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Blood, 1 January 2005, Vol. 105, No. 1, pp. 241-250. Prepublished online as a Blood First Edition Paper on September 2, 2004; DOI 10.1182/blood-2004-06-2482.
IMMUNOBIOLOGY Transition of late-stage effector T cells to CD27+ CD28+ tumor-reactive effector memory T cells in humans after adoptive cell transfer therapyFrom the Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD.
In humans, the pathways of memory T-cell differentiation remain poorly defined. Recently, adoptive cell transfer (ACT) of tumor-reactive T lymphocytes to metastatic melanoma patients after nonmyeloablative chemotherapy has resulted in persistence of functional, tumor-reactive lymphocytes, regression of disease, and induction of melanocyte-directed autoimmunity in some responding patients. In the current study, longitudinal phenotypic analysis was performed on melanoma antigen-specific CD8+ T cells during their transition from in vitro cultured effector cells to long-term persistent memory cells following ACT to 6 responding patients. Tumor-reactive T cells used for therapy were generally late-stage effector cells with a CD27Lo CD28Lo CD45RA- CD62 ligand- (CD62L-) CC chemokine receptor 7- (CCR7-) interleukin-7 receptor  Lo (IL-7RLo) phenotype. After transfer, rapid up-regulation and continued expression of IL-7R in vivo suggested an important role for IL-7R in immediate and long-term T-cell survival. Although the tumor antigen-specific T-cell population contracted between 1 and 4 weeks after transfer, stable numbers of CD27+ CD28+ tumor-reactive T cells were maintained, demonstrating their contribution to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo. At 2 months after transfer, melanoma-reactive T cells persisted at high levels and displayed an effector memory phenotype, including a CD27+ CD28+ CD62L- CCR7- profile, which may explain in part their ability to mediate tumor destruction. (Blood. 2005;105:241-250)
In the initial phase of the adaptive immune response, selection, differentiation, and expansion of antigen-specific naive T cells occurs, followed by a contraction of effector cell numbers and maintenance of a relatively small memory T-cell population.1,2 Considerable effort has been made to characterize definitive pathways of CD8+ T-cell differentiation in humans as well as the precise mechanisms involved in maintenance of antigen-specific CD8+ T-cell memory. In murine studies, longitudinal gene expression profiling of viral epitope-specific T cells in transition from effector to memory cell stages has identified numerous differentially expressed genes, including genes encoding molecules involved in lymphocyte homeostasis and lymphoid organ homing.3 A subset of antigen-specific effector cells expressing high levels of interleukin-7 receptor (IL-7R, CD127) has been shown to preferentially yield long-lived memory CD8+ T cells,4 a finding consistent with the role of IL-7 as a mediator of memory CD8+ T-cell homeostatic maintenance in vivo.5-12 In addition, memory CD8+ T cells can be maintained as 2 phenotypically distinguishable and functionally distinct subsets in mice and humans.13,14 Central memory T cells (CC chemokine receptor 7+ [CCR7+] CD62 ligand+ [CD62L+]) preferentially home to lymph nodes, produce IL-2, and lack immediate effector function, while effector memory T cells (CCR7- CD62L-) circulate in the peripheral blood and tissues, express perforin, and display immediate effector function.13-15 Discrimination between distinct stages of human CD8+ T-cell differentiation has often relied upon the cell surface expression of CD27 and CD28, costimulatory molecules of distinctive function.16,17 Interaction of CD28 with CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APCs) amplifies T-cell receptor (TCR)-mediated T-cell proliferation and activation through a series of direct effects.18 However, prolonged TCR stimulation