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Keywords:

  • CD4+;
  • T-helper cell;
  • melanoma;
  • glioma;
  • shared antigen

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

CD4+ Th cells that are restricted by MHC class II molecules play an important role in the induction of antitumor immune responses. We have established a stable CD4+ Th cell clone (Th35-1A) from the PBMCs of a patient with primary cutaneous melanoma. The Th cell clone is noncytolytic and proliferates specifically in the presence of irradiated autologous melanoma cells or autologous EBV-transformed B cells pulsed with melanoma tumor cell lysates. Th35-1A produces IFN-γ (a Th1-type cytokine) after autologous tumor cell stimulation, and its proliferative reactivity is HLA class II–restricted. Th cells showed helper activity for PWM responses of PBMCs. Using a panel of HLA class II–matched and unmatched EBV-B cells as APCs and allogeneic melanoma tumor cell lysate as stimulant, DR7 was delineated as the HLA class II restriction element used by the Th cell clone. In agreement with these results, transfection of an allogeneic melanoma cell line with HLA-DR7 isolated from autologous EBV-B cells rendered the cell line stimulatory for Th35-1A cells. Specificity studies using autologous EBV-B cells (EBV-B35) pulsed with a panel of allogeneic tumor cell lysates of various tissue origins indicated that the Th cell clone recognizes an antigen shared by melanoma and glioma cells. The availability of the Th cell clone may lead to the development of new therapies against melanoma, using adoptive Th cell transfer and/or active immunization with a shared Th cell antigen. © 2003 Wiley-Liss, Inc.

There is increasing evidence that both CD4+ Th cells and CD8+ CTLs are required for an optimal antitumor response1, 2 and that CD4+ Th cells provide regulatory signals required for priming of CD8+ CTL responses.3 Vaccination of mice with a combination of Th and CTL peptides derived from murine leukemia virus has augmented antitumor immune responses and protected mice against a challenge with MHC class II+ as well as class II tumor cells.4 Similarly, CD4+ Th cells were able to inhibit experimental metastases of murine tumors that do not express MHC class II.5 In this model, Th cells most likely were activated by MHC class II+ antigen-presenting macrophages at the tumor site and the activated Th cells in turn stimulated a variety of both specific (CTL) and nonspecific effectors, such as dendritic cells or neutrophils, leading to tumor lysis.6 These results are important in light of the fact that human tumors often lose HLA class I and II expression in vivo.7, 8, 9, 10

Additional reports demonstrate that adoptive transfer of CD4+ Th cells alone, in the absence of CD8+ CTLs, can inhibit tumor growth in animal models.7, 8, 11, 12, 13 Furthermore, the immunotherapeutic potential of MHC class II–associated, tumor-derived peptides has been demonstrated in experimental animals.14, 15

A novel mechanism of CD4+ T cell–mediated antitumor immunity has been shown to involve inhibition of angiogenesis.16 This report also confirms that other nonspecific effector cells, e.g., macrophages, are needed for complete tumor regression.

In humans, CD4+ Th cells that mediate delayed-type hypersensitivity may be involved in the spontaneous regression of melanoma lesions.17 Spontaneously regressing melanomas had CD4+ T-cell infiltrates.17, 18In vitro induction of CD8+ CTLs by tumor-specific CD4+ T cells and dendritic cells derived from PBMCs, but not by CD4+ T cells alone, has been demonstrated in patients with lung and ovarian adenocarcinomas.19

In light of the important role that CD4+, HLA class II–dependent Th cells play in the control of cancer growth, approaches to augment these immune responses with Th cell–defined antigens or peptides need to be developed. Several human CD4+ Th cell lines and clones directed against various tumors have been described.20, 21, 22, 23, 24, 25, 26, 27, 28, 29 A few of these Th cells recognize antigens shared between tumor cells derived from different patients.20, 21, 22, 24, 29 Seven melanoma antigens/peptides (Tyrosinase, Annexin II, gp100, mutated CDC27, mutated TP1, fusion gene LDLR-FUT and mutated fibronectin) recognized by different tumor-reactive, CD4+, HLA class II–dependent Th lines have been identified.27, 28 Two of these antigens/peptides (Tyrosinase and gp100) are tissue-specific and shared between the melanomas of various patients.26, 30, 31 Annexin II is a major Ca2+-binding protein of the endothelial cell surface abundantly expressed on normal cells. However, the other 4 antigens (mutated CDC27, mutated TP1, fusion gene LDLR-FUT and mutated fibronectin) are individual-specific.27, 28

