southern Biotechnology Associates, Inc., Birmingham, AL).19 In the latter case, Fast Blue BB was first added as an alkaline-phosphatase substrate in the presence of 0.2 mg/ml naphthol As-Mx-phosphate and 1 mmol/L levamizole (Sigma) in 0.1 mol/L Tris-HCl buffer (pH 8.5), followed by 0.25 mg/ml amino ethyl carbazole (Sigma) in 0.1 mol/L NaAc (pH 5.2) as a peroxidase substrate. In single-staining procedures, only amino ethyl carbazole-staining was performed, followed by Mayer’s hematoxylin counterstaining (Dakopatts, Carpinteria, CA) where indicated. Specimens were coverslipped in glycergel (Dakopatts).

FACS Analysis

For membrane antigens, unfixed cells were stained in 10% normal human serum (NHS) in PBS and the primary antibody followed by fluorescein isothiocyanate-labeled anti-mouse Ig antibodies (BioSource, Camarillo, CA) or by biotinylated anti-mouse Ig followed by R-phycoerythrin-labeled streptavidin (Dakopatts). Antibodies used include MEL5, NKI-Beteb, and M2-7 C10, as well as antibodies L243 to HLA-DR antigens (Becton Dickinson) and B9.12.1 to HLA-A, HLA-B, and HLA-C antigens (Immunotech, Marseilles, France). For detection of intracellular antigens, cells were prefixed in 2% paraformaldehyde with 1% fetal bovine serum and 0.1% sodium azide (Sigma) in fluorescent antibody (FA) buffer (Difco, Detroit, MI) and permeabilized in 0.03% saponin in PBS, followed by immunostaining in the presence of 0.3% saponin (Sigma) by primary antibodies and secondary fluorescein isothiocyanate-labeled anti-mouse Ig antibodies (BioSource). A Coulter XL flow cytometer (Miami, FL) and Coulter Elite software were used to determine the mean fluorescence intensity of 5000 cells.

Northern Blot Analysis

RNA was isolated using Tri-Reagent according to the manufacturer’s specifications (Sigma). Fifteen µg per sample of formamide-denatured total RNA was loaded onto a 1% agarose gel in 6% formaldehyde and phosphate buffer and run at 90 V. RNA was transferred to a ZetaBind nylon membrane (Life Science Products Inc., Denver, CO) by capillary transfer overnight. RNA was crosslinked to the membrane by baking. The membrane was hybridized to 1.5 x 107 cpm of 32P-labeled probe prepared using a random primer labeling kit (Life Technologies, Inc.). The probe for MART-1 mRNA was prepared by RT-PCR amplification from total RNA derived from M14 cells with primers 5'-ATGCCAAGAGAAGATGCTCA-3' and 5'-TTAAGGTGAATAAGGTGGTGG-3', and verified by automated sequencing. Likewise, the gp100 probe was prepared with primers 5'-AGTCCCCCTGGATTGTGTTC-3' and 5'-AGCAAGATGCCCACGATCAG-3' and random primer labeled. Hybridization of the blot was performed in the presence of 40 µg/ml of salmon sperm DNA at 65°C overnight. The washed blot was exposed to BioMax film (Kodak, Rochester, NY) at -80°C. After hybridization to the MART-1 probe was analyzed, the blot was stripped in 0.5% sodium dodecyl sulfate at 70°C for 1 hour. Effective stripping was confirmed by autoradiography before hybridization with the radiolabeled probe to gp100.

Cytotoxicity Assays

Cells characterized for HLA-A*0201 expression were plated at 104 cells/well in a 96-well plate and cultured in the presence or absence of 1000 IU/ml of IFN-{gamma} for 72 hours, the same treatment used to compare the consequences of treatment by inflammatory/melanogenic modulators. Where noted, 10 µg/ml of MART-1 peptide AAGIGILTV was subsequently added for 2 hours. Cells were then washed and cultured in the presence of 5 µCi/well of 51Cr (Du Pont NEN Research Products, Boston, MA) at 37°C for 3 hours. The labeling medium was removed, and washed target cells were subsequently reacted with A42 T cells in the medium described for 624.38 cells at 37°C for 24 hours. Cytotoxicity was measured as cpm of 51Cr release compared to spontaneous release (in absence of T cells) and total lysis (in the presence of 1% Triton X-100) according to the formula: percent killing equals cpm (in presence of T cells) - cpm (spontaneous)/cpm(total) - cpm(spontaneous).

Laser Capture Microscopy

Eight-µm frozen sections were cut; followed by treatment with 70% ethanol, acetone, filtered Mayer’s hematoxylin, water, eosin Y, graded ethanol, and xylene; and 30 minutes of drying in a dessicator. Cells within tumor lobes were captured with a PixCell II Laser Capture Microdissection System (Arcturis Engineering Inc., Mountain View, CA) and captured cells were immediately transferred to Tri-Reagent for RNA isolation, performed according to the manufacturer’s specifications.

