Eur. J. Immunol. 1992. 22: 679-687 y/S T cell receptors expressed on TIL 679

y/6 Tcell antigen receptors expressed on tumor-infiltrating lymphocytes from patients with solid tumors*

Masanobu Nanno, Hidetoshi Seki, George Mathioudakis, Ryuzi Suzuki, Kyogo Itoh, Constantin G. Ioannides, Satsuki Suzuki, Pei-Feng Chen and Chris D. Platsoucas

Department of Immunology, The University of Texas, M. D. Anderson Cancer Center, Houston

The expression of y/S Tcell antigen receptors (TcR) in Tcell lines or clones derived from tumor-infiltrating lymphocytes (TIL) from patients with solid tumors was investigated. y/S TcR T cell lines were derived fromTIL from patients with Wilms tumor, sarcoma or metastatic melanoma by stimulation with autologous tumor cells alone and recombinant interleukin 2 and they exhibited nonspecific cytotoxicity against autologous and allogeneic tumor cells, or cells of the K562 or the MEL21 tumor cell lines. Two Tcell lines were derived from a patient with Wilms tumor. One of them expressed a non-disulfide-linked y/S TcR using the 60-kDa y chaiqwhereas, the other expressed a disulfide-linked y/S TcR. A Tcell line was derived from a patient with sarcoma and expressed a disulfide-linked y/S TcR, whereas, a Tcell line derived from a patient with melanoma expressed a non-disulfide-linked y chain of 62 kDa. Several T cell clones were developed from patients with metastatic melanoma or Wilms tumor and expressed either disulfide- or non-disulfide-linked y/S TcR. Northern analysis of RNA from certain of these clones revealed a full-length y chain transcript, whereas, the u or 6 chain transcripts were either absent or truncated.TheseT cell clones exhibited nonspecific cytotoxicity. Both disulfide- and non-disulfide- linked TILTcell lines and clones expressed the S TCSl determinant. y/S TcR+ cells in freshly prepared TIL from these patients were present in low proportions ( < 5 % ) and their STCSl/Sl ratios were within the range observed in the peripheral blood of normal donors. These results demonstrate that both disulfide- and non-disulfide-linked y/S TcR are expressed on T cell lines and clones derived from TIL from solid tumors. Non-disulfide-linked y/S TcR using the 56-66-kDa y chain are frequently found on TIL-derived Tcell lines and clones. These 56-66-kDa y chains are rarely expressed on Tcell lines or clones derived from peripheral blood lymphocytes of normal donors.

1 Introduction

The presence of leukocytic infiltrates in human and animal tumors was first reported by Ehrlich over 100 years ago 111. Tumor-infiltrating lymphocytes (TIL) may represent an active immune response of the host specifically directed to the tumor [2] and their presence in high numbers has been associated in certain tumors with more favorable prognosis 13, 41. Recently, TIL expanded in IL 2 were demonstrated to exhibit potent antitumor activity in vivo in murine tumor models of advanced metastatic disease, when administered with high doses of cyclophosphamide and I L 2 [5 , 61. In these murine systems,TIL were shown to be, on a per cell basis, 50 to 100 times more potent than LAK cells in rejecting established pulmonary metastasis [5].

TIL have also been isolated and expanded in IL 2 f rom a large number of human tumors. We have developed u/@

[I 97381

* Supported in part by grants CA52308, AK41003 and A124669 from the National Institutes of Health.

Correspondence: Chris D. Platsoucas, Department of Immunolo- gy, Box 178, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030, USA

Abbreviation: TIL: Tumor-infiltrating lymphocytes

TcR-expressing Tcell lines and clones from TIL from certain patients with metastatic melanoma 171 or ovarian carcinoma [S, 91 exhibiting autologous tumor-specific activ- ity. These T cell lines and clones exhibited primarily specific cytotoxicity restricted to autologous tumor targets (either melanoma or ovarian) and did not lyse allogeneic tumor cells, normal autologous cells or the K562 targets [7-91. Certain TIL-derived T cell lines exhibited specific prolifer- ative responses and IL 2 production to autologous, but not to allogeneic tumor cells [7]. These cytotoxic Tcells have been propagated in rIL 2 in vitro for a long period of time and have been expanded in numbers by 500-2000-fold, without losing their specific cytotoxic activity [7-91. We demonstrated that the u/p TcR and the CD3 antigen on the effector cells are involved in the specific cytotoxicity and in the proliferation of TIL in response to autologous tumor cells and/or IL 2 [7-91. TIL-derived T cell lines f r o m certain patients with melanoma with autologous tumor-specific cytotoxicity have been also reported by others [lo-121. IL 2-expanded TIL from patients with melanoma have demonstrated antitumor activity in vivo 113, 141 and appear to be localized preferentially to the tumor site [15, 161. In contrast to the generation of TIL-derived Tcell lines and clones with autologous tumor-specific activity in patients with melanoma [7, 10-121 and ovarian carcinoma [8, 91, Tcell lines and clones with only nonspecific lytic activity have been derived so far from other tumors, including renal cell carcinomas [7, 171, sarcomas [7, 181, breast, colon and brain tumors [19], lung carcinomas [20], and others [21].

