Adoptive Transfer of Autologous T Cells Improves T-cell ... Clin Cancer Res. 2012 .pdfStephan A. Grupp, Eline Luning Prak, Jean Boyer, et al. € Neuroblastoma Diversity and Long-term

2012;18:6732-6741. Published OnlineFirst October 23, 2012.Clin Cancer Res Stephan A. Grupp, Eline Luning Prak, Jean Boyer, et al. NeuroblastomaDiversity and Long-term B-cell Function in Pediatric Patients with Adoptive Transfer of Autologous T Cells Improves T-cell Repertoire

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Cancer Therapy: Clinical

Adoptive Transfer of Autologous T Cells Improves T-cellRepertoire Diversity and Long-term B-cell Function inPediatric Patients with Neuroblastoma

Stephan A. Grupp1, Eline Luning Prak4, Jean Boyer4,5, Kenyetta R. McDonald2, Suzanne Shusterman6,Edward Thompson4,5, Colleen Callahan1, Abbas F. Jawad3, Bruce L. Levine4,5, Carl H. June4,5, andKathleen E. Sullivan2

AbstractPurpose: Children with high-risk neuroblastoma have a poor prognosis with chemotherapy alone, and

hematopoietic stem cell transplantation offers improved survival. As a dose-escalation strategy, tandem

transplants have been used, but are associated with persistent immunocompromise. This study evaluated

the provision of an autologous costimulated, activated T-cell product to support immunologic function.

Experimental Design: Nineteen subjects with high-risk neuroblastoma were enrolled in a pilot phase

and 23 subjects were entered in to the randomized study. Immunologic reconstitution was defined by flow

cytometric and functional assays. Next-generation sequencing was conducted to identify changes to the

T-cell repertoire. Twenty-two patients were vaccinated to define effects on antibody responses.

Results: Subjects who received their autologous costimulated T-cell product on day 2 had significantly

superior T-cell counts and T-cell proliferation compared with those who received T cells on day 90. Early

administration of autologous T cells suppressed oligoclonality and enhanced repertoire diversity. The

subjects who received the day 2 T-cell product also had better responses to the pneumococcal vaccine.

Conclusions: The infusion of activated T cells can improve immunologic function especially when given

early after transplant. This study showed the benefit of providing cell therapies during periods of maximum

lymphopenia. Clin Cancer Res; 18(24); 6732–41. �2012 AACR.

IntroductionNeuroblastoma is the most common extracranial solid

tumor in childhood (1). It is remarkable for its clinicalrange, encompassing spontaneous regression to progres-sion and death. Approximately 12% of pediatric deathsfrom malignancy are due to neuroblastoma. The high-riskphenotype is generally characterized by disseminateddisease and increased mortality (2, 3). Additional featuresassociated with the high-risk phenotype include histopath-ologic features and MYCN amplification (2, 4). In thesehigh-risk patients, more than half of the patients relapse

aftermultimodal therapy. Recently, anti-GD2 immunother-apy to treat minimal residual disease after achievingremission has led to additional improvements in survival;however, stem cell transplantation is considered standard-of-care currently for high-risk patients (5, 6).

Autologous stem cell transplantation for neuroblastomahas been under investigation for more than 20 years, and in1997, a large retrospective analysis found improved survivalin patients treated with stem cell transplant as opposed toconventional chemotherapy (7). The single largest random-ized trial of autologous stem cell transplantation was theChildren’s Cancer Group 3891 study (8). In this study, allpatients received a consolidation regimen and then wererandomized to autologous transplantation or additionalchemotherapy. The study established autologous transplan-tation and isotretinoin therapy as the standard-of-care forhigh-risk patients. Despite this, more than half of thepatients relapsed. In recent years, the autologous stem celltransplantation concept has been extended to sequentialcycles. A tandem transplant allows for greater dose intensityand has shown promise as a strategy to improve outcomeswith the largest study showing a 3-year event-free survival(EFS) rate of 55% (9–11).

