L
1165
Expression of Cell Adhesion Molecules in Human Melanoma Cell Lines and Their Role in Cytotoxicity Mediated by Tumor - InfiItrating Lymphocytes Franco Pandolfi, MD, Livio Trenfin, MD, Lenora A. Boyle, BA, Ivan Stamenkovic, MD,* H. Randolph Byers, MD, Robert B. Colvin, MD, and James T. Kurnick, M D
The role of cell adhesion molecules (CAM) LFA1, ICAM1, LFA3, VLA1, VLA4, CD29, CD44, and 0 5 6 in tumorinfiltrating lymphocyte(TIL) and natural killer cell (NK)mediated killing of target cells was studied. Melanoma cell lines and autologous TIL were derived from seven patients with metastatic melanoma, and cytotoxicity assays were done in the presence and absence of monoclonal antibodies (MoAb) to CAM expressed on melanoma cells or TIL. The melanoma cell lines analyzed were all positive for CD29 and LFA3 expression, negative for LFAl expression, but showed variable expression of ICAM-1, VLA1, VLA4, CD44, and CD56. The effects of anti-CAM antibodies on TIL-mediated melanoma killing fell into three categories: (1)consistent inhibition of TILmediated killing was observed when melanoma cells were pretreated with anti-ICAM1 and anti-LFA-3 MoAb or when TIL were pretreated with anti-LFAl; (2)no effect was observed when melanoma cells were pretreated with anti-CDS@or (3) a discreet, but significant, inhibition was observed when target cells were pretreated with anti-CD29, anti-VLAI, anti-VLA4, and anti-CD44. Cytotoxicity was significantly enhanced by pretreatment of
From the Department of Pathology, MassachusettsGeneral Hospital, Harvard Medical School, Boston, Massachusetts. Supported in part by National Institutes of Health (Bethesda, Maryland) grants CA 44324 and AR 39993. * Special Fellow of the Leukemia Society of America, New York, New York. The authors thank the following scientists who donated their monoclonal reagents: Dr. T. A. Springer (Dana Farber Cancer Institute, Boston, Massachusetts) for RRl/l, TS2/9, T11.2, TS1/22, and TS1/18; Dr. M. E. Hemler (Dana Farber Cancer Institute) for A-lA5, TS2/7, and B-5G10; Dr. E. Butcher (Stanford University, Stanford, California)for Hermes 111; Dr. L. Lanier (Becton Dickinson, Mountain View, California) for Leul9; Dr. E. L. Reinherz (Dana Farber Cancer Institute) for T11.2; and Dr. P. Beverly (University of London, London, United Kingdom) for UCHLl. Address for reprints: Franco Pandolfi, MD, Pathology Research, MGH East 7th floor, 149 Thirteenth Street, Charlestown, MA 02129. Accepted for publication May 31, 1991.
target cells with y-interferon (7-IFN),although y-IFN did not augment surface expression of the CAM studied. The NK-mediated killing of K562 cells was blocked by antiLFA1, anti-CD18, and anti-ICAM, and partially inhibited by anti-CD44 MoAb. Together, these results suggest that several accessory CAM may play a role in regulating cellular cytotoxicity. Because cytotoxicity generally correlated with the level of expression of CAM in melanoma cells, weak CAM surface expression may provide a means for melanomas to escape immune surveillance. Cancer 1992; 69:1165-1173.
