- Email: [email protected]
Characterization of anti-HLA class II monoclonal antibody LGII-612.14 reacting with formalin fixed tissues
Journal of Immunological Methods, 161 (1993) 239-256
239
© 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00
JIM 06688
Characterization of anti-HLA class II monoclonal antibody LGII-612.14 reacting with formalin fixed tissues Massimo T e m p o n i a,1 U l a na Kekish a Carl V. H a m b y a Hella Nielsen b Charles C. M a r b o e b and Soldano F e r r o n e a a Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA, and b Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
(Received 3 December 1992, revised received 20 January 1993, accepted 20 January 1993) m A b LGII-612.14 derived from a B A L B / c mouse immunized with interferon-y ( I F N - y ) treated cultured human B lymphoid cells LG-2 has been shown with serological and immunochemical assays to recognize a monomorphic determinant expressed on the /3 chain of H L A - D R , - D Q and -DP antigens. The linear nature of the determinant, which is likely to be formed by residues 19-25, is indicated by the reactivity of m A b LGII-612.14 with H L A - D R , - D Q and -DP /3 chains purified by electrophoresis in presence of SDS. An unusual characteristic of m A b LGII-612.14 is its reactivity with fixed tissue sections. The intensity of staining is affected by the incubation temperature, the incubation time and the fixative used. Maximal intensity of staining of formalin fixed, paraffin embedded tissue sections required an incubation time of 16 h. The intensity of staining of paraffin e m b e d d e d tissues initially fixed with Bouin's solution, formalin or ethanol was similar to that of frozen tissue sections and stronger than that of tissues fixed with B5 solution. No staining was detected of paraffin embedded tissues fixed with glutaraldehyde or Z e n k e r ' s solution. Comparison of the staining patterns with m A b LGII-612.14 of frozen and fixed tissue sections showed that the latter substrates provide a superior detail of tissue architecture and cellular morphology without significant loss of sensitivity. Furthermore, comparison of the characteristics of m A b LGII-612.14 with the few previously published anti-HLA class II mAb reacting with fixed tissues indicates that m A b LGII-612.14 stains formalin fixed, paraffin embedded tissues, while mAb 910D7 and TAL-1B5 stain tissues fixed with less commonly used fixatives. Furthermore, mAb LGII-612.14 is likely to yield more sensitive staining results than a n t i - H L A - D R , - D Q and -DP m A b K U L / 0 5 . The present results indicate that m A b LGII-612.14 represents a useful probe to apply immunohistochemical techniques to the analysis of the distribution of H L A class II antigens in fixed tissues. This will greatly facilitate the use of readily available collections of fixed tissue specimens in retrospective studies to assess the clinical significance of changes in H L A class II antigen expression which occur in various disease states. Key words." HLA class II antigen; Formalin-fixed tissue; Monoclonal antibody; Immunohistochemistry
Correspondence to: S. Ferrone, Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA. Tel.: (914) 993-4175; Fax: (914) 993-4176. 1 Visiting Investigator from the Institute of Chemistry, School of Medicine, University of Brescia, Brescia, Italy.
IFN-y, human recombinant immune interferon; mAb, monoclonal antibody; NP40, Nonidet P-40; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis.
Abbreviations:
240 Introduction
Like their counterparts in other animal species, HLA class II antigens, which include the gene products of HLA-DR, -DQ and -DP loci, consist of two non-covalently associated membrane anchored glycopolypeptides referred to as c~ and /3 chains, with the apparent molecular weight of 33,000 and 29,000, respectively. During their biosynthesis and export, a and /3 chains associate with the invariant polypeptide chain, which dissociates before H L A class II antigen expression on the cell membrane (for review, see Solheim et al., 1986). It has been known for a long time that HLA class II antigens play a key role in the regulation of the immune response by mediating and restricting interactions among immune cells (for review, see Solheim et al., 1986). More recently, H L A class II antigens have been shown to play a broader role in cell economy, since they are involved in the regulatory mechanisms which control cell proliferation (Romagnani et al., 1985; Ruggiero et al., 1987). For many years the analysis of H L A class II antigen expression has been restricted to peripheral blood cells and to cells in long term culture, since the limited specificity of conventional antiHEA class II antisera has hampered the application of immunohistochemicai techniques to analyze the tissue distribution of H L A class II antigens in normal and pathological conditions. These limitations have been overcome by the development of anti-HLA class II monoclonal antibodies (mAb). Immunohistochemical staining of tissues has shown that H L A class II antigens have a broader distribution than originally postulated, since they are expressed also by cells not directly associated with the immune system (for review, see Solheim et al., 1986). Furthermore, malignant transformation of cells may be associated with changes in the expression of H L A class II antigens (for review, see Solheim et al., 1986). These changes may have a clinical significance since expression of H L A class II antigens may aid the differentiation of follicular variants of papillary thyroid carcinomas from follicular thyroid carcinoma (Lloyd et al., 1985) and a relationship has been found between expression of H L A class lI antigens by malignant cells and clinical course of
certain diseases. The latter include breast (Brunner et al., 1991), gastric (Hilton and West, 1990) and laryngeal (Esteban et al., 1989) carcinomas, acute myeloblastic leukemia (El Zimeity et al., 1990), B cell lymphoma (Momburg et al., 1987), chronic lymphocytic leukemia (Orfao et al., 1989), diffuse large cell lymphoma (Miller et al., 1988; Spier et al., 1988; O'Keane et al., 1990; Slymen et al., 1990), and melanoma (Van Duinen et al., 1988). The routine application of immunohistochemical techniques to characterize the tissue distribution of H L A class II antigens in normal and pathological conditions has been hampered by the availability of only a limited number of mAb reacting with H L A class II antigens preserved in formalin fixed, paraffin embedded tissues. To the best of our knowledge, only two anti-HLA class II mAb with this type of reactivity have been described, mAb LK8D3 (Wilson et al., 1984) is no longer available, since a stable hybridoma could not be established (B.S. Wilson, personal communication), mAb K U L / 0 5 (Giacomini et al., 1989) which reacts with the /3 chain of HLA-DR, -DQ and -DP antigens stains formalin fixed, paraffin embedded tissue sections with a weaker intensity than frozen sections. Therefore, the use of fixed tissue sections is likely to cause a reduction of the sensitivity of immunoperoxidase staining. Furthermore, mAb 910D7 which reacts with HLADR antigens (Sorg et al., 1984), and mAb TAL1B5 which reacts with the a chain of H L A - D R antigens and possibly of H L A - D Q antigens (Adams eta[., 1983; Epenetos et al., 1985) do not stain formalin fixed, paraffin embedded tissues, although they stain tissues which had been fixed in 10% non-buffered formol saline and in acid formalin or Bouin's fixative, respectively. The latter three procedures are not routinely used for fixation of tissues. Anti-HLA class II mAb reacting with formalin fixed, paraffin embedded tissues greatly facilitate the analysis of the tissue distribution of these molecules and the assessment in retrospective studies of the clinical significance of changes in their tissue distribution. Therefore, in the present study we have characterized the specificity of mAb LGII-612.14 which stains H L A class II antigens in formalin fixed, paraffin embedded tissues,
241 we have analyzed the effect of various fixatives and incubation conditions on the reactivity of mAb LGII-612.14 with tissues and we have compared the staining patterns of mAb LGII-612.14 with those of the previously described anti-HLA class II mAb.
Materials and methods
Cells Cultured human B lymphoid cells Daudi, LG-2, LKT-13 and WALK, cultured human T lymphoid cells MOLT-4, cultured human melanoma cells Colo 38, FO-1, SK-MEL-33 and 3S5, cultured human ovarian carcinoma cells SK-OV-3, and mouse myeloma cells P3-X63-Ag8.653 were grown in RPMI 1640 medium supplemented with 10% FCS and 2 mM L-glutamine. Tissues Endomyocardial biopsies from heart allograft recipients were obtained by standard transvenous approach. Other tissues were obtained from patients who underwent surgery or at autopsy. Tissues were placed immediately in 10% neutral buffered formalin, routinely processed and paraffin embedded. Some specimens were divided into aliquots and separate portions were frozen and cryostat sectioned or fixed in 10% neutral buffered formalin, alcohol-formalin, absolute ethyl alcohol, B5 (Lillie, 1965), Bouin's solution (Luna, 1968), 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.25, Zamboni's solution (Stefanini et al., 1967) or Zenker's solution (Luna, 1968). Interferon Human recombinant immune interferon (IFN7) was obtained from Hoffman La Roche, Nutley, NJ. mAb and conventional ant&era The mAb LGII-612.14 is secreted by a hybridoma generated from a BALB/c mouse immunized with three intraperitoneal injections on days 0, 10 and 20 of 5 × 106 cultured human B lymphoid cells LG-2 which had been incubated with IFN-y (final concentration 400 U / m l ) for 72
h. 3 days following the last injection, the mouse was killed, splenocytes were isolated and hybridized with mouse myeloma cells P3-X63Ag8.653. Hybridization and subcloning were performed according to standard procedures (K6hler and Milstein, 1975). The isotype of mAb LGII612.14 was determined by testing spent medium of the hybridoma with a mouse monoclonal subisotyping kit (Hyclone Laboratories, Logan, VT) following the manufacturer's instructions. The anti-HLA-DR mAb Q2/70, the antiHLA-DQ mAb SPV-L3, the anti-HLA-DP mAb B7/21, the anti-HLA-DR, -DQ, -DP mAb Q5/13, and the anti-HLA class I mAb TP25.99 and W6/32 were developed and characterized as described elsewhere (Barnstable et al., 1978; Quaranta et al., 1980a,b; Watson et al., 1983; Spits et al., 1984; Temponi et al., 1993). mAb 910D7 and TAL-1B5 were obtained from Dr. W. Ax (Behringwerke, Marburg, Germany) and from Dr. G.J. Bodmer (Imperial Cancer Research Fund, London, England), respectively. mAb were purified from ascitic fluid by sequential precipitation with caprylic acid and ammonium sulfate (Temponi et al., 1989). mAb were labeled with ~25I utilizing the Iodo-gen method (Fraker et al., 1978). The immunoreactive fraction of radiolabeled mAb was at least 38% as determined following Lindmo et al.'s method (Lindmo et al., 1984). The Vectastain Elite ABC kit was purchased from Vector Laboratories (Burlingame, CA). Serological assays The ELISA and the binding assay with ~25Ilabeled mAb were performed in 96-well microtiter plates as described elsewhere (Matsui et al., 1987). Immunoperoxidase staining of frozen and fixed tissues was performed utilizing the Vectastain Elite ABC kit following the manufacturer's instructions. Briefly, when fixed tissue was the substrate, 4 /zm thick tissue sections were mounted on organosilane (3-aminopropyltriethoxysilane, 98%, Aldrich Chemical Company, Milwaukee, WI) coated slides and incubated for 16 h at 57°C. Then, sections were deparaffinized in xylene, rehydrated by passage through decreasing concentrations of ethanol (from 100% to 80%) and
242
washed in phosphate-buffered saline (PBS), pH 7.4. When frozen tissue was the substrate, 4 ~m thick cryostat sections were fixed in the indicated fixative for 10 min. Following one washing in PBS for 20 min, sections were incubated for 20 min at room temperature in 30% normal horse serum to block non-specific binding of mAb. Following removal of horse serum, sections were incubated with an excess of anti-HLA class II mAb LGII612.14 in a humidified chamber for 16 h at 4°C, unless otherwise indicated. Then, slides were washed for 10 min in PBS and incubated with biotinylated anti-mouse IgG xenoantibodies for 30 min at room temperature. Sections were then washed for 10 min in PBS, and incubated for 20 min at room temperature in 0.3% hydrogen peroxide in methanol to block endogenous peroxidase activity. Following washing in PBS, slides were incubated for 30 min at room temperature in avidin-biotin-peroxidase complexes. After an additional washing for 10 min in PBS, slides were incubated in 0.5 mg/ml 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) with 0.01% hydrogen peroxide in 0.05 M Tris-HCl buffer, pH 7.6. Slides were then washed for 5 min in distilled water and dehydrated in increasing concentrations of ethanol (from 80% to 100%). Then, slides were cleaned with xylene and mounted with Permount (Fisher Scientific, Fair Lawn, N J). l m m u n o c h e m i c a l assays
Radiolabeling of cells with 125I utilizing the lactoperoxidase method or with [35S]methionine (Amersham), solubilization with 1% Nonidet P-40 (NP40) (Sigma Chemical Co.), indirect immunoprecipitation, sequential immunoprecipitation, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis (non-equilibrium pH gel electrophoresis in the first dimension and SDS-PAGE in the second dimension) were performed as described (Laemmli, 1970; O'Farrel, 1975; Zweig and Shevach, 1983; Matsui et al., 1987). Gels containing 125I-labeled samples were processed for autoradiography using Kodak XAR-5 films (Eastman Kodak Co., Rochester, NY). Gels containing [35S]methionine labeled samples were processed for fluorography as described (Bonner and Laskey, 1974).
