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THEJOURNAL
OF I"UNOUX;Y
Copyright 0 1987 by The American Assoclatlon of Immunologists
AND
From the *Pigmented Lesion Study Group, the Hematology-Oncology Section, Department
of Medicine. and the 'Department
of Pathology and Laboratory Medicine, University
of Pennsylvania School of Medicine; and the 'Wistar Institute
of Anatomy
and Biology, Philadelphia,P A 191 04
To examine the potential regulatory role of inter- mas contained the highest numbers of activated
feron-y in the cellular immune response to mela- lymphocytes and stained positively for interferonnoma and its precursor lesions, we have tested the ?.
These results suggest that interferon-7 plays a
capacity of this lymphokine to enhance HLA class
I1 antigen-dependent T lymphocyte blastogenesis, central role inthe regulation of the cellular immune
its in vitro production by autologous T cells stimu- response to melanoma. It is produced by T cells,
lated by melanoma, and its presence in melanocyticlikely activated by tumor antigens seen in the conlesions in situ. Cell lines derived from a dysplastic text ofHLA class 11 antigens. In turn, interferon-?
nevus, a radial growth phase primary tumor,a ver- production enhances expression of HLA class 11antical growth phase primary, and metastatic lesions tigens by melanoma and precursor cells, and such
were induced by recombinant interferon-7 to ex- enhancement is associated with additional T cell
press increased amounts of HLA class I1 antigens. activation in a positive feed-back loop.
Such cells were then examined in radioimmunoassay for expression of HLA-DR antigens and in coAnalyses of frozen sections of melanocytic lesions by
culture for their ability to stimulate proliferation of
the
immunoperoxidasetechniquehavedemonstrated
autologous T cells. Interferon-?treatment of melanocytic cells increased their expression of HLA-DR that dysplastic nevi (potential precursors of melanoma)
antigens threefold to sixfold. In parallel with these and biologically early melanoma(radial growth phase
findings, co-culture of T cells with interferon- melanoma, a stage in tumorprogression associated with
treated cells of a dysplastic nevus and a radial phase invasion but without the capacity for local tumorigenic
melanoma ledto augmented T cell incorporation of growth or metastasis) (1) are invariably infiltrated by T
tritiated thymidine, and this stimulation was in- cells,approximately equally distributed between the
hibited with a monoclonal antibodyto HLA-DR an- helper and cytotoxic/suppressor phenotypes (2). In contigens. Despite augmented expressionof HLA class trast, lesions of biologically latemelanoma(vertical
I1 antigens (HLA-DR. -DQ, and -DP),vertical growth growth phase and metastases) are characterized by a
phase and metastatic melanoma cells failed to stim- paucity of infiltrating lymphocytes (2).Additionally, melulate autologous T cells. When T cells wereco- anocytes of melanoma and its precursors commonly excultured with stimulating melanoma cells, culture press class I1 major histocompatibility complex (MHC)
supernatants contained significantly
increased
antigens on their surface (3, 4). These histological findamounts of interferon-? (12 U/ml) in comparison ings are paralleled in functional assays. Melanoma cell
with supernatants of T cells alone (4 U/ml). No inter- lines derived from biologically earlylesionsstimulate
feron wasdetectable in cultures of melanoma cells
coalone. To link these in vitro phenomena to in situ autologous T cells to undergo blastogenesis when
Such stimulation is dependent, in
events, we used murine monoclonal antibodies to cultured in vitro (5,6).
interferon-7, the interleukin 2 receptor, and HLA- part, on the expression ofHLA-DR class I1 antigens by
DR antigens in an immunoperoxidase system to de- the transformed melanocyte, because it is inhibited by
tect interferon production and lymphocyte activa- allospecific antiserum to these HLA antigens (5).Further,
tion in frozen sections of lesions representative of there is a quantitative relationship between the expresmelanocytic tumor progression. In these studies, sion of HLA-DR antigens by tumor cells, as measured in
precursor dysplastic nevi and radial phase melano- radioimmunoassay, and thedegree of lymphocyte prolifReceived for publication July 25,
1986.
Accepted for publlcation April 15, 1987.
The costs of publication of this article were defrayed in part by the
payment of page charges. Thls article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' Thls work was presented in part at the American Association for
Cancer Research meeting. Houston, TX. May 1985 (Proc. Am. Assoc.
Cancer Res. 26:302].This work was supported by Grants CA 29200,CA
25874 and CA25298 from the National Institutes of Health.
'Address correspondence to DuPont Guerry. M.D.. Cancer Center, 7
Sllverstein Bldg., Hospital of the University ofPennsylvania. 3400Spruce
St.. Philadelphia, PA 19104.
