TBS, SS, MR, and PPL designed the study. TBS, SPH, SS, MR, JSW, and PPL analyzed the data. MR provided key validated reagents for the study. AT and JSW enrolled patients. TBS, SPH, SS, AT, MR, JSW, and PPL contributed to writing the paper.
The authors have declared that no competing interests exist.
Melanoma patients vaccinated with tumor-associated antigens frequently develop measurable peptide-specific CD8+ T cell responses; however, such responses often do not confer clinical benefit. Understanding why vaccine-elicited responses are beneficial in some patients but not in others will be important to improve targeted cancer immunotherapies.
We analyzed peptide-specific CD8+ T cell responses in detail, by generating and characterizing over 200 cytotoxic T lymphocyte clones derived from T cell responses to heteroclitic peptide vaccination, and compared these responses to endogenous anti-tumor T cell responses elicited naturally (a heteroclitic peptide is a modification of a native peptide sequence involving substitution of an amino acid at an anchor residue to enhance the immunogenicity of the peptide). We found that vaccine-elicited T cells are diverse in T cell receptor variable chain beta expression and exhibit a different recognition profile for heteroclitic versus native peptide. In particular, vaccine-elicited T cells respond to native peptide with predominantly low recognition efficiency—a measure of the sensitivity of a T cell to different cognate peptide concentrations for stimulation—and, as a result, are inefficient in tumor lysis. In contrast, endogenous tumor-associated-antigen-specific T cells show a predominantly high recognition efficiency for native peptide and efficiently lyse tumor targets.
These results suggest that factors that shape the peptide-specific T cell repertoire after vaccination may be different from those that affect the endogenous response. Furthermore, our findings suggest that current heteroclitic peptide vaccination protocols drive expansion of peptide-specific T cells with a diverse range of recognition efficiencies, a significant proportion of which are unable to respond to melanoma cells. Therefore, it is critical that the recognition efficiency of vaccine-elicited T cells be measured, with the goal of advancing those modalities that elicit T cells with the greatest potential of tumor reactivity.
State-of-the art analysis of patients' response to melanoma vaccines yields lessons about cancer vaccines and rationale vaccine design in general.
Our immune system protects us against infectious diseases. It can also recognize and destroy early cancer cells before they form tumors. Researchers have been trying to find a way to boost the anti-cancer function of the immune system so that it can kill even established tumors. This is the idea behind developing vaccines for treating cancer—the vaccine alerts and boosts the patient's immune system and so helps to fight the cancer. The idea of enlisting the immune system against cancer has been around for a long time. There have been some spectacular successes, but it has proven difficult to find vaccines that work in more than just a few patients. And we don't yet understand why vaccines seem to work in some patients but not in others.
Peter Lee and colleagues are trying to find out why some patients respond to vaccines and others don't by looking at the immune response in vaccinated patients. In this study, using state-of-the art technology, they examined patients who received different vaccines against the skin cancer melanoma. They concentrated on the so-called killer T cells (cytotoxic T cells), which directly attack and kill tumor cells, and analyzed them in great detail.
Most cytotoxic T cells produced by patients after vaccination—including vaccination with so-called heteroclitic peptides that had been specifically designed to provoke a very strong immune response—did not kill tumor cells very well, but a few of them did. These results provide some explanation as to why cancer vaccines haven't been as successful as many had hoped, but also suggest that if it were possible to get more of the potent T cells or to expand the ones that are already produced with the current vaccines, there would be a stronger anti-tumor response.
How to get effective cancer vaccines remains an open question. But at least technologies such as those used in this study now exist that allow researchers to analyze how the immune systems of different patients react to vaccination and hence can guide the development of better vaccines.