Western blotting analysis was performed as described (Towbin et al., 1979). Briefly, NP40 extracts of cultured cells were incubated with 50 /xl of Affi-Gel 10-coupled mAb for 4 h at 4°C on a rotator. Antigens were then eluted by boiling in SDS sample buffer and electrophoresed on a 10% acrylamide gel utilizing the buffer system described by Laemmli (1970). Then, proteins were transferred to a nitrocellulose filter (Bio-Rad Laboratories, Richmond, CA) as described by Towbin et al. (1979). Filters were then blocked with a solution of 30% horse serum in PBS for 1 h at room temperature and incubated with 125Ilabeled mAb LGII-612.14 (2 × 105 cpm/ml) for 4 h at room temperature. After three incubations of 20 min each, at room temperature in PBS containing 0.05% Tween 20 (Sigma Chemical Co.), filters were dried and autoradiographed utilizing Kodak XAR-5 films (Eastman Kodak Co.).
Results
mAb LGII-612.14, an IgG2b, was selected from among those produced by a B A L B / c mouse immunized with IFN-y treated cultured B lymphoid cells LG-2, since it displayed a higher reactivity with the IFN-y treated immunizing cells than with the control cells in ELISA. Testing in a binding assay with a panel of HLA typed cell lines showed that mAb LGII-612.14 displayed a selective reactivity with HLA class II antigen bearing cells. Representative results with HLA class II positive and negative cell lines are shown in Fig. 1. The reactivity of mAb LGII-612.14 with HLA class II antigens was conclusively shown by the characteristic electrophoretic profile of antigens immunoprecipitated from radiolabeled cultured B lymphoid cells LG-2 analyzed by SDSPAGE (Fig. 2). The reactivity of mAb LGII-612.14 with HLA-DR, -DQ and -DP antigens is indicated by three lines of evidence. First, immunodepletion of cultured B lymphoid cell extracts with mAb LGII-612.14 or with the anti-HLA-DR, -DQ, -DP mAb Q5/13 removed the components immunoprecipitated with either mAb. Representative results are shown in Fig. 2. Furthermore, the 2-dimensional gel electrophoretic profile of the components immunoprecipitated by mAb
243
LGII-612.14 is that characteristic of H L A - D R , -DQ and -DP antigens (Fig. 3). Lastly, Western blotting analysis showed that mAb LGII-612.14 reacts with the /3 chain of H L A - D R , -DQ and -DP antigens purified by immunoprecipitation with the a n t i - H L A - D R mAb Q 2 / 7 0 , with the anti-HLA-DQ mAb SPV-L3 and with the antiH L A - D P mAb B 7 / 2 1 from cultured B lymphoid cells. Representative results are shown in Fig. 4. Like the majority of anti-HLA class II mAb described in the literature, mAb LGII-612.14 reacts with H L A class II antigen bearing cells in frozen tissue sections (Fig. 5). Furthermore, mAb LGII-612.14 stained cells in cryostat frozen sec-
Immunoproclpltotlon with HoAb: L611-OI2.14
RBCRBC
615/13
RBC
-
4,3 I
-33Kd - Z6 i
-
121~1
Fig. 2. Structural relationship of antigens immunoprecipitated by mAb LGII-612.14 and by anti-HLA-DR, -DQ, -DP mAb Q5/13 from cultured B lymphoid cells LG-2. Cultured B lymphoid cells LG-2 were radiolabeled with 125Iutilizing the lactoperoxidase method and solubilized with NP-40. The cell extract was incubated 5 times for 4 h with either mAb LGII612.14 (lane B) or mAb Q5/13 (lane C) bound to protein A Sepharose. Each immunodepleted cell extract was then incubated for 16 h at 4°C on a rotator with insolubilized mAb LGII-612.14 and mAb Q5/13. After washing, antigens were eluted from immunoadsorbent beads by boiling with 50 pA of electrophoresis sample buffer and electrophoresed in a 10% SDS-PAGE using the Laemmli buffer system(Laemmli, 1970). After drying, gels were processed for autoradiography using Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) The anti-HLA class I mAb W6/32 (lane A) was used as a specificity control.
m x
gl
Ozdl ~II-11 d-I IT-4 II4I
}I
Nil
I
I-I4
Fig. 1. Reactivity of mAb LGI1-612.14 with cells in long term culture. Cultured B lymphoid cells Daudi, LKT-13 and LG-2, T lymphoid cells MOLT-4, melanoma cells SK-MEL-33, FO-1, Colo 38 and 3S5, and the ovarian carcinoma cells SK-OV-3 (1 X 105 cells/well) were incubated in 96 well V bottom microtiter plates (Dynatech Laboratories, Alexandria, VA) with 12SI-mAb LGII-612.14 (5 × 105 cpm/well) (open bars) for 2 h at 4°C on a rotator. Then, plates were centrifuged, supernatant aspirated and cell bound radioactivity was measured in a gamma counter. Results are expressed as bound cpm. The anti-HLA-DR, -DQ, -DP mAb Q5/13 (stippled bars) and the anti-HLA class I mAb TP25.99 (hatched bars) were used for comparison purposes.
tions which had subsequently been fixed for 10 min in acetone, alcohol-formalin, Bouin's solution, ethanol, formalin or Zamboni's solution. Unexpectedly, mAb LGII-612.14 was found to react also with H L A class II antigen bearing cells in formalin fixed, paraffin embedded tissues (Fig. 6). The intensity of staining of formalin fixed, paraffin embedded tissues was affected by the incubation time and the incubation temperature. The intensity of staining did not change when tissue sections were digested with 0.05-0.1% trypsin for 30-60 min at 37°C prior to incubation with mAb LGII-612.14. On the other hand, the intensity of staining was stronger when tissue sections were incubated with mAb LGII-612.14 for 16 h than for 1 h. Furthermore, the intensity of staining was stronger when tissue sections were
244 incubated with m A b LGII-612.14 at room temperature than at 4°C; however, the increase in intensity was associated with an increase in nonspecific staining. Therefore, the incubation at 4°C for 16 h was selected for staining of fixed tissue sections. Lastly, the intensity of staining was also markedly influenced by the fixation procedure. m A b LGII-612.14 stained strongly cells in paraffin e m b e d d e d tissues which had been fixed in alcohol-formalin, Bouin's solution, ethanol or formalin, weakly cells fixed in B5 or Z a m b o n i ' s solutions, and did not stain cells in glutaraldehyde or Zenker's solution fixed tissues. Representative results are shown in Fig. 5 which compares the immunoperoxidase staining of frozen and fixed sections of a rejected heart allograft. It is noteworthy that in the frozen section the staining of capillary endothelium is strong, but histologic details are not as sharp as in fixed sections. Furthermore, the staining of capillary endothelium and infiltrating cells in formalin fixed sec-
tions is less strong than in sections fixed with absolute ethanol or with Bouin's solution. Additional comparisons of the immunoperoxidase staining patterns of adjacent frozen and formalin fixed sections of a metastatic melanoma lesion and of a rejected renal allograft are shown in Figs. 7 and 8. In both the frozen and the formalin fixed sections of the m e l a n o m a lesion, there is strong staining of cell membranes; the tissue architecture is considerably better preserved in the formalin fixed than in the frozen section. In addition, the staining between tubules is indistinct and only rare cells are stained in the glomerulus in the frozen section of the rejected renal allograft, while infiltrating ceils and interstitial and glomerular capillaries are distinctly stained in the paraffin e m b e d d e d section. The intensity of the staining is similar in the two sections. The staining patterns obtained with mAb LGII-612.14 were then compared with those obtained with the previously described anti-HLA
m
o
DR~ DR~
I I
DP8
DO~
DPo~
"O
•
m NEPHGE
÷
Fig. 3. Two-dimensionalgel electrophoresis analysis of antigens immunoprecipitated by mAb LGII-612.14from labeled cultured B lymphoid cells LG-2. Cultured B lymphoid cells LG-2 were metabolically labeled with [35S]methionine, washed three times with Hanks' Balanced Salt Solution and solubilized with NP-40. The cell extract was then immunoprecipitated with mAb LGII-612.14. Antigens were eluted and analyzed by 2-dimensional gel electrophoresis (non-equilibrium pH gel electrophoresis (NEPHGE) in the first dimension and SDS-PAGE in the second dimension) (O'Farrell, 1975). Gels were then processed for fluorography (Bonner and Laskey, 1974).