In the present study, we established a Th cell clone (Th35-1A) from the PBMCs of a primary melanoma patient. The CD4+, HLA class II–restricted Th clone is noncytolytic and proliferates specifically in the presence of autologous, irradiated melanoma cells. Th35-1A recognizes a shared tumor antigen as the T cells are stimulated by autologous APCs pulsed with lysates of 2 allogeneic melanoma and glioma cell lines. HLA-DR7 is used by Th 35-1A for antigen recognition. The availability of this stable Th clone provides the basic for new therapies against melanoma, using adoptive Th cell transfer and/or active immunization with a shared Th cell antigen.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patient 35

Patient 35 had excision of a “low-risk” primary melanoma of the superficial spreading type. The tumor was 0.69 mm thick and had a brisk lymphocytic infiltrate.32 The primary lesion was excised approximately 23 years ago, and there has been no recurrence or metastasis since. PBMCs were obtained from the patient's blood approximately 6 years ago.

Tumor cell lines

Melanoma cell lines WM98 (HLA-A1, -A3, -B8, -DR3), WM793 [HLA-A1, -A29, -B57(17), -B35, -DRb1 11, -DQb1 0301] and WM902B (HLA-A1, -A29, -B7, -B8, -C0701, -C15, -DRb1 0801, -DRb1 1104, -DQb1 0301, -DQb1 04) were established from vertical growth phase primary lesions, and WM35 (HLA-A2, -B18, -B51, -C2, -C1203, -DR7) was established from the patient's primary melanoma.33 Melanoma cell lines WM9 and WM1158 [HLA-A11, -A24, -B16, -B60(40), -C3, -DR13, -DR4, -DQ3, -DQ6] were derived from metastatic melanoma lesions. Cell line 1205LU [HLA-A1, -A29, -B57(17), -B35, -DRb1 11, -DQb1 0301], the metastatic variant of WM793, was established by repeated passages of WM793 cells both in vitro and in vivo in nude mice.34 All cell lines were maintained in MCDB153-L15 medium (Sigma, St. Louis, MO) containing 2% FBS. Melanocyte cell lines FOM99-1, FOM100-1 and FOM103-1 (obtained from Dr. M. Herlyn, The Wistar Institute) were maintained in MCDB153-L15 medium containing 2% FBS supplemented with insulin (5 μg/ml), epidermal growth factor (5 ng/ml) and TPA (10 ng/ml). Glioma cell line U87MG and colorectal carcinoma cell line SW480 were obtained from the ATCC (Manassas, VA) and maintained in L15 medium supplemented with 10% FBS. Glioma cell lines U251MG and U373MG (obtained from Dr. D. Bigner, Duke University Medical Center) and breast cancer cell line MDA-MB231 (obtained from Dr. D. Santoli, The Wistar Institute) were maintained in RPMI-1640 medium supplemented with 10% FBS.

An EBV-transformed B-cell line (EBV-B35) was established from freshly isolated PBMCs of patient 35 using 2.5 transforming U/cell of B95-8 virus (Tampa Bay Research Institute, Tampa Bay, FL). Similarly, the EBV-BRS cell line was established from the PBMCs of a healthy donor. EBV-B cell lines 793, 888, 1363, 1088, 4226 and 8652 were established from melanoma patients' PBMCs; and EBV-B cell lines D44, D150 and 007 were from colon carcinoma patients' PBMCs. EBV-B cell lines GM08065A and GM08605 were obtained from the National Institute of General Medical Sciences (NIGMS) Human Genetic Mutant Cell Repository (Camden, NJ). The NK cell target K562 (human erythroleukemia cell line) and the lymphokine-activated killer cell target Daudi (human lymphoblastoid cell line) were obtained from the ATCC. All lymphoid cell lines were maintained in RPMI-1640 medium (GIBCO, Grand Island, NY) supplemented with 10% FBS.

