Limitations for TCR gene therapy by MHC-restricted fratricide and TCR-mediated hematopoietic stem cell toxicity.

The clinical use of lymphocytes engineered to express high affinity T-cell receptors (TCRs) specific for two broadly expressed tumor-associated antigens is strongly limited by MHC-restricted fratricide of lymphocytes and TCR-mediated killing of hematopoietic stem cells. Specific clinical applications must therefore be conceived to bypass these limitations.

High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination.

Our previously reported phase I clinical trial with the allogeneic gene-modified tumor cell line RCC-26/CD80/IL-2 showed that vaccination was well tolerated and feasible in metastatic renal cell carcinoma (RCC) patients. Substantial disease stabilization was observed in most patients despite a high tumor burden at study entry. To investigate alterations in immune responses that might contribute to this effect, we performed an extended immune monitoring that included analysis of reactivity against multiple antigens, cytokine/chemokine changes in serum and determination of the frequencies of immune suppressor cell populations, including natural regulatory T cells (nTregs) and myeloid-derived suppressor cell subsets (MDSCs). An overall immune response capacity to virus-derived control peptides was present in 100% of patients before vaccination. Vaccine-induced immune responses to tumor-associated antigens occurred in 75% of patients, demonstrating the potent immune stimulatory capacity of this generic vaccine. Furthermore, some patients reacted to peptide epitopes of antigens not expressed by the vaccine, showing that epitope-spreading occurred in vivo. Frequencies of nTregs and MDSCs were comparable to healthy donors at the beginning of study. A significant decrease of nTregs was detected after vaccination (p = 0.012). High immune response rates, decreased frequencies of nTregs and a mixed T helper 1/T helper 2 (T(H)1/T(H)2)-like cytokine pattern support the applicability of this RCC generic vaccine for use in combination therapies.

Human antitumor CD8+ T cells producing Th1 polycytokines show superior antigen sensitivity and tumor recognition.

Adoptive transfer of T cells expressing transgenic TCR with antitumor specificity provides a hopeful new therapy for patients with advanced cancer. To fulfill a large need for TCR with high affinity and specificity for various tumor entities, we sought to identify parameters for rapid selection of CTL clones with suitable characteristics. Twelve CTL clones displaying different Ag sensitivities for the same peptide-MHC epitope of the melanoma-associated Ag tyrosinase were analyzed in detail. Better MHC-multimer binding and slower multimer release are thought to reflect stronger TCR-peptide-MHC interactions; thus, these parameters would seem well suited to identify higher avidity CTL. However, large disparities were found comparing CTL multimer binding with peptide sensitivity. In contrast, CD8(+) CTL with superior Ag sensitivity mediated good tumor cytotoxicity and also secreted the triple combination of IFN-γ, IL-2, and TNF-α, representing a Th1 pattern often missing in lower avidity CTL. Furthermore, recipient lymphocytes were imbued with high Ag sensitivity, superior tumor recognition, as well as capacity for Th1 polycytokine secretion after transduction with the TCR of a high-avidity CTL. Thus, Th1 polycytokine secretion served as a suitable parameter to rapidly demark cytotoxic CD8(+) T cell clones for further TCR evaluation.

NOD/scid IL-2Rg(null) mice: a preclinical model system to evaluate human dendritic cell-based vaccine strategies in vivo.