One of the significant issues in patients who receive atandem stem cell transplant is prolonged immunosuppres-sion. Posttransplant lymphoproliferative disease and other

Authors' Affiliations: 1Division of Oncology, 2Division of Allergy Immu-nology, 3Department of Pediatrics, The Children's Hospital of Philadelphia;4Department of Pathology and Laboratory Medicine, 5Abramson CancerCenter, Perelman School of Medicine at the University of Pennsylvania,Philadelphia, Pennsylvania; and 6Pediatric Oncology, Dana Farber CancerInstitute, Boston, Massachusetts

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Kathleen E. Sullivan, Division of Allergy Immu-nology, The Children's Hospital of Philadelphia, 3615 Civic Center Blvd.,Philadelphia, PA19104.Phone: 215-590-1697; Fax: 267-426-0363; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-12-1432

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(24) December 15, 20126732



http://clincancerres.aacrjournals.org/

viralmorbidities can occur (12, 13). This suggests that thereis prolonged, medically significant immunosuppression inthis population. To mitigate immunosuppression in othersettings, autologous expanded T cells have been used toprovide defense against viral infections (14–18). We exam-ined immunologic reconstitution in a cohort of childrenwith high-risk neuroblastomawho received a tandem trans-plant and were then randomized to receive either an autol-ogous expanded T-cell product on days 2 or 90.We carefullycharacterized immunologic parameters as well as their

ability to respond to vaccines. The subjects who receivedthe early autologous T-cell product had superior T-cellreconstitution and oligoclonality was suppressed duringthe vulnerable window posttransplant. Significant re-sponses to vaccines were seen almost exclusively in subjectswho received the early autologous T-cell product.

Materials and MethodsStudy design

This study protocol was approved by the InstitutionalReview Boards at Children’s Hospital of Philadelphia (Phi-ladelphia, PA) and Boston Children’s Hospital (Boston,MA), and informed consent was obtained from all partici-pants. The trial was preregistered at ClinicalTrials.gov asNCT00017368. The analysis plan was determined beforestudy initiation, and laboratory assays were conducted in ablinded fashion. In an initial (pilot) safety and feasibilityphase, patients received autologous expanded T cells on day2 (early), day 12 (middle), or day 90 (late) after the secondstem cell infusion. In a second phase of the study, patientswere randomized after enrollment to either the early (day 2)or late (day 90) T-cell infusion group (Fig. 1). The seasonalinactivated influenza vaccine and the conjugated 7-valentpneumococcal vaccine were administered on days 12 and60 after the second stem cell infusion. This protocol did notinclude immunoglobulin administration. In the analyses,baseline refers to samples obtained after diagnosis, butbefore chemotherapy. Days 30, 60, 90, 120, and 365 referto the time after the second transplant.

In all, 22 patients received T cells at day 2, 10 patients atday 12, and 12 patients at day 90. Two patients were

Induction High dose therapy andstem cell rescue

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Figure 1. Protocol description (randomized phase). All patients received the induction regimen and high-dose chemotherapy with stem cell rescue. Patientswere randomized to either the early group (autologous costimulated T-cell product on day 2 after the second stem cell rescue) or the late group (autologouscostimulated T-cell product on day 90 after the second stem cell rescue). The conjugated 7-valent pneumococcal vaccine and the inactivated trivalentinfluenza vaccine were administered on days 12 and 60 after the second transplant. Evaluations of immunologic function were conducted at the time ofdiagnosis and on days 30, 60, 90, 120, and 365. Carbo, carboplatin; CTX, cyclophosphamide; Ifos, ifosfamide; TBI, total body irradiation; VCR, vincristine.

Translational RelevanceNeuroblastoma is one of the most common solid

tumors of childhood. Over half of the high-risk patientsrelapse and the mortality rate is high with multimodaltherapy. Children can tolerate sequential or tandemhematopoietic stem cell transplants but morbidity andmortality are higher for tandem transplants than singletransplants, largely due to prolonged immunosuppres-sion. This study examined the use of an autologous T-cellinfusion to support childrenwhoundergo tandem trans-plants for neuroblastoma. T-cell infusions were showedto increase T-cell counts as expected; however, multiplemeasures of T-cell function and B-cell function wereimproved in a group that received their T-cell infusionsearly after transplant. This study not only shows thepromise of autologous T-cell infusion support aftertransplantation but also shows there is critical timewindow to maximize the effect.

Reconstitution after Tandem Transplant

www.aacrjournals.org Clin Cancer Res; 18(24) December 15, 2012 6733




excluded for early relapse or treatment-related mortality,and the other 42 patients were included in the pilot studyanalysis of the impact of a T-cell infusion, and the timing ofthe infusion, on T-cell recovery. One day 90 patient devel-oped CMV disease after stem cell infusion and received Tcells under a compassionate exception to the protocol at day60 and is included in the analysis of the day 90 group, asassigned. In the analysis of the second randomized phase,22 patients were randomized and included in the immuneresponse analysis, including vaccine response.

