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Cancer Research 66, 11447, December 1, 2006. doi: 10.1158/0008-5472.CAN-06-0998
© 2006 American Association for Cancer Research
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Immunology

Superior Antitumor In vitro Responses of Allogeneic Matched Sibling Compared with Autologous Patient CD8+ T Cells

Sandra Kausche1, Thomas Wehler1, Elke Schnürer1, Volker Lennerz1, Walburgis Brenner2, Sebastian Melchior2, Mark Gröne1, Marion Nonn1, Susanne Strand3, Ralf Meyer1, Elena Ranieri4, Christoph Huber1, Christine S. Falk5 and Wolfgang Herr1

1 Department of Medicine III, Hematology and Oncology, 2 Department of Urology, and 3 Department of Medicine I, University of Mainz, Mainz, Germany; 4 Department of Nephrology, University of Bari, Bari, Italy; and 5 Institute of Molecular Immunology, GSF National Research Center for Environment and Health, Munich, Germany

Requests for reprints: Wolfgang Herr, Department of Medicine III, Hematology and Oncology, Johannes Gutenberg-University of Mainz, Langenbeckstrasse 1, 55101 Mainz, Germany. Phone: 49-6131-17-2710; Fax: 49-6131-17-6678; E-mail: w.herr{at}3-med.klinik.uni-mainz.de.


   Abstract
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Introduction
 Materials and Methods
 Results
 Discussion
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Allogeneic cell therapy as a means to break immunotolerance to solid tumors is increasingly used for cancer treatment. To investigate cellular alloimmune responses in a human tumor model, primary cultures were established from renal cell carcinoma (RCC) tissues of 56 patients. In three patients with stable RCC line and human leukocyte antigen (HLA)-identical sibling donor available, allogeneic and autologous RCC reactivities were compared using mixed lymphocyte/tumor cell cultures (MLTC). Responding lymphocytes were exclusively CD8+ T cells, whereas CD4+ T cells or natural killer cells were never observed. Sibling MLTC populations showed higher proliferative and cytolytic antitumor responses compared with their autologous counterparts. The allo-MLTC responders originated from the CD8+ CD62L(high)+ peripheral blood subpopulation containing naive precursor and central memory T cells. Limiting dilution cloning failed to establish CTL clones from autologous MLTCs or tumor-infiltrating lymphocytes. In contrast, a broad panel of RCC-reactive CTL clones was expanded from each allogeneic MLTC. These sibling CTL clones either recognized exclusively the original RCC tumor line or cross-reacted with nonmalignant kidney cells of patient origin. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles for RCC-reactive sibling CTL clones included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. In one sibling donor-RCC pair, strongly proliferative CD3+CD16+CD57+ CTL clones with non-HLA-restricted antitumor reactivity were established. Our results show superior tumor-reactive CD8 responses of matched allogeneic compared with autologous T cells. These data encourage the generation of antitumor T-cell products from HLA-identical siblings and their potential use in adoptive immunotherapy of metastatic RCC patients. (Cancer Res 2006; 66(23): 11447-54)


   Introduction
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 Introduction
Materials and Methods
 Results
 Discussion
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Treatment options for patients with metastatic renal cell carcinoma (RCC) are limited mainly because this cancer is resistant to conventional chemotherapy. Objective clinical responses are obtained after cytokine-based immunotherapy, with 10% to 20% of patients showing partial or complete tumor remissions (1). This observation established the concept that RCC is an immunogenic tumor. Several in vitro findings support this view. First, RCC tumors are frequently infiltrated by natural killer (NK) and T lymphocytes (2–4). Second, stimulation of tumor-infiltrating lymphocytes (TIL) with autologous RCC cells in vitro generates CD8+ T-cell responders that show human leukocyte antigen (HLA) class I–restricted antitumor activity (5). Third, based on CTLs and TILs expanded from patient blood and tumor tissue samples, a few T-cell-defined RCC antigens have been identified. These epitopes are derived either from nonmutated proteins (6) or from mutated (7, 8) or alternatively processed gene products (9, 10). Reverse immunology strategies were also applied to predict HLA class I–binding and HLA class II–binding peptide epitopes within proteins frequently expressed in RCC. Several predicted epitopes were successfully used to stimulate RCC-reactive CD4+ and CD8+ T cells in vitro (11–15).