Quantitative RT-PCR

RNA (250 ng) was reverse-transcribed in the presence of 62.5 U of Multiscribe reverse transcriptase (RT), 5.5 mmol/L MgCl2, 0.5 mmol/L (per nucleotide) dNTPs, 2.5 µmol/L random hexamers, 20 U RNase inhibitor, and RT buffer (Perkin Elmer, Norwalk, CT) at 48°C for 30 minutes, followed by a 5-minute inactivation of RT activity at 95°C. For quantitative PCR, 10% of the RT reaction was combined (in triplicate) with 62.5% TaqMan PCR Master mix (Perkin Elmer), 1 µmol/L of primers 5'-AGCTCTGCATCGTTTTGGGTT-3' and 5'-GTTCCATTATCCGCTACATCTGAA-3' for IFN-{gamma} or 5'-GGCACCCAGCACAATGAAG-3' and 5'-GCCGATCCACACGGAGTACT-3' for ß-actin, as well as 1 µmol/L of probes FAM-TCTTGGCTGTTACTGCCAGGACCCA-TAMRA or FAM-TCAAGATCATTGCTCCTCCTGAGCGC-TAMRA, respectively.20 Samples were heated 2 minutes at 50°C and 10 minutes at 95°C, followed by 42 cycles of 15 seconds at 95°C, 1 minute at 60°C in an Abi-Prism 7700 Sequence Detection System (Perkin Elmer), and the threshold cycle CT for significant fluorescence was noted.

Relative IFN-{gamma} mRNA levels were calculated following Perkin-Elmer guidelines. Briefly, CT values for actin mRNA were subtracted from IFN mRNA CT values. The difference found for control skin was subtracted from values obtained for melanoma samples, providing a value for {Delta}{Delta}CT. The relative concentration of IFN-{gamma} mRNA is represented by 2-{Delta}{Delta}CT (shown ± {varsigma}n-1).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Immunohistology of M14 Cells Before and After Exposure to Various Treatments

Figure 1 illustrates MART-1 and TRP-1 down-regulation after exposure to IFN-{gamma} for 72 hours, as well as sustained expression of membrane antigen ganglioside D3 (GD3). This is supportive of specific repression of melanosomal antigens. Exposure to other compounds tested did not markedly affect expression as exemplified by MART-1, TRP-1, and GD3 staining after exposure to {alpha}-MSH in Figure 1 . Thus, IFN-{gamma} was chosen as a factor potentially affecting target antigen expression by melanoma cells.



View larger version (165K):
[in this window]
[in a new window]
  		Figure 1. IFN-{gamma}-induced repression of melanosomal antigen expression by M14 cells. M14 cells were exposed to 1000 U/ml of IFN-{gamma} or 10 nmol/L of {alpha}-MSH for 72 hours and stained with antibodies to TRP-1, MART-1, and GD3. Note reduced expression of both melanosomal antigens TRP-1 and MART-1 after exposure to IFN-{gamma}. By contrast, expression of membrane antigen GD3 is maintained in the presence of both IFN-{gamma} and {alpha}-MSH. Original magnification, x36.

 
Effect of IFN-{gamma} on Expression of Melanosomal Antigens at the Protein Level

The effect of IFN-{gamma} on expression of TRP-1, MART-1, gp100, HLA-DR antigens, and HLA-A, HLA-B, and HLA-C antigens was quantified by FACS analysis as shown in Table 1 . Up-regulated expression of HLA-DR and HLA-A, HLA-B, and HLA-C antigens confirms the efficacy of IFN-{gamma} treatment. Simultaneously, it can be observed that expression of MART-1 was reduced by 80%, of TRP-1 by 49%, and of gp100 by 54%. Conversely, expression of HLA-DR and HLA-A, HLA- B, and HLA-C antigens was increased 5.2-fold and 2.2-fold, respectively, by the same treatment.


View this table:
[in this window]
[in a new window]
  		Table 1. FACS Analysis of M14 Cells after Exposure to IFN-{gamma}

 
As the standard concentration of IFN-{gamma} of 1000 U/ml used is higher than considered physiologically relevant, expression of MART-1 as the model antigen was tested after exposure to different IFN-{gamma} concentrations. Mean fluorescence intensities for MART-1 shown in Figure 2 supported the notion that expression is also affected at lower concentrations of the cytokine. Mean fluorescence intensity decreased from 12.8 before treatment by 64.9% to 4.5 at 62.5 IU/ml. The decrease was similar for all concentrations assessed, decreasing by 68.2% to 4.1 at 1000 IU/ml. Thus, the majority of the effect occurs at less than the lowest concentration assessed. In a follow-up experiment, it was determined that suppressed expression was completely maintained at concentrations of 62.5 IU/ml, whereas fluorescence intensities rose to 5.9 at 31.25 U/ml and returned to the untreated value at concentrations of 15.1 IU/ml IFN-{gamma} and below.