T-cell infusionsPatients underwent a single 2 to 6 blood volume leuka-

pheresis at diagnosis (before receiving chemotherapy) tocollect peripheral blood mononuclear cells (PBMC). Thecells were depleted of monocytes and cryopreserved. The Tcells underwent an established GMP costimulated activa-tion and expansion protocol using CD3- and CD28-linkedbeads (19). All infusedT-cell productswere required tomeetU.S. Food and Drug Administration (FDA)-specified safetyand release criteria before infusion. The target number of Tcells for infusion was 2 � 108 to 8 � 108 T cells/kg. As thiswas the first effort at expanding T cells from children, wecompared the expanded product from children and adults(Supplementary Data S1).

Immunologic assaysFor the primary endpoint of T-cell count after stem cell

transplant in the pilot phase, samples were submitted toCLIA-approved clinical laboratories for a complete bloodcount with differential, as well as CD4 percentage and CD8percentage according to standard clinical protocols. For thesecond randomized phase, detailed analyses of lymphocytesubsets were conducted according to research protocols. T-cell subset analyses were conducted after fixation with 1%paraformaldehyde on an LSR II (BD Biosciences) usingFlowJo software (TreeStar). CD4-na€�ve cells were defined asCD45RAþCD31þ, CD4 centralmemory T cells were definedas CD27þCD45ROþCCR7þ, CD4 effector memory T cellswere defined as CD45ROþ/CD27þ/CCR7�, CD4 revertedmemory T cells were defined as CD45RAþ/CD31�/CCR7þ,and regulatory T cells (Treg) were defined as CD4þ/CD25þ/CTLA4þ. CD8-na€�ve cells were defined asCD45RAþCD31þ,CD8 central memory T cells were defined as CD27þ

CD45ROþCCR7þ, CD8 effector memory T cells weredefined as CD45ROþ/CD27þ/CCR7�, and CD8 revertedmemory T cells were defined as CD45RAþ/CD31�/CCR7þ.

For B-cell phenotyping, fresh blood was stained withantibodies (all from BD Pharmingen), as described previ-ously (20) and run on a FACSCalibur (Becton Dickinson)withCellQuest software (Version5.2.1, BectonDickenson).CD19þ lymphocytes were analyzed for CD27 and IgM. Theabsolute B-cell count was obtained by multiplying theabsolute lymphocyte count by the CD19þ fraction.

T-cell proliferation, T-cell repertoire, T-cell ELISPOTs,and B-cell ELISPOTs were conducted as previouslydescribed (18). The assessment of influenza and pneumo-coccal vaccine responses used a standard hemagglutination

inhibition (HAI) assay optimized for the vaccine adminis-tered each year and a commercial laboratory for pneumo-coccal responses (21). Next-generation sequencing wasconducted by Adaptive TCR technologies (www.immuno-seq.com) using DNA from PBMCs, TCRb-specific primers,and a deep level of sequencing on the Illumina platform(22).

Statistical analysisThe linear regression model was used for analyzing

repeated measures based on the generalized estimatingequation (GEE) method using SAS GENMOD. The regres-sion models included group effects, time effects, and theinteraction of group � time. Baseline differences wereexamined to exclude group differences related to random-ization. To define the cumulative differences between the 2groups, the area under the curve was calculated. The inde-pendent t test, Kruskal–Wallis test, or the Mann–Whitneytestwere used for the comparisons of single timepoints. Thesmall size of the patients and the severity of their illnessoccasionally prevented obtaining all study samples. Eachanalysis indicates the number of subjects included. Thestudies reported were predefined in the study design. Sig-nificance was set at P � 0.05. Data analysis was conductedby Drs. Sullivan, Grupp, Boyer, Luning Prak, and Jawad. Allauthors had access to study data.