The Cancer Research Institute:
US Food and Drug Administration page on cancer vaccines:
University of Michigan page on cancer vaccines:
The immunotherapy of cancer holds promise in harnessing the host immune response to specifically target tumor cells without harming normal tissues. Strategies involve adoptive cellular therapy or active immune induction (commonly referred to as “cancer vaccination”). Cancer vaccines may consist of whole tumor cells or tumor lysates, but identification of tumor-associated antigens (TAAs) over the past decade has made possible the use of specific proteins or peptides as cancer vaccines. The anti-tumor potential of TAA-specific CD8+ T cells has been illustrated by the demonstrated capacity of adoptive T cell therapy to reduce tumor size [
Various reasons for the paradoxical coexistence of cancer cells and TAA-specific T cells within patients have been proposed [
While isolated T cell clones with low RE have indeed been generated from melanoma patients following heteroclitic peptide vaccination, the proportion of vaccine-elicited T cell responses these cells represent in vivo is not clear. If predominantly high-RE, tumor-cytolytic T cells are generated, then a small fraction of low-RE T cells generated would be of little consequence. However, if predominantly low-RE T cells are generated, then this low proportion of high-RE T cells may be an important factor in the observed lack of clinical effectiveness of current cancer vaccination strategies. To address this important issue, we undertook a systematic examination of the complexity of T cell responses induced by heteroclitic peptide vaccination, and compared these responses to endogenous anti-tumor T cell responses which develop in some patients. Typically, responses to vaccination are examined following in vitro expansion from patient samples, which may alter the composition of cells and consequently not reveal the proportion of cells in vivo having sufficiently high RE to lyse tumor targets. Although staining with peptide–MHC tetramers provides a direct estimate for the number of TAA-specific T cells present in vivo, and intensity of tetramer staining has been employed as a parameter for isolation of high-RE, tumor-lytic T cells [
To analyze and compare T cell responses in melanoma patients on a single-cell level, we generated and examined a large number of cytotoxic T lymphocyte (CTL) clones derived from post-vaccination or endogenous anti-tumor T cell responses. Each clone was analyzed for T cell receptor (TCR) variable chain beta (VB) expression, RE, and ability to lyse melanoma targets. Importantly, these clones were generated directly ex vivo through tetramer-guided sorting, which minimizes the selection bias that could be introduced by prior in vitro expansion. Therefore, data from these clones could be taken to estimate the complexity of the responses in vivo.
All patients had resected stage III or IV melanoma, as determined by the 1988 modified American Joint Commission on Cancer staging system. They were required to have a magnetic resonance imaging or computed tomographic scan of the head and computed tomographic scans of the chest, abdomen, and pelvis showing no indication of disease within 4 wk of therapy to verify that they were clinically free of melanoma. Eligibility criteria included age 18 y or older, creatinine of less than 180 μmol/l, bilirubin of less than 110 μmol/l, platelet count of 100 × 109/l or more, hemoglobin of 90 g/l or more, and total white blood cell count of 3.0 × 109/l or greater. Tests for human immunodeficiency virus, hepatitis C antibody (Ab), and hepatitis B surface antigen were required to be negative, and all patients were HLA-A2 antigen positive by a microcytotoxicity assay. All patients were required to comprehend and sign an informed consent form approved by the National Cancer Institute (NCI; Bethesda, Maryland, United States) and the Los Angeles County/University of Southern California Institutional Review Board. Analysis of the patient samples was approved by Stanford University's Institutional Review Board. Peripheral blood mononuclear cell (PBMC) samples were isolated from patients after vaccination with the heteroclitic peptides MART 26–35 (27L) (ELAGIGILTV) and gp100 209–217 (210M) (IMDQVPSFV) at the University of Southern California Norris Cancer Center (Los Angeles, California, United States). Clinical-grade peptides used were provided by the Cancer Therapy Evaluation Program of the NCI under an Investigational New Drug application BB 6123 held by the NCI. Immunizations (1 mg of each peptide emulsified with incomplete Freund's adjuvant) were administered every 2 wk for 8 wk, then every 4 wk for 12 wk, and then once 8 wk later. PBMC samples were collected 4 wk after the final immunization and stored at −130 °C. Samples were thawed the day before an experiment for overnight culture in CTL medium. The following morning, viable cells were isolated by ficoll density centrifugation, washed, and resuspended to the appropriate concentration in a solution of 90% Iscove's Modified Dulbecco's Medium (IMDM) and 10% fetal bovine serum (FBS).
For isolation and detection of peptide-specific T cells, patient PBMC samples were stained and analyzed by fluorescence-activated cell sorting (FACS) as previously described [
The HLA-A*0201-positive melanoma lines Malme-3M and A375 and the T2 cell line were purchased from ATCC (Manassas, Virginia, United States) and maintained according to instructions provided by the ATCC. The HLA-A*0201-positive melanoma line mel526 was obtained from the Surgery Branch of the NCI. While Malme-3M and mel526 express both MART and gp100, A375 does not express MART or gp100 and served as a negative control. Expression (or lack thereof) of these antigens by each cell line was further confirmed by immunohistochemical staining. Cells were trypsinized using Trypsin/EDTA solution (GIBCO, San Diego, California, United States) before use. T2 cells were HLA-A2.1+ and were pulsed prior to assays with peptides indicated in the text.