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992 001 4-2980/92/0303-067Y$3.50 + ,2510

680

Recently, a second TcR has been identified on certain populations of human T cells [22-341. We investigated the presence of cells expressing y/S TcR in rIL2-expanded T cell lines and clones derived from TIL from a number of tumor specimens. We observed that certain T cell lines expressing y/6 TcR were derived from TIL from patients with Wilms tumor, sarcoma or metastatic melanoma by stimulation with autologous tumor cells alone and rIL 2 and they exhibited unrestricted cytotoxic activity against auto- logous tumor cells, allogeneic tumor cells and K562 targets. TIL-derived y/6 TcR-expressing T cell lines and clones had either disulfide- or non-disulfide-linked TcR. Non-disul- fide-linked y/6 TcR using the 56-66-kDa y chain were commonly found in these Tcell lines and clones, although they are rarely used in y/6 TcR expressed on T cells derived from the peripheral blood of normal donors.

M. Nanno, H. Seki, G. Mathioudakis et al. Eur. J. Irnmunol. 1992. 22: 679-687

80%. Lymphocytes were distinguished from the tumor cells in these suspensions as described [7].

2 Materials and methods

2.1 mAb

The following mAb were used: anti-Leu-4, PE-conjugated anti-Leu-4, FITC-conjugated WT31 and FITC-conjugated anti-Leu-llb (Becton Dickinson, Sunnyvale, CA); OKT3, OKT4 and OKT8 mAb (Ortho Diagnostics, Raritan, NJ); PF1 (anti-TcR fi chain specific) [35], anti-GTCS1 [36], anti-TcRG1[37] and their FITC conjugates (T Cell Sciences, Cambridge, MA). Cells stained with the anti-GTCS1 mAb were designated in this study GTCSl+. Cells stained with the anti-TcRS1 mAb were designated 61+. A ychain- specific mAb designated 3D5 (IgG1,x) was developed in this laboratory by immunizing mice with a hybrid protein containing a large part of the CI exon of the human C,2 gene segment [38]. A second y chain-specific mAb, desig- nated 907 (IgM,x) was used in the experiment shown in Fig. 6 and it was developed in this laboratory [25] by immunizing mice with a y chain-specific peptide (P13K) selected from the deduced amino acid sequence of the C, of the murine TcR (amino acids 118-130). This C, peptide sequence is highly homologous to the corresponding human C, sequence [25]. The 3D5 and 9D7 mAb recognize both the C,l and C,2 gene products and specifically immunoprecipitate from denatured lysates all three forms of ychain polypeptides [25, 28, 29, 381. They did not immunoprecipitate the alp TcR. An anti-mouse Thy- 1.2 mAb (IgM) was obtained from the American Tissue Culture Collection (Rockville, MD).

2.2 Preparation of cell suspensions

Surgical specimen from 1 patient with Wilms tumor, 5 patients with sarcoma and 26 patients with metastatic melanoma were used in this study. Specimen used for the preparation of cells contained only tumor tissue as deter- mined by macroscopic examination. Normal tissue sur- rounding the tumor or necrotic areas were carefully removed before preparation of the cells. The diagnosis of Wilms tumor or sarcoma or melanoma was confirmed in the tumor specimen by pathology examination performed by the Department of Pathology of the M. D. Anderson Cancer Center. Single-cell suspensions were prepared by treatment for 40 min with collagenase, hyaluronidase and DNase as described 17) and exhibited a viability exceeding

2.3 Development of Tcell lines

Mixtures of TIL and tumor cells were washed twice, resuspended at a concentration of 2 x 105-5 x lo5 celldm1 in RPMI 1640 or AIM V (Gibco, Grand Island, NY), 100 U/ml of highly purified human rIL 2 (Cetus, Emery- ville, CA) [39] and 10% heat-inactivated FCS (Gibco) and cultured in 24-well tissue culture plates (Corning Glass, Corning, NY) at 37°C in a humidified incubator with 5% c02.

2.4 Development of Tcell clones

To develop TIL Tcell clones, mixtures of TIL and tumor cells were cultured under limiting dilution conditions (1,3, 10, or 30 cells/well) in U-bottom microtiter plates in AIM V supplemented with 10% FCS, 100 U/ml of rIL2 and 10 pg/ml PHA (Gibco). T cell clones from normal donors were developed in the same media. A mixture of irradiated (5000 rad) allogeneic PBMC (100 000 cells/well) from two or three normal donors (feeder cells) were added to each well before the addition of theTIL or of normal T cells [28]. Irradiated feeder PBMC were added to the cultures every 10 days. Clones derived from these cultures were pheno- typed and CD3+WT31-81f cells were expanded in 24-well tissue culture plates in AIM V with 10% FCS and 100 U/ml of rIL2.