ResultsPatient characteristics

All patients had high-risk neuroblastoma and were olderthan 1 year. All but 2 patients had stage IV disease. Con-sistent with the known epidemiology of the disease, thechildren were ages 1 to 5 years. The subjects were roughlyhalf female and themajority of patients wereCaucasian. Forthe pilot study of T-cell infusions, 8 patients received theinfusion on day 2 (8 were stage IV and the median age was2.4 years), 8 patients received the infusiononday 12 (8werestage IV and the median age was 3.4 years), and 3 patientsreceived the infusion on day 90 (3 were stage IV and themedian age was 5.1 years). The randomized groups con-sisted of 12 subjects in the early group (day 2 infusion, 11 of12 stage IV;median age, 1.9 years) and10 subjects in the lategroup (day 90 infusion, 9 of 10 stage IV; median age, 2.7years).

This study was not designed or powered to look at theimpact of T-cell infusion on survival. However, we con-ducted a descriptive analysis of EFS in the cohort and amongpatients assigned to days 12, 2, and 90 T-cell infusion. Five-year EFS rate in the cohort was 55%, which was similar toour prior report of 47% (12). No differences between T-cellinfusion subgroups were apparent, with 5-year EFS rates inthedays 2, 12, and90 subgroups being55%,50%, and58%,respectively. One subject in each group experienced a post-transplant opportunistic infection. There was one case ofCMV in the late group at day 30 and one case of EBVlymphoproliferative disease in the early group on day 21.Both of these infections were fatal.

Grupp et al.

Clin Cancer Res; 18(24) December 15, 2012 Clinical Cancer Research6734




Pilot study of T-cell reconstitution, comparing threetime pointsWe hypothesized that autologous, costimulated expan-

ded T cells would alter the composition of the T-cellcompartment significantly. This CD3/CD28 bead–stimu-lated GMP cell therapy product has been previouslyshown to produce CD4:CD8 ratios of approximately1:1 and to maintain the T-cell receptor repertoire of theinput T cells (14, 15, 23). For analysis of the initial pilotphase, clinical measurements of ALC, CD3, CD4, andCD8 counts were used. These data are shown in Fig. 2 andshow a profound impact of T-cell infusion on the T-cellcount. The group receiving T cells at day 2 had improvedCD4 and CD8 T-cell counts compared with the day 90group in the day 2 to 90 interval. Interestingly, the day 2group also had improved T-cell recovery compared with

the day 12 group, suggesting that very early T-cell infusionmight result in even more robust CD4 cell recovery. Asimilar effect across the groups was seen in terms of CD8þ

cell recovery.

T-cell recovery after early or late T-cell infusionsDuring the randomized phase of the study, we expanded

the immunologic reconstitution assessments. We first char-acterized the T cells in the expanded autologous T-cellproduct because there have been no previous studies ofpediatric specimens. We identified the frequency of stem-like T cells (24), follicular helper T cells, Tregs, and othersubsets of biological relevance. Surprisingly, there werealmost no differences between the cell populations in theexpanded products from children and adults, althoughna€�ve CD4 T cells were increased in the expanded product

Figure 2. Schedule-dependentimpact of costimulated activatedT-cell transfer on T-cellreconstitution after tandemstem cell transplant. Patientsreceived T cells at 1 of 3 points in thissafety and feasibility phase: at days2, 12, and 90 after stem cell infusion.CD3þ, CD3þCD4þ (CD4), CD3þ

CD8þ (CD8) T cells were enumeratedon clinical samples at each timepoint. X-axis: pretransplant refers toblood drawn immediately before thefirst transplant; the other time pointsrefer to days after the secondtransplant. The CD4 and CD8 T-cellcount differences between the 3groups are significant at day 30(Kruskal—Wallis: P ¼ 0.0021 and0.0033, respectively). At day 60,CD3, CD4, and CD8 T cells areall significantly different withP ¼ 0.0005, 0.0003, and 0.0007,respectively.

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from the children compared with adults (SupplementaryData S1).

We characterized T-cell subsets using flow cytometry andcompared the day 2 early group (n¼ 12) and the day 90 lategroup (n¼ 7; Fig. 3). Absolute counts of bothCD4 andCD8T cells were higher in the early group than in the late groupin the early posttransplant interval (Fig. 3). We measuredthe area under the curve to compare the advantage of theearly T-cell product over the late T-cell product betweendays0 and 90. The improvement in peripheral blood T-cellcounts was significant with P ¼ 0.0023 for CD4 T cells anda P¼ 0.0010 for the CD8 T cells. To determine whether theexcess cells in the early group were of a specific subset, weanalyzed CD4 and CD8 T-cell na€�ve and memory subsets(Fig. 3). Examples of the na€�ve andmemory flow cytometricgating are given in Supplementary Data S2. Na€�ve CD4 andCD8 T-cell counts were higher in the early group than in the