Antibodies

The following MAbs were used: W6/32 (anti-HLA class I), B33.1 and D1.B6 (anti-HLA class II) were obtained from Dr. B. Perussia (Thomas Jefferson University, Philadelphia, PA); H24B5 to influenza virus hemagglutinin was obtained from Dr. W. Gerhard (The Wistar Institute); M77 to colon carcinoma antigen 17-1A was obtained from Dr. G. Riethmüller (University of Munich, Munich, Germany); 4F-2 and 5AG to IL-4, B133.1.1 and B133.5.1 to IFN-γ and B154.9.1 and B154.9.2 to TNF-α were obtained from Dr. G. Trinchieri (The Wistar Institute); HB-180 (anti-HLA-DR and -DQ) and HB-144 (anti-HLA-DQ) were obtained from the ATCC.

HLA typing

HLA typing of EBV-B and tumor cell lines was performed using tissue typing trays and genomic typing.35

Generation of antimelanoma Th line and clones

A Th cell line was obtained from cocultures of PBMCs (105 cells/well of 96-well round-bottomed microtiter plates; Corning, Corning, NY) of melanoma patient 35 with irradiated (10,000 rads, Cs source) autologous melanoma WM35 cells (105/well) in RPMI-1640 medium containing 10% human AB serum, 10 mm HEPES (Sigma), L-arginine (116 mg/l), L-asparagine (36 mg/l), L-glutamine (216 mg/l, GIBCO) and 2-ME (5 × 10−5 M, Sigma). Cultures were stimulated with autologous WM35 cells and irradiated autologous PBMCs (days 8 and 14) or autologous EBV-B35 cells (from day 21 on) in RPMI-1640 medium containing purified IL-2 (5 U/ml; Virotech, Rockville, MD). After 4–5 weeks, cultures were maintained in 20 U/ml IL-2.

Th clones were obtained by plating a limited number of Th cells (10 cells/well) obtained from a 4-week-old bulk culture in 96-well U-bottomed microtiter plates (Corning). Each well received a mixture of irradiated autologous WM35 cells as stimulators (3 × 103), autologous EBV-B35 cells as feeder cells (5 × 104) and purified IL-2 (20 U/ml). On day 7, fresh feeder cells and IL-2 were added to cultures. Cultures were subcultured on day 14, transferred to 96-well U-bottomed plates and tested for proliferative T-cell responses on day 28. Wells showing positive proliferative responses to stimulation with autologous WM35 cells were transferred to 24-well plates (Corning), propagated further and characterized.

Cell lysate preparation for T-cell proliferation assay

Cell lysates were prepared by freezing and thawing tumor or EBV-B cells (107/ml in RPMI-1640) 3 times, followed by sonication. Microscopic evaluation of cell lysates confirmed the absence of viable intact cells.

T-cell proliferation assay

The T-cell proliferation assay was performed as described previously.36 Briefly, lymphocytes (2 × 104 cultured T cells/well of 96-well U-bottomed microtiter plates) were stimulated in T-cell medium (RPMI-1640, 10% human AB serum) with equal numbers of irradiated (10,000 rads, Cs source) tumor cells in the presence or absence of autologous EBV-B35 cells and IL-2 (purified, 20 U/ml) for 5–6 days. For assays with cell lysates, EBV-B cells (2 to 10 × 104/well of 96-well U-bottomed plate) were pulsed overnight with tumor or EBV-B cell lysates (107 cells/ml, 20 μl/well), washed, irradiated and used as stimulants. Proliferative responses of lymphocytes were determined by 3H-TdR incorporation assay. All determinations were performed in triplicate with SEM < 5%.

Coculture assay

The coculture assay was performed as described previously.37 Briefly, irradiated Th cells were cocultured with equal numbers of allogeneic lymphocytes (5 × 104/well of 96-well U-bottomed microtiter plate) in the presence or absence of PWM (2.5 μg/ml, Sigma). Proliferative responses of lymphocytes were determined as described above.

Blocking of Th cell proliferation with MAb

Tumor cells or EBV-B cells pulsed with lysates were incubated with HLA class I or class II specific antibodies at saturating concentrations (1 μg/ml) for 1 hr at room temperature. Isotype-matched control MAbs were used at similar concentrations. Excess antibody was removed by centrifugation without washing the cells. Following blocking of tumor cells, proliferation assays were performed in the presence of blocking MAb as described above.