Allogeneic hematopoietic stem cell transplantation (HSCT) aims to break autologous immunotolerance toward the host malignancy. This treatment is based on the graft-versus-malignancy effect that is mainly mediated by donor-derived T lymphocytes (16). Allogeneic HSCT is capable of inducing long-term disease control in patients with chemorefractory leukemias. Several groups have translated this therapeutic approach to solid tumors, with a main focus on immunogenic cancer types. Although results with malignant melanoma were disappointing (17), different investigators reported tumor remission rates ranging from 20% to 50% of metastatic RCC patients (18–20). The vast majority of treated RCC patients were refractory to previous cytokine therapy. This raised the idea that tumor regressions were mediated by allogeneic T lymphocytes and were not only caused by a cytokine storm induced by the allotransplantation procedure. Further clinical and experimental observations support this hypothesis. Although associated with acute graft-versus-host disease (GVHD), RCC remissions typically occurred after GVHD when posttransplant immunosuppression was already tapered (18). In addition, responding RCC patients were complete donor T-cell chimeras (18). Clinical tumor remissions following allogeneic HSCT were associated with an expansion of IFN-{gamma}-producing CD8+ T cells in peripheral blood (21). In a further study, CD8+ CTL clones recognizing minor histocompatibility (minor H) antigens on RCC cells were isolated from posttransplant peripheral blood mononuclear cells (PBMC; ref. 22). Nevertheless, the precise effector mechanisms leading to tumor rejection are not yet defined.

Generation and characterization of RCC-reactive T cells require the availability of RCC cell lines with long-term in vitro growth. Such stable tumor lines are only obtained from a minority of patients. In a systematic and prospective effort, we attempted to establish tumor cell lines from primary RCC tissue of patients who underwent nephrectomy. In patients with stable RCC lines and HLA-identical sibling donors available, we investigated autologous and allogeneic T-cell responses against RCC in vitro. We observed a superior capability to generate RCC-reactive CD8+ CTLs from HLA-identical sibling donors compared with their patient counterparts. RCC-reactive sibling CTLs originated from the CD8+ CD62L(high)+ T-cell subset and recognized multiple RCC antigens by either HLA-restricted or non-HLA-restricted mechanisms.


   Materials and Methods
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Results
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Donors and cell lines. The study protocol was approved by the local Ethics Committee. PBMC donors were RCC patients and their healthy siblings who participated in this study after informed consent in accordance with the Helsinki protocol. High-resolution HLA typing was done from genomic DNA using class I–specific and class II–specific primers (Dr. B. Thiele, Institute of Immunology and Genetics, Kaiserslautern, Germany).

Primary cultures were initiated from single-cell suspensions that were processed from RCC and adjacent nonmalignant kidney tissues. Briefly, small tissue pieces were digested in HBSS buffer supplemented with 560 µg/mL collagenase type VIII and 26 µg/mL DNase type IV (both were from Sigma, St. Louis, MO) for 30 minutes at 38°C. Cells were cultured in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 20% FCS, 40 µg/mL fluconazole, and 50 µg/mL gentamicin (both were from Ratiopharm, Ulm, Germany). Cytogenetic analysis was done on early passage cell lines using standard quinacrine staining (V. Bayer, Institute of Human Genetics, Mainz, Germany).

The RCC cell lines MZ1257-RCC, MZ1851-RCC, MZ1846-RCC, and MZ1774-RCC were provided by Prof. A. Knuth (University of Zürich, Zürich, Switzerland). B-lymphoblastoid cell lines (LCL) and phytohemagglutinin-activated PBMC blasts (PHA blasts) were generated according to standard procedures. TILs were expanded from primary RCC single-cell suspensions using 1,000 IU/mL interleukin (IL)-2 (Chiron, Emeryville, CA) and 5 ng/mL IL-7 (R&D Systems, Wiesbaden, Germany) in AIM-V medium (Life Technologies) supplemented with 5% human serum (medium Ma).

Isolation of CD8+ CD62L+ T cells. CD8+ T cells were purified from PBMCs by negative isolation technique using a cocktail of biotin-conjugated non-CD8 monoclonal antibodies (mAb) and anti-biotin microbeads followed by depletion of magnetically labeled cells on LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany). CD62L+ cells were subsequently isolated using CD62L microbeads and MS columns (Miltenyi Biotec).