Recently, considerable interest has been focused on molecules that mediate cell-cell or cell-matrix interactions, commonly referred to as cell adhesion molecules (CAM).'-5 At least four categories of CAM currentIy are recognized, including the integrins?' a subset of molecules of the immunoglobulin supergene family,' and members of the LEC-CAM family.5Cell-surfaceproteoglycans, consisting of a core protein substituted in glycosaminoglycan side chains, constitute the fourth group. The core proteins of most cell-surface proteoglycans characterized currently appear to bear little homology to each other? although some, such as CD44, are related to proteoglycans found in the extracellular matrix.'O In cellular immunology, CAM play a significant role in several lymphocytic functions requiring cell-cell contact. Perhaps the most notable of such functions is cellular cytotoxicity. Two main types of cytotoxicity have been described:" (1)nonmajor histocompatibility complex (MHC)-restricted, such as natural killer (NK) cytotoxicity, in which lymphocytes interact with the target cells by nonspecific CAM-CAM interactions and (2) MHC-restricted, mediated by cytotoxic T-lymphocytes (CTL) that specifically recognize the target through the T-cell receptor (TCR). The role in cellular
1166
CANCER March 1,1992, Volume 69, No. 5
cytotoxicity of several well-characterized CAM has been in~estigated.’~’’-’~ However, little is known about the role in cytotoxicity of several of the more recently discovered CAM, such as the integrins of the bl family or CD44. The presence of a lymphoid infiltrate in malignant melanoma and its correlationwith progn~sis,’~ together with the observation that melanoma-derived tumor-infiltrating lymphocytes (TIL) often have specific autologous CTL activity, stimulated several investigations of TIL-mediated cyt~toxicity.’*-’~ Little is known, however, about the distribution of CAM on melanoma cells and their role in cytotoxicity. To address these questions, we derived pairs of melanoma cell lines and autologous TIL and determined the cytotoxic activities of the TIL against autologous targets. We subsequently investigated the expression of several CAM on the melanoma cells and assessed their role in MHC-restricted cytotoxicity mediated by CTL. Our results suggest that several of the more recently identified CAM are involved in regulating TIL-mediated cytolysis of melanoma cells. The downregulation of CAM molecules in melanoma cells can be related to tumor evasion from TIL-mediated surveillance. Materials and Methods
Cell Lines Seven pairs of melanoma cell lines and TIL were derived from parallel cultures grown from biopsy specimens of patients with metastatic melanoma. The specimens were obtained from the site of cutaneous metastases. Tissues were cut into I-mm fragments and cultured in either RPMI-1640 supplemented with 10% fetal bovine serum (FBS) or RPMI supplemented with 5% human serum and interleukin-2 (IL-2, 100 units/ ml). In this way, parallel cultures of melanoma cells and activated T-lymphocytes were ~btained.’~,’~ During the first 3 to 5 weeks after establishment of cultures, the melanoma cells were propagated from the tissue in the absence of any exogenous stimulus. On reaching confluence, adherent cells were passaged serially. Surface expression of CAM also was assessed in four additional melanoma cell lines for which TIL were not available. To confirm the melanoma origin of our cell lines, we used the S-100 and the HMB-4526monoclonal antibodies (MoAb)in an indirect immunofluorescence assay on fixed cytocentrifuged samples. These data, in conjunction with morphologic observations and the presence of melanin in some cell lines (M3 and M4), confirmed the melanoma origin of the cells. Cultures supplemented with IL-2 propagated activated T-cells in those specimens containing lymphocytic infiltrates. These cultures
were kept in IL-2 and restimulated with phytohemagglutinin (PHA) and irradiated feeder cells every 2 to 4 weeks to enhance proliferation, as previously rep~rted.’~,’~ Cultures are labeled as follows: melanoma cell lines were labeled as M (for melanoma), followed by a patient identification code: 1 for Patient 1, 2 for Patient 2,3 for Patient 3, and so on, to 7 for Patient 7. The TIL cultures were labeled as TIL followed by the patient identification number. For example, M-1 and TIL-1 indicate the melanoma cell line and the TIL cell line, respectively, derived from Patient 1.
Monoclonal Antibodies The CAM were studied with a panel of M o A ~ ~ , ’ ~ - ‘ ~ whose specificities are reported in Table 1. Other reagents used included anti-CD3, anti-CD4, anti-CD8 (Ortho, Raritan, NJ), anti-CD16 and anti-CD57 (Becton Dickinson, Sunnyvale, CA), UCHLl (CD45RO);’ and W6/32, an antibody to histocompatibility antigen (HLA) Class I. The MoAb were stained in an indirect immunofluorescence test and analyzed in a Becton Dickinson Facscan I1 with standard techniques.