BACKGROUND: To date very few systems have been described for preclinical investigations of human cellular therapeutics in vivo. However, the ability to carry out comparisons of new cellular vaccines in vivo would be of substantial interest for design of clinical studies. Here we describe a humanized mouse model to assess the efficacy of various human dendritic cell (DC) preparations. Two reconstitution regimes of NOD/scid IL2Rg(null) (NSG) mice with adult human peripheral blood mononuclear cells (PBMC) were evaluated for engraftment using 4-week and 9-week schedules. This led to selection of a simple and rapid protocol for engraftment and vaccine evaluation that encompassed 4 weeks. METHODS: NSG recipients of human PBMC were engrafted over 14 days and then vaccinated twice with autologous DC via intravenous injection. Three DC vaccine formulations were compared that varied generation time in vitro (3 days versus 7 days) and signals for maturation (with or without Toll-like receptor (TLR)3 and TLR7/8 agonists) using MART-1 as a surrogate antigen, by electroporating mature DC with in vitro transcribed RNA encoding full length protein. After two weekly vaccinations, the splenocyte populations containing human lymphocytes were recovered 7 days later and assessed for MART-1-specific immune responses using MHC-multimer-binding assays and functional assessment of specific killing of melanoma tumor cell lines. RESULTS: Human monocyte-derived DC generated in vitro in 3 days induced better MART-1-specific immune responses in the autologous donor T cells present in the humanized NSG mice. Moreover, consistent with our in vitro observations, vaccination using mature DC activated with TLR3 and TLR7/8 agonists resulted in enhanced immune responses in vivo. These findings led to a ranking of the DC vaccine effects in vivo that reflected the hierarchy previously found for these mature DC variations in vitro. CONCLUSIONS: This humanized mouse model system enables comparisons among different DC vaccine types to be rapidly assessed in vivo. In addition, ex vivo analyses of human CD3+ T cells recovered from the spleens of these mice are also possible, including studies on lymphocyte subsets, Th1/Th2 polarization, presence of regulatory T cells and the impact of DC vaccination on their functions.

TCR-transgenic lymphocytes specific for HMMR/Rhamm limit tumor outgrowth in vivo.

The hyaluronan-mediated motility receptor (HMMR/Rhamm) is overexpressed in numerous tumor types, including acute lymphoid leukemia and acute myeloid leukemia (AML). Several studies have reported the existence of T-cell responses directed against HMMR in AML patients that are linked to better clinical outcome. Therefore, we explored the use of HMMR-specific TCRs for transgenic expression in lymphocytes and their in vivo impact on HMMR(+) solid tumors and disseminated leukemia. We obtained TCRs via an in vitro priming approach in combination with CD137-mediated enrichment. Recipient lymphocytes expressing transgenic TCR revealed the specific tumor recognition pattern seen with the original T cells. Adoptive transfer experiments using a humanized xenograft mouse model resulted in significantly retarded solid tumor outgrowth, which was enhanced using IL-15-conditioned, TCR-transgenic effector memory cells. These cells also showed an increased potency to retard the outgrowth of disseminated AML, and this was further improved using CD8-enriched effector memory cells. To define a safe clinical setting for HMMR-TCR gene therapy, we analyzed transgenic T-cell recognition of hematopoietic stem cells (HSCs) and found on-target killing of HLA-A2(+) HSCs. Our findings clearly limit the use of HMMR-TCR therapy to MHC- mismatched HSC transplantation, in which HLA-A2 differences can be used to restrict recognition to patient HSCs and leukemia.

Third generation dendritic cell vaccines for tumor immunotherapy.

This review summarizes our studies of the past several years on the development of third generation dendritic cell (DC) vaccines. These developments have implemented two major innovations in DC preparation: first, young DCs are prepared within 3 days and, second, the DCs are matured with the help of Toll-like receptor agonists, imbuing them with the capacity to produce bioactive IL-12 (p70). Based on phenotype, chemokine-directed migration, facility to process and present antigens, and stimulatory capacity to polarize Th1 responses in CD4+ T cells, induce antigen-specific CD8+ CTL and activate natural killer cells, these young mDCs display all the important properties needed for initiating good antitumor responses in a vaccine setting.

Humanized tumor mice--a new model to study and manipulate the immune response in advanced cancer therapy.