View larger version (46K):
[in this window]
[in a new window]
  		Figure 2. Loss of MART-1 protein expression in the presence of varying IFN-{gamma} concentrations and its reversibility. FACS analysis of MART-1 expression in M14 cells treated with the indicated concentrations of IFN-{gamma} for 72 hours. Where indicated by /R and for the concentrations shown, the cytokine was withdrawn and cells were cultured another 72 hours before harvesting cells for FACS analysis.

 
To determine whether loss of expression was permanent, cells treated with either 500 IU/ml or 1000 IU/ml of IFN-{gamma} for 3 days were washed and cultured for an additional 3 days in absence of IFN-{gamma}. Subsequent evaluation of MART-1 expression by FACS analysis revealed mean fluorescence intensities of 9.4 and 7.6 (Figure 2) . Such a 63.5% and a 40.9% reversion, respectively, toward the level of MART-1 expression observed before treatment, demonstrated that repression of MART-1 expression by IFN-{gamma} is dependent on the continued presence of IFN-{gamma}.

Consequences of Exposure to IFN-{gamma} for Transcription of Melanosomal Antigens

Figure 3 illustrates reduced expression of MART-1 as well as gp100 at the RNA level in melanoma cells exposed to IFN-{gamma}. The intensity of bands hybridizing to the MART-1 cDNA probe as measured by NIH Image 1.62 software did not decrease in the lane representing 8 hours of treatment, and decreased thereafter by 44.3% and by 78.9% at 16 hours and 32 hours of treatment, respectively, when compared to untreated cells. The stripped blot, exposed similarly to a gp100 cDNA probe exhibited the same tendency, with a decrease in transcript abundance of 51.6% during the last 16 hours of treatment. It thus seems that IFN-{gamma} affects melanosomal antigen expression by reducing mRNA levels.



View larger version (58K):
[in this window]
[in a new window]
  		Figure 3. Northern analysis of MART-1 and gp100 expression in the presence of IFN-{gamma}. Expression of MART-1 and gp100 after exposure to 1000 U/ml of IFN-{gamma} for 0, 8, 16, or 32 hours was demonstrated by sequential hybridization of a Northern blot to appropriate probes. Note reduced expression after prolonged exposure to IFN-{gamma}.

 
IFN-{gamma}-Affecting Immune Recognition of Target Cells

Apart from reducing melanosomal antigen expression, IFN-{gamma} induces expression of MHC molecules as confirmed in Table 1 . Consequently, the recognition of target cells exposed to IFN-{gamma} will depend on the relative importance of HLA up-regulation and target antigen suppression. The prevailing effect was analyzed by reacting target cells with MART-1-responsive cytotoxic T cells as shown in Figure 4A . The percent killing is reduced by up to 78% for melanoma cells pretreated with 1000 U/ml IFN-{gamma} compared to untreated cells. By contrast, it can be observed that recognition of Mf9931 P2 normal melanocytes was not affected by IFN-{gamma} pretreatment. As expected, Mf9807 P11 HLA-mismatched melanocytes were not recognized by A42 MART-1-reactive T cells.



View larger version (19K):
[in this window]
[in a new window]
  		Figure 4. Reduced cytotoxicity of A42 cells toward multiple melanoma cell cultures in the presence of IFN-{gamma} and its restoration by MART-1 peptide. A: 51Cr release by multiple melanocytic cell cultures in the presence or absence of 1000 U/ml of IFN-{gamma} is shown. Note reduced recognition of patient-derived melanoma cells, but not of melanocytes after cytokine treatment. HLA-A2+ melanocytes derived from a vitiligo patient similarly showed a lack of reduced recognition in the presence of IFN-{gamma} (results not shown). Mf9807 normal melanocytes are HLA-A*0201-negative. Effector:target ratios were 10:1. SE of chromium release measurements did not exceed 15%. B: Recognition of the IFN-{gamma}-treated 624.38 cell line is increased after the addition of synthetic MART-1 peptide at 10 µg/ml. Such an increase in the cytotoxic response was absent if target cells were not pretreated with IFN-{gamma} (results not shown). SE of chromium release measurements did not exceed 15%.