Incubation of Melanoma Cells W i t h y-Interferon To evaluate the effect of y-interferon (7-IFN) preincubation on melanoma CAM expression, each melanoma cell line was incubated for 48 hours with 500 units/ml of y-IFN (a gift of Genentech, San Francisco, CA) in RPMI with FBS at 37°C in a 5% CO, atmosphere. These conditions produce optimal enhancement of the expression of HLA-DR antigens on control melanoma cell lines. The CAM expression was assessed by indirect immunofluorescence, and the fluorescence pattern was compared with that observed in melanoma cell lines kept in media without y-IFN. Melanoma cells incubated with y-IFN were washed and used in the cytotoxicity assay as described subsequently. Table 1. Monoclonal Antibodies Directed to Cell Adhesion Molecules Used in the Current Study Monoclonal antibody
CD
Other designation/structure recognized
T11.2 TS1/22 TS1/18 A-lA5 Hermes 111 TS2/7 B-5G10 RRl/l Leu19 TS2/9
CD2 CDl l a CD18 CD29 CD44 CD49a CD49d CD54 CD56 CD58
LFA2 LFAl, a1 chain integrin 2nd subfamily j3 chain integrin 2nd subfamily j31 chain of the VLA Hyaluronic acid VLAI, a1 chain of the VLA VLA4, a 4 chain of the VLA ICAM-1 N-CAM LFA3
Adhesion Molecules and TIL Cytotoxicity/Pandolfi et al.
1167
Cytotoxicity
Table 2. Expression of Cell Adhesion Molecules on Human Melanoma Cell Lines
Cytotoxic activity was assessed by the lysis of 51Cr-labeled target cells in a 4-hour chromium release assay, as previously rep~rted.~' Target cells were represented by autologous or allogeneic melanoma cell lines or the NKsensitive K-562 cell line. Effector cells were TIL kept in IL-2, 8 to 20 days after restimulation with PHA and irradiated feeder cells. Our results are reported as the percent of specific lysis.
Monoclonal antibody
Inhibition of Cellular Cytotoxicity by Preincubation W i t h Monoclonal Antibodies Melanoma cell lines or TIL were incubated for 1hour at 37°C in a CO, atmosphere with MoAb. The TIL were preincubated with TS1/22, T11.2, OKT3, and W6/32; the melanoma cells were preincubated with the other reagents studied. A final dilution of 10 &ml of purified MoAb (OKT3, Hermes 111, and Leul9) or a 1 : l O O dilution of ascites was used. Preincubated cells then were tested in the cytotoxicity assay as reported. Excess antibody was present through the cytotoxicity assay. Our results are expressed as the percent of inhibition of the cytotoxicity observed without a blocking antibody, at a 50:l effector-to-target ratio. Our results are the means of triplicate or sextuplicateexperiments.Because preliminary experiments showed that control ( i e l e vant) MoAb always gave results within k 5% of those obtained without antibodies in the tests, we established an arbitrary cutoff at 10% of inhibition (ix.,considering the effects of a MoAb as inhibitory when inhibition was greater then 10%). Results
Expression of Cell Adhesion Molecules on Melanoma Cell Lines Melanoma cell lines were studied with a panel of MoAb as reported in Table 2. None of the cell lines expressed LFAl and CD18 lymphoid/myeloid markers, but all stained with CD29 and LFA3. Variable expression of the other CAM was observed. CD44 was expressed in six of seven lines; VLA1, in five of seven; VLA4, in six of seven; ICAM-1, in six of seven; and CD56/N-CAM, in three of seven. Four additional melanoma cell lines (for which corresponding TIL were not available)confirmed this heterogeneity of expression with CD44 expressed in three of four lines; VLAl, in two of four; VLA4, in three of four; and ICAM-I, in two of four (data not shown in the table).
CDlla/LFAl CD18 CD29/VLA 81 CD44 CD49a/VLAl CD49d/VLA4 CD54/ICAM1 CD56/N-CAM CD58/LFA3
M-1
M-2
M-3
M-4
M-5
M-6
M-7 ND ND
-
-
-
-
ND
-
-*
-
-
-*
-
-
++ ++ ++ -*
++
-*
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ -
+* ++ ++ ++ ++ t+ ++ ++ ++ ++ ++ ++ ++ ++ ++
+* ++
++ ++ -*
++ ++ -
++
++:
> 60% of cells positive; +: 10-60% of cells positive; -: < 10% of cells positive; ND: not done. * CAM expressionwas tested also after incubation of the cells with y-interferon and found to remain unchanged.