late group at the 60-day time point (P¼ 0.0380 and 0.0344,respectively). The CD4 central memory T-cell count washigher in the early group than in the late group with P ¼0.0311. Tregs were somewhat higher in the early group.After the T-cell infusion in the late group at day 90, theadvantage of the early infusion was lost. The T-cell recon-stitution was variable in both groups. We examined age,number of T cells infused, level of stem-like cells in theexpanded product, and Tregs in the expanded product forassociations with na€�ve CD4 T-cell reconstitution. We didnot identify any associations.

On the basis of data from non-human primates, it isestimated that a typical 2- to 3-year-old child has a totalbody content of approximately 2 � 1011 T cells (25).Therefore, the increase in T-cell count in the peripheralblood was unlikely to be directly due to the addition of2 � 108 to 8 � 108 costimulated, activated T cells/kg

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Figure 3. Accelerated andsustained reconstitution of T cellsin the early group. CD4 centralmemory, CD4-naïve, and CD8-naïve cell counts were significantlyhigher at the 60-day time point inthe early group than in the lategroup (Kruskal–Wallis: P ¼ 0.03,0.04, and 0.006, respectively).�, significance.

Grupp et al.





(approximately 3 � 108 to 12 � 109 cells total). Thelymphocyte count was significantly higher in the earlygroup at day 30 than in the late group (P ¼ 0.007). Todeterminewhether T cells expanded in vivo, as has been seenin other settings (26, 27), we identified dividing CD3-positive T cells in the absence of stimulation by carboxy-fluorescein succinimidyl ester (CFSE; Fig. 4A). We reasonedthat T cells undergoinghomeostatic expansion in vivowouldcomplete the response to that signal in vitro. Cells wereincubated 5 days after staining with CFSE. There was enor-mous variability in the early group, whereas the late groupconsistently showed low cell division in this assay. Never-theless, the early group exhibited significantly more spon-taneous proliferation than the late group, consistent withhomeostatic expansion (Wilcoxon: P¼ 0.0010, early group:n ¼ 12, late group: n ¼ 7). To further examine the mech-anismof expansion, serum interleukin (IL)-7wasmeasuredin both groups. A slow increase in IL-7 was seen in the lategroup (Fig. 4B). Chemotherapy-driven release of cytokinesmay mediate early expansion, whereas IL-7 might beresponsible for the late expansion seen before the day 90infusion (28).To examine proliferative responses after stimulation, we

incubated PBMCs with phytohemagglutinin (PHA). CFSEdilution was defined using flow cytometry and the prolif-eration index (ameasure of average number of cell divisionsa responding cell has undergone) and the percentage divid-ed (a measure of the fraction of cells competent to divide)were calculated. The proliferation index was higher in theearly group, but this difference did not reach statisticalsignificance (P¼ 0.0522); however, the percentage dividedafter PHAwas significantly higher in the early group than inthe late group at day 60 with P ¼ 0.0232 (Fig. 4C). There-fore, provision of an autologous T-cell product at an earlytime point after the second transplantation provided super-ior quantitative and qualitative reconstitution of the T-cellcompartment early posttransplant.

Expansion of T cells could be associated with contractionof the T-cell repertoire. We initially analyzed the diversity ofthe repertoire by spectratyping (29). Unexpectedly, thediversity was impressively restricted in the patients beforeany chemotherapy with more than half of the Vb familiesmissing or having oligoclonal peaks (Fig. 5). A previousstudy of adults, using this technology, found approximately10% abnormal Vb families (30). There was a modestincrease in the frequency of abnormal Vb families at days30 and 60 in the late group, suggesting expansion of a smallnumber of founder clones. To better explore the contribu-tion of the infusion to the T-cell repertoire, 5 patients in theearly group and 4 patients in the late group were examinedusing next-generation sequencing to better identify altera-tions to the repertoire. We hypothesized that baselinerepertoire contraction could be due to expansion of atumor-specific clone; however, at baseline, there were noT-cell receptor (TCR) sequences shared by all patients. Forall 4 patients who had baseline samples available, thenumber of unique reads was higher in the infusion samplethan in the baseline sample, supporting the previous find-ing that in vitro expansion using this method preservespolyclonality (15). There were relatively few clones fromthe infusion that appeared in later time points,�5.5% in allcases. This is consistent with the small contributionexpected from the infusion. Unexpectedly, provision of thissmall number of cells led to increased repertoire diversity(diminished oligoclonality; Fig. 5). For this analysis, weenumerated the number of clones comprising more than5% of the total reads as a marker of oligoclonality. Therewere no oligoclonal sequences at baseline, but at day 30, thelate group had an average of 4 oligoclonal sequences,whereas the early group had an average of 0.25 oligoclonalTCR sequences (P ¼ nonsignificant). Similarly, when thefrequencies of the most common clones were analyzed, theearly group had amean frequency of 3.5%, whereas the lategroup had amean frequency of 13.5% (P¼nonsignificant).