Effector cells, which include clones, cell line, and PBMC samples, were frozen and analyzed in batches. The cells were thawed the day before an experiment for overnight culture in CTL medium. The following morning, viable cells were isolated by ficoll density centrifugation, washed, and resuspended to the appropriate concentration (usually 107/ml) in CTL medium.
All assays were done at least twice, with duplicates for each condition. The effector to target (E:T) ratio used was generally 1:2, with 2 × 105 for clones or 106 for the cell line and patient PBMC samples. To each well, the following was added in order: 1 μl of 2 mM monensin (Sigma, St. Louis, Missouri, United States) in 100% EtOH, 100 μl of target cells, 100 μl of effector cells, and 1 μl of CD107-APC Abs. The cells were mixed well using a multichannel pippetor. The plate was centrifuged at 300
CD8+ T cell clones were derived by FACSorting individual tetramer-positive cells from PBMC samples prepared for flow cytometry as described above. CD8+ tetramer-positive T cells were sorted under sterile conditions into 96-well plates, one cell per well, using a FACS Vantage (Becton Dickinson). Wells contained 100 μl of CTL IMDM, with 10% FBS, 2% human AB sera, and penicillin, streptomycin, and L-glutamine, supplemented with 100 units/ml IL-2. Sorted cells were expanded in vitro using standard protocols. Briefly, irradiated feeder cells (JY cells and fresh PBMCs) were added to wells containing the sorted T cells, and the 96-well plates were incubated at 37 °C with 7% CO2 to allow for growth. Potential clones became visible around day 14 and were then transferred to 24-well plates containing 1 ml of CTL medium with 100 units/ml IL-2. Wells were selected based on cell confluency for expansion and further analysis. Clones confirmed to be tetramer-positive were expanded in T-25 flasks containing irradiated JY cells and fresh PBMCs in 25 ml of CTL medium containing PHA. IL-2 was added to a final concentration of 50 units/ml on day 1 and then every 2 d thereafter for 2 wk.
Target cells were as described above under CD107 Mobilization Assays, and were labeled overnight with 51Chromium, washed, and resuspended to 105 cells/ml. One hundred microliters of target cells were incubated with 100 μl CTL clones at 10:1 E:T ratio for 4 h. Percent specific release of 51Chromium from target cells was calculated from 40-μl cell-free supernatants.
Chromium-labeled T2 targets were pulsed with a range of peptide concentrations, generally starting at 10−7 M and decreasing by log steps to 10−13 M. T cell clones were incubated with T2 targets at 10:1 E:T ratios for 4 h, then chromium release was measured and percentage cytotoxicity calculated by standard methods. Prior to each cytotoxicity assay, clones underwent ficoll-hypaque centrifugation to remove dead feeder cells and were determined to be greater than 80% CD8+ tetramer-positive T cells by FACS. The E:T ratio was based upon live T and target cells. For each T cell clone, percent cytotoxicity was plotted against peptide concentration. The peptide concentration at which the curve crossed 40% cytotoxicity was defined as the RE of that clone [
Cells were isolated directly from PBMCs from patient 422 by FACS as described above. Cells were collected in microfuge tubes containing 1 ml of ice-cold 90% IMDM with 10% FBS. Collected cells were washed and resuspended to 83,300 cells/ml in 90% IMDM with 10% FBS. Targets were prepared as described above and resuspended to 8,300 cells/ml in 90% IMDM with 10% FBS. A total of 2,500 sorted cells (30 μl) and 250 target cells (30 μl) were transferred to a microcentrifuge tube (VWR International, West Chester, Pennsylvania, United States), centrifuged 1 min at 200
RNA was extracted from clones and tetramer-positive cells using TRIzol (Invitrogen, Carlsbad, California, United States) and reverse-transcribed into cDNA using SuperScript II Reverse Transcriptase (Invitrogen). PCR was performed using 34 different 5′ primers that specifically amplify all functional TCR VB genes. Most of the 5′ primers used have been previously described [
A standard software package (SigmaPlot 5.0, Systat Software, Richmond, California, United States) was used to provide descriptive statistical plots. Barcharts were provided with standard errors on them. Linear plots were provided with standard errors computed at each point. A linear regression (using least squares) of percent specific lysis on recognition efficiency is shown in
CTL clones 476.105 and 132.1 were assayed for lysis of T2 cells pulsed with 10-fold dilutions of (A) native or (C) heteroclitc peptide at concentrations ranging from 100 fg/ml to 100 ng/ml. (B) Lysis of Malme-3M melanoma cells by 476.105 and 132.1 CTLs. All assays were performed in triplicate, and each clone was assayed twice. Error bars reflect variation between two separate assays.