 
In Figure 4B , reduced recognition of IFN-{gamma}-treated 624.38 melanoma cells was restored in the presence of the synthetic MART-1 peptide AAGIGILTV added at 10 µg/ml 2 hours before incubation with A42 T cells. Thus, reduced recognition of target cells in the presence of IFN-{gamma} is because of loss of MART-1 expression.

IFN-{gamma} Expression in Melanocytic Lesions

Local expression of IFN-{gamma} in primary and metastatic melanoma lesions was analyzed by quantitative RT-PCR. Of three specimens investigated, one contained 10-fold elevated concentrations of IFN-{gamma} transcripts whereas others contained levels similar to control skin (Table 2) . By contrast, metastatic melanoma tissue contained an elevated IFN-{gamma} concentration of 188 times that detected in control skin.


View this table:
[in this window]
[in a new window]
  		Table 2. Quantitative RT-PCR Analysis of IFN-{gamma} Expression in Melanocytic Lesions

 
Tumor cells are clustered in tumor lobes separated by tumor stroma. To analyze whether IFN-{gamma} concentrations are elevated within tumor lobes or solely within the tumor stroma, the former was isolated by laser capture microscopy as shown in Figure 5 and RNA from the captured tissue was again used for quantitative RT-PCR. Comparing IFN-{gamma} mRNA in captured tissue to control skin, it appeared that the majority of IFN-{gamma} is encoded by cells present in the stroma, as levels were elevated 4.5-fold in the captured tissue compared to the control skin values. Focally however, IFN-{gamma}-producing T cells will generate high levels of the cytokine.



View larger version (87K):
[in this window]
[in a new window]
  		Figure 5. Laser capture of melanoma cells. A hematoxylin-stained, 10-µm frozen section of metastatic melanoma tissue is shown, together with captured tissue showing selective capture of tissue from a tumor lobe and exclusion of the tumor stroma. The resulting section lacking captured cells is also represented. Original magnification, x50.

 
A Potential Source for Elevated IFN-{gamma} Expression

An explanation for the differences in IFN-{gamma} transcript levels presented in Table 2 may be found in differential abundance of T cells as the source of this cytokine. T-cell infiltration of the tissue samples represented in Table 2 is shown by immunohistochemistry in Figure 6, A to E . It can be observed, that infiltrating T cells were indeed relatively abundant in metastatic melanoma, with fewer T cells in melanoma in situ specimens. Among the latter, sample C displayed the highest T-cell frequencies, in concordance with the observed increase in IFN-{gamma} transcripts observed in this sample.



View larger version (146K):
[in this window]
[in a new window]
  		Figure 6. T-cell infiltration of primary and metastatic tumors. Six-µm frozen sections of the same control and tumor tissues used for quantitative RT-PCR analysis as shown in Table 2 were immunostained with antibodies to CD3 to compare pan-T cell infiltration. A–C: Melanoma in situ samples presented in the order shown in Table 2 . D: Control skin. E: Melanoma metastasis. F: MART-1 (blue) and CD3 (red) immuno-double staining of metastatic tissue. It can be observed that infiltrates are present in all tumor samples, yet T cells are more abundant in (sections of) metastatic tumor tissue. F: A halo of apparently viable cells with reduced MART-1 expression can be observed surrounding tumor-infiltrating T cells, possibly as a consequence of IFN-{gamma} secretion by these T cells. Control stainings performed in the absence of one and both of the primary antibodies confirmed these staining patterns (results not shown). Original magnifications: x100 (A–E); x132 (F).

 
In the immuno-double staining performed to simultaneously observe MART-1 expression by melanoma cells and their proximity to T cells shown in Figure 6F, a halo of reduced MART-1 expression was observed in tumor cells in close proximity to tumor-infiltrating T cells, suggesting that IFN-{gamma}-generating T cells may be actively suppressing MART-1 expression in adjacent tumor cells.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Reduced expression of melanosomal antigens poses a risk to melanoma patients, as dedifferentiation leaves melanoma cells less recognizable to the immune system.2 The microanatomy of melanocytic lesions seems to affect the level of expression of differentiation antigens. This was suggested by expression analysis performed on frozen tissue and cultured cells as previously reported.13 As melanocytic cells encounter an altered microenvironment on entering the dermal compartment, it was likely that factors encountered within this environment affect the expression pattern of differentiation antigens. Candidate compounds used for the present study were chosen for their known involvement in melanogenesis or inflammation.

Among factors tested, IFN-{gamma} most markedly altered expression of differentiation markers expressed in the melanosomal compartment of the cell. Comparable expression of lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) in cells treated with and without IFN-{gamma} has since indicated that down-modulation is not associated with reduced melanosome content (results not shown). Because of its frequent administration to tumor patients, it should also be noted that exposure to 1000 U/ml of IFN-{alpha} did not affect melanosomal antigen expression (results not shown).