results in the subsequent down-regulation of CD28 expression.19 Interaction of CD27 with its ligand, CD70, augments TCR-stimulated proliferation of CD8+ T cells.20 Changes in CD27 expression are less well characterized, although expression is known to increase on naive T cells upon TCR stimulation.21,22 Prolonged T-cell stimulation results in loss of CD27 expression, which appears to be irreversible, and the loss of CD27 expression is thought to define a population of terminally differentiated effector T cells.16,21 CD27 may also be required in part for the generation and maintenance of T-cell memory.23 Based on human viral infection studies, a linear model of T-cell differentiation has been proposed wherein CD27+ CD28+ CD45RA+ naive cells progress through a CD27+ CD28+ CD45RA- early antigen-experienced phenotype to a CD27+ CD28- CD45RA-/+ intermediate phenotype and finally to a CD27- CD28- CD45RA+/- late antigen-experienced phenotype that parallels an increased cytotoxic potential and reduced ability to proliferate.24 Our recent clinical studies have demonstrated the efficacy of nonmyeloablative chemotherapy and subsequent adoptive transfer of ex vivo-expanded tumor-infiltrating lymphocytes (TILs) for the treatment of HLA-A*0201 (HLA-A2) patients with refractory metastatic melanoma.25 Of 13 heavily pretreated patients, refractory to standard therapies, 6 (42%) experienced an objective tumor response. Some patients with significant tumor regression experienced dramatic lymphocytosis after infusion of tumor-reactive TIL. Following an effector cell-like contraction phase, cell therapy resulted in the long-term persistence of a clonal melanoma-reactive CD8+ T-cell population in the peripheral blood. Other patients responding to therapy have experienced similar persistence of circulating tumor antigen-specific CD8+ T-cell clones. The transfer of characterized tumor antigen-specific T cells into patients and the monitoring of their progeny over time in vivo have provided a unique opportunity to study the dynamics of phenotypic change in humans occurring during effector-to-memory transition. In the current study, longitudinal phenotypic analysis was performed on tumor-reactive CD8+ T-cell populations from 6 patients who experienced immunotherapy-associated tumor regression. The data presented here demonstrate the in vivo transition of tumor-reactive CD8+ T-cell clones from a late-stage effector to an effector memory phenotype. The dynamics of homeostatic cytokine receptor and costimulatory molecule expression by persistent tumor antigen-reactive CD8+ T cells in the peripheral blood after adoptive cell transfer (ACT) suggest that expression of these receptors is important for the maintenance and long-term survival of adoptively transferred melanoma-reactive CD8+ T cells in vivo.
Patient cell samples All patients whose cells were used in this study had metastatic melanoma and were entered on institutional review board-approved protocols in the Surgery Branch of the National Cancer Institute. Informed consent was obtained from all subjects. TIL cultures were established as previously described.25,26 Briefly, TIL cultures were grown for 2 to 4 weeks in IL-2, screened to select tumor antigen-reactive cultures, and subsequently expanded using a rapid expansion protocol27 using 30 ng/mL OKT3 (anti-CD3) antibody (Ortho Biotech, Bridgewater, NJ) and 6000 IU/mL IL-2 in the presence of irradiated (50 Gy), allogeneic feeder cells at a 200:1 ratio of feeder cells to TIL cells. After about 14 days, cells were harvested from culture bags and prepared for patient treatment, and aliquots were cryopreserved for future experimental analysis. Peripheral blood lymphocytes were purified on Ficoll-Hypaque step gradients (LSM Lymphocyte Separation Medium; ICN Biochemicals, Aurora, OH) and cryopreserved. Tetramers, monoclonal antibodies, and flow cytometric immunofluorescence analysis