Effect of y-Interferon on the Expression of Cell Adhesion Molecules The cytokine y-IFN stimulates Class I1 antigen expression and enhances cellular cytotoxicity. However, the underlying mechanism by which y-IFN augments killing is unclear. In an attempt to determine whether yIFN induces CAM expression in target cells, melanoma cells that expressed some of the CAM weakly or not at all were incubated with y-IFN (Table 2). Among the CAM found to be variably expressed were CD18 (tested in two cell lines), CD44 (negative in M-2), VLAl (negative in M-2), VLA4 (weak in M-1 and M-4), and ICAM1 (negative in M-2). Although y-IFN induced or enhanced the expression of HLA-DR antigens in M-2 and M-4 cell lines, respectively, (data not shown) in the experimental conditions we used, no y-IFN effect on the expression of the CAM tested was observed.
Expression of Surface Molecules on TumorInfiltrating Lymphocytes We analyzed TIL maintained in IL-2 for the expression of several T-cell-related surface markers and for markers associated with some cytotoxic functions and CAM expression. In seven TIL cultures studied, more than 95% of cells were positive for: CD3, CD2, CD1la/ LFAl, CD29, CD44, CD45R0, VLA4, and LFA3. However, these cultures were negative consistently(< 5%of positive cells) for CD16 and CD57 reagents. Four MoAb (CD4, CD8, VLA1, and CD56) gave a more variable pattern, and these results are reported in Table 3. Of the seven TIL cell lines, two were almost pure populations of CD4 cells; two, CD8 cells; and the other three cultures, mixed. Expression of CD56 and VLAl was also variable.
CANCER March 1,1992, Volume 69, No. 5
1168
Table 3. Surface Markers Expressed by TumorInfiltrating Lymphocytes ~
J 80 -
~~
Monoclonal antibody CD4 CD8 CD49a/VLAl CD56
.............
TIL-1 TIL-2 TIL-3 TIL-4 TIL-5 TIL-6 TIL-7
1165
Expression of Cell Adhesion Molecules in Human Melanoma Cell Lines and Their Role in Cytotoxicity Mediated by Tumor - InfiItrating Lymphocytes Franco Pandolfi, MD, Livio Trenfin, MD, Lenora A. Boyle, BA, Ivan Stamenkovic, MD,* H. Randolph Byers, MD, Robert B. Colvin, MD, and James T. Kurnick, M D
The role of cell adhesion molecules (CAM) LFA1, ICAM1, LFA3, VLA1, VLA4, CD29, CD44, and 0 5 6 in tumorinfiltrating lymphocyte(TIL) and natural killer cell (NK)mediated killing of target cells was studied. Melanoma cell lines and autologous TIL were derived from seven patients with metastatic melanoma, and cytotoxicity assays were done in the presence and absence of monoclonal antibodies (MoAb) to CAM expressed on melanoma cells or TIL. The melanoma cell lines analyzed were all positive for CD29 and LFA3 expression, negative for LFAl expression, but showed variable expression of ICAM-1, VLA1, VLA4, CD44, and CD56. The effects of anti-CAM antibodies on TIL-mediated melanoma killing fell into three categories: (1)consistent inhibition of TILmediated killing was observed when melanoma cells were pretreated with anti-ICAM1 and anti-LFA-3 MoAb or when TIL were pretreated with anti-LFAl; (2)no effect was observed when melanoma cells were pretreated with anti-CDS@or (3) a discreet, but significant, inhibition was observed when target cells were pretreated with anti-CD29, anti-VLAI, anti-VLA4, and anti-CD44. Cytotoxicity was significantly enhanced by pretreatment of
From the Department of Pathology, MassachusettsGeneral Hospital, Harvard Medical School, Boston, Massachusetts. Supported in part by National Institutes of Health (Bethesda, Maryland) grants CA 44324 and AR 39993. * Special Fellow of the Leukemia Society of America, New York, New York. The authors thank the following scientists who donated their monoclonal reagents: Dr. T. A. Springer (Dana Farber Cancer Institute, Boston, Massachusetts) for RRl/l, TS2/9, T11.2, TS1/22, and TS1/18; Dr. M. E. Hemler (Dana Farber Cancer Institute) for A-lA5, TS2/7, and B-5G10; Dr. E. Butcher (Stanford University, Stanford, California)for Hermes 111; Dr. L. Lanier (Becton Dickinson, Mountain View, California) for Leul9; Dr. E. L. Reinherz (Dana Farber Cancer Institute) for T11.2; and Dr. P. Beverly (University of London, London, United Kingdom) for UCHLl. Address for reprints: Franco Pandolfi, MD, Pathology Research, MGH East 7th floor, 149 Thirteenth Street, Charlestown, MA 02129. Accepted for publication May 31, 1991.