245
class II mAb 910D7 (Sorg et al., 1984) and TAL1B5 (Adams et al., 1983; Epenetos et al., 1985). To this end, serial sections from frozen tissues or fixed, paraffin embedded tissues were stained with optimal concentrations of the three mAb. Each of the mAb stained cells in cryostat frozen sections which had been subsequently fixed in acetone, alcohol-formalin, Bouin's solution, ethanol, formalin, glutaraldehyde or Zamboni's solution (Fig. 9). At variance with mAb LGII612.14, neither mAb 910D7 nor mAb TAL-1B5 stained cells in paraffin embedded tissues which
IMMUNOPRECIPITATED
had been fixed in 10% neutral buffered formalin (Fig. 10). mAb 910D7 stained cells in tissues fixed in alcohol-formalin (Fig. 11), ethanol or Zamboni's solution, although less intensely than did mAb LGII-612.14. On the other hand, mAb 910D7 did not stain cells in tissues fixed in Bouin's solution or in glutaraldehyde, mAb TAL-1B5 stained cells in paraffin embedded tissues fixed in alcohol-formalin, Bouin's solution or ethanol with an intensity similar to that obtained with mAb LGII-612.14. Furthermore, mAb TAL-1B5 stained cells in tissues fixed in glutaraldehyde or
WITH
CELL
mAb:
EXTRACT
II
LG]]-612.14 W 6 / 3 2
tp 2s.99
LGn-612.14
Q2/70
SPV-L3
Iq
B7/21
L~ ~- 612.14 PROBED
WITH
W6/32
WALK
1
MOLT-4
I
12SI-mAb
Fig. 4. Western blot analysis of the reactivity of m A b LGII-612.14 with the/3 chains of H L A - D R , -DQ and -DP antigens. An NP40 cell extract of B lymphoid cells W A L K (2 × 106 cells/lane) was incubated with 50 txl of Affi-Gel 10-coupled a n t i - H L A - D R m A b Q 2 / 7 0 , a n t i - H L A - D Q m A b SPV-L3 and anti-HLA-DP m A b B 7 / 2 1 for 6 h at 4°C on a rotator. Antigens were eluted and electrophoresed on a 10% S D S - P A G E using the Laemmli buffer system (Laemmli, 1970). Then, proteins were transferred to a nitrocellulose filter (Towbin et al., 1979). Following a 1 h incubation in PBS supplemented with 30% horse serum to block protein binding sites, the filter was incubated for 4 h at room temperature with 125I labeled m A b LGII-612.14 ( 2 × 105 c p m / m l ) . After washing and drying, the nitrocellulose filter was autoradiographed utilizing a Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY). Specificity controls included the NP40 extract of cultured B lymphoid cells W A L K immunoprecipitated with mAb LGII-612.14 or with anti-HLA class I m A b W 6 / 3 2 and probed with 125I-labeled anti-HLA class I m A b TP25.99 and NP40 extracts of cultured B lymphoid cells W A L K and of cultured T lymphoid cells MOLT-4 resolved by S D S - P A G E in a 10% gel and probed with 125I-labeled m A b LGII-612.14.
B
LI
~w
t' ~
o
•
p
1 ¢ " ""
4
"IL w
O
PdlVq
.
8
|
f
ii
I
Fig. 5. Effect of the fixative on the staining of tissue sections by m A b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction. A rejected cardiac allograft was divided into multiple portions. One was frozen and fixed in acetone (panel A), one was fixed in absolute ethanol (panel B), one in 10% neutral buffered formalin (panel C) and one in Zamboni's solution (panel D). Tissue sections were then stained with m A b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not counterstained ( × 200).
LI
b
q
247 Zenker's solution and paraffin embedded, while m A b LGII-612.14 did not.
Discussion
Serological and immunochemical studies have shown that m A b LGII-612.14 recognizes a determinant expressed on the /3 chain of H L A - D R , - D Q and -DP antigens. The expression of the
determinant recognized by m A b LGII-612.14 on the gene products of H L A - D R , - D Q and -DP loci is indicated by the characteristic 2-dimensional gel electrophoretic profile of the molecules immunoprecipitated from cultured B lymphoid cells by mAb LGII-612.14 and by its specific reactivity with H L A - D R , - D Q and -DP antigens purified from lymphoid cells by immunoprecipitation with the appropriate specificity. The determinant is monomorphic, since m A b
Fig. 6. Immunoperoxidase staining with mAb LGII-612.14 of sections from a formalin fixed paraffin embedded tonsil. A tonsil was fixed in 10% neutral buffered formalin. Tissue sections were then stained with mA_b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not counterstained (A ×40; B × 200).
248 L G I I - 6 1 2 . 1 4 r e a c t s with cells expressing d i f f e r e n t H L A class II p h e n o t y p e s a n d with all H L A - D R allospecificities i s o l a t e d from H L A - D R h o m o z y gous cell lines as r e p o r t e d e l s e w h e r e ( K a u f m a n et al., 1990). T h e d e t e r m i n a n t r e c o g n i z e d by m A b L G I I - 6 1 2 . 1 4 has b e e n m a p p e d to t h e 1[~1 d o m a i n of H L A class II a n t i g e n s ( K a u f m a n et al., 1990) a n d is e x p r e s s e d by H L A class II /3 chains purified by e l e c t r o p h o r e s i s in the p r e s e n c e of SDS.
C o m p a r i s o n of the p u b l i s h e d a m i n o acid seq u e n c e s o f H L A - D R , - D Q a n d - D P /3 chains ( M a r s h a n d B o d m e r , 1991) a n d o f rat (Eccles and M c M a s t e r , 1985) a n d chicken ( B o u r l e t et al., 1988) M H C class II /3 chains ( T a b l e I) which also express the d e t e r m i n a n t d e f i n e d by m A b L G I I 612.14 ( u n p u b l i s h e d results) suggests that this d e t e r m i n a n t is e x p r e s s e d by t h e a m i n o acid stretch 19-25, which is s h a r e d by all t h e reactive /3
Fig. 7. Comparison of the staining patterns of a frozen section and of an adjacent formalin fixed, paraffin embedded section of a metastatic melanoma lesion with mAb LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction. One metastatic melanoma lesion was divided into two portions. One was frozen and fixed in acetone (panel A) and the other one was fixed in 10% neutral buffered formalin (panel B). Tissue sections were then stained with mAb LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not countertained ( x 250).
249
chains. This possibility is corroborated by differences in two residues in the stretch 19-25 between H L A - D R /3 chain and B A L B / c mouse MHC class II /3 chain (Saito et al., 1983) which does not react with mAb LGII-612.14. Of the two differences, the change of histidine at position 23 in B A L B / c mouse MHC class II /3 chain with arginine in H L A - D R /3 chain is likely to play the
major role in the expression of the determinant defined by mAb LGII-612.14, since the change of glutamic acid at position 22 in HLA-DR /3 chain with glutamine in B A L B / c mouse MHC class II /3 chain also occurs in HLA-DP/3 chain without affecting its reactivity with mAb LGII-612.14. mAb LGII-612.14 was elicited with an unusual immunogen, i.e., cultured human B lymphoid cells
Fig. 8. Comparison of the staining patterns of a frozen section and of an adjacent formalin fixed, paraffin embedded section of a renal allograft undergoing acute cellular rejection with m A b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction. The rejected kidney was divided into two portions. O n e was frozen and fixed in acetone (panel A) and the other one was fixed in 10% neutral buffered formalin (panel B). Tissue sections were then stained with m A b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not counterstained (A x250; B × 200).
251
incubated with IFN-y. Whether the development of an anti-HLA class II mAb with an unusual specificity is a fortuitous event or reflects changes in the immunogenicity of HLA class II antigens because of their upregulation by IFN-y, remains to be determined. We favor the latter possibility since, in previous studies we have developed antiHLA class I antigens and anti-intercellular adhesion molecule-1 mAb with a unique specificity from mice immunized with IFN-y treated cells (Matsui et al., 1987; Temponi et al., 1993). Like HLA class II antigens, the latter two antigens are upregulated by IFN-y. If our hypothesis is correct, IFN-y treated cells may represent useful immunogens to broaden the specificity of mAb to antigenic structures whose expression is susceptible to enhancement by this cytokine. An unexpected characteristic of mAb LGII612.14 is its ability to stain cells in formalin fixed, paraffin embedded tissue sections. The intensity of staining is in most cases equivalent to that of frozen tissue sections, provided that the incubation time is prolonged to 16 h. These results suggest that use of formalin fixed, paraffin embedded tissue sections as substrates in the immunoperoxidase reaction does not affect the sensitivity of the immunohistochemical technique, provided that the appropriate incubation conditions are used. B5, glutaraldehyde and Zenker's solution which all prevent the reactivity of fixed tissues with mAb LGII-612.14 share the property to react with the sulfhydryl group of cysteine. It is of interest that cysteine is close to the amino acid stretch which is likely to carry the determinant recognized by mAb LGII-612.14. In addition, the differential effect of formaldehyde and glutaraldehyde on the reactivity of fixed tissues with mAb LGII-612.14 may reflect the more extensive and stable crosslinking caused by glutaraldehyde.
Comparison of the characteristics of mAb LGII-612.14 with those of the previously described anti-HLA class II mAb reacting with fixed tissues showed that mAb 910D7 (Sorg et al., 1984) and TAL-1B5 (Adams et al., 1983) do not stain formalin fixed-paraffin embedded tissues, but stain tissues fixed with fixatives which are not routinely used. This characteristic markedly reduces the usefulness of the two mAb to analyze the tissue distribution of HLA class II antigens in normal and pathological conditions. Furthermore, the intensity of fixed tissue sections with mAb 910D7 was lower than that with mAb LGII612.14 and TAL-1B5; the latter two mAb did not differ in their intensity of staining of alcohol/formalin and ethanol fixed tissue sections, mAb KUL/05 was not made available to us by the investigators who had described it (Giacomini et al., 1989); therefore, its staining pattern of fixed tissues could not be compared to that of mAb LGII-612.14. Since mAb KUL/05 has been reported to stain formalin fixed, paraffin embedded tissues with a weaker intensity than frozen tissues, even when the incubation time is prolonged, the sensitivity of immunoperoxidase staining of formalin fixed, paraffin embedded tissues with mAb KUL/05 is likely to be lower than with mAb LGII-612.14. The results we have described clearly indicate that the staining of fixed tissue sections provides a superior detail of tissue architecture and cellular morphology than frozen tissue sections. This improves the analysis of cellular localization of HLA class II antigens in normal tissues and malignant lesions. Furthermore, mAb LGII-612.14 allows the study of HLA class II antigen expression in diagnostic biopsies too small to be divided into separate portions for fixation or snap-freezing, and in lymphoid lesions too small to be divided into separate portions to be disaggre-
Fig. 9. Effect of fixation and paraffin embedding on the staining of tissue sections by m A b LGII-612.14 in an avidin-biotin immunoperoxidase reaction. A normal h u m a n tonsil was divided into two portions. O n e was frozen and cryostat sections cut and fixed in acetone (panel A ) or 10% buffered formalin (panel B) and one was fixed in 10% buffered formalin and e m b e d e d in paraffin (panel C). Tissue sections were then stained with m A b LGII-612.14 in an avidin biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. A lymphoid follicle is at the top of each field. Tissue sections were not counterstained ( :x: 100).
252
(A)
Fig. 10. Comparison of staining of formalin fixed, paraffin embedded tissue sections by m A b LGII-612.14, and by anti-HLA class I1 m A b 910D7 and TAL-1B5 in an avidin-biotin immunoperoxidase reaction. Normal h u m a n tonsil was fixed in 10% buffered formalin and tissue sections stained with m A b LGII-612.14 (panel A), 910D7 (panel B), or TAL-1B5 (panel C) in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. T h e tonsillar epithelium is at the left side of each panel. Tissue sections were not counterstained ( x 100).
253
Fig. 11. Comparison of staining of ethanol formalin-fixed, paraffin embedded tissue sections by mAb LGII-612.14 and by anti-HLA class II mAb 9107D and TAL-1B5 in an avidin-biotin immunoperoxidase reaction. Normal human tonsil was fixed in ethanol-formalin (70% ethanol, 0.25% formalin, 0.1 M NaCI). Tissue sections were then stained with mAb LGII-612.14 (panel A), 910D7 (panel B), or TAL-1B5 (panel C) in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not counterstained ( x 100).