2.5 Immunofluorescence

This was carried out as described [40] using an Epics Profile Analyzer (Coulter Corp.). FITC-conjugated F(ab'h frag- ments of goat anti-mouse IgG were purchased from Cooper Biomedical Inc. (Malvern, PA).

2.6 Immunoprecipitation

Cells 1 x 107-2 x lo7 were labeled with NalZ5I (sp. act. 14.8 mCi/yg of iodine; Amersham Corp., Arlington Heights, IL) by the lacto-peroxidase method. The cells were lysed in 0.01 M Tris-HC1 (pH 8.0)-buffered 0.15 M NaCl supple- mented with 20 kallikrein-inhibiting unitdm1 of aprotinin, 2 mg/ml ovomucoid trypsin inhibitor, 1 mM PMSF, 0.01% NaN3, and either 5 mM Chaps or 1% NP40 (all from Sigma, St. Louis, MO). Detergent lysates were centrifuged for 30 min at 16000 x g to remove insoluble materials, and were precleared by two incubations with 10 mg of Pansor- bin (Calbiochem Behring Diagnostics, San Diego, CA) coated with normal mouse serum and by a subsequent incubation with 10 mg of Pansorbin coated with normal mouse IgG (Cooper). Specific immunoprecipitation was carried out with Pansorbin coated with 2-5 yg of specific mAb. For immunoprecipitation with the TcR y chain- specific mAb 3D5 or 9D7 (experiment of Fig. 6 only), the lysates were denatured by heating at 56 "C for 30 min in the presence of 1% SDS, diluted by adding 4 volumes of 1.5% NP40 in 0.01 M Tris-HC1 (pH 7.4) containing 0.15 M NaCl and alkylated with 20 mM of iodoacetamide for 20 min at

Eur. J. Immunol. 1992. 22: 679-687

room temperature [25]. lmmunoprecipitates were washed seven times with 0.01 M Tris-HC1 (pH 8.0) containing 0.5 M NaCl, solubilized in SDS sample buffer with 2-ME (reduc- ing conditions; R) or without (nonreducing conditions; NR) and analyzed by SDS-PAGE using 10% (NR) or 12.5% (R) gels. Gels were dried and visualized by autora- diography at - 70°C on Kodak XAR-5 film using inten- sifying screens.

w1

y/6 Tcell receptors expressed on TIL 681

Table 1. y/b TcR+ cells in TIL from patients with solid tumors

NT

2.7 Northern blotting

Northern blotting was carried out by standard methods [41, 421, The human TcR u chain-specific pY 1.4 [41] and the humanTcR fi chain-specific Jur-fi2 [42] cDNA probes were provided by Dr. T. W. Mak (Ontario Cancer Institute, Toronto). The human TcR ychain pTy1 probe [43] was provided by Drs.T. Quertermous and J. G. Seidman (Har- vard Medical School, Boston).

2.8 Cell-mediated cytotoxicity

Cell-mediated cytotoxicity was determined by a 51Cr- release assay [44].

2.9 Amplification, cloning and sequencing of y chain TcR cDNA

The type of the y/6 TcR expressed on six Tcell clones derived from the peripheral blood of normal donors was identified by amplification of the y chain cDNA using polymerase chain reaction (PCR; [45]), followed by cloning and sequencing of the amplified cDNA. Total RNA was isolated from theTcell clones as described [46] and cDNA was synthesized using a Cy-specific primer according to the method of Gubler and Hoffmann [47]. The cDNA was then amplified by PCR, using 5' end amplification primers specific for V-1,V-I1,V-111, and V-IV y chain subgroups and a 3' end C, primer (Mathioudakis, Chen, Li, Chernajovsky and Platsoucas, manuscript in preparation). The PCR product was purified by centrifugation using Centricon-100 filtering units (Amicon, Danvers, MA), and cloned into pUC18 vector. The plasmids were used to transform DHSa competent cells and white bacterial colonies were screened with a C,probe (5' end 32P-labeled) located 5' to the C, amplification primer. Positive clones were sequenced using the SequenaseV2.0 system (U.S. Biochemicals, Cleveland, OH). All recombinant DNA procedures were carried out as described [47]. V, D, J gene segments were identified by comparison to those in the Genbank Data Base.