In a separate study, it was shown by quantitative RT-PCR that exposure to 20 µmol/L of {alpha}-MSH can elevate TRP-1 and TRP-2 transcript abundance ~4.5-fold and 1.5-fold, respectively, in F002 melanoma cells (not shown). M14 cells have a much higher baseline expression of TRP-1 than F002 cells, so that such induction cannot be well appreciated in Figure 1 . The apparent antagonism between {alpha}-MSH and IFN-{gamma} is of interest in the light of the opposing role of these compounds in the immune response, {alpha}-MSH being anti-inflammatory whereas IFN-{gamma} supports inflammation.21,22

Dedifferentiation is poorly understood at present. Expression of melanosomal antigens is clearly not essential to the viability of melanoma cells. It has been suggested that depigmentation observed in malignant melanoma cells is the consequence of aberrant intracellular trafficking of melanosomal tyrosinase.23 It was thus of interest to define the gene expression level affected by IFN-{gamma} treatment. The cytokine clearly affected expression at the RNA level. The effect of IFN-{gamma} was not permanent, as suppression of MART-1 expression could be reversed as shown in Figure 3 . This suggests that additional mechanisms contribute to the formation of tumors that persistently lack expression of one or more differentiation antigens, even in absence of modulating factors. It is possible that epigenetic modulation eventually consolidates antigen loss. Yet importantly, reduced expression of MART-1 after exposure of melanoma cells to IFN-{gamma} was sufficient to reduce the efficacy of MART-1-reactive T cells by up to 78%. This is of particular interest in light of elevated MHC class I and II expression known to occur after exposure to IFN-{gamma}.24,25 Preliminary experiments suggested that reduced MART-1 expression can similarly suppress T-cell activation, as HLA-matched dendritic cells pulsed with antigenic lysates prepared from melanocytic cells induced T-cell activation to an extent correlating with MART-1 content of the lysate (not shown). Such results are compatible with high antigen-expression level requirements for effective pulsing of dendritic cells.26 It should be noted that IFN-{gamma} exposure can modulate expression of hundreds of genes in a cell-type-dependent manner.25 Among these, some may contribute to immune escape of tumors through pathways other than through reduced target antigen expression. For example, IFN-{gamma} exposure modulates expression of cellular adhesion molecules, thereby augmenting tumor dissemination and metastasis, and potentially providing a safe haven for tumor cells entering immune-privileged sites.25,27 Moreover, IFN-{gamma} may contribute to reduced tumor recognition by inducing expression of the immunoproteasome at the expense of the standard proteasome, as only the latter seems capable of cleaving MART-1 to render immunogenic peptides.28

Although a suppressive effect of IFN-{gamma} on melanoma recognition was previously noted, such effect was not assigned to reduced antigen expression by tumor cells.29 In fact, reduced expression of gp100 was not substantiated by a study reported by Takechi and colleagues,30 demonstrating increased expression of this antigen after IFN-{gamma} exposure. As we have presently expanded our studies for the most dramatically affected antigen MART-1 at the expense of studies related to other melanosomal antigens, we cannot exclude the possibility that gp100 is differentially affected in melanoma cells of different origins. It will be important to further define the consequences of cytokine exposure for tumor cell recognition with T-cell clones to additional melanosomal antigens. Also, it should be taken into account that others have reported an augmented humoral response to melanosomal antigens after overexpression of IFN-{gamma} by melanoma cells.31 Augmenting cell-mediated immunity has been the primary focus in tumor immunology as it can be directed to a greater arsenal of antigens than humoral immunity, the latter being effective only for antigens expressed at the cell surface.32 Nevertheless, the humoral response may well compensate, at least in part, for reduced cellular cytotoxicity. In fact, our results demonstrating sustained expression of membrane antigen GD3 in the presence of {alpha}-MSH as well as IFN-{gamma} is supportive of this concept, as antibodies to GD3 have been proposed for the treatment of melanoma patients.33

Interestingly, a suppressive effect of IFN-{gamma} on tumor antigen gp70 expression and subsequent tumor killing was also recently reported for murine colon carcinoma.34 Taking into account that T cells are the most likely source for IFN-{gamma}, it is likely that within a MART-1-expressing tumor, infiltrating activated T cells can both eliminate part of the tumor cells, and provide an effective immune escape mechanism to others. Even a few remaining tumor cells can endanger the patient, particularly if such tumor cells loose expression of target antigens and can thus no longer be recognized by tumor-infiltrating lymphocytes. Such extrapolation to the in vivo situation is of particular interest considering the high concentrations of IFN-{gamma}, as well as the abundance of T cells detectable in metastatic melanoma lesions. Despite a more elaborate immune response, T cells do not effectively eliminate the tumor. By contrast, in vitiligo minute infiltrates effectively eliminate all pigment-producing cells from the skin, leading to progressive depigmentation.35,36 This is understandable, as levels of IFN-{gamma} generated in the course of this immune response will not affect recognition of normal melanocytes (see Figure 5 ). Given the threshold level of antigen expression required for effective recognition by T cells, cytotoxicity toward normal melanocytes after IFN-{gamma} exposure is likely explained by relatively high levels of remaining target antigen expression in these cells.37 The apparent sustained recognition of IFN-{gamma}-exposed melanocytes by CTL may explain how some patients develop extensive vitiligo with concomitant growth of metastatic tumor sites apparently escaping T-cell recognition.