Cytokine determinations

Cultured Th cells were washed twice with human AB serum–containing medium, incubated in human AB serum–containing medium (without stimulants or IL-2) for 10–12 hr at 37°C in a 5% CO2 incubator and washed once. Th cells (2 × 104/well) were stimulated with irradiated stimulator cells (2 × 104/well) in 96-well U-bottomed microtiter plates. Supernatants obtained from cultured Th cells after 24 hr were tested for the presence of TNF-α and IFN-γ, and supernatants obtained from Th cells after 48 hr were tested for the presence of IL-4.36

IL-4, IFN-γ and TNF-α were measured by radioimmunoassay. T-cell supernatants at various dilutions were placed in antibody-coated wells (MAb 4F-2 to IL-4, MAb B133.1.1 to IFN-γ and MAb B154.9.2 to TNF-α), and binding was determined using 125I-labeled MAb specific for different determinants on the cytokine molecule (MAb 5AG to IL-4, MAb B133.5.1 to IFN-γ and MAb B154.9.1 to TNF-α). Concentrations of IL-4, IFN-γ and TNF-α were determined using standard recombinant preparations of the respective cytokines (Genzyme, Cambridge, MA). Sensitivity of the assays was 0.01–0.1 U of cytokine/ml.

Phenotyping of T cells

Cultured T cells were incubated with saturating concentrations (5 μg/ml) of fluoresceinated or phycoerythrin-labeled MAbs detecting human lymphocyte markers (CD3, CD4, CD8, CD40, CD40L and CD25) in RPMI-1640 medium supplemented with 5% human AB serum for 45 min at 4°C. Binding of MAbs was analyzed in the cytofluorograph (Ortho Diagnostics, Raritan, NJ). All values are corrected for irrelevant, isotype-matched control antibody binding.

TCR analysis by RT-PCR

TCR was analyzed as described previously.36 In brief, mRNA was extracted from 3 × 106 T cells using the mRNA DIRECT kit (Dynal Biotech, Lake Success, NY). RT-PCR and cDNA synthesis were performed using the SuperScript One-Step RT-PCR kit (Invitrogen, Carlsbad, CA), specific 5′ primers encoding variable Vα (1–22) and Vβ (1–24) and a common C region (Cα and Cβ) 3′ primer. PCR products were run on 2% agarose gels and analyzed.

HLA-DR7 transfection of melanoma cells

RNA from EBV-B35 cells was isolated using the RNAqueous-4PCR kit (Ambion, Austin, TX). RT-PCR was carried out using forward primer 5′-GGGAGAATTCCTGGTCCTGTCCTGTTCTCC-3′ and reverse primer 5′-GGGATCTAGAAGCGTTAGGTAAAGGGGAGC-3′ flanking the DRB1*07011 gene.38 A 1 kb band was isolated and cloned into pTracer expression vector (Invitrogen). Clones with inserts of the predicted size were sequenced using the dideoxynucleotide chain termination method. The insert was identified as HLA DRB1*07011.38

WM793 melanoma cells expressing HLA-DR (>90% of cells positive) were transfected with HLA-DRB1*07011 cDNA in the eukaryotic expression vector pTracer under the control of the cytomegalovirus immediate early promoter (Invitrogen). Cells were transfected using FuGENE 6 transfection reagent (Roche, Indianapolis, IN) and transfectants selected with 6 μg/ml blasticidin S (Invitrogen). The percentage of transfectants expressing HLA-DRB1*07011 was determined in a complement-dependent lysis assay using HLA typing trays (One Lambda, Canoga Park, CA). As a control, WM793 cells were transfected with pTracer expression vector (without HLA-DRB1*07011 cDNA).

Statistical analyses

Differences between experimental and control values were analyzed for significance by Student's t-test.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Characteristics of Th cells

The phenotype of the Th cell line derived from patient 35 was 98% CD3+, 85% CD4+ and 5% CD8+ at 4 weeks in culture, when it was cloned successfully. Three clones of CD4 phenotype (approx. 98% of the cells CD4+) with proliferative activities against autologous WM35 cells were isolated, of which 1 (Th35-1A) was studied in detail.

Th35-1A clone was 98% CD4+, 85% CD25+, 98% CD40+, 40% CD40L+, 98% HLA class I+ and 98% HLA class II+. Fewer than 3% of the cells bound the irrelevant, isotype-matched control MAbs. Th35-1A expressed Vα8 and Vβ6 chains as determined by RT-PCR. It was not cytolytic against 51Cr-labeled autologous WM35 cells (data not shown).