3 Results

3.1 y/6 TcR+ TIL in patients with solid tumors

Patient Lymphocyte/ CD3+ GTCSl+ 61+ GTCSlI tumor ratio (%) (%) (%) 81 ratio

Wilms 0.40 ND 0.2 0.7 0.28 tumor Sarcoma S6 0.35 35 0.7 2.5 0.28 Melanoma M40 0.30 70 0.3 2.0 0.15 TXMSO ND ND 1.2 3.1 0.38 M73 ND 78 0.9 3.1 0.29

3.2 y/6 TcR TIL Tcell lines and clones from Wilms tumor

Two T cell lines, designated W1 and W2 were developed by stimulating TIL from a patient with Wilms tumor with autologous tumor cells alone and rIL 2. Both these Tcell lines expressed the y/6 TcR. The W1 line (CD3+CD4-CD8+WT31-6TCSl+) (Fig. 1) was pheno- typically homogeneous (CD2: 99%; CD3: 95%; CD4: 0%;

Leu-19: 95%; and CD16: 2%). The W2 line was pheno- typically heterogeneous and contained approximately equal proportions of cells expressing either alp TcR or y/6

38%) (Fig. 1). Two-color immunofluorescence analysis revealed that the W2 cell line was a mixture of CD4-CD8high WT31f (54%). CD4-CD8d"1161+ (14%), and CD4-CD8-61+ (24%), populations (data not shown), demonstrating that the 61 population contained CD8+ and CD8- cells. The W1 and W2 Tcell lines were the only lines (y/6+ or u/fi+) developed from TIL from this patient with Wilms tumor. Also, twoT cell clones expressing the y/6 TcR were developed from TIL from this patient by limiting dilution, the W3-1 (CD3+CD4-CD8-WT31-6TCS1+61+) and the W3-7 (CD3+CD4-CD8-WT31-6TCS1-61+) (Fig. 1). Immunoprecipitation of W1 lysates with the anti-6TCS1 mAb followed by SDS-PAGE analysis under

CD8: 94%; WT31: 2%; 6TCS1: 97%; HLA-DR: 92%;

TcR (CD3: 94%; CD4: 2%; CD8: 69%; WT31: 45%; 61:

Control CD3 CD4 CD8 WT31 bTCS1 TCR61

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The proportions of 61+ and 6TCSl+ cells in freshly prepared TIL from patients with solid tumors that were subject to this study are shown in Table 1. These cells were present in low proportions (< SYO) in TIL. Their 6TCS1/61 ratios were in the range of 0.15 to 0.38.

Fluorescence Intensity

Figure 1. Immunofluorescence analysis of the W1, W2 and S6 y /6 Tcell lines and of the W3-1 and W3-7 T cell clones was carried out using an Epics Profile Analyzer. Mouse IgG was used as control anti body.

682

either nonreducing or reducing conditions revealed two polypeptide chains of 40 kDa and 57-60 kDa suggesting that these cells expressed a non-disulfide-linked y/G TcR employing the 55-60-kDa y chain constant region gene segment (Fig. 2). The broad band of the y chain in Fig. 2 is probably due to the presence of two distinct y chain polypeptides with molecular masses in the range of 55-60 kDa that may utilize different V, polypeptides. These polypeptides are expressed on different cells of the W1 cell 1ine.We have previously observed [25] the expression of two different y chain polypeptides on different cells of a single IL 2-dependent Tcell line. The anti-GTCS1 mAb did not immunoprecipitate any polypeptide chains from the con- trol TILT cell line designated TXMSO (CD3+CD4-CD8+- WT31fGl-) derived from a patient with metastatic mela- noma. The y chain polypeptides expressed on the W1 cells are similar to the one that we have previously observed [25] onT cell lines derived from the peripheral blood of patients with primary immunodeficiency disorders. These 55-60- kDa y chain polypeptides are rarely found onT cells derived from the peripheral blood from normal donors (see bel-

M. Nanno, H. Seki, G. Mathioudakis et al.

ow).

In contrast, the W2 T cell line exhibited a disulfide-linked y/G heterodimer (Fig. 3). Immunoprecipitation of dena- tured nonreduced W2 lysates with the y chain-specific 3D5 mAb followed by SDS-PAGE analysis under nonreducing conditions revealed an 80-kDa band which was resolved under reducing conditions into a broad band in the range of 39-42 kDa. Immunoprecipitation by the anti-Leud mAb followed by SDS-PAGE analysis under reducing conditions detected two polypeptides of 43 kDa and 40 kDa (Fig. 3). Immunoprecipitation of W2 cell lysates with the anti-TcRG1 mab followed by SDS-PAGE analysis under reducing conditions revealed a broad band in the range of 37-42 kDa (Fig. 3).These results demonstrate that cells of the W2 cell line express a disulfide-linked y /6 TcR.