target cells with y-interferon (7-IFN),although y-IFN did not augment surface expression of the CAM studied. The NK-mediated killing of K562 cells was blocked by antiLFA1, anti-CD18, and anti-ICAM, and partially inhibited by anti-CD44 MoAb. Together, these results suggest that several accessory CAM may play a role in regulating cellular cytotoxicity. Because cytotoxicity generally correlated with the level of expression of CAM in melanoma cells, weak CAM surface expression may provide a means for melanomas to escape immune surveillance. Cancer 1992; 69:1165-1173.
Recently, considerable interest has been focused on molecules that mediate cell-cell or cell-matrix interactions, commonly referred to as cell adhesion molecules (CAM).'-5 At least four categories of CAM currentIy are recognized, including the integrins?' a subset of molecules of the immunoglobulin supergene family,' and members of the LEC-CAM family.5Cell-surfaceproteoglycans, consisting of a core protein substituted in glycosaminoglycan side chains, constitute the fourth group. The core proteins of most cell-surface proteoglycans characterized currently appear to bear little homology to each other? although some, such as CD44, are related to proteoglycans found in the extracellular matrix.'O In cellular immunology, CAM play a significant role in several lymphocytic functions requiring cell-cell contact. Perhaps the most notable of such functions is cellular cytotoxicity. Two main types of cytotoxicity have been described:" (1)nonmajor histocompatibility complex (MHC)-restricted, such as natural killer (NK) cytotoxicity, in which lymphocytes interact with the target cells by nonspecific CAM-CAM interactions and (2) MHC-restricted, mediated by cytotoxic T-lymphocytes (CTL) that specifically recognize the target through the T-cell receptor (TCR). The role in cellular
1166
CANCER March 1,1992, Volume 69, No. 5
cytotoxicity of several well-characterized CAM has been in~estigated.’~’’-’~ However, little is known about the role in cytotoxicity of several of the more recently discovered CAM, such as the integrins of the bl family or CD44. The presence of a lymphoid infiltrate in malignant melanoma and its correlationwith progn~sis,’~ together with the observation that melanoma-derived tumor-infiltrating lymphocytes (TIL) often have specific autologous CTL activity, stimulated several investigations of TIL-mediated cyt~toxicity.’*-’~ Little is known, however, about the distribution of CAM on melanoma cells and their role in cytotoxicity. To address these questions, we derived pairs of melanoma cell lines and autologous TIL and determined the cytotoxic activities of the TIL against autologous targets. We subsequently investigated the expression of several CAM on the melanoma cells and assessed their role in MHC-restricted cytotoxicity mediated by CTL. Our results suggest that several of the more recently identified CAM are involved in regulating TIL-mediated cytolysis of melanoma cells. The downregulation of CAM molecules in melanoma cells can be related to tumor evasion from TIL-mediated surveillance. Materials and Methods
Cell Lines Seven pairs of melanoma cell lines and TIL were derived from parallel cultures grown from biopsy specimens of patients with metastatic melanoma. The specimens were obtained from the site of cutaneous metastases. Tissues were cut into I-mm fragments and cultured in either RPMI-1640 supplemented with 10% fetal bovine serum (FBS) or RPMI supplemented with 5% human serum and interleukin-2 (IL-2, 100 units/ ml). In this way, parallel cultures of melanoma cells and activated T-lymphocytes were ~btained.’~,’~ During the first 3 to 5 weeks after establishment of cultures, the melanoma cells were propagated from the tissue in the absence of any exogenous stimulus. On reaching confluence, adherent cells were passaged serially. Surface expression of CAM also was assessed in four additional melanoma cell lines for which TIL were not available. To confirm the melanoma origin of our cell lines, we used the S-100 and the HMB-4526monoclonal antibodies (MoAb)in an indirect immunofluorescence assay on fixed cytocentrifuged samples. These data, in conjunction with morphologic observations and the presence of melanin in some cell lines (M3 and M4), confirmed the melanoma origin of the cells. Cultures supplemented with IL-2 propagated activated T-cells in those specimens containing lymphocytic infiltrates. These cultures
were kept in IL-2 and restimulated with phytohemagglutinin (PHA) and irradiated feeder cells every 2 to 4 weeks to enhance proliferation, as previously rep~rted.’~,’~ Cultures are labeled as follows: melanoma cell lines were labeled as M (for melanoma), followed by a patient identification code: 1 for Patient 1, 2 for Patient 2,3 for Patient 3, and so on, to 7 for Patient 7. The TIL cultures were labeled as TIL followed by the patient identification number. For example, M-1 and TIL-1 indicate the melanoma cell line and the TIL cell line, respectively, derived from Patient 1.