Helper function of the Th cells was determined in a coculture assay after 8 weeks in culture. Irradiated Th cells significantly (p < 0.001) enhanced the proliferative responses of PBMCs to PWM compared to PBMC responses in the absence of Th cells (data not shown).

Cytokine secretion by Th cells

Supernatants from Th35-1A after 24 or 48 hr of stimulation with autologous irradiated WM35 melanoma cells contained significant amounts of IFN-γ (4–6 U/ml) but not IL-2, TNF-α or IL-4 (<0.1 U/ml).

Proliferative response of Th35-1A to stimulation with autologous whole tumor cells or cell lysates

The proliferative response of Th35-1A cells (8 weeks in culture) to stimulation with irradiated autologous whole tumor cells or autologous tumor cell lysates presented by autologous EBV-B35 cells was tested in a 3H-TdR-incorporation assay (Fig. 1). Significant proliferation (p < 0.05) of Th35-1A was noted in the presence of autologous irradiated WM35 cells alone. This is not surprising as the melanoma cells express HLA-class II (23% of cells positive by FACS analysis). This response was significantly (p < 0.05) augmented by addition of autologous EBV-B35 cells, probably due to presentation of tumor antigen by the EBV-B cells to the T cells (EBV cells were >96% positive for HLA-class II by FACS analysis). Th35-1A showed the highest (p < 0.001) proliferative activity after stimulation with autologous tumor lysates presented by autologous EBV-B35 cells.

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Figure 1. Proliferative activity of Th35-1A clone to stimulation with autologous, irradiated whole tumor cells, EBV-B35 cells or autologous EBV-B35 cells pulsed with autologous tumor cell lysates. Th35-1A was established in a coculture by stimulating the PBMCs of primary melanoma patient 35 with autologous irradiated WM35 melanoma cells, as described in Material and Methods. Th35-1A was obtained from 4-week-old bulk culture by plating limiting numbers of Th35-1A cells. Th35-1A clone at 6 weeks in culture (2 × 104 cells/well of 96-well U-bottomed microtiter plate) was incubated with equal numbers of irradiated autologous EBV-B35 cells (negative control), autologous WM35 melanoma cells (negative control), autologous tumor and EBV-B35 cells or autologous EBV-B35 cells pulsed with the optimal concentration (20 μl, corresponding to 2 × 105 cells) of tumor lysate. Proliferative activity was determined by 3H-TdR incorporation assay. *Values differ significantly (p < 0.05 to < 0.001) from each other.

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Proliferative response and IFN-γ secretion by Th35-1A to stimulation with lysates of allogeneic tumor or normal cells presented by autologous EBV-B35 cells

Th35-1A (10–12 weeks in culture) was evaluated for its reactivity with allogeneic tumor cell lysates presented by autologous EBV-B35 cells as APCs (Table I). Tumor cell lysates from 6 allogeneic melanoma cell lines, 3 glioma cell lines and 1 lysate each of lymphoma, leukemia and carcinomas of the bladder, breast and colon were included in the assays. Th35-1A proliferated significantly (p < 0.001) in the presence of lysates of allogeneic melanoma WM793 cells and the metastatic variant 1205LU (Table I, footnote 4), indicating that the T cells recognize a shared melanoma antigen. In addition, allogeneic glioma cell lysates (U87MG and U373MG) significantly (p < 0.05) stimulated proliferation of Th35-1A.

Table I. Proliferative Responses of Th35-1A to Stimulation With Whole, Irradiated Cells or Cell Lysates Presented by Autologous EBV-B Cells
DesignationStimulator cell originCell preparationStimulation index1IFN-γ release2 (U/ml)
  • 1

    Stimulation index is derived by the following formula: mean cpm in T cells with stimulant/mean cpm in T cells without stimulant.

  • 2

    Th supernatants obtained after 24 hr of stimulation and IFN-γ release determined by radioimmunoassay.

  • 3

    Mean cpm of 3H-TdR incorporation by T cells in the presence of irradiated autologous tumor cells and autologous EBV-B cells or tumor cell lysates presented by autologous EBV-B cells is significantly (p < 0.05) higher than the mean cpm obtained in the presence of T cells plus EBV-B cells only.

  • 4

    Metastatic variant 1205LU cells were derived from primary WM793 cells by serial passage of these cells both in vitro and in vivo in nude mice, (Herlyn et al.34).