254
51,, 5
~ o
Z
~ °
I I
gLL~
g l l l
I I
I I
I -J I
.J I -J
I I I I
I I >Z
I I-I I
E W I
,,, Z
5 5 ~
~
''
~ o
5
~
~
M
~ °
<
I I
I I
Q.
a. I I I
~
©
I I I
I
~
I Z
5
I
m
I •~
''
I I Z
I I
z~
<
I
z
I
I
I I I
I I
0
Z
I I
5 1 1 u..J
0
I I I I I I
Z
<
~ °
=
Z
I
~
5
I
I
,I g
~ 5 5 Z l l
5
5
1
~ ° LI.
© z
1
J
~
~
J
l
J
I I I I I I I
~ °
©
I
>
I
>-
I I I I
m rl~
""
~
Z
~r
< [-
_J
I
I---
I---
I I
I
0
m N z
~
Acknowledgements T h i s w o r k w a s s u p p o r t e d by N I H G r a n t s A I 2 1 3 8 4 a n d C A 3 9 5 5 9 a n d by N e w Y o r k H e a r t Association Grant-in-Aid 91017G. T h e a u t h o r s w i s h to a c k n o w l e d g e t h e e x c e l l e n t secretarial a s s i s t a n c e o f Mrs. E d w i n a L. J o n e s , Mrs. H a r r i e t t V. H a r r i s o n , Mrs. D o n n a D. James a n d Ms. G a i l D . Price, as w e l l as the e x c e l l e n t p h o t o g r a p h i c a s s i s t a n c e o f Mr. A l f r e d L a m m e , FBPA.
References
u-
1
gated for cell suspension staining, snap-freezing, and fixation. Lastly, mAb LGII-612.14 will facilitate the application of immunohistochemicai techniques to the analysis of the tissue distribution of HLA class II antigens in various disease states through retrospective studies with readily available collections of formalin-fixed, paraffin embedded tissue specimens. The resulting information will contribute to our understanding of the role of HLA class II antigens in the pathogenesis of diseases and will assess the practical value of HLA class II antigens as diagnostic and prognostic markers.
Adams, T.E., Bodmer, J.G. and Bodmer, W.F. (1983) Production and characterization of monoclonal antibodies recognizing the s-chain subunits of human Ia alloantigens. Immunology 50, 613. Barnstable, C.J., Bodmer, W.F., Brown, G., Galfr~, G., Milstein, C., Williams, A.F. and Ziegler, A. (1978) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens - New tools for genetic analysis. Cell 14, 9. Bonnet, W.M. and Laskey, R.A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83. Bourlet, Y., Behar, G., Guillemot, F., Fr~chin, N., Billault, A., Cbauss~, A.-M., Zoorob, R. and Auffray, C. (1988) Isolation of chicken major histocompatibility complex class II (B-L) /3 chain sequences: comparison with mammalian /3 chains and expression in lymphoid organs. EMBO J. 7, 1031. Brunner, C.A., Gokel, J.M., Riethmuller, G. and Johnson, J.P. (1991) Expression of HLA-D subloci DR and DQ by breast carcinomas is correlated with distinct parameters of favourable prognosis. Eur. J. Cancer 27, 411.
255 Eccles, S.J. and McMaster, W.R. (1985) DNA sequence analysis of a rat RT1 Class II At3 gene. Immunogenetics 22, 653. El Zimeity, M., Bayle, C. and Sabbour, M.S. (1990) Immunologic and morphologic typing as prognostic factor in M1 acute myeloblastic leukemia. J. Chemother. 2, 136. Epenetos, A.A., Bobrow, L.G., Adams, T.E., Collins, C.M., Isaacson, P.G. and Bodmer, W.F. (1985) A monoclonal antibody that detects HLA-D region antigen in routinely fixed, wax embedded sections of normal and neoplastic lymphoid tissues. J. Clin. Pathol. 38, 12. Esteban, F., Concha, A., Huelin, C., Perez-Ayala, M., Pedrinaci, S., Ruiz-Cabello, F. and Garrido, F. (1989) Histocompatibility antigens in primary and metastatic squamous cell carcinoma of the larynx. Int. J. Cancer 43, 436. Fraker, P.J. and Speck, Jr., J.C. (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem. Biophys. Res. Commun. 80, 849. Giacomini, P., Tecce, R., Nicotra, M.R., Cohen, B.B., Mazzilli, M.C. and Natali, P.G. (1989) mAb KUL/05 identifies a denaturation-resistant determinant shared by Class II MHC products DR, DQ and DP. J. Immunogenet. 1989, 16, 203. Hilton, D.A. and West, K.P. (1990) An evaluation of the prognostic significance of HLA-DR expression in gastric carcinoma. Cancer 66, 1154. Kaufman, J., Ferrone, S., Flajnik, M., Kilb, M., Volk, H. and Parisot, R. (1990) MHC-like molecules in some nonmammalian vertebrates can be detected by some cross-reactive monoclonal antibodies. J. Immunol. 144, 2273. K6hler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Lillie, R.D. (1965) Histopathologic Technic and Practical Histochemistry, 3rd edn. McGraw-Hill, New York. Lindmo, T., Boven, E., Cuttitta, F., Fedorko, J. and Bunn, Jr., P.A. (1984) Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J. Immunol. Methods 72, 77. Lloyd, R.V., Johnson, T.L., Blaivas, M., Sisson, J.C. and Wilson, B.S. (1985) Detection of HLA-DR antigens in paraffin-embedded thyroicl epithelial cells with a monoclonal antibody. Am. J. Pathol. 120, 106. Luna, L. (1968) Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd edn. McGrawHill, New York. Marsh, S.G.E. and Bodmer, J.G. (1991) HLA class II nucleotide sequences, 1991. Immunogenetics 33, 321. Matsui, M., Temponi, M. and Ferrone, S. (1987) Characterization of a monoclonal antibody-defined human melanoma-associated antigen susceptible to induction by immune interferon. J. Immunol. 139, 2088. Miller, T.P., Lippman, S.M., Spier, C.M., Slymen, D.J. and
Grogan, T.M. (1988) HLA-DR (Ia) immune phenotype predicts outcome for patients with diffuse large cell lymphoma. J. Clin. Invest. 82, 370. Momburg, F., Herrmann, B., Moldenhauer, G. and Moiler, P. (1987) B-cell lymphomas of high-grade malignancy frequently lack HLA-DR, -DP and -DQ antigens and associated invariant chain. Int. J. Cancer 40, 598. O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007. O'Keane, J.C., Mack, C., Lynch, E., Harrington, D. and Neiman, R.S. (1990) Prognostic correlation of HLA-DR expression in large cell lymphoma as determined by LN3 antibody staining. An Eastern Cooperative Oncology Group (ECOG) Study. Cancer 66, 1147. Orfao, A., Gonzalez, M., San Miguel, JF., Rios, A., Canizo, M.C., Hernandez, J., Maricato, M.L. and Lopez Borrasca, A. (1989) B-cell chronic lymphocytic leukaemia: prognostic value of the immunophenotype and the clinico-haematological features. Am. J. Hematol. 31, 26. Quaranta, V., Indiveri, F., Glassy, M.C., Ng, A.K., Russo, C., Molinaro, G.A., Pellegrino, M.A. and Ferrone, S. (1980a) Serological, functional, and immunochemical characterization of a monoclonal antibody (MoAb Q2/70) to human Ia-like antigens. Hum. Immunol. 1,211. Quaranta, V., Walker, L.E., Pellegrino, M.A. and Ferrone, S. (1980b) Purification of immunologically functional subsets of human Ia-like antigens on a monoclonal antibody (Q5/13) immunoadsorbent. J. Immunol. 125, 1421. Romagnani, S., Almerigogna, F., Biagiotti, R., Giudizi, M.G., Alessi, A., Damiani, G., Ricci, M. and Ferrone, S. (1985) Role of Class II histocompatibility antigens in Staphylococcus aureus protein A-induced activation of human T lymphocytes. Cell. Immunol. 90, 52. Ruggiero, G., Manzo, C., Fontana, S., Scala, G., Pirozzi, G., Ferrone, S. and Zappacosta, S. (1987) Inhibition by antiHLA class II monoclonal antibodies of monocyte-dependent T cell proliferation induced by monoclonal antibody OKT3. Eur. J. Immunol. 17, 1585. Saito, H., Maki, R.A., Clayton, L.K. and Tonegawa, S. (1983) Complete primary structures of the E/3 chain and gene of the mouse major histocompatibility complex. Proc. Natl. Acad. Sci. USA 80, 5520. Slymen, D.J., Miller, T.P., Lippman, S.M., Spier, C.M., Kerrigan, D.P., Rybski, J.A., Rangel, C.S., Richter, L.C. and Grogan, T.M. (1990) Immunobiologic factors predictive of clinical outcome in diffuse large cell lymphoma. J. Clin. Oncol. 8, 986. Solheim, B.G., Moiler, E. and Ferrone. S. (1986) HLA Class II Antigens. A Comprehensive Review of Structure and Function. Springer-Verlag, Berlin, Heidelberg, New York, Tokyo. Sorg, C., Brocker, E.B., Zwadlo, G., Redmann, K., Feige, U., Ax, W. and Feller, A.C. (1984) A monoclonal antibody to a formaldehyde-resistant epitope on the nonpolymorphic constant part of the HLA-DR antigens. Transplantation 39, 90. Spier, C.M., Grogan, T.M., Lippman, S.M., Slymen, D.J., Rybski, J.A. and Miller, T.P. (1988) The aberrancy of
256 immunophenotype and immunoglobulin status as indicators of prognosis in B cell diffuse large cell lymphoma. Am. J. Pathol. 133, 118. Spits, H., Borst, J., Giphart, M., Coligan, J., Terhorst, C. and De Vries, J.E. (1984) HLA-DC antigens can serve as recognition elements for human cytotoxic T lymphocytes. Eur. J. Immunol. 14, 299. Stefanini, M., De Martino, C. and Zamboni, L. (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216, 173. Temponi, M., Kageshita, T., Perosa, F., Ono, R., Okada, H. and Ferrone, S. (1989) Purification of murine IgG monoclonal antibodies by precipitation with caprylic acid: Comparison with other methods of purification. Hybridoma 8, 85. Temponi, M., Kekish, U., Cozzi, E. and Ferrone, S. (1993) Selective expression on /32-microglobulin (/32-/z) free HLA-B and C heavy chains of the determinant recognized by mAb TP25.99 on /32-/x associated HLA-A, -B and -C heavy chains. To be submitted for publication.
Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350. Van Duinen, S.G., Ruiter, D.J., Broecker, E.B., Van der Velde, E.A., Sorg, C., Welvaart, K. and Ferrone, S. (1988) Level of HLA antigens in locoregional metastases and clinical course of the disease in patients with melanoma. Cancer Res. 48, 1019. Watson, A.J., DeMars, R., Trowbridge, I.S. and Bach, F.H. (1983) Detection of a novel human class II HLA antigen. Nature 304, 358. Wilson, B.S., Herzig, M.A. and Lloyd, R.V. (1984) Immunoperoxidase staining for Ia-like antigens in paraffinembedded tissues from human melanoma and lung carcinoma. Am. J. Pathol. 115, 102. Zweig, S.E. and Shevach, E.M. (1983) Production and properties of monoclonal to guinea pig la antigens. Methods Enzymol. 92, 66.