Figure 4. Preservation of peripheralblood T-cell proliferation by earlyT-cell transfer. A, at day 30, T-cellproliferation occurs in the absenceof stimulation in the early group,consistent with homeostaticproliferation. The difference betweenthe early and late Groups wassignificant at day 30 (Wilcoxon:P < 0.001). B, IL-7 serum levels,detectedbyELISA, increase slowly inthe late group. C, the proliferationindex measures the competence tocycle after PHA stimulation and thiswas not different between the 2groups. The percentage dividedmetric measures the fraction of cellsthat entered into cell cycle after PHAstimulation. The early group had ahigher fraction of responding cellsthan the late group at day 30(P ¼ 0.02). PI, propidium iodide.

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As a final measure of diversity, we enumerated the numberof distinct Vb-J combinations at each time point. The earlygrouphad significantlymorediversity at day30 than the lategroup (P ¼ 0.008; Fig. 5).

The suppression of oligoclonality could be due to greater"fitness" of the ex vivo expanded cells or could be due toenhancement of thymic output, such as has been previouslysuggested (31). Clones that were identified in both baselineand day 150 samples comprised an average of 1.1% of thetotal day 150 clones and clones that were identified in boththe infusion and the day 150 samples comprised anaverage of 0.65% of the total day 150 clones, suggestingthat the final composition of the repertoire was modestlyderived from the infused cells. Thymic export of new T cellswould be consistent with the na€�ve phenotype identifiedby flow cytometry (Fig. 3). To explore this, we conductedanalyses of T-cell receptor excision circles (TREC). Four of9 subjects in the early group had increased TRECs at day 30compared with their baseline, whereas 1 of 7 of the lategroup did. At day 150, 5 of 7 of the late group and 2 of 6 ofthe early group had increased TRECs compared withbaseline.

Responses to vaccinesImproved T-cell reconstitution could improve B-cell

function. To analyze this, we examined the response to theinfluenza vaccine for each of the 3 serotypes present in theinactivated vaccine. There was wide variability in the early

group, whereas the late group exhibited very little variabil-ity. There were no significant differences in the mean titerbetween the 2 groups and overall the responses wereextremely low. We also calculated the seroconversionfrequency at each time point by defining a 4-fold increasefrom the baseline titer. Two patients in the early group(n¼ 14) produced a 4-fold titer increase after vaccination toall 3 serotypes. Three additional patients produced a 4-foldincrease in titer to at least one serotype in the early group.In contrast, only one patient in the late group (n ¼ 8)produced a 4-fold response to a single serotype (P ¼ notsignificant). The low titers at baseline suggest that thispopulation was largely na€�ve to influenza, which mayhave compromised their ability to respond to the vaccine.We therefore examined responses to the conjugated pneu-mococcal vaccine, which the patients should havereceived as part of their childcare before developing neu-roblastoma.

Pneumococcal responses were evaluated as representa-tive of an antigen previously encountered and a typicalmemory response (Fig. 6). Fourteen serotypes were evalu-ated: 7 contained in the conjugate vaccine and 7 serotypesnot in the vaccine. There were no responses to the serotypesthat are not in the vaccine, as expected. For the serotypes inthe vaccine, the baseline titers were higher than the non-vaccine serotypes, consistent with prior vaccination. Therewas a dichotomous response at the 1-year time point withthe early group having significantly higher responses than