Monoclonal Antibodies The CAM were studied with a panel of M o A ~ ~ , ’ ~ - ‘ ~ whose specificities are reported in Table 1. Other reagents used included anti-CD3, anti-CD4, anti-CD8 (Ortho, Raritan, NJ), anti-CD16 and anti-CD57 (Becton Dickinson, Sunnyvale, CA), UCHLl (CD45RO);’ and W6/32, an antibody to histocompatibility antigen (HLA) Class I. The MoAb were stained in an indirect immunofluorescence test and analyzed in a Becton Dickinson Facscan I1 with standard techniques.
Incubation of Melanoma Cells W i t h y-Interferon To evaluate the effect of y-interferon (7-IFN) preincubation on melanoma CAM expression, each melanoma cell line was incubated for 48 hours with 500 units/ml of y-IFN (a gift of Genentech, San Francisco, CA) in RPMI with FBS at 37°C in a 5% CO, atmosphere. These conditions produce optimal enhancement of the expression of HLA-DR antigens on control melanoma cell lines. The CAM expression was assessed by indirect immunofluorescence, and the fluorescence pattern was compared with that observed in melanoma cell lines kept in media without y-IFN. Melanoma cells incubated with y-IFN were washed and used in the cytotoxicity assay as described subsequently. Table 1. Monoclonal Antibodies Directed to Cell Adhesion Molecules Used in the Current Study Monoclonal antibody
CD
Other designation/structure recognized
T11.2 TS1/22 TS1/18 A-lA5 Hermes 111 TS2/7 B-5G10 RRl/l Leu19 TS2/9
CD2 CDl l a CD18 CD29 CD44 CD49a CD49d CD54 CD56 CD58
LFA2 LFAl, a1 chain integrin 2nd subfamily j3 chain integrin 2nd subfamily j31 chain of the VLA Hyaluronic acid VLAI, a1 chain of the VLA VLA4, a 4 chain of the VLA ICAM-1 N-CAM LFA3
Adhesion Molecules and TIL Cytotoxicity/Pandolfi et al.
1167
Cytotoxicity
Table 2. Expression of Cell Adhesion Molecules on Human Melanoma Cell Lines
Cytotoxic activity was assessed by the lysis of 51Cr-labeled target cells in a 4-hour chromium release assay, as previously rep~rted.~' Target cells were represented by autologous or allogeneic melanoma cell lines or the NKsensitive K-562 cell line. Effector cells were TIL kept in IL-2, 8 to 20 days after restimulation with PHA and irradiated feeder cells. Our results are reported as the percent of specific lysis.