Mixed lymphocyte/tumor cell culture and T-cell cloning. PBMCs (2 x 106 per well) were cocultured in 24-well plates with irradiated RCC cells (105 per well) in 2 mL medium Ma. On day 3, 150 IU/mL IL-2 and 5 ng/mL IL-7 were added. Responder lymphocytes (106 per well) were weekly stimulated with 105 irradiated tumor cells in medium Ma containing IL-2 and IL-7. Day 28 mixed lymphocyte/tumor cell culture (MLTC) responders were cloned by limiting dilution in round-bottomed 96-well plates preseeded with irradiated RCC stimulator (3 x 103 per well) and allogeneic LCL feeder cells (4 x 104 per well) in medium Ma supplemented with IL-2 and IL-7. Growing CTL clones were expanded in 24-well plates by weekly addition of stimulator (5 x 104 per well) and feeder (2 x 105 per well) cells.

Flow cytometry analysis. Cells were incubated for 15 minutes at 4°C with FITC/phycoerythrin (PE)-conjugated mAbs. Antibodies were from Immunotech (Marseille, France), except for anti-CCR7 (R&D Systems), anti-CD69, anti-CD54, and anti-CD94 (BD Biosciences, San Jose, CA). Analysis was done on flow cytometer EPICS ALTRA (Beckman Coulter, Fullerton, CA).

Cytokine assays. After stimulation with tumor cells for 24 hours, cytokine secretion of CTLs was measured using the multiplex protein array system technology (Bio-Rad Laboratories, Hercules, CA).

IFN-{gamma} enzyme-linked immunospot assay. Twenty-hour IFN-{gamma} enzyme-linked immunospot (ELISPOT) assays were done as recently described (23). Spot numbers were automatically counted using a computer-assisted video image analysis system (Zeiss, Jena, Germany).

51Cr-release assay. Target cells were incubated for 90 minutes with 100 µCi Na251CrO4 (Amersham Buchler, Braunschweig, Germany). After washing, labeled targets (103 per well) were plated in conical 96-well plates. CTLs were added in duplicates in a total volume of 160 µL/well. After 4 to 6 hours of incubation, 80 µL supernatant/well was collected for counting in a gamma counter.

Antibody blocking test. The following murine mAbs were used at 10 and 100 µg/mL as blocking reagents: W6/32, an anti-HLA class I IgG2a; MA2.1, an anti-HLA-A2 IgG1; GAP-A3, an anti-HLA-A3 IgG2a; B1.23.2, an anti-HLA-B and HLA-C IgG2a; L243, an anti-HLA-DR IgG2a (all hybridomas were from the American Type Culture Collection, Manassas, VA);6 C7709A2, an anti-HLA-A24 IgG2a (Ludwig Institute for Cancer Research, Brussels, Belgium); OKT3, an anti-CD3 IgG2a (Janssen-Cilag, Neuss, Germany); OKT8, an anti-CD8 IgG1 (BD Biosciences); anti-NKG2D IgG1 (Beckman Coulter); anti-CD1d IgG1 (Biozol, Eching, Germany); and NOK-1, an anti-CD95L IgG1 (BD Biosciences).

Statistical evaluation. The Student's t test for paired samples was used to evaluate statistical differences between allogeneic and autologous MLTC results. The same test was applied to compare data obtained from CD62L(high)+ and CD62L(low)+/negative CD8+ T-cell populations. Values of P < 0.05 were considered statistically significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishment and characterization of RCC cell lines and nonmalignant kidney cells. Primary cultures were initiated from single-cell suspensions that were processed from tumor and adjacent nonmalignant kidney tissues of 56 consecutive RCC patients who underwent nephrectomy. A total of 13 RCC cell lines was obtained that maintained stable in vitro growth beyond 20 culture passages. Nonmalignant kidney cells (NKC) were cultured up to a maximum of seven passages. In a comprehensive analysis, RCC cell lines and corresponding NKCs were characterized using cytogenetic, HLA typing, and flow cytometry procedures. Furthermore, all siblings of patients with established RCC cell lines underwent HLA typing. Altogether, three sibling donor/RCC pairs with complete HLA match were obtained for subsequent analysis. The respective RCC cell lines were clear cell carcinomas and showed complex chromosomal abnormalities (Table 1 ) typically observed in this cancer entity (24). Compared with NKC counterparts, RCC cells expressed higher levels of surface HLA class I complexes under basal conditions. Expression levels of adhesion molecules were either comparable (CD58) or higher on NKC (CD54). Both RCC and NKC did not express HLA class II or the costimulatory molecules CD80 and CD86. Using these three well-characterized RCC/NKC pairs, we compared autologous and allogeneic cellular immune responses against RCC occurring in vitro.