239
© 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00
JIM 06688
Characterization of anti-HLA class II monoclonal antibody LGII-612.14 reacting with formalin fixed tissues Massimo T e m p o n i a,1 U l a na Kekish a Carl V. H a m b y a Hella Nielsen b Charles C. M a r b o e b and Soldano F e r r o n e a a Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA, and b Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
(Received 3 December 1992, revised received 20 January 1993, accepted 20 January 1993) m A b LGII-612.14 derived from a B A L B / c mouse immunized with interferon-y ( I F N - y ) treated cultured human B lymphoid cells LG-2 has been shown with serological and immunochemical assays to recognize a monomorphic determinant expressed on the /3 chain of H L A - D R , - D Q and -DP antigens. The linear nature of the determinant, which is likely to be formed by residues 19-25, is indicated by the reactivity of m A b LGII-612.14 with H L A - D R , - D Q and -DP /3 chains purified by electrophoresis in presence of SDS. An unusual characteristic of m A b LGII-612.14 is its reactivity with fixed tissue sections. The intensity of staining is affected by the incubation temperature, the incubation time and the fixative used. Maximal intensity of staining of formalin fixed, paraffin embedded tissue sections required an incubation time of 16 h. The intensity of staining of paraffin e m b e d d e d tissues initially fixed with Bouin's solution, formalin or ethanol was similar to that of frozen tissue sections and stronger than that of tissues fixed with B5 solution. No staining was detected of paraffin embedded tissues fixed with glutaraldehyde or Z e n k e r ' s solution. Comparison of the staining patterns with m A b LGII-612.14 of frozen and fixed tissue sections showed that the latter substrates provide a superior detail of tissue architecture and cellular morphology without significant loss of sensitivity. Furthermore, comparison of the characteristics of m A b LGII-612.14 with the few previously published anti-HLA class II mAb reacting with fixed tissues indicates that m A b LGII-612.14 stains formalin fixed, paraffin embedded tissues, while mAb 910D7 and TAL-1B5 stain tissues fixed with less commonly used fixatives. Furthermore, mAb LGII-612.14 is likely to yield more sensitive staining results than a n t i - H L A - D R , - D Q and -DP m A b K U L / 0 5 . The present results indicate that m A b LGII-612.14 represents a useful probe to apply immunohistochemical techniques to the analysis of the distribution of H L A class II antigens in fixed tissues. This will greatly facilitate the use of readily available collections of fixed tissue specimens in retrospective studies to assess the clinical significance of changes in H L A class II antigen expression which occur in various disease states. Key words." HLA class II antigen; Formalin-fixed tissue; Monoclonal antibody; Immunohistochemistry
Correspondence to: S. Ferrone, Department of Microbiology and Immunology, New York Medical College, Valhalla, NY 10595, USA. Tel.: (914) 993-4175; Fax: (914) 993-4176. 1 Visiting Investigator from the Institute of Chemistry, School of Medicine, University of Brescia, Brescia, Italy.
IFN-y, human recombinant immune interferon; mAb, monoclonal antibody; NP40, Nonidet P-40; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis.
Abbreviations:
240 Introduction
Like their counterparts in other animal species, HLA class II antigens, which include the gene products of HLA-DR, -DQ and -DP loci, consist of two non-covalently associated membrane anchored glycopolypeptides referred to as c~ and /3 chains, with the apparent molecular weight of 33,000 and 29,000, respectively. During their biosynthesis and export, a and /3 chains associate with the invariant polypeptide chain, which dissociates before H L A class II antigen expression on the cell membrane (for review, see Solheim et al., 1986). It has been known for a long time that HLA class II antigens play a key role in the regulation of the immune response by mediating and restricting interactions among immune cells (for review, see Solheim et al., 1986). More recently, H L A class II antigens have been shown to play a broader role in cell economy, since they are involved in the regulatory mechanisms which control cell proliferation (Romagnani et al., 1985; Ruggiero et al., 1987). For many years the analysis of H L A class II antigen expression has been restricted to peripheral blood cells and to cells in long term culture, since the limited specificity of conventional antiHEA class II antisera has hampered the application of immunohistochemicai techniques to analyze the tissue distribution of H L A class II antigens in normal and pathological conditions. These limitations have been overcome by the development of anti-HLA class II monoclonal antibodies (mAb). Immunohistochemical staining of tissues has shown that H L A class II antigens have a broader distribution than originally postulated, since they are expressed also by cells not directly associated with the immune system (for review, see Solheim et al., 1986). Furthermore, malignant transformation of cells may be associated with changes in the expression of H L A class II antigens (for review, see Solheim et al., 1986). These changes may have a clinical significance since expression of H L A class II antigens may aid the differentiation of follicular variants of papillary thyroid carcinomas from follicular thyroid carcinoma (Lloyd et al., 1985) and a relationship has been found between expression of H L A class lI antigens by malignant cells and clinical course of
certain diseases. The latter include breast (Brunner et al., 1991), gastric (Hilton and West, 1990) and laryngeal (Esteban et al., 1989) carcinomas, acute myeloblastic leukemia (El Zimeity et al., 1990), B cell lymphoma (Momburg et al., 1987), chronic lymphocytic leukemia (Orfao et al., 1989), diffuse large cell lymphoma (Miller et al., 1988; Spier et al., 1988; O'Keane et al., 1990; Slymen et al., 1990), and melanoma (Van Duinen et al., 1988). The routine application of immunohistochemical techniques to characterize the tissue distribution of H L A class II antigens in normal and pathological conditions has been hampered by the availability of only a limited number of mAb reacting with H L A class II antigens preserved in formalin fixed, paraffin embedded tissues. To the best of our knowledge, only two anti-HLA class II mAb with this type of reactivity have been described, mAb LK8D3 (Wilson et al., 1984) is no longer available, since a stable hybridoma could not be established (B.S. Wilson, personal communication), mAb K U L / 0 5 (Giacomini et al., 1989) which reacts with the /3 chain of HLA-DR, -DQ and -DP antigens stains formalin fixed, paraffin embedded tissue sections with a weaker intensity than frozen sections. Therefore, the use of fixed tissue sections is likely to cause a reduction of the sensitivity of immunoperoxidase staining. Furthermore, mAb 910D7 which reacts with HLADR antigens (Sorg et al., 1984), and mAb TAL1B5 which reacts with the a chain of H L A - D R antigens and possibly of H L A - D Q antigens (Adams eta[., 1983; Epenetos et al., 1985) do not stain formalin fixed, paraffin embedded tissues, although they stain tissues which had been fixed in 10% non-buffered formol saline and in acid formalin or Bouin's fixative, respectively. The latter three procedures are not routinely used for fixation of tissues. Anti-HLA class II mAb reacting with formalin fixed, paraffin embedded tissues greatly facilitate the analysis of the tissue distribution of these molecules and the assessment in retrospective studies of the clinical significance of changes in their tissue distribution. Therefore, in the present study we have characterized the specificity of mAb LGII-612.14 which stains H L A class II antigens in formalin fixed, paraffin embedded tissues,
241 we have analyzed the effect of various fixatives and incubation conditions on the reactivity of mAb LGII-612.14 with tissues and we have compared the staining patterns of mAb LGII-612.14 with those of the previously described anti-HLA class II mAb.
Materials and methods
Cells Cultured human B lymphoid cells Daudi, LG-2, LKT-13 and WALK, cultured human T lymphoid cells MOLT-4, cultured human melanoma cells Colo 38, FO-1, SK-MEL-33 and 3S5, cultured human ovarian carcinoma cells SK-OV-3, and mouse myeloma cells P3-X63-Ag8.653 were grown in RPMI 1640 medium supplemented with 10% FCS and 2 mM L-glutamine. Tissues Endomyocardial biopsies from heart allograft recipients were obtained by standard transvenous approach. Other tissues were obtained from patients who underwent surgery or at autopsy. Tissues were placed immediately in 10% neutral buffered formalin, routinely processed and paraffin embedded. Some specimens were divided into aliquots and separate portions were frozen and cryostat sectioned or fixed in 10% neutral buffered formalin, alcohol-formalin, absolute ethyl alcohol, B5 (Lillie, 1965), Bouin's solution (Luna, 1968), 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.25, Zamboni's solution (Stefanini et al., 1967) or Zenker's solution (Luna, 1968). Interferon Human recombinant immune interferon (IFN7) was obtained from Hoffman La Roche, Nutley, NJ. mAb and conventional ant&era The mAb LGII-612.14 is secreted by a hybridoma generated from a BALB/c mouse immunized with three intraperitoneal injections on days 0, 10 and 20 of 5 × 106 cultured human B lymphoid cells LG-2 which had been incubated with IFN-y (final concentration 400 U / m l ) for 72
h. 3 days following the last injection, the mouse was killed, splenocytes were isolated and hybridized with mouse myeloma cells P3-X63Ag8.653. Hybridization and subcloning were performed according to standard procedures (K6hler and Milstein, 1975). The isotype of mAb LGII612.14 was determined by testing spent medium of the hybridoma with a mouse monoclonal subisotyping kit (Hyclone Laboratories, Logan, VT) following the manufacturer's instructions. The anti-HLA-DR mAb Q2/70, the antiHLA-DQ mAb SPV-L3, the anti-HLA-DP mAb B7/21, the anti-HLA-DR, -DQ, -DP mAb Q5/13, and the anti-HLA class I mAb TP25.99 and W6/32 were developed and characterized as described elsewhere (Barnstable et al., 1978; Quaranta et al., 1980a,b; Watson et al., 1983; Spits et al., 1984; Temponi et al., 1993). mAb 910D7 and TAL-1B5 were obtained from Dr. W. Ax (Behringwerke, Marburg, Germany) and from Dr. G.J. Bodmer (Imperial Cancer Research Fund, London, England), respectively. mAb were purified from ascitic fluid by sequential precipitation with caprylic acid and ammonium sulfate (Temponi et al., 1989). mAb were labeled with ~25I utilizing the Iodo-gen method (Fraker et al., 1978). The immunoreactive fraction of radiolabeled mAb was at least 38% as determined following Lindmo et al.'s method (Lindmo et al., 1984). The Vectastain Elite ABC kit was purchased from Vector Laboratories (Burlingame, CA). Serological assays The ELISA and the binding assay with ~25Ilabeled mAb were performed in 96-well microtiter plates as described elsewhere (Matsui et al., 1987). Immunoperoxidase staining of frozen and fixed tissues was performed utilizing the Vectastain Elite ABC kit following the manufacturer's instructions. Briefly, when fixed tissue was the substrate, 4 /zm thick tissue sections were mounted on organosilane (3-aminopropyltriethoxysilane, 98%, Aldrich Chemical Company, Milwaukee, WI) coated slides and incubated for 16 h at 57°C. Then, sections were deparaffinized in xylene, rehydrated by passage through decreasing concentrations of ethanol (from 100% to 80%) and
242
washed in phosphate-buffered saline (PBS), pH 7.4. When frozen tissue was the substrate, 4 ~m thick cryostat sections were fixed in the indicated fixative for 10 min. Following one washing in PBS for 20 min, sections were incubated for 20 min at room temperature in 30% normal horse serum to block non-specific binding of mAb. Following removal of horse serum, sections were incubated with an excess of anti-HLA class II mAb LGII612.14 in a humidified chamber for 16 h at 4°C, unless otherwise indicated. Then, slides were washed for 10 min in PBS and incubated with biotinylated anti-mouse IgG xenoantibodies for 30 min at room temperature. Sections were then washed for 10 min in PBS, and incubated for 20 min at room temperature in 0.3% hydrogen peroxide in methanol to block endogenous peroxidase activity. Following washing in PBS, slides were incubated for 30 min at room temperature in avidin-biotin-peroxidase complexes. After an additional washing for 10 min in PBS, slides were incubated in 0.5 mg/ml 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) with 0.01% hydrogen peroxide in 0.05 M Tris-HCl buffer, pH 7.6. Slides were then washed for 5 min in distilled water and dehydrated in increasing concentrations of ethanol (from 80% to 100%). Then, slides were cleaned with xylene and mounted with Permount (Fisher Scientific, Fair Lawn, N J). l m m u n o c h e m i c a l assays
Radiolabeling of cells with 125I utilizing the lactoperoxidase method or with [35S]methionine (Amersham), solubilization with 1% Nonidet P-40 (NP40) (Sigma Chemical Co.), indirect immunoprecipitation, sequential immunoprecipitation, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-dimensional gel electrophoresis (non-equilibrium pH gel electrophoresis in the first dimension and SDS-PAGE in the second dimension) were performed as described (Laemmli, 1970; O'Farrel, 1975; Zweig and Shevach, 1983; Matsui et al., 1987). Gels containing 125I-labeled samples were processed for autoradiography using Kodak XAR-5 films (Eastman Kodak Co., Rochester, NY). Gels containing [35S]methionine labeled samples were processed for fluorography as described (Bonner and Laskey, 1974).