Phycoerythrin (PE)- or allophycocyanin-labeled MART-1:26-35(27L) (ELA-GIGILTV) peptide/HLA-A*0201 tetramer complexes were obtained from Beckman Coulter (Fullerton, CA). Antibodies specific for the Cytokine release assay
Cryopreserved TIL or PBL samples were thawed and cultured overnight in complete medium (CM) plus recombinant human IL-2 (rhIL-2, 300 IU/mL). Responder cells were washed twice and 105 cells cocultured overnight in CM with 105 HLA-A2+ T2 APCs unpulsed or pulsed with 1 µM or 10 µM MART-1:27-35, gp100:209-217 or gp100:280-288 peptides, or melanoma cell lines 526, 624, 888, and 2098. Coculture supernatants were harvested and assessed for the presence of gamma-interferon (IFN-
Patient selection and treatment
Selected for study were 6 HLA-A2+ patients with metastatic melanoma who had received immunodepleting chemotherapy with cyclophosphamide and fludarabine for 7 days prior to adoptive transfer of highly selected tumor-reactive TIL (Table 1). Patient selection was based upon the ability to identify tumor antigen-specific T-cell populations in TIL and peripheral blood after ACT with melanoma-associated peptide-loaded HLA-A2 tetramer complexes or antibodies specific for the
Characteristics of tumor antigen-specific CD8+ TIL populations in vitro
Antigen specificity of TIL was determined by cytokine release assay prior to patient infusion (Table 2). TIL from 5 patients secreted IFN-
Clonal populations of MART-1-reactive T cells were identified in the TIL of patients 9, 10, and 21 using TCR V(D)J region-specific nucleotide sequence analysis. MART-1-specific V
The frequency of tumor antigen-specific CD8+ T cells in TIL was measured using MART-1:26-35(27L) peptide-loaded HLA-A2 tetramer complexes or V
Flow cytometric analysis was performed to measure the expression of molecules associated with CD8+ T-cell differentiation, homeostatic proliferation and maintenance, and lymph node homing on tumor antigen-specific CD8+ T cells from ex vivo-expanded TIL (Figure 1B). Expression of CD27 and CD28 costimulatory molecules on tumor antigen-specific CD8+ T cells in TIL revealed frequencies of 3% to 70% and 10% to 61%, respectively. Despite variability in the frequency of costimulatory molecule-expressing cells in TIL, the level at which CD27 and CD28 were expressed on patients' tumor antigen-specific CD8+ T cells was generally low to absent, compared with pretreatment CD8+ T cells. The frequency of tumor-reactive T cells that expressed the migration-associated markers, CD62L and CCR7, was low (0%-7% and 1%-32%, respectively). Consistent with ex vivo TCR stimulation, CD45 isoforms were differentially expressed. CD45RO was expressed at a high level on all cells, while the frequency of CD45RA-expressing TIL was low (1%-36%).
The expression level of alpha chain receptor subunits for common Fate of adoptively transferred TIL in vivo Immediately following lymphodepleting chemotherapy conditioning and prior to ACT, few lymphocytes were detectable in the peripheral blood and thus adoptively transferred T cells could be detected without difficulty (Figure 2A). Approximately 1 week after transfer, when lymphocytes were first detectable in the blood, lymphocytosis developed rapidly in patients 9 and 10. Patients 20 and 23 also demonstrated a rapid elevation in ALC. ALCs from patients 21 and 28 peaked at nearly 1400 and 1100 cells/mm3, respectively. Despite these lower cell numbers, patients 21 and 28 underwent objective tumor regressions in vivo. Over the next 2 months, all patients' ALCs normalized to homeostatic levels. Approximately 1 week after transfer, more than 80% of circulating lymphocytes were CD8+ in 4 patients, recapitulating frequencies observed in the transferred TIL (Figure 2B). Circulating CD8+ T-cell frequencies generally remained elevated over the course of study compared with pretreatment levels, which ranged between 11% and 32% of lymphocytes (Figure 2B).
Tumor antigen-specific CD8+ T-cell frequencies generally peaked 1 week after infusion, ranging from 18% to 97% of CD8+ T cells (Figure 2C). Compared with the transferred TIL, this frequency was elevated in patients 10, 21, 23, and 28 and decreased in patients 20 and 9. Following early peaks, the percentage of CD8+ T cells that were tumor antigen-specific remained relatively stable with only minor diminution in most patients over time. Absolute numbers of circulating tumor antigen-specific CD8+ T cells generally peaked approximately 1 week after transfer (Figure 2D). High numbers of tumor antigen-specific T cells persisted in the peripheral blood of all patients nearly 2 months after ACT (Figure 2D). At this and later time points, PBLs from treated patients maintained tumor antigen-specificity and reactivity as demonstrated by cytokine secretion following MART-1 peptide (Figure 2E) and HLA-matched, MART-1-expressing tumor stimulation, except for PBL from patient 21 that recognized a unique antigen only on the autologous tumor, 2098mel (Figure 2F). The high number of persistent, clonal CD8+ T cells of known antigen specificity observed in these patients is virtually unprecedented in human immunology and offered a unique opportunity to study their phenotype. Persistent tumor antigen-specific memory CD8+ T cells express an effector memory phenotype in vivo Melanoma-reactive CD8+ T cells were evaluated for the expression of markers associated with memory T-cell differentiation. Prior to administration, tumor-reactive T cells from ex vivo-expanded TIL were uniformly CD45RO+ and had little to no CD45RA cell surface expression (Figure 3A-B). At 2 months after transfer, persisting tumor-reactive cells from all 6 patients showed marked CD45RA up-regulation with concomitant maintenance of CD45RO expression (Figure 3A). Over time, CD45RO expression was sustained, but the intensity of CD45RO staining was modestly reduced in parallel with the observed increase in CD45RA (Figure 3B). Tetramer-negative CD8+ T cells, which represent the reconstituting endogenous repertoire, also displayed a CD45RO+ memory phenotype 2 months after transfer in these patients (not shown). Expression of CD62L and CCR7, which was not detectable in most TIL, was similarly absent on persisting tumor antigen-specific T cells from all 6 patients in vivo (Figure 3C). In these PBL samples, expression of CD62L and CCR7 was readily evident on tetramer-negative CD8+ T cells confirming the activity and sensitivity of these antibodies (Figure 3D). The lack of detectable CD62L and CCR7 expression by persistent transferred TIL is consistent with the long-term maintenance of effector memory CD8+ T cells with tumor antigen specificity in metastatic melanoma patients responding to immunotherapy.
Immediate and continued expression of IL-7R
To determine the early and long-term impact of ACT on tumor antigen-specific CD8+ T-cell differentiation, longitudinal cell surface phenotype was evaluated. At the earliest detection of lymphocytes in the blood nearly 1 week after transfer, IL-7R
Similar to IL-7R CD27 expression on tumor antigen-specific T cells at the peak of the inflammatory response is predictive for long-term persistence
The costimulatory molecule CD27 has been implicated in T-cell differentiation and memory generation,23 therefore CD27 expression was evaluated on persisting melanoma-specific CD8+ T cells. Unlike CD28, a high frequency of CD27-expressing tumor antigen-specific CD8+ T cells was not detected in the peripheral blood 1 week after infusion (Figure 5). Rather, the frequency of melanoma-specific T cells expressing CD27 was minimally increased compared with TIL in 3 patients' PBLs immediately after transfer and slightly diminished in the remaining patients. Figure 5A demonstrates the near absence of CD27 expression on the clonal V
Calculating the number of cells with distinct phenotypic characteristics can provide a significantly different picture than that observed when studying cell frequency. To evaluate temporal alterations in T-cell number, the absolute number of blood-derived tumor antigen-specific T cells that expressed IL-7R
The number of circulating tumor antigen-specific CD8+ T cells that expressed IL-7R Coexpression of CD27 and CD28 on persistent tumor-specific memory T cells Although CD28 signaling amplifies TCR-mediated T-cell proliferation and activation,28 stable numbers of CD28-expressing melanoma-reactive T cells did not persist over time. Prior to chemotherapeutic conditioning, the majority of circulating CD8+ T cells concomitantly expressed cell surface CD27 and CD28 molecules, with smaller frequencies of CD27+ CD28- and CD27- CD28- cells (Figure 6A). Few CD27- CD28+ CD8+ T cells were detected in pretreatment PBL samples. Two months after chemotherapy, the reconstituting endogenous CD8+ T-cell population was composed of CD27 and CD28 expression subsets similar to pretreatment PBLs. In accordance with robust TCR stimulation and expansion ex vivo, the majority of tumor antigen-specific CD8+ T cells in TIL displayed a CD27- CD28- effector phenotype (Figure 6B). However about 1 week after infusion, the preponderance of the transferred T-cell population had transitioned to a CD27- CD28+ phenotype, with small frequencies of CD27+ CD28+ cells. Expression of the CD27- CD28+ phenotype by tumor-reactive T cells was transient as the frequency of CD27+ CD28+ cells generally increased in parallel with a reduction in CD27- CD28+ frequency over time (Figure 6B). Longitudinal determination of T-cell phenotypic frequency measures the changes within a distinct cell population yet fails to adequately represent the effects on circulating T-lymphocyte number. Enumeration of phenotypic subsets revealed temporal reductions in the number of CD27- CD28+ tumor antigen-specific CD8+ T-cell clones with concomitant maintenance of relatively stable numbers of CD27+ CD28+ cells, therefore the high frequency of CD28+ cells that persist over time reflects, in part, the maintenance of stable numbers of CD28+ cells that coexpress CD27 in patients undergoing objective tumor responses (Figure 6C). High numbers of CD27+ CD28- and CD27- CD28- tumor antigen-specific T cells were not detected at late time points after ACT. In summary, these data demonstrate that the long-term melanoma-reactive memory T-cell population is largely composed of CD27+ CD28+ cells. These data further imply that CD27+ CD28+ melanoma-specific CD8+ T-cell clones detectable in the blood soon after ACT may represent precursors to long-term tumor antigen-specific memory T cells in vivo.
Recent data from our lab have demonstrated a significant correlation between the persistence of transferred lymphocyte clones and cancer regression in ablated patients receiving cell transfer therapy.29 Based on these findings, we hypothesized that tumor-reactive clones that persist more than one month after ACT are the cells or progeny of cells mediating therapy-based tumor regressions in vivo. The ability to longitudinally monitor and examine autologous tumor antigen-reactive CD8+ T-cell clones after ACT in the context of tumor regression in vivo provided a novel opportunity to characterize the transition of functional tumor-reactive effector CD8+ T cells to a long-lived memory T-cell population. The results herein implicate a series of signature markers for immediate and long-term survival of antigen-reactive CD8+ T cells, which are present in the peripheral blood following ACT and endure effector cell contraction to give rise to a persistent memory T-cell population, and illustrate a model of in vivo memory T-cell development in parallel with ongoing antigen-specific immune responses.
The common
Since TCR-mediated signals alone are generally insufficient for proliferation of T cells during immune responses, secondary costimulatory signals are required.38 Compared with TIL, whose CD28 expression was commonly low as measured by fluorescence intensity, the frequency of circulating CD28+ melanoma epitope-reactive CD8+ T cells was augmented 1 week after ACT. Reports of CD28 re-expression by T cells have been few and limited to in vitro studies.39,40 Elevated CD28 expression by persisting transferred cells suggests that CD28 up-regulation can occur in the conditions of ACT after lymphodepletion in vivo. Furthermore, the presence of CD28+ T cells and the lack of CD28- cells immediately following cell infusion in vivo suggest an early survival advantage for CD28+ CD8+ T cells. Preferential expansion of CD28+ melanoma-reactive cells in vivo may also account in part for their increased prevalence. Despite the known prosurvival effects of CD28 and IL-7R Signaling through tumor necrosis factor (TNF) receptor family molecules, including CD27, OX-40 (CD134), and 4-1BB (CD137), in association with TNF receptor-associated factor (TRAF) family adaptor proteins, may promote cell survival.41-43 CD27 was generally expressed at low intensity levels on tumor antigen-specific T cells in TIL, consistent with their recent ex vivo activation. Compared with TIL, a reduction in CD27+ T-cell frequency was detected within the circulating melanoma-reactive T-cell populations from 3 patients immediately after ACT. Since prolonged TCR stimulation is known to result in the loss of CD27,45,46 reductions in CD27+ cell frequencies after ACT might reflect recent antigenic stimulation in these patients undergoing immune-mediated tumor regressions. While CD27- melanoma-reactive CD8+ T cells predominated during the inflammatory response in the early aftermath of ACT, CD27- cell numbers severely declined over the following weeks, suggesting that these are terminally differentiated T cells with the ability to eliminate tumor but not persist. Studies have shown that CD27- CD8+ T cells are primarily composed of cytolytic effector T cells, preferentially expressing perforin and exhibiting cytotoxic activity.16 Conversely, the level of CD27 fluorescence intensity was augmented on T-cell clones from patients 9 and 21 in the interim between cell infusion and 1 week after infusion. Thus, it appears that adoptive transfer of effector CD8+ T cells under lymphopenic conditions may promote CD27 re-expression in vivo. Within the confines of this study, it remains uncertain what mechanisms account for the preferential expression of CD27 by the memory population developing in the peripheral blood of lymphodepleted patients receiving ACT. CD27 loss has been reported to be irreversible in in vitro studies and serves as a marker for terminal differentiation.16,21 Our data are thus most consistent with the selective survival of infused effector T cells that immediately express CD27 after transfer, resulting in the continued maintenance of relatively stable numbers CD27+ T-cell clones in vivo that give rise to a long-lived memory population. Moreover, CD27 signals may function to support the generation of the tumor-reactive memory CD8+ T cells in vivo. The significance of CD27-mediated signals for the generation of memory responses after primary stimulation has been demonstrated in knock-out mice and contributes by stimulating the survival of activated T cells in vivo, even in the absence of CD28.23,41 Alternatively, enduring CD27- effector CD8+ T cells might temporally reacquire CD27 expression in vivo, a phenomenon not previously described. In the transition to memory, transferred tumor-reactive T cells from these 6 patients generally underwent similar phenotypic dynamics, progressing from a primarily CD27- CD28- CD45RA- effector stage in TIL to a CD27+ CD28+ CD45RAINT memory status in PBLs. CD27- CD28- CD45RA- cells, which failed to persist at high numbers, appear to represent a terminally differentiated T-cell population. Immediately after ACT, melanoma-reactive CD8+ T cells in the blood primarily displayed a distinct CD27- CD28+ CD45RA- phenotype, present at minimal frequencies in healthy individuals,47 which was generally short-lived and underwent pronounced contraction over the ensuing weeks. In contrast, CD27+ CD28+ melanoma-reactive clones were largely stable in number, persisted, and concomitantly augmented CD45RA expression. At 2 months after transfer, most persistent melanoma antigen-specific CD8+ T-cell clones re-expressed CD45RA in vivo, albeit at intermediate levels, with a modest diminishment in CD45RO intensity, a transition described previously.48 Reacquisition of CD45RA expression is postulated to denote terminal CD8+ T-cell differentiation.16,49 The CD27+ CD28+ CD45RAINT phenotype displayed by melanoma-reactive memory CD8+ T cells is consistent with the early antigen-experienced phenotype reported in virus-specific T-cell populations.24 The cause of this preferential phenotype observed in transferred melanoma-reactive memory CD8+ T cells may be multifactoral. Virus-specific memory CD8+ T cells that result from transient influenza infection primarily express CD27 and CD28, while CD27-CD28- virus-specific memory cells predominate in individuals infected with persistent human cytomegalovirus (HCMV),50 suggesting that duration of antigen exposure may influence phenotype. Immunotherapeutic targeting of MART-1 was associated with melanocyte-related autoimmune manifestations in 4 of 5 studied patients, illustrating MART-1-directed immune activity in vivo. Despite the potential for chronic MART-1 antigen exposure, the majority of circulating melanoma-reactive memory CD8+ T cells continued to maintain the early antigen-experienced phenotype, reminiscent of an Epstein-Barr virus (EBV)-specific CD8+ T-cell phenotype observed during acute and chronic infections.24 This suggests that beyond chronic antigen exposure other phenot | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
chain variable regions, V
) by enzyme-linked immunosorbent assay (ELISA) in accordance with manufacturer's protocol. Values reflect the average of triplicate measurements and are reported in pg/mL. 
), 10 (
), 20 (
), 21 (
), 23 (x), and 28 (
). The x-axis represents days relative to cell transfer, with day 0 representing the day of cell infusion. Latest time points analyzed for patients 9, 10, and 23 were days 124, 524, and 161 after infusion, respectively. (B) CD8+ T-cell frequencies are generally elevated after cell transfer and diminish temporally. The frequency of CD8+ cells was measured on gated viable lymphocytes. (C) High frequencies of tumor antigen-specific CD8+ T cells persist in the peripheral blood of select patients. Tumor antigen-specific CD8+ T cells were identified using MART-1: 27-35(27L) peptide containing HLA-A*0201 tetramer complexes or V
), 21 (
), 23 (x), and 28 (
), and the average of all values is shown (