Eur. J. Immunol. 1992. 22: 679-687

Biochemical analysis of the two T cell clones derived from the same patient with Wilms tumor is shown in Fig. 4. Immunoprecipitation of denatured nonreduced lysates of the W3-1 Tcell clone by the y chain-specific 3D5 mAb followed by SDS-PAGE analysis under either reducing or nonreducing conditions, revealed that this clone expressed

8.. k D 2 f

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94-

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43-

30-

w2 kD

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-67

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Figure 3. Cells of the W2 T cell line were labeled with 1251 and lyscd in S mM Chaps (lanes 1-2; reducing) or 1% NP40 lysis buffer (lanes 1 and 2; nonreducing; 3, 4, 5 and 6 reducing). Lysates were immunoprecipitated with mouse IgG (1 , nonreducing; 1, 3, S reducing); anti-y chain 3DS mAb (from denatured nonreduced lysates; lanes 2, NR; 4, R), anti-Leu-4 mAb (lane 2, R) or anti-TcRG1 mAb (lane 6, R). Samples were solubilized in SDS sample buffer with 2-ME (R) or without (NR) and electrophoresed on 10% (NR) or 12.5% (R) SDS-PAGE gels.

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Figure 2. Cells of the W1 (CD3+WT31VGTCSl+) (lanes 3 and 4) and TXMSO (CD3+WT31+6TCS1-61-) (lanes 1 and 2) Tcell lines were labeled with 1251 and lysed in 1% NP40 lysis buffer. Lysates were immunoprecipitated with mouse IgG (lanes 1 and 3) or anti-GTCS1 mAb (lanes 2 and 4), solubilized in SDS sample buffer with f3-mercaptoethanol (reducing conditions; R) or without (nonreducing conditions: NR) and subjected to electrophoresis on 10% (NR) or 12.5% (R) SDS-PAGE gels.

20.1 - -20.1

1 2 3 4 5 6 1 2 3 4 5 6

INRI - R l Figure 4. Cells of the W3-1 (lanes 1-2) and W3-7 (lanes 3-6) Tcell clones were labeled with 1251 and lysed in 1% NP40 (lanes 1 , 2, 5 and 6) or S mM Chaps lysis buffer (lanes 3 and 4). The lysates were immunoprecipitated with mouse IgG (1,3 and 5 ) , anti-y chain 3DS mAb (from denatured NR lysates; lanes 2 and 6) or anti-Leu-4 mAb (lane 4). Samples were solubilized in SDS sample buffer with 2-ME (R) or without (NR) and electrophoresed on 10% (NR) or 12.5% (R) SDS-PAGE gels.

Eur. J. Immunol. 1992. 22: 679-687 y/S Tcell receptors expressed on TIL 683

a non-disulfide-linked $6 TcR employing a 66-kDa y chain polypeptide. In contrast, the W3-7 Tcell clone exhibited a disulfide-linked $6 TcR of 82-kDa, which was resolved in reducing SDS-PAGE into two polypeptides in the range of 38-41 kDa (Fig. 4). Additional Tcell lines and clones to those described above were not obtained from this patient with Wilms tumor.

kD S6 kD

94- -94

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LNRJ LRJ Figure 5 . Cells of the S6 Tcell line were labeled with lZSI and lysed in 1% NP40 lysis buffer.The nonreduced lysates were denatured as described in Sect. 2.6 and immunoprecipitated with mouse IgG (lane 1) or anti-y chain 3D5 mAb (lane 2). Samples were solubi- lized in SDS sample buffer with 2-ME (R) or without (NR) and were electrophoresed on 10% (NR) or 12.5% (R) SDS-PAGE gels.

- 97

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Figure 6. Cells from a melanoma TILTcell line were labeled with lZSI and lysed in 1% NP40 lysis buffer.The nonreduced lysates were denatured as described in Sect. 2.6 and immunoprecipitated with the y chain-specific anti-peptide 9D7 mAb (IgM) (lane 1) or with control antibody anti-mouseThy-1.2 mAb (IgM) (lane 2). Samples were solubilized in SDS sample buffer without 2-ME (NR) and electrophoresed on 10% SDS-PAGE gels.

3.3 y/6 TcR TIL Tcell line from sarcoma

A CD3+CD4-CDS-WT31-6TCS1+61+ T cell line, desig- nated S6, was developed from TIL of a patient with sarcoma, by stimulating TIL with autologous tumor cells alone and rIL 2. The phenotype of these cells was (CD3: 98%; CD4: 2%; CD8: 7%; 6TCS1: 51%; 61: 86%) (Fig. 1). These cells expressed a disulfide-linked y/6 TcR. Immuno- precipitation of denatured nonreduced S6 lysates by the y chain-specific 3D5 mAb followed by SDS-PAGE analysis under nonreducing conditions revealed a band of 82-kDa that was resolved into a broad band of 40-44 kDa under reducing conditions (Fig. 5) . This T cell line was the only one (y/6+ or a/p+) developed from this patient with sarcoma. Stimulation of TIL from four other patients with sarcoma with autologous tumor cells and rIL 2 resulted in the development of CD3+a/B-TcR+ or CD3-CD16+ lines which exhibited nonspecific cytotoxicity (data not shown).