Western blotting analysis was performed as described (Towbin et al., 1979). Briefly, NP40 extracts of cultured cells were incubated with 50 /xl of Affi-Gel 10-coupled mAb for 4 h at 4°C on a rotator. Antigens were then eluted by boiling in SDS sample buffer and electrophoresed on a 10% acrylamide gel utilizing the buffer system described by Laemmli (1970). Then, proteins were transferred to a nitrocellulose filter (Bio-Rad Laboratories, Richmond, CA) as described by Towbin et al. (1979). Filters were then blocked with a solution of 30% horse serum in PBS for 1 h at room temperature and incubated with 125Ilabeled mAb LGII-612.14 (2 × 105 cpm/ml) for 4 h at room temperature. After three incubations of 20 min each, at room temperature in PBS containing 0.05% Tween 20 (Sigma Chemical Co.), filters were dried and autoradiographed utilizing Kodak XAR-5 films (Eastman Kodak Co.).
Results
mAb LGII-612.14, an IgG2b, was selected from among those produced by a B A L B / c mouse immunized with IFN-y treated cultured B lymphoid cells LG-2, since it displayed a higher reactivity with the IFN-y treated immunizing cells than with the control cells in ELISA. Testing in a binding assay with a panel of HLA typed cell lines showed that mAb LGII-612.14 displayed a selective reactivity with HLA class II antigen bearing cells. Representative results with HLA class II positive and negative cell lines are shown in Fig. 1. The reactivity of mAb LGII-612.14 with HLA class II antigens was conclusively shown by the characteristic electrophoretic profile of antigens immunoprecipitated from radiolabeled cultured B lymphoid cells LG-2 analyzed by SDSPAGE (Fig. 2). The reactivity of mAb LGII-612.14 with HLA-DR, -DQ and -DP antigens is indicated by three lines of evidence. First, immunodepletion of cultured B lymphoid cell extracts with mAb LGII-612.14 or with the anti-HLA-DR, -DQ, -DP mAb Q5/13 removed the components immunoprecipitated with either mAb. Representative results are shown in Fig. 2. Furthermore, the 2-dimensional gel electrophoretic profile of the components immunoprecipitated by mAb
243
LGII-612.14 is that characteristic of H L A - D R , -DQ and -DP antigens (Fig. 3). Lastly, Western blotting analysis showed that mAb LGII-612.14 reacts with the /3 chain of H L A - D R , -DQ and -DP antigens purified by immunoprecipitation with the a n t i - H L A - D R mAb Q 2 / 7 0 , with the anti-HLA-DQ mAb SPV-L3 and with the antiH L A - D P mAb B 7 / 2 1 from cultured B lymphoid cells. Representative results are shown in Fig. 4. Like the majority of anti-HLA class II mAb described in the literature, mAb LGII-612.14 reacts with H L A class II antigen bearing cells in frozen tissue sections (Fig. 5). Furthermore, mAb LGII-612.14 stained cells in cryostat frozen sec-
Immunoproclpltotlon with HoAb: L611-OI2.14
RBCRBC
615/13
RBC
-
4,3 I
-33Kd - Z6 i
-
121~1
Fig. 2. Structural relationship of antigens immunoprecipitated by mAb LGII-612.14 and by anti-HLA-DR, -DQ, -DP mAb Q5/13 from cultured B lymphoid cells LG-2. Cultured B lymphoid cells LG-2 were radiolabeled with 125Iutilizing the lactoperoxidase method and solubilized with NP-40. The cell extract was incubated 5 times for 4 h with either mAb LGII612.14 (lane B) or mAb Q5/13 (lane C) bound to protein A Sepharose. Each immunodepleted cell extract was then incubated for 16 h at 4°C on a rotator with insolubilized mAb LGII-612.14 and mAb Q5/13. After washing, antigens were eluted from immunoadsorbent beads by boiling with 50 pA of electrophoresis sample buffer and electrophoresed in a 10% SDS-PAGE using the Laemmli buffer system(Laemmli, 1970). After drying, gels were processed for autoradiography using Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) The anti-HLA class I mAb W6/32 (lane A) was used as a specificity control.
m x
gl
Ozdl ~II-11 d-I IT-4 II4I
}I
Nil
I
I-I4
Fig. 1. Reactivity of mAb LGI1-612.14 with cells in long term culture. Cultured B lymphoid cells Daudi, LKT-13 and LG-2, T lymphoid cells MOLT-4, melanoma cells SK-MEL-33, FO-1, Colo 38 and 3S5, and the ovarian carcinoma cells SK-OV-3 (1 X 105 cells/well) were incubated in 96 well V bottom microtiter plates (Dynatech Laboratories, Alexandria, VA) with 12SI-mAb LGII-612.14 (5 × 105 cpm/well) (open bars) for 2 h at 4°C on a rotator. Then, plates were centrifuged, supernatant aspirated and cell bound radioactivity was measured in a gamma counter. Results are expressed as bound cpm. The anti-HLA-DR, -DQ, -DP mAb Q5/13 (stippled bars) and the anti-HLA class I mAb TP25.99 (hatched bars) were used for comparison purposes.
tions which had subsequently been fixed for 10 min in acetone, alcohol-formalin, Bouin's solution, ethanol, formalin or Zamboni's solution. Unexpectedly, mAb LGII-612.14 was found to react also with H L A class II antigen bearing cells in formalin fixed, paraffin embedded tissues (Fig. 6). The intensity of staining of formalin fixed, paraffin embedded tissues was affected by the incubation time and the incubation temperature. The intensity of staining did not change when tissue sections were digested with 0.05-0.1% trypsin for 30-60 min at 37°C prior to incubation with mAb LGII-612.14. On the other hand, the intensity of staining was stronger when tissue sections were incubated with mAb LGII-612.14 for 16 h than for 1 h. Furthermore, the intensity of staining was stronger when tissue sections were
244 incubated with m A b LGII-612.14 at room temperature than at 4°C; however, the increase in intensity was associated with an increase in nonspecific staining. Therefore, the incubation at 4°C for 16 h was selected for staining of fixed tissue sections. Lastly, the intensity of staining was also markedly influenced by the fixation procedure. m A b LGII-612.14 stained strongly cells in paraffin e m b e d d e d tissues which had been fixed in alcohol-formalin, Bouin's solution, ethanol or formalin, weakly cells fixed in B5 or Z a m b o n i ' s solutions, and did not stain cells in glutaraldehyde or Zenker's solution fixed tissues. Representative results are shown in Fig. 5 which compares the immunoperoxidase staining of frozen and fixed sections of a rejected heart allograft. It is noteworthy that in the frozen section the staining of capillary endothelium is strong, but histologic details are not as sharp as in fixed sections. Furthermore, the staining of capillary endothelium and infiltrating cells in formalin fixed sec-
tions is less strong than in sections fixed with absolute ethanol or with Bouin's solution. Additional comparisons of the immunoperoxidase staining patterns of adjacent frozen and formalin fixed sections of a metastatic melanoma lesion and of a rejected renal allograft are shown in Figs. 7 and 8. In both the frozen and the formalin fixed sections of the m e l a n o m a lesion, there is strong staining of cell membranes; the tissue architecture is considerably better preserved in the formalin fixed than in the frozen section. In addition, the staining between tubules is indistinct and only rare cells are stained in the glomerulus in the frozen section of the rejected renal allograft, while infiltrating ceils and interstitial and glomerular capillaries are distinctly stained in the paraffin e m b e d d e d section. The intensity of the staining is similar in the two sections. The staining patterns obtained with mAb LGII-612.14 were then compared with those obtained with the previously described anti-HLA
m
o
DR~ DR~
I I
DP8
DO~
DPo~
"O
•
m NEPHGE
÷
Fig. 3. Two-dimensionalgel electrophoresis analysis of antigens immunoprecipitated by mAb LGII-612.14from labeled cultured B lymphoid cells LG-2. Cultured B lymphoid cells LG-2 were metabolically labeled with [35S]methionine, washed three times with Hanks' Balanced Salt Solution and solubilized with NP-40. The cell extract was then immunoprecipitated with mAb LGII-612.14. Antigens were eluted and analyzed by 2-dimensional gel electrophoresis (non-equilibrium pH gel electrophoresis (NEPHGE) in the first dimension and SDS-PAGE in the second dimension) (O'Farrell, 1975). Gels were then processed for fluorography (Bonner and Laskey, 1974).