CTL clones representing different tetramer-positive populations in each patient expressing different VB were assayed for lysis of T2 cells pulsed with various dilutions of G209n, G209–2M, M27, or M26 peptides in 51Chromium release cytotoxicity assays as described in
(A and B) RE scores for both (A) MART-specific and (B) gp100-specific clones from all patients were correlated with efficiency in lysing melanoma cells. Correlation coefficients were 0.66 for MART-specific clones and 0.81 for gp100-specific clones.
(C–F) Comparison of RE scores for endogenous (patients 461 and 132) and vaccine-induced (patients 517, 520, 422 and 476) responses.
(C and D) RE analysis with native peptides (C) M27 and (D) G209n. Mean RE (weighted) for each response is indicated with horizontal bars. Weighted means were based on all clones, not only those assayed, and were estimated by summing the RE of each analyzed clone multiplied by the number of total clones expressing the same VB, in each patient. Weighted means were as follows: patient 517, 5.7; patient 520, 7.0; patient 461, 7.9; patient 422, 9.7; patient 476, 9.9; and patient 132, 11.2. One-tailed T-tests demonstrated that endogenous responses had significantly higher RE than vaccine-induced responses: patient 461 versus patient 517,
(E and F) RE analysis with heteroclitic peptides (E) M26 and (F) G209–2M. Weighted means were as follows: patient 517, 10.6; patient 520, 11.1; patient 461, 11.2; patient 422, 10.5; patient 476, 11.6; and patient 132, 11.3.
To address the complexity of T cell responses against melanoma in vivo, patients with vaccine-induced or endogenous TAA-specific responses were selected. In recent cancer vaccine trials [
(A) Six patients with T cell responses reactive with for M26 or G209–2M tetramers were selected for analysis. PBMCs from each patient were stained with PE-conjugated peptide–MHC tetramers, G209–2M-tet PE or M26-tet PE, and co-stained with anti-CD8 fluorescein isothiocyanate and anti-CD14, -CD19, and -CD4 Cy5PE. The plots shown are gated for CD8+, CD14−, CD19−, and CD4− cells. Tetramer-positive cells are boxed and estimated for percent of total CD8+ cells: patient 422, 2.5%; patient 476, 0.31%; patient 132, 0.22%; patient 517, 0.23%; patient 520, 0.12%, and patient 461, 0.50%.
(B) Microcytotoxicity 51Chromium release assay with tetramer-positive cells isolated by FACS from the CD8+ PBMC population from patient 422. Isolated cells were assayed for lysis of T2 cells treated with relevant or irrelevant peptide, or mel526 melanoma cells. Sorted cells were combined with 250 target cells at 13:1 E:T ratios for 4 h, and supernatants were assayed for percent specific release of radiolabel.