3.4 y/6 TcR TIL Tcell lines and clones from malignant melanoma

A T cell line comprised of approximately 50% CD3+WT31- and 50% CD3+WT31f cells was developed by stimulating TIL from a patient with metastatic melano- ma with autologous tumor cells and rIL 2. This Tcell line was the only line (y/6+ or a/B+) developed from TIL from this patient with malignant melanoma. Immunoprecipita- tion of denatured nonreduced lysates from this line by the 9D7 y chain-specific mAb, followed by SDS-PAGE analysis under non-reducing conditions detected a y chain polypep- tide of 62 kDa, suggesting that this line expressed a non-disulfide-linked y/6 TcR (Fig. 6).

Two T cell clones (M40-4 and M40-5), expressing y/6 TcR, were established from the TIL from a second patient with metastatic melanoma. Both clones were phenotypically homogeneous and exhibited the CD3+CD4-CDS-WT31-- GTCSl+ phenotype (Fig. 7). Biochemical analysis revealed that both these clones expressed a disulfide-linked hetero-

M40-4 M40-5 M40-8 I I I

L - - L - - u Control

6TCS WT31 1 k Fluamcmce h t m i t y (Log)

Figure 7. Immunofluorescence analysis of the M40-4, M40-5 and M40-8 TIL Tcell clones. Mouse IgG was used as control anti- body.

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M. Nanno, H. Seki, G. Mathioudakis et al.

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Figure 8. Cells of the M40-4 T cell clone were labeled with Iz5I and lysed in 5 mM Chaps (lanes 1 and 2) or 1% NP40 lysis buffer (lanes 3-6). The lysates were immunoprecipitated with mouse IgG (lanes 1 , 3 and 5), anti-Leu-4 mAb (lane 2), anti-y chain 3D5 mAb (from denatured nonreduced lysates; lane 4) or anti-GTCS1 mAb (lane 6). Samples were solubilized in SDS sample buffer with 2-ME (R) or without (NR) and were electrophoresed on 10% (NR) or 12.5% (R) SDS-PAGE gels.

dimer. Results from the M40-4 clone are shown in Fig. 8. Immunoprecipitation experiments using the anti-Leu-4, the anti-y chain 3D5 and the anti-TCR 61 mAb revealed a disulfide-linked y/6 TcR of 80-kDa, which was resolved under reducing conditions to two polypeptide chains of 37 kDa and 40 kDa (Fig. 8).

Northern blotting analysis demonstrated that the M40-4 T cell clone expressed no c1 chain mRNA, very low level of truncated chain mRNA, and full-length y chain transcript (Fig. 9). Stimulation of TIL from the remaining 25 patients with melanoma with autologous tumor cells and rIL 2 resulted in the development of CD3+a/PTcR+y/6TcR- T cell lines and clones, exhibiting autologous tumor specific cytotoxicity [7, 211.

3.5 Cytolytic activity of y/6 TIL T cell lines/clones

y/6 TcR TIL T cell lines/clones exhibited nonspecific cyto- lytic activity against autologous or allogeneic tumor cells, and K562 and MEL21 (melanoma) targets (Table 2).

a

P

Eur. J. Immunol. 1992. 22: 679-687

-1.6kb

- 1.3kb

-1.7kb Y

4

Figure 9. Northern blotting analysis of RNA isolated from the M40-4 TIL Tcell clone, the PEER, HPB-ALL, and EBV- transformed B cell line (EBV-B). a-, fi-, and y-chain specific cDNA probes are described in Sect. 2.7. Equal amounts of mRNA were added in each lane.

3.6 y/6 TcR expressed on T cell clones from normal donors

We have studied the structure of the y/6 TcR on 16 CD3+CD4-CD8-WT31-61f T cell clones developed from the peripheral blood of eight different normal donors. Fourteen of sixteen of these T cell clones expressed disulfide-linked y/6 TcR. The structure of the y/6 TcR in 10 of these Tcell clones was investigated by immuno- precipitation, using the 3D5 anti-y chain mAb or the anti41 mAb and/or the anti-Leu-4 mAb, followed by SDS-PAGE analysis. This analysis revealed that eight of these T cell clones expressed disulfide-linked y/6 TcR and two non-disulfide-linked y/6 TcR. One of these two clones (CD3+WT3l-CD4-CD8-6TCSl-61+) utilized the C,2abc constant region gene segment (60-kDa y chain polypeptide), whereas the other (CD3+WT31-- CD4-CD8-6TCSlf61+) the C,2bc (37-kDa y chain). The structure of the $6 TcR of the remaining six Tcell clones

Table 2. Cytotoxic activity of y16 TcR TIL Tcell lines and clones

Patient TIL E :T Percent JICr release ratio

Autologous Allogeneic K562 MEL21 tumor tumor

Wilms tumor W1 cell line 10: 1 15.0 16.2 33.4 11.5 W3-1 clone 20: 1 19.3 ND 49.0 2.4 W3-7 clone 20 : 1 21.3 ND 74.6 9.8