245
class II mAb 910D7 (Sorg et al., 1984) and TAL1B5 (Adams et al., 1983; Epenetos et al., 1985). To this end, serial sections from frozen tissues or fixed, paraffin embedded tissues were stained with optimal concentrations of the three mAb. Each of the mAb stained cells in cryostat frozen sections which had been subsequently fixed in acetone, alcohol-formalin, Bouin's solution, ethanol, formalin, glutaraldehyde or Zamboni's solution (Fig. 9). At variance with mAb LGII612.14, neither mAb 910D7 nor mAb TAL-1B5 stained cells in paraffin embedded tissues which
IMMUNOPRECIPITATED
had been fixed in 10% neutral buffered formalin (Fig. 10). mAb 910D7 stained cells in tissues fixed in alcohol-formalin (Fig. 11), ethanol or Zamboni's solution, although less intensely than did mAb LGII-612.14. On the other hand, mAb 910D7 did not stain cells in tissues fixed in Bouin's solution or in glutaraldehyde, mAb TAL-1B5 stained cells in paraffin embedded tissues fixed in alcohol-formalin, Bouin's solution or ethanol with an intensity similar to that obtained with mAb LGII-612.14. Furthermore, mAb TAL-1B5 stained cells in tissues fixed in glutaraldehyde or
WITH
CELL
mAb:
EXTRACT
II
LG]]-612.14 W 6 / 3 2
tp 2s.99
LGn-612.14
Q2/70
SPV-L3
Iq
B7/21
L~ ~- 612.14 PROBED
WITH
W6/32
WALK
1
MOLT-4
I
12SI-mAb
Fig. 4. Western blot analysis of the reactivity of m A b LGII-612.14 with the/3 chains of H L A - D R , -DQ and -DP antigens. An NP40 cell extract of B lymphoid cells W A L K (2 × 106 cells/lane) was incubated with 50 txl of Affi-Gel 10-coupled a n t i - H L A - D R m A b Q 2 / 7 0 , a n t i - H L A - D Q m A b SPV-L3 and anti-HLA-DP m A b B 7 / 2 1 for 6 h at 4°C on a rotator. Antigens were eluted and electrophoresed on a 10% S D S - P A G E using the Laemmli buffer system (Laemmli, 1970). Then, proteins were transferred to a nitrocellulose filter (Towbin et al., 1979). Following a 1 h incubation in PBS supplemented with 30% horse serum to block protein binding sites, the filter was incubated for 4 h at room temperature with 125I labeled m A b LGII-612.14 ( 2 × 105 c p m / m l ) . After washing and drying, the nitrocellulose filter was autoradiographed utilizing a Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY). Specificity controls included the NP40 extract of cultured B lymphoid cells W A L K immunoprecipitated with mAb LGII-612.14 or with anti-HLA class I m A b W 6 / 3 2 and probed with 125I-labeled anti-HLA class I m A b TP25.99 and NP40 extracts of cultured B lymphoid cells W A L K and of cultured T lymphoid cells MOLT-4 resolved by S D S - P A G E in a 10% gel and probed with 125I-labeled m A b LGII-612.14.
B
LI
~w
t' ~
o
•
p
1 ¢ " ""
4
"IL w
O
PdlVq
.
8
|
f
ii
I
Fig. 5. Effect of the fixative on the staining of tissue sections by m A b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction. A rejected cardiac allograft was divided into multiple portions. One was frozen and fixed in acetone (panel A), one was fixed in absolute ethanol (panel B), one in 10% neutral buffered formalin (panel C) and one in Zamboni's solution (panel D). Tissue sections were then stained with m A b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not counterstained ( × 200).
LI
b
q
247 Zenker's solution and paraffin embedded, while m A b LGII-612.14 did not.
Discussion
Serological and immunochemical studies have shown that m A b LGII-612.14 recognizes a determinant expressed on the /3 chain of H L A - D R , - D Q and -DP antigens. The expression of the
determinant recognized by m A b LGII-612.14 on the gene products of H L A - D R , - D Q and -DP loci is indicated by the characteristic 2-dimensional gel electrophoretic profile of the molecules immunoprecipitated from cultured B lymphoid cells by mAb LGII-612.14 and by its specific reactivity with H L A - D R , - D Q and -DP antigens purified from lymphoid cells by immunoprecipitation with the appropriate specificity. The determinant is monomorphic, since m A b
Fig. 6. Immunoperoxidase staining with mAb LGII-612.14 of sections from a formalin fixed paraffin embedded tonsil. A tonsil was fixed in 10% neutral buffered formalin. Tissue sections were then stained with mA_b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not counterstained (A ×40; B × 200).
248 L G I I - 6 1 2 . 1 4 r e a c t s with cells expressing d i f f e r e n t H L A class II p h e n o t y p e s a n d with all H L A - D R allospecificities i s o l a t e d from H L A - D R h o m o z y gous cell lines as r e p o r t e d e l s e w h e r e ( K a u f m a n et al., 1990). T h e d e t e r m i n a n t r e c o g n i z e d by m A b L G I I - 6 1 2 . 1 4 has b e e n m a p p e d to t h e 1[~1 d o m a i n of H L A class II a n t i g e n s ( K a u f m a n et al., 1990) a n d is e x p r e s s e d by H L A class II /3 chains purified by e l e c t r o p h o r e s i s in the p r e s e n c e of SDS.
C o m p a r i s o n of the p u b l i s h e d a m i n o acid seq u e n c e s o f H L A - D R , - D Q a n d - D P /3 chains ( M a r s h a n d B o d m e r , 1991) a n d o f rat (Eccles and M c M a s t e r , 1985) a n d chicken ( B o u r l e t et al., 1988) M H C class II /3 chains ( T a b l e I) which also express the d e t e r m i n a n t d e f i n e d by m A b L G I I 612.14 ( u n p u b l i s h e d results) suggests that this d e t e r m i n a n t is e x p r e s s e d by t h e a m i n o acid stretch 19-25, which is s h a r e d by all t h e reactive /3
Fig. 7. Comparison of the staining patterns of a frozen section and of an adjacent formalin fixed, paraffin embedded section of a metastatic melanoma lesion with mAb LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction. One metastatic melanoma lesion was divided into two portions. One was frozen and fixed in acetone (panel A) and the other one was fixed in 10% neutral buffered formalin (panel B). Tissue sections were then stained with mAb LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not countertained ( x 250).
249
chains. This possibility is corroborated by differences in two residues in the stretch 19-25 between H L A - D R /3 chain and B A L B / c mouse MHC class II /3 chain (Saito et al., 1983) which does not react with mAb LGII-612.14. Of the two differences, the change of histidine at position 23 in B A L B / c mouse MHC class II /3 chain with arginine in H L A - D R /3 chain is likely to play the
major role in the expression of the determinant defined by mAb LGII-612.14, since the change of glutamic acid at position 22 in HLA-DR /3 chain with glutamine in B A L B / c mouse MHC class II /3 chain also occurs in HLA-DP/3 chain without affecting its reactivity with mAb LGII-612.14. mAb LGII-612.14 was elicited with an unusual immunogen, i.e., cultured human B lymphoid cells
Fig. 8. Comparison of the staining patterns of a frozen section and of an adjacent formalin fixed, paraffin embedded section of a renal allograft undergoing acute cellular rejection with m A b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction. The rejected kidney was divided into two portions. O n e was frozen and fixed in acetone (panel A) and the other one was fixed in 10% neutral buffered formalin (panel B). Tissue sections were then stained with m A b LGII-612.14 in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not counterstained (A x250; B × 200).
251
incubated with IFN-y. Whether the development of an anti-HLA class II mAb with an unusual specificity is a fortuitous event or reflects changes in the immunogenicity of HLA class II antigens because of their upregulation by IFN-y, remains to be determined. We favor the latter possibility since, in previous studies we have developed antiHLA class I antigens and anti-intercellular adhesion molecule-1 mAb with a unique specificity from mice immunized with IFN-y treated cells (Matsui et al., 1987; Temponi et al., 1993). Like HLA class II antigens, the latter two antigens are upregulated by IFN-y. If our hypothesis is correct, IFN-y treated cells may represent useful immunogens to broaden the specificity of mAb to antigenic structures whose expression is susceptible to enhancement by this cytokine. An unexpected characteristic of mAb LGII612.14 is its ability to stain cells in formalin fixed, paraffin embedded tissue sections. The intensity of staining is in most cases equivalent to that of frozen tissue sections, provided that the incubation time is prolonged to 16 h. These results suggest that use of formalin fixed, paraffin embedded tissue sections as substrates in the immunoperoxidase reaction does not affect the sensitivity of the immunohistochemical technique, provided that the appropriate incubation conditions are used. B5, glutaraldehyde and Zenker's solution which all prevent the reactivity of fixed tissues with mAb LGII-612.14 share the property to react with the sulfhydryl group of cysteine. It is of interest that cysteine is close to the amino acid stretch which is likely to carry the determinant recognized by mAb LGII-612.14. In addition, the differential effect of formaldehyde and glutaraldehyde on the reactivity of fixed tissues with mAb LGII-612.14 may reflect the more extensive and stable crosslinking caused by glutaraldehyde.
Comparison of the characteristics of mAb LGII-612.14 with those of the previously described anti-HLA class II mAb reacting with fixed tissues showed that mAb 910D7 (Sorg et al., 1984) and TAL-1B5 (Adams et al., 1983) do not stain formalin fixed-paraffin embedded tissues, but stain tissues fixed with fixatives which are not routinely used. This characteristic markedly reduces the usefulness of the two mAb to analyze the tissue distribution of HLA class II antigens in normal and pathological conditions. Furthermore, the intensity of fixed tissue sections with mAb 910D7 was lower than that with mAb LGII612.14 and TAL-1B5; the latter two mAb did not differ in their intensity of staining of alcohol/formalin and ethanol fixed tissue sections, mAb KUL/05 was not made available to us by the investigators who had described it (Giacomini et al., 1989); therefore, its staining pattern of fixed tissues could not be compared to that of mAb LGII-612.14. Since mAb KUL/05 has been reported to stain formalin fixed, paraffin embedded tissues with a weaker intensity than frozen tissues, even when the incubation time is prolonged, the sensitivity of immunoperoxidase staining of formalin fixed, paraffin embedded tissues with mAb KUL/05 is likely to be lower than with mAb LGII-612.14. The results we have described clearly indicate that the staining of fixed tissue sections provides a superior detail of tissue architecture and cellular morphology than frozen tissue sections. This improves the analysis of cellular localization of HLA class II antigens in normal tissues and malignant lesions. Furthermore, mAb LGII-612.14 allows the study of HLA class II antigen expression in diagnostic biopsies too small to be divided into separate portions for fixation or snap-freezing, and in lymphoid lesions too small to be divided into separate portions to be disaggre-
Fig. 9. Effect of fixation and paraffin embedding on the staining of tissue sections by m A b LGII-612.14 in an avidin-biotin immunoperoxidase reaction. A normal h u m a n tonsil was divided into two portions. O n e was frozen and cryostat sections cut and fixed in acetone (panel A ) or 10% buffered formalin (panel B) and one was fixed in 10% buffered formalin and e m b e d e d in paraffin (panel C). Tissue sections were then stained with m A b LGII-612.14 in an avidin biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. A lymphoid follicle is at the top of each field. Tissue sections were not counterstained ( :x: 100).
252
(A)
Fig. 10. Comparison of staining of formalin fixed, paraffin embedded tissue sections by m A b LGII-612.14, and by anti-HLA class I1 m A b 910D7 and TAL-1B5 in an avidin-biotin immunoperoxidase reaction. Normal h u m a n tonsil was fixed in 10% buffered formalin and tissue sections stained with m A b LGII-612.14 (panel A), 910D7 (panel B), or TAL-1B5 (panel C) in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. T h e tonsillar epithelium is at the left side of each panel. Tissue sections were not counterstained ( x 100).
253
Fig. 11. Comparison of staining of ethanol formalin-fixed, paraffin embedded tissue sections by mAb LGII-612.14 and by anti-HLA class II mAb 9107D and TAL-1B5 in an avidin-biotin immunoperoxidase reaction. Normal human tonsil was fixed in ethanol-formalin (70% ethanol, 0.25% formalin, 0.1 M NaCI). Tissue sections were then stained with mAb LGII-612.14 (panel A), 910D7 (panel B), or TAL-1B5 (panel C) in an avidin-biotin complex immunoperoxidase reaction, using 3,3'-diaminobenzidine as chromogen. Tissue sections were not counterstained ( x 100).