WM35Autologous primary melanomaWhole tumor cells20.334
WM35Autologous primary melanomaTumor cell lysate26.036
WM793Allogeneic primary melanomaTumor cell lysate22.836
1205LU4Allogeneic metastatic variantTumor cell lysate24.632
WM98Allogeneic metastatic melanomaTumor cell lysate2.0<0.01
WM9Allogeneic metastatic melanomaTumor cell lysate2.1<0.01
WM1158Allogeneic metastatic melanomaTumor cell lysate0.9<0.01
WM902BAllogeneic primary melanomaTumor cell lysate1.2<0.01
U87MGAllogeneic gliomaTumor cell lysate14.834
U251MGAllogeneic gliomaTumor cell lysate1.4<0.01
U373MGAllogeneic gliomaTumor cell lysate5.434
U937Allogeneic bladder carcinomaTumor cell lysate1.1<0.01
MDAMB231Allogeneic breast carcinomaTumor cell lysate0.9<0.01
SW480Allogeneic colon carcinomaTumor cell lysate1.7<0.01
FOM99-1Allogeneic melanocytesCell lysate1.1<0.01
FOM100-1Allogeneic melanocytesCell lysate1.2<0.01
FOM103-1Allogeneic melanocytesCell lysate1.4<0.01
EBV-B35Autologous B cellsCell lysate2.1<0.01
EBV-B793Allogeneic B cellsCell lysate0.7<0.01
EBV-B RSAllogeneic B cellsCell lysate0.6<0.01
EBV-B888Allogeneic B cellsCell lysate0.2<0.01
EBV-B1363Allogeneic B cellsCell lysate1.1<0.01
EBV-B1088Allogeneic B cellsCell lysate0.7<0.01
EBV-B4226Allogeneic B cellsCell lysate0.8<0.01
K562Allogeneic erythroleukemia cellsCell lysate0.6<0.01
DaudiAllogeneic lymphoma cellsCell lysate0.8<0.01

The T cells did not proliferate after stimulation with lysates derived from 4 additional allogeneic melanoma cell lines, 3 allogeneic melanocyte cell lines any of the carcinoma cell lines (the autologous EBV-B cell line, 6 allogeneic EBV-B cell lines, 1 leukemia or 1 lymphoma cell line) (Table I).

Proliferating Th35-1A cells secreted significant amounts (4–6 U/ml) of IFN-γ when stimulated with autologous WM35 cells or autologous EBV-B35 cells pulsed with autologous or allogeneic tumor cell lysates (Table I). The same lysates/irradiated tumor cells that induced Th35-1A proliferation also induced IFN-γ release in the Th cells, and preparations that were negative in proliferation assay were also negative in cytokine release assay (Table I).

HLA restriction of Th35-1A

To map the HLA restriction element of Th35-1A, a panel of partially HLA-matched and -unmatched EBV-B cells were used as APCs for presentation of tumor lysates to Th35-1A cells in T-cell proliferation assays (Fig. 2). Allogeneic melanoma cell lysate WM793 was used to pulse the EBV-B cells for the following reasons: (i) this lysate consistently stimulated Th35-1A cells and (ii) WM793 are HLA-unmatched with WM35 cells; therefore, even if live tumor cells were present in the WM793 lysate, these cells would not stimulate Th35-1A. In contrast, when autologous WM35 lysates are used and presented to Th35-1A cells by various allogeneic, HLA-matched and -unmatched EBV-B cells, direct stimulation of Th cells by live WM35, HLA class II+ tumor cells that may be present in the WM35 lysate preparation cannot be excluded with certainty (though live tumor cells could not be detected in these lysates microscopically). The panel of EBV-B cells used for lysate presentation (Fig. 2) expressed high levels of HLA class II, as determined by FACS or HLA microcytotoxicity assay (not shown).

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Figure 2. Mapping of HLA restriction of Th35-1A. Proliferative activity of Th35-1A clone to stimulation with a panel of HLA class II-matched (matched HLA in bold in parentheses), partially matched and unmatched EBV-B cells pulsed with WM793 cell lysate. Th35-1A clone (2 × 104 cells/well of 96-well U-bottomed microtiter plate) was incubated with EBV-B cells that were pulsed with tumor cell lysates as described in Figure 1 legend. Th35-1A clone only, irradiated autologous EBV-B35 cells only or Th35-1A cultured with autologous irradiated EBV-B35 cells served as controls. Proliferative activity was determined by 3H-TdR incorporation assay. Values obtained with Th35-1A in the presence of EBV-B pulsed with WM35 lysate are significantly (p < 0.001) higher than each of the 3 control values in (a–d). NS, not significant.