Sarcoma S6 cell line 20: 1 ND 76.0 45.0 Melanoma M40-4 clone 20 : 1 17.2 20.0 79.1 28.5

M40-5 clone 20: 1 12.5 2.3 13.7 3.5

Eur. J. Immunol. 1992. 22; 679-687 685

Table 3. Comparison of y / 6 TcR types expressed onTcell lines/clones derived fromTIL from patients with solid tumors to those derived from peripheral blood lymphocytes from normal donors

y/S T cell receptors expressed on TIL

Peripheral blood-derived TIL-derived

Disulfide-linked Non-disulfide-linked q 2 a b c q 2 b c

14/16 (87.5%) 1/16 ( 6.25%) 1/16 (6.25%) 5/ 8 (62.5%) 3/ 8 (37.5 %) 0

was studied by amplification of the y chain cDNA by PCR, followed by cloning and sequencing of the amplified cDNA. All these six Tcell clones expressed aV,9.J,1.2.Cy1 y chain and therefore exhibited disulfide-linked y/6 TcR. Six of fourteen T cell clones that exhibited disulfide-linked y/6 TcR were 6TCSlf (42.8%). These results demonstrate that 87.5% (14 of 16) of Tcell clones derived from peripheral blood lymphocytes of normal donors expressed disulfide- linked y/6 TcR. In contrast, 62.5% (5 of 8) TIL derived T cell lines/clones exhibited disulfide-linked y/6 TcR. Fur- thermore, only 1 of 16 (6.25%) Tcell clones from the peripheral blood of normal donors expressed y/b TcR utilizing the C,2abc gene segment, whereas, 3 of 8 (37.5%) TIL-derived T cell linedclones expressed y/6 TcR utilizing the Cy2abc gene segment. These results are summarized in Table 3.These results demonstrate significant differences in utilization of the C, TcR polypeptides in T cell lines/clones derived fromTIL from patients with solid tumors vs. those derived from the peripheral blood.

4 Discussion

y/6 TcR cells were present in low proportions (< 5 % ) in TIL from certain solid tumors and the 6TCS1/61 ratios were within the range found in the peripheral blood of normal donors [36,48-511. Tcell lines expressing the y/6 TcR were developed from TIL from certain patients with Wilms tumor, sarcoma, and malignant melanoma in response to autologous tumor cells alone and rIL 2. Additionally, several T cell clones were developed from TIL by limiting dilution using autologous tumor cells, rIL 2 and PHA. Certain of these Tcell lines or clones expressed a non- disulfide-linked y/6 TcR using a 55-66-kDa y chain, whereas, others expressed a disulfide-linked y/6 TcR using a 40-kDa y chain. Both disulfide- and non-disulfide-linked y/6 TcR have been reported in the literature [22-341. Although all these y/S TcR use a single 6 chain constant region gene segment [52-54], they use three different y chain constant region gene segments, resulting in three different y/6 TcR forms: (a) a disulfide-linked y/6 TcR, comprised of a y chain polypeptide of approximately 40 kDa, covalently bound through a disulfide bond with a 6 chain polypeptide. This y chain is encoded by the C,l gene segment and is expressed on Tcell lines and clones derived from peripheral blood lymphocytes or thymocytes from normal donors [26-28, 311, certain patients with primary immunodeficiency disorders [29] or cerebrospinal fluid [30]. (b) a non-disulfide-linked y/6 TcR, comprised of a ychain of 55-60 kDa utilizing a C,2 gene segment containing three copies of the CII exon [31-33].This y chain

is noncovalently associated with the 6 chain and is expressed on T cells derived from the peripheral blood of patients with primary immunodeficiency [22, 251 and on certain tumor Tcell lines [26, 341. (c) a non-disulfide-linked y/S TcR comprised of 40-kDa y chain polypeptide noncov- alently associated with the 6 chain. The constant region of this y chain is encoded by the C,2 gene segment with only two copies of the CII exon. This receptor is expressed on T cells derived from the peripheral blood of patients with primary immunodeficiency [25], normal donors [26,28,31] and thymocytes [23].

Certain TIL-derived T cell lines or clones expressed disul- fide-linked y/6 TcR which appear to be similar to those of T cell derived from the blood of normal donors, thymocytes [24, 26, 27, 311 or certain patients with immunodeficiency disorders [29]. Our observations ([28] and this report) and those of others [55-57] suggest that the majority (80%-95%) of the y/6 TcR Tcell lines or clones derived from peripheral blood lymphocytes from normal donors expressed the disulfide-linked y/6 TcR. These disulfide- linked y/6 TcR are often comprised of two well-resolved (by SDS-PAGE) polypeptide chains, usually in the range of 37 kDa and 40 kDa [22, 281. In certainTILTcel1 lines (Figs. 3 and 5) or clones (Fig. 4) these polypeptide chains were only partially resolved and they migrated into a higher molecular mass range (40-43 kDa). We have observed this pattern in certain T cell clones derived from the peripheral blood of patients with immunodeficiency disorders [29]. At least in the case of the TIL Tcell clones (Fig. 4) these partially resolved y and 6 chain polypeptides may indicate the preferential use of different V, and Vb gene segments than those usually found in certainTcel1 clones derived from the peripheral blood of normal donors. Similarly, the use of different V, may account for the higher molecular mass found (range 60-66 kDa) in the non-disulfide-linked y chain polypeptides in certainTILTcell lines (Figs. 2 and 4). Whether particular V, or Vb gene segments are preferen- tially used inTIL remains to be determined. The melanoma TIL-derived T cell clones (Fig. 7) exhibited two completely resolved y/6 TcR polypeptides similar to those that we have observed onT cell clones derived from the peripheral blood P81.