254
51,, 5
~ o
Z
~ °
I I
gLL~
g l l l
I I
I I
I -J I
.J I -J
I I I I
I I >Z
I I-I I
E W I
,,, Z
5 5 ~
~
''
~ o
5
~
~
M
~ °
<
I I
I I
Q.
a. I I I
~
©
I I I
I
~
I Z
5
I
m
I •~
''
I I Z
I I
z~
<
I
z
I
I
I I I
I I
0
Z
I I
5 1 1 u..J
0
I I I I I I
Z
<
~ °
=
Z
I
~
5
I
I
,I g
~ 5 5 Z l l
5
5
1
~ ° LI.
© z
1
J
~
~
J
l
J
I I I I I I I
~ °
©
I
>
I
>-
I I I I
m rl~
""
~
Z
~r
< [-
_J
I
I---
I---
I I
I
0
m N z
~
Acknowledgements T h i s w o r k w a s s u p p o r t e d by N I H G r a n t s A I 2 1 3 8 4 a n d C A 3 9 5 5 9 a n d by N e w Y o r k H e a r t Association Grant-in-Aid 91017G. T h e a u t h o r s w i s h to a c k n o w l e d g e t h e e x c e l l e n t secretarial a s s i s t a n c e o f Mrs. E d w i n a L. J o n e s , Mrs. H a r r i e t t V. H a r r i s o n , Mrs. D o n n a D. James a n d Ms. G a i l D . Price, as w e l l as the e x c e l l e n t p h o t o g r a p h i c a s s i s t a n c e o f Mr. A l f r e d L a m m e , FBPA.
References
u-
1
gated for cell suspension staining, snap-freezing, and fixation. Lastly, mAb LGII-612.14 will facilitate the application of immunohistochemicai techniques to the analysis of the tissue distribution of HLA class II antigens in various disease states through retrospective studies with readily available collections of formalin-fixed, paraffin embedded tissue specimens. The resulting information will contribute to our understanding of the role of HLA class II antigens in the pathogenesis of diseases and will assess the practical value of HLA class II antigens as diagnostic and prognostic markers.
Adams, T.E., Bodmer, J.G. and Bodmer, W.F. (1983) Production and characterization of monoclonal antibodies recognizing the s-chain subunits of human Ia alloantigens. Immunology 50, 613. Barnstable, C.J., Bodmer, W.F., Brown, G., Galfr~, G., Milstein, C., Williams, A.F. and Ziegler, A. (1978) Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens - New tools for genetic analysis. Cell 14, 9. Bonnet, W.M. and Laskey, R.A. (1974) A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83. Bourlet, Y., Behar, G., Guillemot, F., Fr~chin, N., Billault, A., Cbauss~, A.-M., Zoorob, R. and Auffray, C. (1988) Isolation of chicken major histocompatibility complex class II (B-L) /3 chain sequences: comparison with mammalian /3 chains and expression in lymphoid organs. EMBO J. 7, 1031. Brunner, C.A., Gokel, J.M., Riethmuller, G. and Johnson, J.P. (1991) Expression of HLA-D subloci DR and DQ by breast carcinomas is correlated with distinct parameters of favourable prognosis. Eur. J. Cancer 27, 411.
255 Eccles, S.J. and McMaster, W.R. (1985) DNA sequence analysis of a rat RT1 Class II At3 gene. Immunogenetics 22, 653. El Zimeity, M., Bayle, C. and Sabbour, M.S. (1990) Immunologic and morphologic typing as prognostic factor in M1 acute myeloblastic leukemia. J. Chemother. 2, 136. Epenetos, A.A., Bobrow, L.G., Adams, T.E., Collins, C.M., Isaacson, P.G. and Bodmer, W.F. (1985) A monoclonal antibody that detects HLA-D region antigen in routinely fixed, wax embedded sections of normal and neoplastic lymphoid tissues. J. Clin. Pathol. 38, 12. Esteban, F., Concha, A., Huelin, C., Perez-Ayala, M., Pedrinaci, S., Ruiz-Cabello, F. and Garrido, F. (1989) Histocompatibility antigens in primary and metastatic squamous cell carcinoma of the larynx. Int. J. Cancer 43, 436. Fraker, P.J. and Speck, Jr., J.C. (1978) Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril. Biochem. Biophys. Res. Commun. 80, 849. Giacomini, P., Tecce, R., Nicotra, M.R., Cohen, B.B., Mazzilli, M.C. and Natali, P.G. (1989) mAb KUL/05 identifies a denaturation-resistant determinant shared by Class II MHC products DR, DQ and DP. J. Immunogenet. 1989, 16, 203. Hilton, D.A. and West, K.P. (1990) An evaluation of the prognostic significance of HLA-DR expression in gastric carcinoma. Cancer 66, 1154. Kaufman, J., Ferrone, S., Flajnik, M., Kilb, M., Volk, H. and Parisot, R. (1990) MHC-like molecules in some nonmammalian vertebrates can be detected by some cross-reactive monoclonal antibodies. J. Immunol. 144, 2273. K6hler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680. Lillie, R.D. (1965) Histopathologic Technic and Practical Histochemistry, 3rd edn. McGraw-Hill, New York. Lindmo, T., Boven, E., Cuttitta, F., Fedorko, J. and Bunn, Jr., P.A. (1984) Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J. Immunol. Methods 72, 77. Lloyd, R.V., Johnson, T.L., Blaivas, M., Sisson, J.C. and Wilson, B.S. (1985) Detection of HLA-DR antigens in paraffin-embedded thyroicl epithelial cells with a monoclonal antibody. Am. J. Pathol. 120, 106. Luna, L. (1968) Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd edn. McGrawHill, New York. Marsh, S.G.E. and Bodmer, J.G. (1991) HLA class II nucleotide sequences, 1991. Immunogenetics 33, 321. Matsui, M., Temponi, M. and Ferrone, S. (1987) Characterization of a monoclonal antibody-defined human melanoma-associated antigen susceptible to induction by immune interferon. J. Immunol. 139, 2088. Miller, T.P., Lippman, S.M., Spier, C.M., Slymen, D.J. and
Grogan, T.M. (1988) HLA-DR (Ia) immune phenotype predicts outcome for patients with diffuse large cell lymphoma. J. Clin. Invest. 82, 370. Momburg, F., Herrmann, B., Moldenhauer, G. and Moiler, P. (1987) B-cell lymphomas of high-grade malignancy frequently lack HLA-DR, -DP and -DQ antigens and associated invariant chain. Int. J. Cancer 40, 598. O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007. O'Keane, J.C., Mack, C., Lynch, E., Harrington, D. and Neiman, R.S. (1990) Prognostic correlation of HLA-DR expression in large cell lymphoma as determined by LN3 antibody staining. An Eastern Cooperative Oncology Group (ECOG) Study. Cancer 66, 1147. Orfao, A., Gonzalez, M., San Miguel, JF., Rios, A., Canizo, M.C., Hernandez, J., Maricato, M.L. and Lopez Borrasca, A. (1989) B-cell chronic lymphocytic leukaemia: prognostic value of the immunophenotype and the clinico-haematological features. Am. J. Hematol. 31, 26. Quaranta, V., Indiveri, F., Glassy, M.C., Ng, A.K., Russo, C., Molinaro, G.A., Pellegrino, M.A. and Ferrone, S. (1980a) Serological, functional, and immunochemical characterization of a monoclonal antibody (MoAb Q2/70) to human Ia-like antigens. Hum. Immunol. 1,211. Quaranta, V., Walker, L.E., Pellegrino, M.A. and Ferrone, S. (1980b) Purification of immunologically functional subsets of human Ia-like antigens on a monoclonal antibody (Q5/13) immunoadsorbent. J. Immunol. 125, 1421. Romagnani, S., Almerigogna, F., Biagiotti, R., Giudizi, M.G., Alessi, A., Damiani, G., Ricci, M. and Ferrone, S. (1985) Role of Class II histocompatibility antigens in Staphylococcus aureus protein A-induced activation of human T lymphocytes. Cell. Immunol. 90, 52. Ruggiero, G., Manzo, C., Fontana, S., Scala, G., Pirozzi, G., Ferrone, S. and Zappacosta, S. (1987) Inhibition by antiHLA class II monoclonal antibodies of monocyte-dependent T cell proliferation induced by monoclonal antibody OKT3. Eur. J. Immunol. 17, 1585. Saito, H., Maki, R.A., Clayton, L.K. and Tonegawa, S. (1983) Complete primary structures of the E/3 chain and gene of the mouse major histocompatibility complex. Proc. Natl. Acad. Sci. USA 80, 5520. Slymen, D.J., Miller, T.P., Lippman, S.M., Spier, C.M., Kerrigan, D.P., Rybski, J.A., Rangel, C.S., Richter, L.C. and Grogan, T.M. (1990) Immunobiologic factors predictive of clinical outcome in diffuse large cell lymphoma. J. Clin. Oncol. 8, 986. Solheim, B.G., Moiler, E. and Ferrone. S. (1986) HLA Class II Antigens. A Comprehensive Review of Structure and Function. Springer-Verlag, Berlin, Heidelberg, New York, Tokyo. Sorg, C., Brocker, E.B., Zwadlo, G., Redmann, K., Feige, U., Ax, W. and Feller, A.C. (1984) A monoclonal antibody to a formaldehyde-resistant epitope on the nonpolymorphic constant part of the HLA-DR antigens. Transplantation 39, 90. Spier, C.M., Grogan, T.M., Lippman, S.M., Slymen, D.J., Rybski, J.A. and Miller, T.P. (1988) The aberrancy of
256 immunophenotype and immunoglobulin status as indicators of prognosis in B cell diffuse large cell lymphoma. Am. J. Pathol. 133, 118. Spits, H., Borst, J., Giphart, M., Coligan, J., Terhorst, C. and De Vries, J.E. (1984) HLA-DC antigens can serve as recognition elements for human cytotoxic T lymphocytes. Eur. J. Immunol. 14, 299. Stefanini, M., De Martino, C. and Zamboni, L. (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216, 173. Temponi, M., Kageshita, T., Perosa, F., Ono, R., Okada, H. and Ferrone, S. (1989) Purification of murine IgG monoclonal antibodies by precipitation with caprylic acid: Comparison with other methods of purification. Hybridoma 8, 85. Temponi, M., Kekish, U., Cozzi, E. and Ferrone, S. (1993) Selective expression on /32-microglobulin (/32-/z) free HLA-B and C heavy chains of the determinant recognized by mAb TP25.99 on /32-/x associated HLA-A, -B and -C heavy chains. To be submitted for publication.
Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350. Van Duinen, S.G., Ruiter, D.J., Broecker, E.B., Van der Velde, E.A., Sorg, C., Welvaart, K. and Ferrone, S. (1988) Level of HLA antigens in locoregional metastases and clinical course of the disease in patients with melanoma. Cancer Res. 48, 1019. Watson, A.J., DeMars, R., Trowbridge, I.S. and Bach, F.H. (1983) Detection of a novel human class II HLA antigen. Nature 304, 358. Wilson, B.S., Herzig, M.A. and Lloyd, R.V. (1984) Immunoperoxidase staining for Ia-like antigens in paraffinembedded tissues from human melanoma and lung carcinoma. Am. J. Pathol. 115, 102. Zweig, S.E. and Shevach, E.M. (1983) Production and properties of monoclonal to guinea pig la antigens. Methods Enzymol. 92, 66.