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  		Table 1. RCC cell lines and corresponding NKCs of patients with available HLA-identical sibling donor

 
Low or absent RCC reactivity in autologous peripheral blood and TILs. To analyze preexisting antitumor immunity in these three RCC patients, TILs were cultured from six different primary RCC tissue samples. Rapidly expanding TILs were obtained in all cases. None of them recognized the autologous RCC tumor line (data not shown). Furthermore, autologous PBMCs were stimulated with RCC cells in six independent MLTC experiments. Resulting MLTC responders were CD3+ CD8+ T cells with low or absent reactivity against RCC stimulator cells as determined in cytotoxicity (Fig. 1B ) and IFN-{gamma} ELISPOT assays (data not shown). Only in the ELTHEM-RCC model, significant anti-RCC CTL activity was detectable in autologous MLTCs (Fig. 1B). However, extensive efforts to clone these T-cell specificities failed due to the inability of autologous CTL populations to expand in vitro.


Figure 1
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  		Figure 1. Superior antitumor CTL responses obtained from matched sibling compared with patient PBMCs. MLTCs were established using PBMCs of either autologous patients MZ3114, MZ3126, and ELTHEM or their allogeneic HLA-identical siblings, respectively. RCC cell lines were weekly added as stimulator cells. Representative data from one of three experiments. A, weekly cell counts of autologous patient ({lozenge}) and allogeneic sibling ({blacklozenge}) MLTC responders. B, autologous and allogeneic MLTC responders isolated between d45 and d55 of culture were simultaneously analyzed for cytolytic activity in 51Cr-release assays. Targets were patient-derived RCC cell lines ({blacksquare}), LCLs (MZ3114, ELTHEM) or PHA blasts (MZ3126; {blacktriangleup}), and K562 ({circ}). E/T, effector to target ratio. All differences of RCC reactivity between sibling donor and patient PBMCs were statistically significant (P < 0.05), except for the proliferative response observed in the MZ3114 model.

 
RCC-reactive CD8+ CTL clones can be isolated from HLA-identical sibling donors. We simultaneously investigated the primary cellular immune responses of HLA-identical sibling PBMCs against RCC. After two to three stimulations with allogeneic RCC cells, a vigorous proliferation of MLTC bulk populations was observed in eight independent experiments. Weekly cell counts obtained from allogeneic MLTCs were generally higher compared with their autologous counterparts (Fig. 1A). Allogeneic MLTC responder proliferation depended on the addition of tumor cells, thereby excluding antigen-nonspecific cytokine-induced T-cell expansion (data not shown). Responding lymphocytes were exclusively CD8+ T cells, and expansion of CD4+ T cells or NK cells was never observed. Without exception, allogeneic MLTC populations recognized RCC cells in cytotoxicity assays (Fig. 1B). The levels of anti-RCC lysis were significantly higher compared with those obtained in autologous MLTCs. Sibling MLTC responders showed only low or absent reactivity against LCLs or PHA blasts that were generated from the related RCC patients as surrogate targets of hematopoietic cells (Fig. 1B). Significant cross-reactivity against patient-derived NKC was only observed in allogeneic ELTHEM-MLTCs (data not shown).

To analyze target specificity, MLTC responder lymphocytes derived from all three different sibling donors were cloned by limiting dilution. From 5,000 wells initially seeded, >400 RCC-reactive CD8+ CTL clones were isolated. Of them, 65 CTL clones could be expanded to cell numbers that allowed their detailed characterization. This included a comprehensive analysis of phenotypic markers, MHC restriction elements, and cross-reactivity pattern using patient-derived NKC and hematopoietic cells as well as HLA-matched allogeneic RCC and non-RCC tumors as targets. The non-RCC target panel contained stable tumor cell lines previously established from melanomas and from breast, colon, lung, pancreatic, hepatocellular, and cholangiocellular carcinomas.