Certain other TIL-derived T cell lines and clones expressed non-disulfide-linked y/6 TcR using a 56-66-kDa y chain. This type of y/6 TcR is rarely found on T cells derived from peripheral blood lymphocytes of normal donors, but appear to be common on TIL-derived Tcell lines and clones. Moretta et al. [ S S ] reported that 2% of their y/6 TcR expressing T cell clones derived from the peripheral blood of normal donors used the 55-66-kDa y chain.We have also

686

found only one such ylS TcR+ T cell clone developed from peripheral blood lymphocytes from a normal donor. In contrast to the observations that there was a correlation in the expression of the CD8 antigen and the 55-60-kDa non-disulfide-linked y chain [55], our clone was CD8-. Certain cells of the W2 T cell line were CD8+, whereas they expressed a disulfide-linked y/6 TcR.

A y/6 T cell line developed from a patient with sarcoma and two melanoma Tcell clones were STCSl+ and expressed disulfide-linked y/6 TcR. Similarly, a Tcell line and two Tcell clones developed from the peripheral blood from a patient with DiGeorge syndrome [29] and six Tcell clones developed from the peripheral blood of normal donors ([28] and this report) were STCSl+ and expressed a disulfide-linked y/6 TcR. In contrast, it has been reported [56,57] that the STCSl+ determinant is expressed only on non-disulfide-linked y/6 TcR. Our findings [28,29,58] and those of others [59-611 suggest that there is no association between the expression of the 6TCS1 determinant and disulfide- or non-disulfide-linked $6 TcR.

M. Nanno, H. Seki, G. Mathioudakis et al Eur. J. Immunol. 1992. 22: 679-687

blood of normal donors. Different C, gene segments may be associated with different variable and joining region segments and may exhibit different functions.

Two Tcell lines were developed from the patient with Wilms tumor studied here, by stimulating TIL with autol- ogous tumor cells alone and rIL 2. One of theseTcell lines was y/6 TcR+ (GTCS1+: 97%),whereas the other contained approximately equal proportions of alp TcR+ or y/6 TcR+ cells (Fig. 1). Only oneT cell line was obtained from patient S6 with sarcoma, by stimulating TIL with autologous tumor cells alone and rIL 2, and it was y/6 TcR+. For reasons not known at the present, alp TcR+ Tcell lines were not obtained from this patient. However, stimulation of TIL with autologous tumor cells alone plus rIL2 from four other patients with sarcoma resulted in the development of CD3+ u/f3TcR+ yl6TcR- or CD3- CD16+ lines with nonspecific cytotoxic activity (data not shown). Results on the characterization of this type of T cell lines from patients with sarcoma are presented elsewhere [7, 211.

TIL-derived Tcell lines and clones from patients with metastatic melanoma expanded in culture with rIL 2 in the presence of autologous tumor cells alone exhibited autolo- gous tumor-specific cytotoxicity [7,10-12,621. TIL-derived Tcell lines and clones from 24 of 26 patients were CD3+ a@+ y/6-. They expressed both Tac and non-Tac I L 2 receptors [63], which were induced after stimulation with autologous tumor cells [7]. We demonstrated that the alp TcR and the CD3 antigen on the effector cells were involved in t h e cytolytic process and in t h e proliferation of TIL in response to autologous tumor cells and/or IL 2 [7 ] . In contrast, y/6 TcR+ TIL-derived Tcell lines and clones in patients with metastatic melanoma appear to be infre- quent. Only in 2 patients of 26 did we observe the development of y/6 TcR+ T cell lines or clones.

These studies demonstrate that both disulfide- and non- disulfide-linked y/6 TcR are expressed on different T cell lines and clones derived from TIL from patients with certain solid tumors. Although non-disulfide-linked y/6 TcR using the 40-kDa y chain were not found in the lines and clones investigated in this study, they may be expressed in certainTIL-derived T cells. Non-disulfide-linked y/6 TcR employing the 60-66-kDa y chain polypeptide are com- monly found onT cells derived from TIL, whereas, they are rarely expressed on T cells derived from the peripheral

Received July 11, 1991; in final revised form October 15, 1991.

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