The immunological impact on antibody-based anticancer therapies remains incompletely understood due to the lack of appropriate animal models for in vivo analysis. Here, we present a novel humanized tumor mouse (HTM) model, generated by concurrent transplantation of human hematopoietic stem cells (HSCs) and human breast cancer cells in neonatal NOD-scid IL2Rγ(null) mice. Five weeks after intrahepatic transplantation, a functional human immune system was developed in all organs, and, in addition, tumor cells were detectable in lung and bone marrow (early dissemination). After 3 months posttransplant, tumor-cell effusions and macroscopic tumors associated with liver or spleen were found. Furthermore, disseminated cells in different lymphoid and nonlymphoid organs were measurable. Tumor growth was accompanied by specific T-cell maturation and tumor cell-specific T-cell activation. In addition, Natural-Killer cell accumulation and activation were observed in HTM, which was further enhanced upon IL-15 treatment facilitating the possibility of immune cell modulation in, e.g., antibody-dependent cellular cytotoxicity-based immunotherapeutic approaches. This novel mouse model makes it possible to combine transfer of MHC mismatched tumor cells together with human HSCs resulting in a solid coexistence and interaction without evidence for rejection. Overall, humanized tumor mice represent a powerful in vivo model that for the first time permits the investigation of human immune system-related target cancer therapy and resistance.

Three-day dendritic cells for vaccine development: antigen uptake, processing and presentation.

BACKGROUND: Antigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility. METHODS: In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date. RESULTS: Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivt)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivtRNA. CONCLUSIONS: This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivtRNA as sources of specific antigen.

MHC-restricted fratricide of human lymphocytes expressing survivin-specific transgenic T cell receptors.

The apoptosis inhibitor protein survivin is overexpressed in many tumors, making it a candidate target molecule for various forms of immunotherapy. To explore survivin as a target antigen for adoptive T cell therapy using lymphocytes expressing survivin-specific transgenic T cell receptors (Tg-TCRs), we isolated HLA-A2-allorestricted survivin-specific T cells with high functional avidity. Lymphocytes expressing Tg-TCRs were derived from these T cells and specifically recognized HLA-A2+ survivin+ tumor cells. Surprisingly, HLA-A2+ but not HLA-A2- lymphocytes expressing Tg-TCRs underwent extensive apoptosis over time. This demise was caused by HLA-A2-restricted fratricide that occurred due to survivin expression in lymphocytes, which created ligands for Tg-TCR recognition. Therefore, survivin-specific TCR gene therapy would be limited to application in HLA-A2-mismatched stem cell transplantation. We also noted that lymphocytes that expressed survivin-specific Tg-TCRs killed T cell clones of various specificities derived from HLA-A2+ but not HLA-A2- donors. These results raise a general question regarding the development of cancer vaccines that target proteins that are also expressed in activated lymphocytes, since induction of high-avidity T cells that expand in lymph nodes following vaccination or later accumulate at tumor sites might limit themselves by self-MHC-restricted fratricide while at the same time inadvertently eliminating neighboring T cells of other specificities.

Generation of Th1-polarizing dendritic cells using the TLR7/8 agonist CL075.

In this paper, we describe a new method for preparation of human dendritic cells (DCs) that secrete bioactive IL-12(p70) using synthetic immunostimulatory compounds as TLR7/8 agonists. Monocyte-derived DCs were generated using a procedure that provided mature cells within 3 d. Several maturation mixtures that contained various cytokines, IFN-gamma, different TLR agonists, and PGE(2) were compared for impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation. Mixtures that included the TLR7/8 agonists R848 or CL075, combined with the TLR3 agonist polyinosinic:polycytidylic acid, yielded 3-d mature DCs that secreted high levels of IL-12(p70), showed strong chemotaxis to CCR7 ligands, and had a positive costimulatory potential. They also had excellent capacity to activate NK cells, effectively polarized CD4(+) and CD8(+) T cells to secrete IFN-gamma and to induce T cell-mediated cytotoxic function. Thereby, mature DCs prepared within 3 d using such maturation mixtures displayed optimal functions required for vaccine development.

Phase 1 trial of allogeneic gene-modified tumor cell vaccine RCC-26/CD80/IL-2 in patients with metastatic renal cell carcinoma.