In flow cytometry, RCC-reactive sibling CTL clones expressed CD27–, CD45RO+, CD62L–, CD25+, and CD69+, consistent with a phenotype of activated mature effector memory cells (Table 2 ). Most CTL clones exclusively recognized the original RCC tumor line but did not react with patient-derived NKC and hematopoietic cells as well as other tumor targets (Fig. 2A , clone D24). This suggested tumor-associated or even tumor-specific antigens as their target structures. A considerable proportion of CTL clones cross-reacted with NKC of patient origin (Fig. 2B, clone G179). Apparently, these CTLs were directed against kidney lineage antigens. A minority of CTL clones also recognized patient-derived hematopoietic cells or other allogeneic tumor targets. The MHC-restricting alleles of CTLs included HLA-A2, HLA-A3, HLA-A11, HLA-A24, and HLA-B7. Results obtained from all RCC-reactive sibling CTL clones are summarized in Table 3 .


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  		Table 2. Flow cytometry analysis of RCC-reactive sibling CTL clones

 

Figure 2
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  		Figure 2. Characterization of RCC-reactive CTL clones isolated from HLA-identical healthy sibling donors. A and B, MZ3126 sibling CTL clones D24 and G179 were tested for cytolytic activity against MZ3126-RCC ({blacksquare}), MZ3126-NKC ({square}), MZ3126-PHA blasts ({blacktriangleup}), and K562 ({circ}) in 51Cr-release assay. The effect of anti-HLA blocking antibodies on recognition of MZ3126-RCC and the cross-reactivity pattern against various HLA-A11+ tumor cell lines were determined in IFN-{gamma} ELISPOT assays. Both CTL clones failed to recognize any other target from a large panel of tumor lines matched with MZ3126-RCC for HLA-A2 or HLA-B/HLA-C alleles (data not shown). Mel, melanoma. C, ELTHEM sibling CTL clone 27B11 was tested for cytolytic activity against ELTHEM-RCC ({blacksquare}), ELTHEM-NKC ({square}), and K562 ({circ}) in 51Cr-release assay. This clone was further analyzed at an effector to target ratio of 60/1 for anti-RCC lysis in the presence of mAbs that can block the interaction between CTLs and tumor cells. Cytolysis was also determined at the same effector to target ratio against various cell lines of which several were not matched with ELTHEM-RCC for any HLA allele. CO, colon carcinoma; PC, pancreatic carcinoma.

 

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  		Table 3. Summary of HLA class I–restricted CD8+ CTL clones isolated from healthy sibling donors in RCC models MZ3126, MZ3114, and ELTHEM

 
Identification of matched sibling CTLs with non-HLA-restricted RCC reactivity. In the ELTHEM-RCC model, allogeneic MLTC responder lymphocytes showed an antigen-dependent in vitro proliferation at extraordinarily high levels (Fig. 1A). After limiting dilution cloning of these responders in 2,000 wells, we obtained >200 CTL clones that could be expanded much faster than most HLA-restricted sibling CTLs. Twenty of these clones were characterized in detail. These CTLs recognized ELTHEM-RCC tumor and, to a lower extent, ELTHEM-NKC (Fig. 2C, clone 27B11). They lacked reactivity against ELTHEM-LCL or LCLs of other individuals. Whereas several CTL clones showed minor but significant cross-recognition of the NK target K562 (Fig. 2C), others had no K562 reactivity (data not shown). In addition, CTLs recognized various RCC and non-RCC tumor cell lines. Several of them were not matched with ELTHEM-RCC for any HLA allele (Fig. 2C).