Monoclonal antibody
Inhibition of Cellular Cytotoxicity by Preincubation W i t h Monoclonal Antibodies Melanoma cell lines or TIL were incubated for 1hour at 37°C in a CO, atmosphere with MoAb. The TIL were preincubated with TS1/22, T11.2, OKT3, and W6/32; the melanoma cells were preincubated with the other reagents studied. A final dilution of 10 &ml of purified MoAb (OKT3, Hermes 111, and Leul9) or a 1 : l O O dilution of ascites was used. Preincubated cells then were tested in the cytotoxicity assay as reported. Excess antibody was present through the cytotoxicity assay. Our results are expressed as the percent of inhibition of the cytotoxicity observed without a blocking antibody, at a 50:l effector-to-target ratio. Our results are the means of triplicate or sextuplicateexperiments.Because preliminary experiments showed that control ( i e l e vant) MoAb always gave results within k 5% of those obtained without antibodies in the tests, we established an arbitrary cutoff at 10% of inhibition (ix.,considering the effects of a MoAb as inhibitory when inhibition was greater then 10%). Results
Expression of Cell Adhesion Molecules on Melanoma Cell Lines Melanoma cell lines were studied with a panel of MoAb as reported in Table 2. None of the cell lines expressed LFAl and CD18 lymphoid/myeloid markers, but all stained with CD29 and LFA3. Variable expression of the other CAM was observed. CD44 was expressed in six of seven lines; VLA1, in five of seven; VLA4, in six of seven; ICAM-1, in six of seven; and CD56/N-CAM, in three of seven. Four additional melanoma cell lines (for which corresponding TIL were not available)confirmed this heterogeneity of expression with CD44 expressed in three of four lines; VLAl, in two of four; VLA4, in three of four; and ICAM-I, in two of four (data not shown in the table).
CDlla/LFAl CD18 CD29/VLA 81 CD44 CD49a/VLAl CD49d/VLA4 CD54/ICAM1 CD56/N-CAM CD58/LFA3
M-1
M-2
M-3
M-4
M-5
M-6
M-7 ND ND
-
-
-
-
ND
-
-*
-
-
-*
-
-
++ ++ ++ -*
++
-*
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ -
+* ++ ++ ++ ++ t+ ++ ++ ++ ++ ++ ++ ++ ++ ++
+* ++
++ ++ -*
++ ++ -
++
++:
> 60% of cells positive; +: 10-60% of cells positive; -: < 10% of cells positive; ND: not done. * CAM expressionwas tested also after incubation of the cells with y-interferon and found to remain unchanged.
Effect of y-Interferon on the Expression of Cell Adhesion Molecules The cytokine y-IFN stimulates Class I1 antigen expression and enhances cellular cytotoxicity. However, the underlying mechanism by which y-IFN augments killing is unclear. In an attempt to determine whether yIFN induces CAM expression in target cells, melanoma cells that expressed some of the CAM weakly or not at all were incubated with y-IFN (Table 2). Among the CAM found to be variably expressed were CD18 (tested in two cell lines), CD44 (negative in M-2), VLAl (negative in M-2), VLA4 (weak in M-1 and M-4), and ICAM1 (negative in M-2). Although y-IFN induced or enhanced the expression of HLA-DR antigens in M-2 and M-4 cell lines, respectively, (data not shown) in the experimental conditions we used, no y-IFN effect on the expression of the CAM tested was observed.
Expression of Surface Molecules on TumorInfiltrating Lymphocytes We analyzed TIL maintained in IL-2 for the expression of several T-cell-related surface markers and for markers associated with some cytotoxic functions and CAM expression. In seven TIL cultures studied, more than 95% of cells were positive for: CD3, CD2, CD1la/ LFAl, CD29, CD44, CD45R0, VLA4, and LFA3. However, these cultures were negative consistently(< 5%of positive cells) for CD16 and CD57 reagents. Four MoAb (CD4, CD8, VLA1, and CD56) gave a more variable pattern, and these results are reported in Table 3. Of the seven TIL cell lines, two were almost pure populations of CD4 cells; two, CD8 cells; and the other three cultures, mixed. Expression of CD56 and VLAl was also variable.
CANCER March 1,1992, Volume 69, No. 5
1168
Table 3. Surface Markers Expressed by TumorInfiltrating Lymphocytes ~
J 80 -
~~
Monoclonal antibody CD4 CD8 CD49a/VLAl CD56
.............
TIL-1 TIL-2 TIL-3 TIL-4 TIL-5 TIL-6 TIL-7