Patient 422 had the largest detectable TAA-specific CD8+ T cell response (2.5% G209–2M-tetramer-positive) and thus sufficient numbers for examination of lytic function immediately following isolation. To test whether peptide-vaccine-induced T cell responses were functionally active directly ex vivo, T cells isolated by G209–2M-tetramer-guided cell sorting from patient 422 were tested for lysis of peptide-pulsed and melanoma target cells in microcytotoxic assays (
To assess the functional status of the smaller TAA-specific CD8+ T cell responses in the other five patients—which were too small for direct cytotoxicity assays after sorting—we utilized a novel FACS assay for degranulation based on CD107 mobilization [
Functional response was determined by percent of G209–2M- and M26-tetramer-positive cells that mobilized CD107 and/or downregulated CD3 complex in response to incubation with T2 cells pulsed with 100 ng/ml of cognate peptide (G209–2M or M26), mel526, or Malme-3M melanoma cells. A375 melanoma cells served as negative control
To substantiate the generality of these findings, we analyzed four additional patients with vaccine-elicited responses. One subject responded to G209–2M only (patient 722), one to M26 only (patient 713), and two to both G209–2M and M26 (patients 721 and 735). Similar to the first four vaccine-elicited patients, these four additional patients (six TAA-specific responses in total) exhibited variable reactivity to melanoma targets, ranging from 13% to 49.6% (
To confirm and further investigate the differences in tumor reactivity between endogenous and vaccine-elicited responses, we reasoned that analysis of a set of clonal CTL lines that represented the tetramer-positive population would provide an accurate estimate of the complexity of the TAA-specific T cell response in each patient. A large number of clonal CTL lines (more than 200) were generated by FACS of individual G209–2M- and M26-tetramer-positive cells directly from PBMC samples (
aClonal CTL lines were established from each patient
bNumber of clonal CTL lines from each patient expressing the same TCR VB chain
cPercent of G209–2M- and M26-tetramer-reactive CD8+ T cells in each patient expressing the indicated TCR VB chain
PCR fragment length was determined for selected clones and for sorted G209–2M- or M26-tetramer-positive populations from which the clones were derived. Numbers indicate length in base-pairs of fragments generated by PCR with VB14 or VB17 5′ primer and BC63 constant region 3′ primer followed by a run-off reaction with VB-specific nested primers and FAM-labeled BC63 primer. Fragments were analyzed using an Applied Biosystems 377 automated sequencer and GeneScan software
The TCR VB usage of each CTL clone was determined using a panel of 19 anti-VB monoclonal Abs by flow cytometry or by PCR with 34 VB-specific primers. All clones selected for functional analysis were assayed for lysis of melanoma cells. Some clones were also subjected to RE analysis
M, assay for lysis of melanoma cells; MR, RE analysis
Peptide specificity and CD8 expression of each clone was confirmed by staining with G209–2M- and M26-tetramers and anti-CD8 monoclonal Ab (data not shown). To obtain an accurate reflection of the total T cell population detected with tetramer in each patient, we decided to rigorously examine at least one representative clone for each subpopulation expressing a different TCR VB (
To determine the effectiveness of tumor lysis by the different TAA-specific T cell clones that were propagated, clones were analyzed for their ability to lyse melanoma cell lines mel526 and Malme-3M. A375 cells served as a control for antigen-specific killing. In addition, each CTL clone was examined for antigen-specific lysis of T2 cells pulsed with high levels (1μg/ml) of G209–2M or M26 peptides. “Efficient lysis” in these experiments was defined as 40% or greater specific release of radiolabel from the target cells; 10% or less specific release was categorized as “low or no lysis,” and 10% to 40% was termed “intermediate lysis.” All but two of the CTL clones elicited from endogenous anti-tumor responses (from patients 132 and 461) exhibited “efficient lysis” of both the mel526 and Malme-3M melanoma cell lines (
Cells from 87 clonal CTL lines were assayed for lysis of melanoma cells mel526, Malme-3M, and A375 in 51Chromium release cytotoxicity assays. Mel526 and Malme-3M are HLA-A2.1+ and express both gp100 and MART-1. A375 cells are HLA-A2.1+ but do not express either gp100 or MART-1 and served as a negative control. T2 cells treated with 1 μg/ml G209–2M or M26 peptides served as controls for antigen-specific lysis. The CTL clones assayed were selected to represent different tetramer-positive subsets expressing different VB. Dominating tetramer-positive populations in each patient were represented with two or more clones. Each CTL clone was assayed in triplicate wells, and the data displayed are averages of two different experiments. Clones from the same patient expressing similar VB while exhibiting different lysis potential were viewed as separate subsets. Each assay was performed at 10:1 E:T ratio as detailed in Methods. The height of each bar represents percent specific lysis, while the width represents the relative size of the tetramer-positive subpopulations (defined by VB expression) in each patient. Population size was defined as the percent of clones from each patient expressing the same VB. Error bars show standard deviation between two experiments within each clone and/or between different clones where more than one clone was analyzed.