In addition to CD3 and CD8, the ELTHEM sibling CTL clones coexpressed the NK-associated molecules CD16, CD56, CD57, and CD94dim (Table 2). Compared with HLA class I–restricted CTLs, they expressed higher levels of CCR7. After stimulation with RCC tumor cells, ELTHEM sibling CTL clones secreted IFN-{gamma}, granulocyte macrophage colony-stimulating factor, IL-6, IL-8, and macrophage inflammatory protein 1ß but not IL-1ß, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-13, IL-17, monocyte chemoattractant protein-1, granulocyte colony-stimulating factor, and tumor necrosis factor-{alpha} (data not shown). To further characterize the ELTHEM sibling CD8+ CTL clones, cytolytic activity was analyzed after addition of mAbs specific for HLA class I, CD1d, CD3, CD8, NKG2D, CD95L (all Fig. 2C), and ILT2 (data not shown). These antibodies failed to block target cell lysis. From these findings, we concluded that a significant proportion of ELTHEM sibling CTL clones represent non-HLA-restricted T cells that show several phenotypic and functional similarities with NK cells.

RCC-reactive matched sibling CTLs derive from CD8+ CD62L+ precursors. Sibling donors were healthy individuals and had no history of previous priming against RCC antigens. To investigate whether RCC-reactive CTLs originated from naive or memory T-cell precursors in vitro, ex vivo–isolated sibling CD8+ T cells were separated based on their CD62L expression profile. After selection, we obtained an almost pure CD62L(high)+ population (Fig. 3A ), which coexpressed CCR7, CD27, and CD28 (data not shown). Thus, this fraction contained naive and central memory CD8+ T cells (25). The CD62L-depleted fraction included few CD62L(low)+ cells and was negative for CCR7, CD27, and CD28, consistent with an effector memory phenotype. After stimulation of both fractions with HLA-identical RCC cells in MLTCs, the median proliferation was 2-fold higher in the CD8+ CD62L(high)+ compared with the CD8+ CD62L(low)+/negative subpopulations (data not shown). In addition, the CD8+ CD62L(high)+ responder cells showed superior antitumor cytolytic and IFN-{gamma} release activities (Fig. 3B and C). Phenotypically, this population down-regulated CD62L expression during the first 2 weeks of in vitro culture (data not shown). Taken together, these observations suggest that in HLA-identical healthy siblings RCC-reactive mature CTLs develop from the CD8+ CD62L(high)+ pool containing both naive and central memory T lymphocytes.


Figure 3
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  		Figure 3. Tumor-reactive sibling CTLs originate from CD8+ CD62L(high)+ precursors. A, after negative isolation of CD8+ T cells from MZ3126 sibling PBMCs, CD62L(high)+ cells were selected using CD62L microbeads. Resulting positive and negative fractions were designated CD62L(high)+ and CD62L(low)+/negative, respectively. B, CD62L(high)+ and CD62L(low)+/negative subsets were weekly stimulated with HLA-identical MZ3126-RCC cells in allogeneic MLTCs. Day 39 responder populations were analyzed for cytolytic activity against MZ3126-RCC ({blacksquare}), MZ3126-NKC ({square}), MZ3126-PHA blasts ({blacktriangleup}), and K562 ({circ}) in 51Cr-release assay. C, specificity of d40 MLTC responders was determined in IFN-{gamma} ELISPOT assay. Representative results of three different experiments. The difference of RCC reactivity between the CD62L(high)+ and CD62L(low)+/negative subsets was statistically significant (P < 0.05).

 

   Discussion
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 Materials and Methods
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References
 
We have recently shown that RCC-reactive CTL clones can be isolated from unrelated healthy donors matched with previously established RCC stimulator cell lines for HLA class I (23). After successful generation of stable RCC lines in a representative patient cohort, we extended our studies to healthy sibling donors with complete HLA class I and II match. In addition, we included autologous patient PBMCs and TILs in the analysis. Our results provide clear evidence that RCC-reactive CTL clones can be generated from naive HLA-identical siblings in vitro. Comparison with autologous patient PBMCs and TILs showed superior antitumor CD8+ T-cell responses using allogeneic sibling PBMCs. Only weak if any RCC reactivity was observed with patient-derived lymphocytes. Consistent with this result, autologous tumor-reactive T cells have been successfully isolated from peripheral blood of only a minority of cancer patients, predominantly in those with melanoma and favorable disease course (26–28).