Preclinical studies showed that the allogeneic tumor cell line RCC-26 displayed natural immunogenic potential that was enhanced through expression of CD80 costimulatory molecules and secretion of interleukin-2. Here we report the study of RCC-26/CD80/IL-2 cells in a phase 1 vaccine trial of renal cell carcinoma patients with metastatic disease (mRCC). Fifteen patients of the HLA-A*0201 allotype, with at least one metastatic lesion, were included. Irradiated vaccine cells were applied in increasing doses of 2.5, 10, and 40 x 10(6) cells over 22 weeks. Primary study parameters included safety and toxicity. Sequential blood samples were analyzed by interferon-gamma enzyme-linked immunospot assays to detect tumor antigen-associated (TAA) effector cells. The vaccine was well tolerated and the designated vaccination course was completed in 9 of 15 patients. Neither vaccine-induced autoimmunity nor systemic side effects were observed. Delayed-type hypersensitivity skin reactions were detected in 11 of 12 evaluated patients and were particularly strong in patients with prolonged survival. In parallel, vaccine-induced immune responses against vaccine or overexpressed TAA were detected in 9 of 12 evaluated patients. No tumor regressions occurred according to RECIST (Response Evaluation Criteria in Solid Tumors) criteria; however, median time to progression was 5.3 months and median survival was 15.6 months, indicating substantial disease stabilization. We conclude that vaccine use was safe and feasible in mRCC. Clinical benefits were limited in these patients with advanced disease; however, immune monitoring revealed vaccine-induced responses against multiple TAAs in the majority of study participants. These results suggest that this vaccine could be useful in combination therapies and/or minimal residual disease.

Dendritic cells pulsed with RNA encoding allogeneic MHC and antigen induce T cells with superior antitumor activity and higher TCR functional avidity.

Adoptive transfer of T cells expressing transgenic T-cell receptors (TCRs) with antitumor function is a hopeful new therapy for patients with advanced tumors; however, there is a critical bottleneck in identifying high-affinity TCR specificities needed to treat different malignancies. We have developed a strategy using autologous dendritic cells cotransfected with RNA encoding an allogeneic major histocompatibility complex molecule and a tumor-associated antigen to obtain allo-restricted peptide-specific T cells having superior capacity to recognize tumor cells and higher functional avidity. This approach provides maximum flexibility because any major histocompatibility complex molecule and any tumor-associated antigen can be combined in the dendritic cells used for priming of autologous T cells. TCRs of allo-restricted T cells, when expressed as transgenes in activated peripheral blood lymphocytes, transferred superior function compared with self-restricted TCR. This approach allows high-avidity T cells and TCR specific for tumor-associated self-peptides to be easily obtained for direct adoptive T-cell therapy or for isolation of therapeutic transgenic TCR sequences.

Prostate cancer lesions are surrounded by FOXP3+, PD-1+ and B7-H1+ lymphocyte clusters.

The immune response against prostate cancer seems to be inefficient although tumour cells show an over-expression of tumour-associated antigens suggesting that regulatory networks inhibit immune cell function locally. To address this proposition, lymphocytes within prostate cancer-inflicted tissue were analysed for the expression of markers associated with negative regulatory function and exhaustion. Prostate cancer, benign prostatic hyperplasia and healthy prostate tissues were investigated by immunohistology for CD25, FOXP3, PD-1 and B7-H1. We had previously documented that prostate cancer islets are surrounded by clustered accumulations of CD3+ lymphocytes, which lack perforin and interferon-gamma (IFNgamma) expression, thus are apparently quiescent. Here, we report that these clusters contain numerous CD25+ and FOXP3+ cells. These markers are associated with regulatory T cells, and their presence in lymphocyte clusters near prostate cancer regions indicates an environment with negative impact on immune response against cancer cells. Consistent with this hypothesis, cells expressing PD-1 and its ligand B7-H1, which are markers associated with exhaustion of lymphocyte function, were also detected in the lymphocyte clusters. Expression of molecules associated with inhibition and exhaustion of lymphocytes may reflect events contributing to ineffective immune responses against cancer cells.

Harnessing innate and adaptive immunity for adoptive cell therapy of renal cell carcinoma.