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Figure 5. TCR repertoire analysis. A, the fraction of abnormal (oligoclonal or absent) Vb families is displayed on the y-axis. A total of 23 Vb families wereexamined by PCR analysis (spectratyping). There were no differences between the early and the late group; however, the baseline diversity was poor. B, toassess oligoclonality, next-generation sequencing was used. As a measure of oligoclonality, the number of clones that represented at least 5% of the readswere enumerated. The infused product itself was polyclonal and oligoclonality in the late group seen at day 30 was corrected after infusion at day 90.C, the frequency of the most abundant clone was also assessed as a measure of oligoclonality. The late group has a higher mean frequency of the mostabundant clone at day 30 than the early group, with normalization by day 150. D, the absolute number of Vb-J combinations detected across the time points isplotted as a measure of diversity. There are 705 potential Vb-J combinations. At day 30, the early group has significantly more Vb-J combinations thanthe late group (P ¼ 0.0082).

Grupp et al.





the late group at 1 year. To determinewhether this effect wasdue to a single serotype, the serotypes were individuallyevaluated. All serotypes individually were significantlyhigher at 1 year in the early group than in the late groupand all patients exhibited the same pattern. These datasupport thebeneficial effect of the autologous T-cell producton host humoral memory responses.

B-cell maturationTo better understand how the early autologous T-cell

infusion led to greater antibody production, we exam-ined the B-cell subsets in the peripheral blood at baseline

and various time points after transplantation. Neithertotal B cells nor any of the subsets differed between theearly and late Groups, although B-cell maturationappeared to be modestly accelerated in the early group(Supplementary Data S3). To examine B-cell function, weused B-cell ELISPOTs, measuring both the total IgG andthe influenza-specific IgG produced. There were no dif-ferences between the early group and the late group, butresponses were universally low using this assay (data notshown).

DiscussionThis study was designed to test the efficacy of an adoptive

transfer of a costimulated, activated autologous T-cell prod-uct as an approach to regenerate cellular immunity afterhigh-dose cytotoxic therapy. Patients who receive a tandemtransplant for high-risk neuroblastoma are rendered severe-ly immunodeficient for a prolonged period of time(5, 9, 13, 32, 12). Consequences of the immunocompro-mise are viral morbidity and mortality related to infection(13). The provision of an autologous T-cell product canprovide support during the vulnerable posttransplant peri-od. In adults, autologous T-cell products have been usedwith a significant improvement in immunologic function asmeasured by T-cell counts and vaccine responses (18, 33–36). To our knowledge, no studies have tested the regener-ative potential of adoptive T-cell transfer in nonhematolo-gicmalignancies. Furthermore, no studies have assessed thisin children where the kinetics of immune reconstitutionmay differ due to increased thymic potential (37).

This study clearly showed a greater effect the earlier the T-cell infusion was administered in the pilot phase, althoughthe improvement was greater than what we could accountfor by the infusion itself. Patients receiving infusions on day12 had the least marked T-cell reconstitution, and wehypothesize that this time point fell between inflammatorycytokine-induced expansion and IL-7–induced expansion.We found evidence of homeostatic expansion of the trans-ferred cells at day 30, supporting a role for this process inexpansion and reconstitution in the cytokine-rich, lympho-penic environment (28). There was a significant increase inna€�ve cells. Although strong homeostatic signals drive ana€�ve tomemory conversion, not all homeostatic expansionis associated with conversion to a memory phenotype andwe hypothesize that both thymic production and homeo-static expansion contributed to quantitative reconstitution(38–40). The TREC data were not uniform among subjects,but the data were suggestive of the induction of thymopoi-esis in some patients after the T-cell infusion. Overall, theearly (day 2) T-cell infusions resulted in superior T-cellreconstitution as assessed by quantitative and qualitativemeasures and we believe this will improve the defenseagainst opportunistic infections.

To probe the breadth of improvement in immunologicfunction, we vaccinated children after their second trans-plant. We anticipated that most children would be na€�ve toinfluenza because of their young age and that nearly all