The "hyperresponsiveness" of T cells isolated from healthy sibling donors might be explained by the lack of immunosuppressive mechanisms that can impair the function of tumor-reactive immune cells from cancer patients. Defective antitumor immune responses have been described at the levels of antigen-presenting cells (APC; ref. 29), effector T cells (30), and regulatory T cells (Treg; ref. 31) in tumor-bearing patients. Recent studies have focused on CD4+CD25+Foxp3+ Treg cells that were capable of suppressing the function of tumor-reactive T cells in several human cancer types (32–35). We determined the proportion of CD4+CD25+Foxp3+ Treg cells in autologous PBMC and TIL samples of RCC patients MZ3126, MZ3114, and ELTHEM and compared these results with those from PBMCs of related HLA-identical siblings. The fraction of Treg cells ranged from 5.0% to 8.9% of total CD4+ T cells with no consistent increase in patient-derived material (data not shown). Thus, in this small study population, our data on Treg cells do not provide a reasonable explanation for the enhanced RCC reactivity observed in sibling PBMCs. The isolated sibling CTL clones did not recognize known HLA class I–associated RCC peptide epitopes (6–14). This precluded analyzing the autologous T-cell repertoire for anergy or deletion of defined antitumor specificities. Further comparative studies in HLA-identical donor-patient pairs are needed to elucidate the immunologic basis for the enhanced RCC reactivity of tumor-naive donor PBMCs.

Because of their superior antitumor in vitro responses, allogeneic sibling PBMCs would be a powerful source to generate tumor-reactive CTLs for adoptive immunotherapy trials. However, the in vivo efficacy of infused allogeneic CTLs may be limited by the immunosuppressive milieu that exists in tumor-bearing patients (31). Clinical studies in metastasized melanoma patients have shown that preceding lymphodepletion chemotherapy can increase the antitumor efficacy of adoptively transferred autologous TILs (36). This chemotherapy is thought to act mainly by depleting Treg cells and by generating space for subsequent repopulation of lymphoid and tumor tissues with infused tumor-reactive T cells through cytokine-mediated homeostatic mechanisms. Similarly, lymphodepletion chemotherapy should enhance the in vivo efficacy of adoptively transferred antitumor sibling CTLs.

Although the combination of cytoreductive conditioning therapy and autologous TIL infusion increased the rate of clinical melanoma remissions, it also resulted in higher incidences of vitiligo and uveitis due to autoimmune reactions against melanocytes (36). This observation highlights the strong link between the immunologic mechanisms of tumor regression and autoimmune attack against normal tissues. Translation of this finding to RCC patients would mean that adoptive T-cell therapy could induce autoimmune kidney disease. However, a significant increase in the incidence of nephritis has not been observed in RCC patients responding to previous cytokine or cell-based immunotherapies (37). Interestingly, a considerable proportion of RCC-reactive sibling CTL clones isolated in our study cross-reacted with NKC of patient origin. Shared antigens expressed by normal and malignant kidney cells might represent unaltered renal differentiation antigens. Alternatively, these antigens could be polymorphic between donor and tumor. The latter antigen category would include minor H antigens that induce potent graft-versus-host and graft-versus-leukemia reactions in leukemia patients (38). The adoptive transfer of sibling CTLs that recognize renal lineage-expressed antigens carries the risk of breaking immunotolerance toward normal kidney tissue. Nevertheless, as the kidney did not show major clinical symptoms of immune destruction in previous transplantation studies (18–20), the in vivo relevance of renal tissue-reactive CTLs remains elusive.

With the in vivo administration of sibling-derived CTLs, GVHD mediated by contaminating alloreactive T cells occurs as a further possible complication. Because RCC patients who developed GVHD after allogeneic transplantation showed a higher rate of objective tumor remissions (18), the induction of alloreactivity in vivo seems tolerable, if not desirable. However, severe and refractory forms of GVHD were accompanied with significant morbidity and mortality in tumor patients receiving allogeneic transplant therapy (18) or donor lymphocyte infusions (39). Sufficient clinical activity in conjunction with absent or moderate GVHD might be most likely achieved by transferring short-term cultured oligoclonal donor T-cell lines as has been suggested from studies in posttransplant cytomegalovirus infection and EBV-induced lymphoproliferation (40, 41). Repeated in vitro stimulations with RCC cells have the advantage of expanding tumor-reactive T-cell precursors that are present at very low frequency in naive PBMCs. The residual alloreactive T cells could be removed in vitro by an immunomagnetic depletion approach targeting the activation-induced antigen CD137 (42). For severe autoimmune kidney disease following sibling CTL transfer, we propose to integrate primary patient-derived NKC as allogeneic APCs during the depletion step. In our hands, NKC can be readily isolated from adjacent normal kidney tissue of tumor nephrectomy samples by short-term in vitro culture.