Taken together, these novel findings indicate that despite its stimulatory effect on MHC molecule expression, IFN-{gamma} can reduce target antigen expression and recognition of melanoma cells by CTL. Consequently, this aspect of IFN-{gamma} action should be considered in the development of melanoma therapy.


   Footnotes
 
Address reprint requests to I. Caroline Le Poole Ph.D., Loyola University Medical Center, Bldg. 112, Rm. 303, 2160 S. 1st Ave., Maywood, IL 60153. E-mail: ilepool{at}lumc.edu

Supported by NIH funding to W.M.K. (R01-CA/AI 78399) and to B.J.N. (P01-CA 59327).

Accepted for publication October 27, 2001.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sakai C, Kawakami Y, Law LW, Furumura M, Hearing VJ: Melanosomal proteins as melanoma-specific immune targets. Melanoma Res 1997, 7:83-95[Medline]
  2. Restifo NP, Rosenberg SA: Developing recombinant and synthetic vaccines for the treatment of melanoma. Curr Opin Oncol 1999, 11:50-57[Medline]
  3. Salgaller ML, Weber JS, Koenig S, Yanelli JR, Rosenberg SA: Generation of specific anti-melanoma reactivity by stimulation of human tumor-infiltrating lymphocytes with MAGE-1 synthetic peptide. Cancer Immunol Immunother 1994, 9:105-116
  4. Valmori D, Lienard D, Waanders G, Rimoldi D, Cerottini JC, Romero P: Analysis of MAGE-3-specific cytolytic T lymphocytes in human leukocyte antigen-A2 melanoma patients. Cancer Res 1997, 57:735-741[Abstract/Free Full Text]
  5. Rosenberg SA, Mule JJ, Spiess PJ, Riechert CM, Schwarz SL: Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2. J Exp Med 1985, 161:1169-1188[Abstract/Free Full Text]
  6. Bakker AB, Schreurs MW, de Boer AJ, Kawakami Y, Rosenberg SA, Adema GJ, Figdor CG: Generation of antimelanoma cytotoxic T lymphocytes from healthy donors after presentation of melanoma-associated antigen-derived epitopes by dendritic cells in vitro. Int J Cancer 1995, 62:97-102[Medline]
  7. Chang AE, Li Q, Bishop DK, Normolle DP, Redman BD, Nickoloff BJ: Immunogenetic therapy of human melanoma utilizing autologous tumor cells transduced to secrete granulocyte-macrophage colony stimulating factor. Hum Gene Ther 2001, 15:2231-2241
  8. Dobrzanski MJ, Reome JB, Dutton RW: Immunopotentiating role of IFN-gamma in early and late stages of type 1 CD8 effector cell-mediated tumor rejection. Clin Immunol 2001, 98:70-84[Medline]
  9. Ferrone S, Marincola FM: Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance Immunol Today 1995, 16:487-494[Medline]
  10. Kageshita T, Hirai S, Ono T, Hicklin DJ, Ferrone S: Down-regulation of HLA class I antigen-processing molecules in malignant melanoma: association with disease progression. Am J Pathol 1999, 154:745-754[Abstract/Free Full Text]
  11. Ren S, Smith MJ, Louro ID, McKie-Bell P, Bani MR, Wagner M, Zochodne B, Redden DT, Grizzle WE, Wang ND, Smith DI, Herbst RA, Bardenheuer W, Opalka B, Schutte J, Trent JM, Ben-David Y, Ruppert JM: The p44S10 locus, encoding a subunit of the proteasome regulatory particle, is amplified during progression of cutaneous malignant melanoma. Oncogene 2000, 19:1419-1427[Medline]
  12. Slingluff CL, Colelle TA, Thompson L, Graham DD, Skipper JC, Caldwell J, Brinckerhoff L, Kittlesen DJ, Deacon DH, Oei C, Harthun NL, Huczko EL, Hut DF, Darrow TL, Engelhard VH: Melanomas with concordant loss of multiple melanocytic differentiation antigens: immune escape that may be overcome by targeting unique or undefined antigens. Cancer Immunol Immunother 2000, 48:661-672[Medline]
  13. Herlyn M, Shih IM: Interactions of melanocytes and melanoma cells with the micro-environment. Pigment Cell Res 1994, 7:81-88[Medline]
  14. Meije CB, Mooi WJ, Le Poole IC, van Muijen GNP, Das PK: Micro-anatomy related antigen expression in melanocytic lesions. J Pathol 2000, 190:572-578[Medline]