305
306
307
INTERFERON-y ANDMELANOMA
TABLE I
rlFN-y treatment of line 81 4 (dysplastic nevus): effecton MHC antigen expression and autologous lymphocyte
stimulation
Line 814
Treated with
Antigen Expression
(cpm RIA + 1 SD)
in
Class F
Class I I ~
Lymphocyte
Stimulation
(S.I./cpmf 1 SD)
P3
Medium alone
374f 20
6.542f 109
349
f3.41630
13
f 110
IFN-r
1,154f
7.933
126
f 156
416f 10
8.0/1501f 405
a Cell line 814 was incubated with medium alone or with rIFN-7 for 48 hr and then wasassayed by radioimmunoassay
and the anti-HLA class I antibody, W6/32.The
(RIA) for the binding of two Mab: the anti-DR antibody. 691-13-17,
background binding of the P3 control was not subtracted here or in Table 11. Aliquots of medium and IFN-y-treated cells
were also mitomycin C treated andcultured with autologous T cells. After 6 days, lymphocyte proliferation was assessed
by [3H]thymidine incorporationand then reported as the S.I. and raw cpm f 1 SD (cpm of T cells alone= 187 f 39).
b691-13-17.
W6132.
TABLE I1
rlFN-y treatment of line WM35 (primary melanoma): effecton MHC antigen expression and autologous lymphocyte
stimulation
Antigen Expression
(cpm in RIA f 1 SD)
Line WM35
Treated with:
Class I I ~
Experiment 1
Medium alone
IFN-7
489
IFN-y f 128.4d
Experiment 2
Medium alone
IFN-y
IFN-7 + 128.4
Class F
426 f 268
1,152
1,897f 3503,859
f 210
1.281
195f 1 1
1,238f 82
219f 21
1,440
P3
107
194
175303
f 140
353
f 1.31219
14
f 1722213.643
f 14
2.01324
k
k
1.006 f 160
5.825f 442
f 149
Lymphocyte
Stimulation
(S.I./cpm2 1 SD)
26
f 754
f
80
f
6
2.71531 f
41.517.922f 1.204
9.7f 1,857f 207
184f 24
219f 9
216 f 13
Cell line WM35 was incubated with medium alone, with rIFN-y preincubated with P3.or with rIFN-y preincubated
with the neutralizing anti-IFN-y antibody. 128.4 PI(50
(5000U) of rIFN-y were incubated for 18 hr at 4C with 10 PI 128.4
ascites (20.000neutralizing U)). This mixture was brought up to 10 ml and added to the preconfluent cells for 48 hr and
then was assayedby radioimmunoassay (RIA)for the binding of the same HLA class I1 and class I antibodies as in Table
I. The capacity of such cells to stimulateautologous T cellswas measured as described in Table I (cpm of T cells alone in
Expt. 1 = 164 f 7,in Expt. 2 = 191 f 15).
b691-13-17.
W6f32.
128.4= Anti-IFN-y.
LYMPHOCYTE
STIMULATION
cpm
40000-
30000.
20000
10 000.
p3
Anti-
Anti-
DR Class I
Figure 1 . Inhibition of melanoma cell stimulation of T cells by antiHLA-DR Mab. Line WM35 melanoma cells were incubated with medium
or rIFN-y. and their relative expression ofHLA-DR antigens (using the
anti-HLA-DR antibody L243) and class I antigens (using the anti-HLA
class I antibody W6/32)was measured in radioimmunoassays (right
panel, mean cpm f 1 SD).IFN-y-treated melanoma cells were then
incubated for 1 hr with each Mab at a final concentration of 1 d m 1 or
in medium alone, and were then co-cultured with autologous T cells.
Stimulation was measured as described in Table I (mean cpm f 1 SD)
(left panel).
308
TABLE I11
Generatlon of ZFN-y by lymphocytes respondlng to autologous melanoma"
S.I./cpm i 1 SD
Stlmulating
Preincubatlon
CellResponding
Cell
Melanoma WM 35
None
um
None
Medium
IFN-7
Medium
IFN-y
T cells
None
Rail
3415,258
6219.694
324
IFN-I
Wmlt
<4
f 1.162
f 1.858
<4
12
12
4
997/155.464 f 15.983
~~~
"Cell line WM35 or the allogeneic lymphoblast Raji was incubated ln the presence or absence of rlFN-y and then
washed. After mitomycin C treatment. these cells were cultured with T cells autologous to line WM35. Lymphocyte
proliferation was assessed by [3H]thymidine incorporatlon
and then reported as the S.1. and raw cpm f 150 (cpmof T cells
alone = 156 2 44). In addltion. Supernatants wereassayed for IFN-y as described In Materials and Methods, and then
reported as units/mllliliter.
MELANOMA
AND
309
INTERFERON-7
TABLE IV
Immunoperoxldase analysls of lymphocytes ln melanocytlc leslons
Type of Leslon
[no. leslonswlth posltive lymphocytes/no. tested]
Antlbody
Dysplastlc nevl
Antl-IFN-y
113.46
128.4*
Both'
EitheP
Anti-TAC'
Anti-DRg
Prlmary melanoma
(50. 20-80)
7/10. (70. 10-100)
519
911 1
(60.2-90)
Metastatlc melanoma'
415. ( 10. 3- 10)
315. (50. 10-90)
214
414
315. (50. 20-80)
515.5-100)
(75.
310
AND
INTERFERON-7
MELANOMA
31 1
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
312
21.
22.
23.
24.
25.
26.
MELANOMA
INTERFERON-? AND