Sibling-derived PBMCs also contained CTL precursors that did not cross-react with NKC. The nature of antigens recognized by these CTLs is certainly of greatest interest. They might be potential targets of single RCC regression responses that were observed in the absence of severe GVHD (18). In case these antigens are not expressed in normal tissues, they would be ideal candidates for tumor immunotherapy. We further show that the antitumor in vitro response of sibling PBMCs can be dominated by CTLs that recognize tumor targets independently of HLA. The latter CTLs lysed various tumor cell lines but spared the corresponding LCL counterparts. They coexpressed the NK-associated markers CD16, CD56, CD57, and CD94dim. Further studies are needed to elucidate the specificity and potential clinical use of this type of tumor-reactive effector cells.

Unfractionated sibling PBMCs were stimulated in allogeneic MLTCs with RCC tumor cells. Interestingly, MLTC responder lymphocytes were exclusively CD8+, whereas no CD4+ T-cell or NK cell responses were observed. The failure to generate antitumor CD4 reactivity might be explained by the lack of HLA class II expression on RCCs under basal conditions (Table 1). Although IFN-{gamma} pretreatment of RCC cells induces HLA class II (43), we were unable to generate CD4 responses using IFN-{gamma}-exposed RCC cells as MLTC stimulators (data not shown). As previously reported, tumor-reactive CD4+ T cells can be efficiently expanded when professional APCs prepulsed with apoptotic or necrotic tumor preparations are used (44–46).

Our in vitro studies show that in matched healthy sibling donors tumor-reactive CTLs derive from the CD8+CD62L+ PBMC subset containing naive precursors and central memory cells. CD62L and CCR7 are involved in the tracking of T cells to secondary lymphoid organs (47). Both molecules are down-regulated on mature effector memory CD8+ T cells. Based on their enhanced proliferative and migratory abilities, CD62L+CCR7+ naive and central memory CD8+ T cells were found to mediate superior antitumor immunity compared with CD62L–CCR7– effector memory CD8+ T cells in murine adoptive transfer studies (48). In our in vitro model, the most conclusive explanation is that CD62L(high)+ naive CD8+ T cells were primed and activated by professional APCs capable of cross-presenting RCC antigens. We cannot exclude that RCC-reactive CTLs have developed from CD62L(low)+ central memory CD8+ T cells recognizing antigens similar or identical to those presented by the RCC.

Our data encourage the generation of tumor-reactive CD8+ CTLs from HLA-identical sibling PBMCs in vitro. Because sibling donors seem to have an interindividually different capability of generating distinct antitumor effector cell types, protocols should implement patients' tumor cells as stimulators. This strategy ensures that the full spectrum of potentially relevant antigens and immunostimulatory target structures are included (49). A further challenge is to develop in vitro methods allowing the successful large-scale expansion of RCC tumor lines. Alternatively, RCC single-cell suspensions or professional APCs preloaded with apoptotic or necrotic RCC preparations could serve as stimulator cells. Finally, sibling-derived antitumor CTLs might be transferred into RCC patients either alone or in combination with allogeneic HSCT. Such trials would answer the question whether allogeneic cell therapy on an individualized donor/tumor basis is feasible and results in significant graft-versus-tumor responses beyond severe GVHD.


   Acknowledgments
 
Grant support: Deutsche Forschungsgemeinschaft grants SFB432/A13 (W. Herr), SFB571/B7 (C.S. Falk), and SFB432/B6 (S. Strand) and Deutsche Krebshilfe grants 70-2428 (W. Herr) and 70-3344 (C.S. Falk).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank B. Mosetter and M. Brkic for excellent technical assistance.


   Footnotes
 
6 http://www.atcc.org. Back

Received 3/16/06. Revised 8/22/06. Accepted 9/18/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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