The development of immunotherapies for renal cell carcinoma (RCC) has been the subject of research for several decades. In addition to cytokine therapy, the benefit of various adoptive cell therapies has again come into focus in the past several years. Nevertheless, success in fighting this immunogenic tumor is still disappointing. RCC can attract a multitude of different effector cells of both the innate and adaptive immune system, including natural killer (NK) cells, gammadelta T cells, NK-like T cells, peptide-specific T cells, dendritic cells (DC), and regulatory T cells (Tregs). Based on intensive research on the biology and function of different immune cells, we now understand that individual cell types do not act in isolation but function within a complex network of intercellular interactions. These interactions play a pivotal role in the efficient activation and function of effector cells, which is a prerequisite for successful tumor elimination. This review provides a current overview of the diversity of effector cells having the capacity to recognize RCC. Aspects of the functions and anti-tumor properties that make them attractive candidates for adoptive cell therapies, as well as experience in clinical application are discussed. Improved knowledge of the biology of this immune network may help us to effectively harness various effector cells, placing us in a better position to develop new therapeutic strategies to successfully fight RCC.

A polyvalent cellular vaccine induces T-cell responses against specific self-antigens overexpressed in chronic lymphocytic B-cell leukemia.

B cell-derived chronic lymphocytic leukemia (CLL) is an incurable disease that requires innovative therapeutic regimens. Experimental approaches of immunotherapy aiming at induction of systemic T-cell responses have been developed. Trioma cells provide a potent vaccine derived from malignant B cells that allows multiple antigens (Ags) from the parental tumor to be ingested by Ag-presenting cells. Like other strategies using modified whole tumor cells, this approach induces polyvalent responses. Using trioma cell-pulsed dendritic cells (DCs) for T-cell activation in vitro, we asked whether specific Ags overexpressed by CLL can be identified as target structures of such responses and what is the nature of these Ags. Expression levels of several genes in CLL samples were quantitated by reverse transcriptase-polymerase chain reaction. T lymphocytes were polyvalently stimulated by trioma-pulsed DCs and specificities were tested by determining cytokine secretion in the presence of target cells transfected with RNA coding for those Ags that were found to be overexpressed. We demonstrate that DCs pulsed with the modified tumor cells efficiently activate T lymphocytes against CLL and that overexpressed Ags related to leukemogenesis, such as BCL-2, MDM2, and ETV5, serve as targets for those T cells. Immune escape by Ag loss or mutation is less likely to occur if immunity is directed against altered self-proteins that are involved in malignant transformation. Therefore, vaccines based on modified tumor cells such as triomas hold promise for immunotherapy of CLL and other malignancies. Polyvalent vaccines originally designed as individualized therapeutics may be more broadly applicable, at least in patients showing similar Ag patterns.

Antitumor effector functions of T cells are dependent on in vivo priming and restricted T-cell receptor expression.

Tumor-specific T cells are crucial for immunologic control of malignant disease. T cells can be induced in vivo by vaccination or adoptively transferred after activation ex vivo. We investigated the requirements for generating T cells with optimal antitumor effector functions in a murine lymphoma model. Using adoptive transfer, we show that in vivo efficacy of T cells cannot be predicted by tumor reactivity in vitro. A restricted T-cell receptor beta chain repertoire of T-cell populations stimulated ex vivo against tumor cells was necessary but not sufficient for tumor protectivity. Tumor elimination furthermore required vaccination of donor mice, hence in vivo priming. The in vivo priming step may allow tumor-specific T cells to accumulate in vitro more rapidly and to survive for longer periods after withdrawal of the antigenic stimulus and adoptive transfer. A possible survival benefit of in vivo induced T cells may be ascribed to the responsiveness to homeostatic cytokines and to unique cytokine milieus encountered in vivo. Most importantly, monoclonal T cells cannot inhibit tumor growth. A prerequisite of tumor rejection was the expression of at least 2 T-cell receptor beta chains by transferred T-cell populations. This finding has implications for designing adoptive transfer strategies for the clinic.

Allorestricted T cells with specificity for the FMNL1-derived peptide PP2 have potent antitumor activity against hematologic and other malignancies.

Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor leukocyte infusion has curative potential, especially in hematologic malignancies. However, this approach is severely restricted due to graft-versus-host disease (GvHD). This limitation may be overcome if target antigens are molecularly defined and effector cells are specifically selected. We chose formin-related protein in leukocytes 1 (FMNL1) as a target antigen after intensive investigation of its expression profile at the mRNA and protein levels. Here, we confirm restricted expression in peripheral blood mononuclear cells (PBMCs) from healthy donors but also observe overexpression in different leukemias and aberrant expression in transformed cell lines derived from solid tumors. We isolated allorestricted T-cell clones expressing a single defined TCR recognizing a particular HLA-A2-presented peptide derived from FMNL1. This T-cell clone showed potent antitumor activity against lymphoma and renal cell carcinoma cell lines, Epstein-Barr virus (EBV)-transformed B cells, and primary tumor samples derived from patients with chronic lymphocytic leukemia (CLL), whereas nontransformed cells with the exception of activated B cells were only marginally recognized. Allorestricted TCRs with specificity for naturally presented FMNL1-derived epitopes may represent promising reagents for the development of adoptive therapies in lymphoma and other malignant diseases.

Immune suppression in renal cell carcinoma.

The clear evidence that tumor-infiltrating lymphocytes with anti-tumor activity exist in situ raises the question why renal cell carcinomas (RCCs) progress in vivo. A complex array of factors and pathways has been identified that impinges on innate and adaptive effector cells thereby inhibiting their activity against RCCs. The current picture of suppressive mechanisms that contribute to the failure of the immune system to control RCCs is reviewed here. Understanding these complex host-tumor interactions has broad implications for successful application of cytokine therapy and other forms of immunotherapy for RCC.

Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70.

BACKGROUND: For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4+ T helper 1 cells, which then support the development of CD8+ cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development. METHODS: After elutriation of leukapheresis products in a closed bag system, enriched monocytes were cultured with GM-CSF and IL-4 for six days to generate immature DCs that were then matured with cocktails, containing cytokines, interferon-gamma, prostaglandin E2, and a ligand for Toll-like receptor 8, with or without poly (I:C). RESULTS: Mature DCs expressed appropriate maturation markers and the lymph node homing chemokine receptor, CCR7. They retained full maturity after culture for two days without maturation cocktails and following cryopreservation. TLR ligand stimulation induced DCs capable of secreting IL-12p70 in primary cultures and after one day of coculture with CD40L-expressing fibroblasts, mimicking an encounter with T cells. DCs matured with our new cocktails containing TLR8 ligand, with or without poly (I:C), induced alloresponses and stimulated virus-specific T cells after peptide-pulsing. DCs matured in cocktails containing TLR8 ligand without poly (I:C) could also be loaded with RNA as a source of antigen, whereas DCs matured in cocktails containing poly (I:C) were unable to express proteins following RNA transfer by electroporation. CONCLUSION: Our new maturation cocktails allowed easy DC harvesting, stable maturation and substantial recoveries of mature DCs after cryopreservation. Our procedure for generating DCs is easily adaptable for GMP-compliance and yields IL-12p70-secreting DCs suitable for development of cancer vaccines using peptides or RNA as sources of immunizing antigens.

Activated B cells mediate efficient expansion of rare antigen-specific T cells.

Potent professional antigen-presenting cells (APC) are essential tools to activate and expand antigen-specific T cells in vitro for use in adoptive immunotherapy. CD40-activated B cells can be easily generated and propagated from human donors and have been successfully used to generate antigen-specific T-cell cultures. Here we show that CD40-activated B cells strongly and specifically expand rare populations of antigen-specific CD8 T cells, with frequencies of less than 1 in 20,000 CD8 T cells in peripheral blood. We focused on T cells recognizing an epitope from the human papillomavirus 16 (HPV-16) E7 protein. In 6 of 6 healthy donors, epitope-specific CD8+ T cells were found to be "rare" by this criterion, as shown by staining with human leukocyte antigen (HLA)/peptide multimers. Using peptide-loaded CD40-activated B cells, epitope-specific T cells could be selectively expanded in all donors up to 10(6) fold, and the resulting T-cell cultures contained up to 88% specific T cells. These results strongly encourage the use of CD40-stimulated B cells as APCs in immunotherapy.