  15. Katano M, Saxton RE, Cochran AJ, Irie RF: Establishment of an ascitic melanoma cell line that metastasizes to lung and liver in nude mice. J Cancer Res Clin Oncol 1984, 108:197-203[Medline]
  16. Riker AI, Panelli MC, Kamulla US, Wang E, Wunderlich J, Abati A, Fetsch P, Rosenberg SA, Marincola FM: Development and characterization of melanoma cell lines by fine-needle aspiration biopsy: advances in the monitoring of patients with metastatic melanoma. Cancer Detect Prev 1999, 23:387-396[Medline]
  17. Cole DJ, Weil DP, Shamamian P, Rivoltini L, Kawakami Y, Topalian S, Jennings C, Eliyahu S, Rosenberg SA, Nishimura MI: Identification of MART-1 specific T-cell receptors: T cells utilizing distinct T-cell receptor variable and joining regions recognize the same tumor epitope. Cancer Res 1994, 54:5265-5268[Abstract/Free Full Text]
  18. Le Poole IC, van den Berg FM, van den Wijngaard RMJGJ, Galloway DA, van Amstel PJ, Buffing AA, Smits HL, Westerhof W, Das PK: Generation of a human melanocyte cell line by introduction of HPV16 E6 and E7 genes. In Vitro Cell Dev Biol Anim 1997, 33:42-49[Medline]
  19. Van der Loos CM, Becker AE, van den Oord JJ: Practical suggestions for successful immunoenzyme double-staining experiments. Histochem J 1993, 25:1-13[Medline]
  20. Kamulla US, Lee KH, Riker AI, Wang E, Ohnmacht GA, Rosenberg SA, Marincola FM: Functional analysis of gene expression in peripheral blood mononuclear cells and tumor specimens. J Immunol 1999, 163:6867-6875[Abstract/Free Full Text]
  21. Carrel S, Schmidt-Kessen A, Giuffre L: Recombinant interferon-gamma can induce the expression of HLA-ABC antigens and tumor-associated antigens. Eur J Immunol 1985, 15:118-123[Medline]
  22. Luger TA, Kalden D, Scholtzen TE, Brzoska T: Alpha-melanocyte stimulating hormone as a mediator of tolerance induction. Pathobiology 1999, 67:318-321[Medline]
  23. Halaban R, Cheng E, Zhang Y, Moellmann G, Hanlon D, Michalak M, Setaluri V, Hebert DN: Aberrant retention of tyrosinase in the endoplasmic reticulum mediates accelerated degradation of the enzyme and contributes to the dedifferentiated phenotype of amelanotic melanoma cells. Proc Natl Acad Sci USA 1997, 94:6210-6215[Abstract/Free Full Text]
  24. Giacomini P, Fisher PB, Duigou GJ, Gambari R, Natali PG: Regulation of class II MHC gene expression by interferons: insights into the mechanism of action of interferon. Anticancer Res 1988, 8:1153-1161[Medline]
  25. Boehm U, Klamp T, Groot M, Howard JC: Cellular responses to interferon-{gamma}. Annu Rev Immunol 1997, 15:749-795[Medline]
  26. Rawson P, Hermans IF, Huck SP, Roberts JM, Pircher H, Ronchese F: Immunotherapy with dendritic cells and tumor major histocompatibility complex class I-derived peptides requires a high density of antigen on tumor cells. Cancer Res 2000, 60:4493-4498[Abstract/Free Full Text]
  27. Creyghton WM, de Waard-Siebenga I, Danen EH, Luyten GP, van Muijen GN, Jager MJ: Cytokine-mediated modulation of integrin, ICAM-1 and CD44 expression on human uveal melanoma cells in vitro. Melanoma Res 1995, 5:235-242[Medline]
  28. Morel S, Levy F, Burlet-Schiltz O, Brassear F, Probst-Kepper M, Peitrequin AL, Monsarrat B, Van Velthoven R, Cerrotini JC, Boon T, Gairin JE, van den Eynde BJ: Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells. Immunity 2000, 12:107-117[Medline]
  29. Kirkin AF, thor Straten P, Zeuthen J: Differential modulation by interferon gamma of the sensitivity of human melanoma cells to cytolytic T cell clones that recognize differentiation or progression antigens. Cancer Immunol Immunother 1996, 42:203-212[Medline]
  30. Takechi Y, Hara I, Naftzger C, Xu Y, Houghton AN: A melanosomal membrane protein is a cell surface target for melanoma therapy. Clin Cancer Res 1996, 2:1837-1842[Abstract]