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Th35-1A cells showed significant proliferative responses to stimulation with allogeneic WM793 melanoma lysates when the lysates were presented by EBV-B cells matched with WM35 cells for HLA-DR7, as determined by serotyping (EBV-B: RS, D150, D44 and GM08065A; Fig. 2a–d). In contrast, pulsed, HLA-nonmatched EBV-B cells did not stimulate Th35-1A (Fig. 2e,f). The relatively high 3H-TdR incorporation into EBV-BD44 cells cannot be explained by insufficient irradiation of these cells as higher doses of irradiation lysed the cells, rendering them incapable of presenting antigen. Despite the relatively high background 3H-TdR incorporation by the EBV-B cells, addition of melanoma cell lysate to irradiated EBV-B cells induced significant T-cell proliferation. The relatively high proliferative activity of Th35-1A in the presence of EBV-B8652 cells cannot be explained by the presence of EBV-B-specific T cells in the Th35-1A population as this population is monoclonal and therefore tumor-specific (see above).

The proliferative activity of Th35-1A to stimulation with autologous, irradiated WM35 cells was tested in the presence of saturating concentrations (1 μg/ml) of anti-HLA class I or class II antibodies in 2 independent experiments (Table II). The T-cell proliferative response was significantly (p < 0.001) inhibited by anti-HLA class II MAbs (B33.1 and D1.B6), whereas there was no significant inhibition in the presence of anti-HLA class I antibody W6/32.

Table II. Blocking of Th35-1A Proliferation by Anti-HLA MAbs
MAbStimulant/EBV-B1cmp incorporated (means ± SD)% Inhibition1
DesignationSpecificityIsotype
  • 1

    Th35-1A cells were cultured with irradiated, autologous WM35 melanoma cells and EBV-B35 cells or WM793 lysates and allogenic HLA-matched or autologous EBV-B cells, and inhibition of T-cell proliferative activity by MAb was determined by 3H-TdR incorporation assay relative to buffer controls, as described in Material and Methods.

  • 2

    Values (means of triplicate determinations) are significantly (p < 0.05 to < 0.001) different from values obtained with cultures that received isotype-matched control MAb or normal mouse Ig. This experiment was repeated once with similar results.

NoneWM35 cells/EBV-B3534,285 ± 2,142
W6/32HLA class IIgG2aWM35 cells/EBV-B3529,347 ± 3,68414.4
B33.1HLA class IIIgG2aWM35 cells/EBV-B354,518 ± 1,07186.822
H24B5Influenza virusIgG2aWM35 cells/EBV-B3530,925 ± 1,1419.8
D1.B6HLA class IIIgG2bWM35 cells/EBV-B3515,215 ± 2,45655.622
M77Colon carcinomaIgG2bWM35 cells/EBV-B3532,159 ± 3,4216.2
HB180DR and DQIgG2aWM793 lysate/EBV-B3511,416 ± 1,83266.72
Normal mouse IgUnknownMixtureWM793 lysate/EBV-B3530,753 ± 3,73010.3

Presentation of WM35 cells by EBV-B35 to Th35-1A was inhibited by 66.7% (p < 0.05) with anti-DR/DQ MAb HB-180, whereas there was no significant (p > 0.05) inhibition with normal mouse IgG (Table II). Furthermore, antigen presentation by EBV-B GM08065A (DR7-matched with WM35 tumor cells) was completely (97.9%, p < 0.001) blocked by anti-DR/DQ MAb HB-180 (results not shown). These results suggest DR7 restriction of Th35-1A.

Proliferative response of Th35-1A clone to stimulation with WM793 melanoma cells transfected with HLA-DR7 (DRB1*07011) cDNA

Th35-1A cells showed a low but significant (p < 0.0001) proliferative response (stimulation index = 3.57) to stimulation with allogeneic WM793 transfected with HLA-DR7 cDNA (Table III). Low proliferative response may be due to low expression of HLA-DR7 by the transfected cells (25% of cells were HLA-DR7 surface-positive, as determined in complement-dependent lysis assay in HLA tissue typing trays).