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Figure 6. Early T-cell reconstitution promotes B-cell memory responsesfollowing pneumococcal vaccination. A and B, aggregate pneumococcalmean titers were calculated by summing the antibody responses to the 7serotypes not in the vaccine (A) and for the vaccine (B) serotypes (earlygroup: n¼ 9; late group: n¼ 7). At baseline, the differences between thevaccine serotypes and nonvaccine serotypes were significant for boththe early group (Mann–Whitney:P¼0.020) and the late group (P¼0.002),confirming prior vaccination. At 1 year, the difference between the earlyand the late groups comparing the vaccine serotypes was significant(Kruskal–Wallis: P ¼ 0.002), whereas serologic response to nonvaccineserotypes did not differ. The vertical arrows denote the vaccineadministration time points. C, when each serotype was examinedseparately, the effect at day 365 was consistent across all vaccineserotypes. The vertical arrows denote the vaccine administration timepoints. Each black line indicates a single vaccine serotype response andeach gray line indicates a single nonvaccine serotype response.






children would have had the conjugated pneumococcalvaccine as part of routine childcare. We noted that thebaseline aggregate titers to pneumococcal serotypes weresignificantly different between the vaccine serotypes and thenon-vaccine serotypes, confirming that this cohort waspreviously vaccinated. There was a modest increase in titersat the day 30 time point, but themost remarkable result wasthe dramatic increase in vaccine-specific pneumococcaltiters at the 1-year time point, indicating that long-termB-cell immunity is dependent on antigen-specific T-cellfunction.

The delayed kinetics were surprising; however, similarstudies in adults show that priming before the T-cell harvestmarkedly improves subsequent vaccine responses (18, 19).In those studies, the conditioning regimen did not depletememory B cells, and it could not be determinedwhether thepriming of B cells was critical for the vaccine responses. Inthis study, the conditioning before transplant led to anabsence of peripheral blood B cells on day 2, suggestingthat the infused primed T cells were largely responsible forthe pneumococcal responses in the early group. The earlygroup received the vaccines after infusion of the T cells,whereas the late group received the vaccines before the cellinfusion. The presence of primed T cells at the time ofvaccination had a salutary if undefined effect on subsequentantibody responses to the vaccine. The delayed kineticsmight reflect the recovery kinetics of the B-cell compart-ment, which achieved normal numbers only at the 1-yeartime point. The T cells may have had a trophic effect on B-cell development (41).

Harnessing the immune system for an antitumor effect isa long-term goal of immune reconstitution and infusion ofgenetically modified T cells expanded using CD3- andCD28-linked beads recently show dramatic antitumorresponses (26, 27). The current data suggest that primingis important, for humoral responses and a study of adultsreceiving a similar product also concluded that primingoptimized vaccine responses (34). The most importantlesson is that the autologous T-cell product can provideimportant immunological support but it must be admin-istered early. These results are also the first indication that

the kinetics of immune reconstitution in the pediatricimmune systemhas profound effects on long-termhumoralimmunity. These results may guide future studies to recon-stitute immunity after high-dose chemotherapy and per-haps for the induction of antitumor immunity.

Disclosure of Potential Conflicts of InterestE.L. Prak is a consultant/advisory board member of Genentech. B.L.

Levine and C.H. June are inventors on patents related to the T-cellmanufacturing process that was used for this protocol. This conflict hasbeen disclosed and was managed in accordance with the policies of theUniversity of Pennsylvania. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: S.A. Grupp, B.L. Levine, C.H. June, K.E. SullivanDevelopmentofmethodology:S.A.Grupp, E. Thompson, B.L. Levine,C.H.JuneAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S.A. Grupp, E.L. Prak, J. Boyer, K.R. McDonald, S.Shusterman, E. Thompson, B.L. LevineAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S.A. Grupp, E.L. Prak, A. Jawad, B.L.Levine, C.H. JuneWriting, review, and/or revisionof themanuscript: S.A.Grupp, E.L. Prak,K.R. McDonald, A. Jawad, B.L. Levine, C.H. JuneAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.A. Grupp, C. Callahan, A. Jawad,B.L. LevineStudy supervision: S.A. Grupp, K.R. McDonald

AcknowledgmentsThe authors thank the expert technical contributions of Kelly Maurer, the

staff at theHuman ImmunologyCore facility, the staff of theClinical Cell andVaccine Production Facility (Julio Cotte, Zhaohui Zheng, Andrea Brennan),Yang-Zhu Du, Noah Goodman, and Xiaoling Hou. They also thank thenurses andphysicians caring for these patients aswell asDan Schullery for hisorganizational contributions.

Grant SupportThis work was supported in part by NIH NO1-AI-50 024 and R01-

CA105216.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received May 2, 2012; revised September 14, 2012; accepted September28, 2012; published OnlineFirst October 23, 2012.

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