CTL clones derived from each patient were classified as “efficient” (greater than 40%), “intermediate” (between 10% and 40%), or “low/no” (less than 10%) in lysis of melanoma cells based on data displayed in
We hypothesized that CTL clones that did not efficiently lyse melanoma targets may be incapable of recognizing the relatively low surface densities of native peptide present on tumor cells. CTL clones selected for analysis of tumor lysis were also assessed for RE for the native and heteroclitic peptides via a ten-log range of dilutions. This is illustrated with clones 132.1 and 476.105 (
Similar RE assays were performed for the remaining clones from each patient selected for analysis. In order to compare REs of various CTL lines, each clone was assigned an RE score expressed as the negative log10 value of the peptide concentration required for 40% specific lysis at an E:T ratio of 10:1. For clones 132.1 and 476.105, these scores were 11.1 and 8.3 for assays with G209n peptide (
To achieve maximal clinical responses, the majority of T cells elicited by vaccination in cancer patients should be capable of responding to tumor targets. We have undertaken the most detailed analysis to date, on a single-cell level, of T cell responses elicited by cancer vaccination and have compared these with endogenous anti-tumor responses. To evaluate the full spectrum of T cells elicited in each patient by vaccination, we utilized tetramers made with the vaccine peptides (heteroclitic M26 and G209–2M) to isolate such cells. CTL clones were selected directly from patient PBMC samples without enrichment in culture to closely reflect the composition of the antigen-specific T cell response in vivo at the time of isolation.
Our data revealed that T cell populations induced by vaccination were significantly different from endogenous responses: while some CTLs elicited by vaccination could kill melanoma targets, most were inefficient in tumor cell lysis. In contrast, nearly all clones from endogenous responses were efficient at melanoma cell lysis. This difference was related to RE for the native peptide. Clones that did not lyse tumor cells required up to 103-fold higher concentration of peptide for similar levels of lysis of targets compared to T cell clones that were tumor-lytic. Side-by-side comparison of endogenous responses and vaccine-induced responses suggests that low RE TAA-specific T cell responses may be preferentially driven by heteroclitic peptide vaccination. Thus, high doses of peptide and/or the higher levels of expression of heteroclitic peptide on APCs may induce and actively propagate predominantly T cells with RE too low for recognition of physiological levels of the native peptide present on tumor targets. These data suggest an inverse relationship between antigen density and the RE of T cells elicited. This would be an important consideration in design of future vaccine strategies.
Differential recognition of native and heteroclitic peptides by many T cells may also account for the induction of non-tumor-lytic clones by heteroclitic peptide vaccines, which has been suggested previously [
Another implication of this study is that the number of cells measured by current methods, including ELISPOT or staining with MHC tetramers, may not correlate directly with the RE or tumor reactivity of T cell responses to vaccination. For example, of the nine clones analyzed from patient 517, none were efficient in tumor cell lysis, yet these cells were detectable by MHC tetramer staining. T cells with low RE for native TAA do not efficiently lyse tumor, and therefore are unlikely to have an impact on clinical outcome. Furthermore, it may be possible that low-RE TAA-specific T cells may interfere with elicitation of high-RE T cells, either by direct competition for antigen on APC surface [
Our data support the notion that not only quantity, but quality, of the T cell response elicited by vaccination may be important for clinical efficacy. There are a number of strategies to increase the magnitude of T cell responses to peptide vaccines. These include using various adjuvants, such as incomplete Freund's adjuvant and immunomodulatory agents, such as IL-12 [
In summary, we have demonstrated that vaccination with heteroclitic peptide at high concentrations may drive T cell responses of variable tumor-cytolytic potential in cancer patients—and that the ability to lyse tumor cells correlates with the T cell's RE for native peptides. This represents an important—but not sole—factor in explaining the lack of correlation between immunological and clinical responses after vaccination for cancer. Importantly, the situation is different in endogenous responses, in which cells are predominantly of high RE. This suggests that the manner in which T cells are elicited in vivo are different in these two settings and may underlie their differences in biology.
This work was supported by National Institutes of Health R01 CA 090809 (PL) and the Damon Runyon Cancer Research Foundation (Scholar Award to PL). Part of this work was supported by research grant NSF DMS 0241246 to SPH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
antibody
antigen-presenting cell
cytotoxic T lymphocyte
effector to target
fluorescence-activated cell sorting
fetal bovine serum
Iscove's Modified Dulbecco's Medium
major histocompatibility complex
National Cancer Institute
phycoerythrin
peripheral blood mononuclear cell
recognition efficiency
tumor-associated antigen
T cell receptor
variable chain beta