Table III. Proliferative Responses of Th35-1A to Stimulation with WM793 Melanoma Cells Transfected with HLA DR7 cDNA
Stimulator cellsTransfected cDNAcpm incorporated (means ± SD)Stimulation index1
  • 1

    See Table I, footnote 1.

  • 2

    p < 0.0001 compared to stimulator cells transfected with vector only.

NoneNone6,259 ± 49
WM793None6,950 ± 9161.11
WM793-DR7DRB1*0701122,3632 ± 5093.572
WM793-pTracerVector only10,980 ± 5241.76
WM35None68,324 ± 1,80310.91

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

CD4+ MHC class II–dependent Th cells play an important role in the control of cancer. The frequency of tumor-reactive CD4+ Th cells in cancer patients is low compared to that of CD8+ T cells.39 Furthermore, vaccinations of cancer patients with peptides40, 41, 42 or antigens43 defined by CD8+ CTLs have not induced CD4+ Th cell responses despite expression of Th epitopes recognized by CD4+ T cells on CTL antigens. Thus, there is a need for boosting CD4+ T-cell responses in patients. Well-characterized CD4+ Th cells provide the basis for achieving this goal.

In the present study, we isolated a CD4+ Th cell clone of Th1-type (Th35-1A) from the PBMCs of a primary melanoma patient following stimulation of the PBMCs with irradiated, autologous, primary melanoma cells. Th cells showed helper activity for PWM responses of PBMCs. Th35-1A recognized lysates of both primary melanomas tested and the metastatic variant cells isolated from one of these melanomas in nude mice. The Th clone also recognized lysates of 2 glioma cell lines. However, additional metastatic melanoma cell lysates (4 lines), 3 melanocyte cell lysates and lysates of 1 cell line each of bladder, breast and colon carcinoma, glioma and leukemia/lymphoma did not stimulate the Th cells. Furthermore, lysates of autologous and allogeneic EBV-B cells did not stimulate the Th clone. Thus, the Th cells recognized an antigen shared by at least some primary melanomas and gliomas. As both melanocytes and glial cells are derived from the neural crest, antigen sharing by these 2 malignancies is not surprising. CD4+ Th cells recognizing tumor-derived peptides shared between tumors derived from different patients or between tumors of different histologic types have been described.20, 21, 22, 24, 26, 29, 43

Th35-1A cells were isolated from the patient's PBMCs 18 years after surgical excision of the primary lesion. This suggests that the Th cells are memory T cells, though they were isolated at a relatively late time point compared to other memory cells.44, 45 Tumor antigen cross-presentation by APCs is a requirement for memory T-cell induction, whereas optimal induction of effector T cells requires direct antigen presentation by the tumor cells.46, 47 Thus, in our study, addition of APCs to irradiated tumor cells as stimulants significantly enhanced the activity of the Th35-1A clone (Fig. 1).

The proliferative activity of Th35-1A is HLA class II-dependent. This was determined in proliferation inhibition assays using MAb to HLA class II. Proliferation assays performed with melanoma lysate-pulsed, allogeneic, HLA class II–matched and nonmatched EBV-B cells suggested involvement of DR7 in the proliferative activity of Th35-1A. Additional experiments with HLA-DR7-transfected allogeneic melanoma cells (Table III) confirmed the involvement of HLA-DR7 in antigen recognition by the Th cell clone.

The antigen/peptide recognized by T35-1A is a candidate vaccine for melanoma patients of HLA-DR7 type. Approximately 12% of the human population expresses DR7 (Geigy Scientific Table, vol. 3).48 DR7+ melanoma patients are candidates for receiving the Th35-1A-defined peptide or antigen that will be identified in our future studies using biochemical30 and molecular27 approaches. Once the full-length Th35-1A antigen is cloned, extensive studies on its tissue distribution will become possible. Furthermore, the Th35-1A-defined antigen may also express CTL- or B cell-defined epitopes, analogous to several tumor antigens originally defined by CD8+ CTLs or B cells and later found to coexpress epitopes defined by CD4+ Th cells.26, 30, 31, 49, 50 Interestingly, some of these antigens were recognized not only by the Th cells from cancer patients but also by the Th cells from healthy individuals.51

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Dr. M. Alexander for providing lymphocytes from patient 35.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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