  31. Fujii S, Huang S, Fong TC, Ando D, Burrows F, Jolly DJ, Nemunaitis J, Hoon DS: Induction of melanoma-associated antigen systemic immunity upon intratumoral delivery of interferon-gamma retroviral vector in melanoma patients. Cancer Gene Ther 2000, 7:1220-1230[Medline]
  32. Berard F, Blanco P, Davoust J, Neidhart-Berard EM, Nouri-Shirazi M, Taquet N, Rimoldi D, Cerottini JC, Banchereau J, Palucka AK: Cross-priming of naïve CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells. J Exp Med 2000, 192:1535-1544[Abstract/Free Full Text]
  33. Kirkwood JM, Mascari RA, Edington HD, Rabkin MS, Day RS, Whiteside TL, Vlock DR, Shipe-Spotloe JM: Analysis of therapeutic and immunologic effects of R(24) anti-GD3 monoclonal antibody in 37 patients with metastatic melanoma. Cancer 2000, 88:2693-2702[Medline]
  34. Beatty GL, Paterson Y: IFN-{gamma} can promote tumor evasion of the immune system by down-regulating cellular levels of an endogenous tumor antigen. J Immunol 2000, 165:5502-5508[Abstract/Free Full Text]
  35. van den Wijngaard R, Wankowicz-Kalinska A, Le Poole C, Tigges B, Westerhof W, Das P: Local immune response in skin of generalized vitiligo patients. of melanocytes is associated with the prominent presence of CLA+ T cells at the perilesional site. Lab Invest 2000, 80:1299-1309[Medline]
  36. Yee C, Thompson JA, Roche P, Byrd DR, Lee PP, Piepkorn M, Kenyon K, Davis MM, Riddell SR, Greenberg PD: Melanocyte destruction after antigen-specific immunotherapy of melanoma. Direct evidence of T cell mediated vitiligo. J Exp Med 2000, 192:1637-1644[Abstract/Free Full Text]
  37. Riker AI, Kammula US, Panelli MC, Wang E, Ohnmacht GA, Steinberg SM, Rosenberg SA, Marincola FM: Threshold levels of gene expression of the melanoma antigen gp100 correlate with tumor cell recognition by cytotoxic T lymphocytes. Int J Cancer 2000, 86:818-826[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
I. S. Dunn, T. J. Haggerty, M. Kono, P. J. Durda, D. Butera, D. B. Macdonald, E. M. Benson, L. B. Rose, and J. T. Kurnick
Enhancement of Human Melanoma Antigen Expression by IFN-beta
J. Immunol., August 15, 2007; 179(4): 2134 - 2142.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
D. Liu, S. J. O'Day, D. Yang, P. Boasberg, R. Milford, T. Kristedja, S. Groshen, and J. Weber
Impact of Gene Polymorphisms on Clinical Outcome for Stage IV Melanoma Patients Treated with Biochemotherapy: An Exploratory Study
Clin. Cancer Res., February 1, 2005; 11(3): 1237 - 1246.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. J. Dobrzanski, J. B. Reome, J. A. Hollenbaugh, and R. W. Dutton
Tc1 and Tc2 Effector Cell Therapy Elicit Long-Term Tumor Immunity by Contrasting Mechanisms That Result in Complementary Endogenous Type 1 Antitumor Responses
J. Immunol., February 1, 2004; 172(3): 1380 - 1390.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. G. Carrabba, C. Castelli, M. J. Maeurer, P. Squarcina, A. Cova, L. Pilla, N. Renkvist, G. Parmiani, and L. Rivoltini
Suboptimal Activation of CD8+ T Cells by Melanoma-derived Altered Peptide Ligands: Role of Melan-A/MART-1 Optimized Analogues
Cancer Res., April 1, 2003; 63(7): 1560 - 1567.
[Abstract] [Full Text] [PDF]

Home page
 Mol Cancer ResHome page
P. J. Durda, I. S. Dunn, L. B. Rose, D. Butera, E. M. Benson, F. Pandolfi, and J. T. Kurnick
Induction of "Antigen Silencing" in Melanomas by Oncostatin M: Down-Modulation of Melanocyte Antigen Expression
Mol. Cancer Res., April 1, 2003; 1(6): 411 - 419.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Le Poole, I. C.
Right arrow Articles by Nickoloff, B. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Le Poole, I. C.
Right arrow Articles by Nickoloff, B. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS