Transferred melanoma-specific CD8+ T cells persist,mediate tumor regression, and acquire centralmemory phenotypeAude G. Chapuisa, John A. Thompsonb, Kim A. Margolinb, Rebecca Rodmyrea, Ivy P. Laia, Kaye Dowdya, Erik A. Farrara,Shailender Bhatiab, Daniel E. Sabathc, Jianhong Caoa, Yongqing Lia, and Cassian Yeea,1

aProgram in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; bGeneral Oncology and Hematology, Seattle Cancer Care Alliance andUniversity of Washington, Seattle, WA 98109; and cDepartment of Laboratory Medicine, University of Washington, Seattle, WA 98195

Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research, Ontario Cancer Institute at Princess Margaret Hospital, University HealthNetwork, Toronto, ON, Canada, and approved February 3, 2012 (received for review August 30, 2011)

Adoptively transferred tumor-specific T cells offer the potential fornon–cross-resistant therapy and long-term immunoprotection.Strategies to enhance in vivo persistence of transferred T cellscan lead to improved antitumor efficacy. However, the extrinsic(patient conditioning) and intrinsic (effector cell) factors contribut-ing to long-term in vivo persistence are not well-defined. Asa means to enhance persistence of infused T cells in vivo and limittoxicity, 11 patients with refractory, progressive metastatic mela-noma received cyclophosphamide alone as conditioning beforethe infusion of peripheral blood mononuclear cell-derived, anti-gen-specific, CD8+ cytotoxic T-lymphocyte (CTL) clones followedby low-dose or high-dose IL-2. No life-threatening toxicities oc-curred with low-dose IL-2. Five of 10 evaluable patients had stabledisease at 8 wk, and 1 of 11 had a complete remission that con-tinued for longer than 3 y. On-target autoimmune events with theearly appearance of skin rashes were observed in patients withstable disease or complete remission at 4 wk or longer. In vivotracking revealed that the conditioning regimen provided a favor-able milieu that enabled CTL proliferation early after transfer andlocalization to nonvascular compartments, such as skin and lymphnodes. CTL clones, on infusion, were characterized by an effectormemory phenotype, and CTL that persisted long term acquiredphenotypic and/or functional qualities of central memory typeCTLs in vivo. The use of a T-cell product composed of a clonalpopulation of antigen-specific CTLs afforded the opportunity todemonstrate phenotypic and/or functional conversion to a centralmemory type with the potential for sustained clinical benefit.

adoptive transfer | immunotherapy

Strategies aimed at increasing the number and quality of au-tologous T cells targeting melanoma-associated antigens havebeen effective in reducing tumor burden in a limited numberof patients, and clinical responses have been correlated withthe duration of in vivo persistence of transferred T cells (1–4).Adoptive transfer of ex vivo-expanded melanoma-specific cyto-toxic T-lymphocyte (CTL) clones can increase the in vivo fre-quency of melanoma-reactive T cells in a setting in which theelicited response can be tracked and the transferred cells char-acterized (5, 6). Improving the benefit of infused T cells hasinvolved either extrinsic modulation of the host environment orintrinsic manipulation of the infused product to promote ulti-mate in vivo survival of T cells (7).Nonmyeloablative chemotherapy conditioning regimens [high-

dose (HD) cyclophosphamide (CY) and fludarabine] or ablativedoses of total body irradiation (TBI; 1,200 rad) administeredbefore T-cell infusions have been shown to facilitate the in vivoengraftment and expansion of adoptively transferred cells at thecost of serious and potentially life-threatening toxicities (8). Wehypothesized that administration of HD CY given alone mayinduce a transient lymphopenia sufficient to allow engraftmentand persistence of transferred T cells without incurring prolongedimmunosuppression (8–10). Concurrent HD IL-2 (600,000–

720,000 U/kg thrice daily) may contribute to increased toxicity(8), and although low-dose (LD) IL-2 (250,000 U/m2 s.c. twicedaily) can enhance T-cell survival by several days, it is not suffi-cient to prolong the clinical efficacy of adoptively transferred cellssignificantly (3). We sought to determine in a phase I/II clinicaltrial whether HD CY (4 g/m2) followed by LD or HD IL-2 wassufficient to promote survival of melanoma-specific T-cell clonesand induce measurable clinical responses.Previous studies in mice and nonhuman primates have shown

that infusion of T cells derived from a central memory (Tcm)population exhibit greater replicative potential in response toantigen and prolonged in vivo persistence compared with thosederived from an effector memory (Tem) source (11, 12), andmay likely be poised to eliminate tumor more effectively. How-ever, it remains unclear if cytotoxic T cells can be establishedfrom a Tcm pool in patients with high tumor burden, wherein themajority of endogenous circulating T cells are likely Tem; thus,T-cell clones generated from these patients in vitro are likely tobe Tem or naive T cell-derived. In this study, particular attentionwas given to the analysis of the phenotype and function of T-cellclones that had prolonged in vivo survival because studies in thenonhuman primate model demonstrated that after in vitro ex-pansion, T cells derived from Tcm or Tem share a Tem phenotypebut their behavior is ultimately representative of their source,reverting to their original phenotype in vivo (11).Here, we show that conversion from a Tem to Tcm phenotype

occurred in the setting of minimal, nonablative, lymphodepletingconditioning in two patients, wherein long-term in vivo persis-tence of transferred CTLs was observed. Furthermore, in thislimited number study, HD CY alone accompanied by LD IL-2postinfusion allowed for a transient expansion of the transferredcells, on-target autoimmune skin toxicity, disease stabilization inat least half of the patients at 8 wk, and a continuous completeresponse in one patient.

ResultsAdoptive Transfer of Melanoma-Specific CTL Clones Preceded by HDCY and Followed by LD IL-2 Is Well-Tolerated and Safe. Elevenpatients with bulky metastatic melanoma who had failed previoustherapy (Table 1) received CTL clones specific for Mart1/A2,Tyr/A2, gp100/A2, gp100/A3, and Tyr/B44. Characteristics of theinfused melanoma-specific CTL clones are described in Fig. S1,Tables S1, S2, and the treatment plan in Fig. S2. Of the patientswho received LD IL-2 following CY and T cells (cohort 1), all

Author contributions: C.Y. designed research; A.G.C., J.A.T., K.A.M., R.R., I.P.L., K.D., E.A.F.,S.B., D.E.S., J.C., Y.L., and C.Y. performed research; A.G.C., J.A.T., K.A.M., R.R., and C.Y.analyzed data; and A.G.C. and C.Y. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: cyee@fhcrc.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113748109/-/DCSupplemental.

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experienced transient (36mo after CTL infusion without further intervention. In patient6, there was a 42% decrease in all target lesions 19 wk afterinfusion (a growing paraspinal lesion was surgically removed

before imaging was performed). Except for patient 1 (CR), allpatients eventually progressed by 12–19 wk. Patients who expe-rienced stabilization or regression of disease ≥4 wk after infu-sions experienced skin rashes within 7 d of CTL infusion. Patient1’s rash (Fig. 1B) was biopsied at day 5, and the T-cell receptor-γ(TCR-γ) rearrangement found in DNA extracted from the skinbiopsy was identical to the infused CTL clone (Fig. 1C), dem-onstrating that infused CTLs were present in the skin and werethe likely cause of the inflammatory rash. Although not all rasheswere biopsied, these results suggest that infused CTLs have thepotential to migrate to the skin consistent with our previousfindings (13).

Melanoma-Specific CTL Clones Isolated from Patients with MetastaticMelanoma Can Persist in Vivo Following Transfer. Tracking of theinfused cells in vivo was performed by clone-specific TCR Vβquantitative PCR and, where possible, corresponding multimerstains (3) (Fig. 2). These methods have different limits of de-tection (1 of 105 cells for quantitative TCR-specific PCR and0.1% for multimer stains), and although they have previouslybeen individually used to quantitate in vivo frequencies of in-fused CTL clones (1, 14), they were compared for patient 1 (Fig.2A). The frequency of multimer-positive CTLs reached 70.4%,2.2%, and 1.5% compared with 86.9%, 2.6%, and 1.4% by TCRVβ-specific PCR at days 4, 56, and 508, respectively, thus yieldingnear-coincident results for frequencies ≥0.1% of CD8+ T cellsand validating the use of either method for tracking purposes.

Table 1. Patient characteristics

Patient no. Age, y, (gender) Previous treatments Disease site

LD IL-21 57 (M) Surgery, radiation, HD IL-2, anti–CTLA-4 Mesenteric LN, scapular, s.c.2 38 (F) Surgery, radiation, carboplatin, and paclitaxel Cervical, supraclavicular LN, chest wall,

breast, pulmonary nodes3 65 (M) Surgery, radiation, MEK inhibitor Lung, mediastinal and mesenteric LN4* 60 (M) Ocular enucleation, surgery, Mel48 vaccine Kidney, pancreas, liver, muscle5 68 (M) Surgery, radiation, gamma-knife, temozolomide Mediastinal LN, pulmonary nodes6 40 (F) Surgery, radiation, anti–CTLA-4, peptide-based vaccine Mediastinal, supraclavicular, mammary

chain, periportal, portacaval nodes.7 61 (F) Surgery, radiation, bevacizumab, paclitaxel Right and left kidneys, adrenal, liver8 50 (M) Surgery, radiation, IFN-α, HD IL-2, paclitaxel Pulmonary, inguinal, s.c.

HD IL-29 75 (M) HD IL-2 Liver, peritoneum, bone10* 35 (M) Brachytherapy, lipiodol, 90Y microsphere liver embolization Liver, mesenteric LN, sacrum11* 55 (M) Surgery, radiation, cisplatin, vinblastine, dacarbazine, IFN-α HD IL-2 Liver, mesenteric LN, sacrum

F, female; LN, lymph node; M, male.*Choroidal melanoma.

Table 2. Assessment of clinical response

Patient no. Cohort T-cell specificity HLA type

On study Follow-up

Rash4 wk 8 wk 12 wk 16 wk 19 wk 24 wk

1 1 Mart1 A2 CR (−100%) CR (−100%) CR (−100%) CR (−100%) Y2 1 Tyr B44 ND SD (−13%) PD (NT) PD (new) Y3 1 gp100 A2 SD (+0.6%) ND* PD* (+52%) Y4 1 gp100 A3 SD (+0.6%) SD (+8%) PD (+24%) Y5 1 Tyr A2 SD (+10%) PD (new) Y6 1 Tyr A2 SD (−7%) SD (−2%) NE† Y7 1 Tyr B44 SD (+6.3%) PD (+28%) PD (+61%) Y8 1 Mart1 A2 PD (+37%) PD (+47%) PD (+64%) N9 2 Mart1 A2 SD (+13%) SD (+15%) PD (new) Y10 2 Mart1 A2 SD (−9.1%) SD (−4.6%) SD (+9%) PD (+21%) Y11 2 Mart1 A2 PD (+36%) PD (+31%) PD (+51%) N

N, no; ND, not done; new, new metastasis; NT, nontarget lesion progression; Y, yes.*Patient initiated alternate treatment modality.†Not evaluable: 42% reduction in all lesions except growing paraspinal mass surgically removed before scans.

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Patient 2 was infused with an HLA-B44/Tyr CTL clone for whichan MHC multimer could not be synthesized. Clone frequencyby TCR Vβ-specific PCR revealed frequencies of 2.6% and 4.8%at days 56 and 117, respectively. Clones for Patients 3 and 4persisted at levels below multimer but above quantitative PCRdetection levels for 49 d and ≥98 d, respectively. Overall, forpatients in cohort 1, persistence beyond 42 d was observed in 4 of8 patients (Fig. 2 A and B). Infused CTL could not be detectedbeyond 28 d in cohort 2 (Fig. 2C). The frequency of specific CTLreached peak levels at 7 ± 3 d (range: 0.49–95%, mean of 23.8%for cohort 1; range: 0.98–80%, mean of 32.5% for cohort 2). Nocorrelation could be established between the phenotype, avidityof infused clones to cognate antigen (Fig. S1), and in vivo per-sistence or clinical response.

CTL Clones That Persist Sustain Function in Peripheral Blood andTissues. Enzyme-linked immunospot (ELISpot) assays were per-formed by stimulating whole peripheral blood mononuclear cells(PBMCs) obtained before and after infusions with patient-spe-cific peptide-pulsed autologous monocytes (Table S1). IFN-γ wasdetected (Fig. 3A) at the same time points the infused clonesreached frequencies >0.1% in vivo by multimer stains (patient 1)and/or TCR PCR (patients 1 and 2) (Fig. 2A). Patient 2 de-veloped enlargement of a lymph node in the neck 51 d after T-cell infusion. The node was excised for diagnostic purposes.Tumor microinvasion with an associated lymphocytic infiltratewas identified within connective tissue (Fig. 3B, Left), and ex-pression of tyrosinase was confirmed on the melanoma tumorcells (Fig. 3B, Right). Specific IFN-γ secretion in response topeptide-pulsed autologous monocytes was detected by ELISpot

in fresh cells isolated from the lymph node (Fig. 3C). Takentogether, this suggests the infused clones from patients 1 and 2had the capacity to extravasate from peripheral blood and tosustain functional capacity in vivo in peripheral tissue, includingskin (patient 1) and tumor-infiltrated lymph nodes (patient 2).

Transferred CTLs Acquire Phenotypic Characteristics of Tcm in Vivowith Complete Tumor Regression. Immediately before infusion,patient 1’s clone expressed CD45RO (100%), moderate levels ofCD27 [53.8%, median fluorescence intensity (MFI) = 757], butrelatively low or absent levels of CD28 (10.2%, MFI = 114),CD127 (0.5%), CD62L (0.0%), and CCR7 (0.0%) (Fig. 4A,

Fig. 1. Complete response associated with a skin rash infiltrated with theadoptively transferred CTL clone. (A) Abdominal computed tomography (CT)(Upper) and PET scan combined with low-resolution noncontrast CT (Lower)before infusion (Left) and 28 d (Center) and 56 d after infusion (Right).Arrows indicate radiological image and active fluorodeoxyglucose (FDG)uptake before treatment, and circled areas show absence of detectableimage and FDG uptake. (B) Back (Left), front chest (Center), and close-up ofthe front chest lesion (Right) of patient 1’s rash 5 d after CTL infusion. (C)Superposition of TCR-γ length analysis of DNA extracted from the CTL clonepreinfusion (Upper) and paraffin-embedded skin biopsy (Lower).

Fig. 2. In vivo persistence of melanoma-specific CTL clones. (A) TCR copiesper 100 CD8+ T-cell DNA equivalents (Left, x axis) and percentage of multi-mer-positive (%Mult.) CD8+ T cells (Right, x axis) in PBMCs collected 7 d (±2d) before and after infusions for four of eight patients in cohort 1 whoseclones showed persistence (≥48 d). Inset dot plots above the graph for pa-tient 1 show percentages of multimer-positive CD8+ T cells at days 4, 56, and508. (B) Same analysis performed for four of eight patients in cohort 1 whoseclones demonstrated persistence for

Left). MFI was not calculated for markers with a surface ex-pression of ≤1% compared with isotype controls. However, thephenotype of the infused clone based on the gating of multimer-positive CD8+ T cells at days 6, 34, and 508 demonstrated thatthe infused clone maintained CD45RO; further accumulatedexpression of CD27 consistent with previous studies (15) (Fig.4A, Upper); and, additionally, accumulated expression of CD28,CD127, CD62L, and CCR7 in vivo over time to reach 93.6%,87.4%, 9.88%, and 7.47%, respectively, at day 508 (Fig. 4A,Lower). CD62L- and CCR7-expressing cells were comprisedwithin a CD27hi subset of the infused CTLs (Fig. 4B). A similaranalysis could not be performed for patient 2 because nomultimerwas available (Table S1). Although an expansion of an endoge-nous CTL contributing to the Tcm phenotype observed cannotformally be excluded, concurrent detection of the clone-specificTCR renders this alternative unlikely and suggests that a subset ofinfused cells for patient 1 acquired a Tcm phenotype in vivo.

CTL Clones That Persist Express Functional Characteristics Shared withTcm in Vivo. Further determination of the functional profile ofboth infused CTL clones that persisted and remained detectableby multimer in the peripheral blood could be assessed by gatingon IFN-γ+–reactive cells. Clones for patient 1 (CR) and patient2 (progressive disease by 19 wk) secreted IFN-γ and variableTNF-α but no IL-2 in response to peptide-pulsed, HLA-matched,B-lymphoblastoid cell lines, and expressed little or no CD28 (Fig. 4C and D, Left). Consistent with a low frequency of melanoma-reactive cells, IFN-γ secretion was below detection levels beforeinfusions (0.1%) (Fig. 4 C and D, second column). However,CD8+ IFN-γ+–reactive cells obtained on days 20, 56, and 508(patient 1) or 117 (patient 2) after CTL infusion demonstratedacquisition of a polyfunctional phenotype characterized by con-current expression of IFN-γ, TNF-α, and IL-2 (Fig. 4 C and D,Right). IL-2–secreting cells were contained within the CD28+fraction of antigen-reactive IFN-γ+ cells (Fig. 4 C and D, Lower),compatible with the associated functional capacity of CD28+ cellsto secrete IL-2 in response to cognate antigen (16, 17). Together,these data suggest that on the basis of both functional (patients

1 and 2) and phenotypic (patient 1) criteria, infused cells thatpersisted long term expressed features associated with CD8+ Tcm.No evidence of persistence by multimer analysis or antigen-specificIFN-γ secretion was observed in the peripheral blood of theremaining patients.

CY Conditioning Followed by Exogenous IL-2 Fosters a FavorableEnvironment for Replication of Infused CTLs. Because cells thatpossess the potential to divide or replicate are likely betterequipped to persist, we investigated whether infused CTL clonesexpressed Ki-67 in vivo (18). Before infusion, clones exhibiteda resting state (3–12% Ki-67

+) for the most part. However,a majority of infused multimer-positive CTLs (>90%) enteredthe cell cycle early after transfer (day 7). By day 14, the numberof detectable CTLs in the peripheral blood contracted and ei-ther persisted in a Ki-67

− quiescent state (patient 1) or haddisappeared from peripheral blood (patients 9, 10, and 11) (Fig.5A). Localization of transferred CTLs to nonvascular compart-ments (i.e., lymph node, tumor sites) cannot be excluded. Pro-liferation of infused cells was distinct from the recovery ofendogenous CD8+ T cells after lymphodepletion because thepeak percentage of replicating endogenous cells occurred later(day 14) and involved a lower fraction of total CD8+ T cells(mean of 51%). Plasma IL-15 levels (Fig. 5B, Upper) were in-creased in patients postinfusion. Significantly higher levels wereobserved in patients who had received HD IL-2 (Fig. 5B, Lower).However, there was no correlation between peak levels or cu-mulative levels (area under the curve) of IL-15 and clone per-sistence. Similarly, plasma levels of other cytokines tested in vivo(Fig. S3) did not correlate with clone persistence or survival.Regulatory T-cell (T-reg) dynamics were also monitored be-cause murine and human studies suggested a role for CY inreducing the circulating T-reg populations (19, 20). Aftera transient decrease in absolute numbers of T-regs coincidingwith lymphodepletion induced by CY, both percentages andabsolute numbers of T-regs peaked 14 d after infusions; theythen declined and reached pretreatment levels at 21 and 49 d,respectively (Fig. S4 A and B). Ki-67 expression by T-regs wassignificantly higher before and after treatment compared withCD4+ T-effector cells, suggesting a replicative advantage of T-regs compared with CD4+ T-effector cells (Fig. S4C). Overall,we observed that CY lymphodepletion followed by LD or HDIL-2 fostered an environment that enabled infused cells toproliferate early after transfer. However, neither early pro-liferation detected in the peripheral blood or plasma IL-15 northe dynamics of T-regs translated into long-term peripheral invivo persistence.

DiscussionThe optimal host conditions and effector cells favoring the es-tablishment of a long-lived, transferred, tumor-specific pop-ulation have not been determined. In this study, we set out toevaluate prospectively the effects of HD CY followed by LD orHD IL-2 on the persistence of transferred, antigen-specificCTLs in patients with progressive metastatic melanoma. Adop-tive therapy using antigen-specific CTL clones for patients re-ceiving HD CY conditioning alone followed by LD s.c. IL-2 iswell-tolerated, nontoxic, and sufficient to render a favorablemilieu enabling infused T cells to proliferate early after trans-fer. CTL persistence beyond 42 d was observed in 4 of 11patients, of whom 2 presented with prolonged T-cell persistenceand establishment of an antigen-specific Tcm clonal T-cellpopulation, with 1 of these 2 patients experiencing objectivesustained CR.Evidence supporting the critical role of Tcm comes from

murine lymphocytic choriomeningitis virus infection models,where protective immunity provided by Tcm was more robustcompared with Tem after in vivo challenge. The greater effec-tiveness of Tcm in this setting likely reflects the distinct ability ofTcm to secrete and use IL-2 as an autocrine growth factor,rendering these cells “helper-independent” (12). Furthermore,

Fig. 3. Adoptively transferred melanoma-specific CTL clones show sus-tained functional capacity in peripheral blood and tumor-infiltrated lymphnodes. (A) IFN-γ spot-forming cells per 105 PBMCs for patient (Pt) 1 (Left) andpatient 2 (Right) after stimulation with unpulsed (empty circles) or HLA-A*0201-Mart1- or HLA-B*4402-tyrosinase-pulsed monocytes (filled circles),respectively, over time (y axis). (B) Stain of a cervical lymph node from pa-tient 2 51 d after infusion stained with H&E (Left) and tyrosinase (Right).White arrows indicate lymphocyte infiltrates, and black arrows indicate in-vasive tyrosinase-positive melanoma. Images were collected with an objec-tive with a magnification of 10×. (C) IFN-γ spot-forming cells per 105 cells (yaxis). (Left to Right) Cells isolated from patient 2’s infiltrated lymph node(LN) with unpulsed autologous monocytes (M); unpulsed M and infusedclone cells; HLA-B*4402-tyrosinase-pulsed M only (negative controls); HLA-B*4402-tyrosinase-pulsed M and infused clone (positive control); and HLA-B*4402-tyrosinase-pulsed M and LN.

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studies in nonhuman primates have demonstrated that cells de-rived from Tcm, differentiated to Tem ex vivo, can reacquireinherent features shared with Tcm in vivo (11). In this study,analysis of the transferred CTLs in two patients (patients 1 and2) demonstrated long-term persistence, up-regulation of pheno-typic markers associated with Tcm, and/or acquisition of helperindependence (antigen-driven IL-2 production), suggesting thatthese melanoma-specific CTL clones may have originated froma Tcm population. Although the infused CTLs were not geneti-cally marked, the established specific Vβ clonal identity arguesalmost unequivocally in favor of phenotypic reexpression ofmarkers shared with Tcm and against the outgrowth of a Tcmsubset in the infused population as might be conceivable wheninfusing polyclonal T cells. For patient 1, CTL persistence co-incided with tumor regression by 4 wk and continuous remissionfor 3+ years. Patient 2 demonstrated extended in vivo persistenceof the transferred CTLs but eventually progressed by 19 wk.Studies performed by the Surgery Branch at the National

Cancer Institute (NCI) have also demonstrated the significantimpact of lymphodepletion conditioning on in vivo persistenceof adoptively transferred tumor-infiltrating lymphocytes (TILs);in these seminal studies, CR rates as high as 16% were observedusing myeloablative regimens combining HD CY, fludarabine,and 12-Gy TBI, followed by HD IL-2 postinfusion (8, 9, 21).However, there are marked differences in the configuration andtoxicity profile of these NCI trials: A surgical procedure wasrequired to acquire TILs, TILs were propagated in vitro withhigh doses of IL-2, and the regimens led to acute morbidity andpotentially life-threatening toxicities, thereby limiting the poolof eligible patients. In the present study, preinfusion condi-tioning with a regimen of HD CY alone when used in con-junction with postinfusion LD IL-2 is safe and well-tolerated andrepresents a clinically feasible alternative with a broader patienteligibility profile. HD CY alone achieved a transient lympho-depleted condition that induced measurable circulating levels ofthe “prosurvival” cytokine IL-15 consistent with previous studies(8, 10) and enabled transferred cells to undergo early cell di-vision as demonstrated by Ki-67 expression (22). On-target tox-icities (skin rashes) appeared within 1 wk of infusion in patientswho achieved SD or CR at ≥4 wk, suggesting that the environ-ment fostered by HD CY enabled transferred T cells to reachperipheral tissue. In our study, the administration of HD IL-2after T-cell infusion yielded unacceptable toxicities and addeduncertain benefit.The use of CTL clones in this study provided very strong evi-

dence that transferred CTLs that persist in vivo (a reported pre-dictor of response) acquire characteristics of Tcm. In contrast toa previous study using a transferred polyclonal T-cell product, theuse of CTL clones eliminates the possibility of an outgrowth ofa preexisting Tcm subpopulation (4). However, the selection ofpersistent T-cell clones used for patients 1 and 2 was stochastic. Ameans to generate Tcm-like effector cells for adoptive therapyprospectively would be desirable. More precise control over a T-cell product with a Tcm phenotype may be achieved through theuse of in vitro cytokine modulation (23, 24), Tcm selection andTCR gene transfer, or infusion of a polyclonal T-cell populationwith an increased statistical probability of comprising a Tcmpopulation. Such Tcm-derived effectors become highly responsiveto homeostatic cytokines, such as IL-15, which is up-regulatedin vivo following CY lymphodepletion. In future studies, it is

Fig. 4. Adoptively transferred melanoma-specific CTLs show phenotypicand functional characteristics shared with CD8+ Tcm in vivo. (A) Patient (Pt)

1: Expression of CD45RO, CD27, CD28, CD127 CD62L, and CCR7 (bold lines)on the HLA*0201-Mart1-specific CTL clone preinfusion (Pre-inf.; Left), and ondays 6, 34, and 508 after transfer (Right). MFIs are shown for surface markerswith >1% positivity compared with isotype controls (gray areas). (B) Ex-pression of CD27 (y axis), CD62L (x axis) (Upper), and CCR7 (Lower) gatedon multimer-positive CTLs 508 d after transfer. Expression of CD28; secretionof TNF-α and IL-2 within IFN-γ+ cells on infused clones; and PBMCs pre-infusion and on days 20, 56, and 508 (C; patient 1) and days 20, 56, and 117(D; patient 2).

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conceivable that the combination of adoptively transferred Tcmeffectors, together with a source of LD exogenous cytokines, mayeliminate the requirement for a conditioning regimen altogetherwhile providing long-term antitumor control.

MethodsAll clinical investigations were conducted according to the Declaration ofHelsinki principles. Protocol 2140 was approved by the Fred HutchinsonCancer Research Center Institutional Review Board and the Food and DrugAdministration. All patients provided written informed consent, had meta-

static melanoma (Table 1), and had an historical estimated survival of lessthan 40% at 1 y (25).

Detailed descriptions of the materials and methods used, including se-lection of targets, treatment plan, cytotoxicity assays, T-cell tracking assays,and flow cytometry, are provided in SI Methods.

ACKNOWLEDGMENTS. This work was supported by National Institutes ofHealth/National Cancer Institute Grant R21 CA128283. A.G.C. was supportedby the Oncology Fellowship T32 Grant of the University of WashingtonMedical Center. C.Y. is supported by a Burroughs Wellcome Fund Trans-lational Scientist Award TS60637.

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Fig. 5. CY conditioning followed by exogenous IL-2 fosters a favorable environment for replication of infused CTLs. (A) Intranuclear Ki-67 expression onclones (Left) and CD8+ multimer-positive or -negative cells within PBMCs of patients 1, 9, 10, and 11 (rows) before CY and on days 0, 7, 14, 28, 56, and 508(patient 1 only) after T-cell infusion (columns). Multimer (y axis) and Ki-67 (x axis). (B) IL-15 plasma levels (pg/mL) (y axis) plotted over time (x axis). Gray linesrepresent individual patients, and filled circles show mean and SD. Patients in cohort 1 (Upper) and cohort 2 (Lower).

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