Journal of Dermatological Science 54 (2009) 31–37
Immunoregulatory T cells in the peripheral blood of melanoma patientstreated with melanoma antigen-pulsed mature monocyte-deriveddendritic cell vaccination
Noriaki Nakai *, Norito Katoh, Tomoko Kitagawa, Eiichiro Ueda, Hideya Takenaka, Saburo Kishimoto
Department of Dermatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan
A R T I C L E I N F O
Article history:
Received 13 August 2008
Received in revised form 17 November 2008
Accepted 22 November 2008
Keywords:
Regulatory T cells
Forkhead box protein 3
Dendritic cells vaccination
Melanoma
A B S T R A C T
Background: Regulatory T cells (Treg) may inhibit monocyte-derived melanoma antigen-pulsed
dendritic cells (DC) vaccination in treatment of melanoma. However, the Treg level in peripheral
blood mononuclear cells (PBMCs) following DC vaccination has not been examined in melanoma
patients in Japan.
Objective: To evaluate differences in the helper T cell and Treg population and mRNA levels of Treg in
pre- and post-DC vaccination PBMCs obtained from melanoma patients.
Methods: Levels of intracellular forkhead box protein 3 (Foxp3) mRNA as well as levels of
CD4+CD25+Foxp3+ and CD4+CD25+ T cells were examined by real-time PCR and flow cytometry using
PBMCs from 9 patients who received DC vaccination.
Results: Eight of the 9 cases and 7 of the 9 cases showed increased populations of CD4+CD25+ T cells and
CD4+CD25+Foxp3+ T cells, respectively after repeated DC vaccination. Five of 8 cases showed an increase
of Foxp3 mRNA after treatment. Four of these 5 cases also had increased CD4+CD25+ and
CD4+CD25+Foxp3+ T cells, but the fifth case showed a decrease in CD4+CD25+Foxp3+ T cells. Three
cases showed a decrease of Foxp3 mRNA. One of these 3 cases showed decreased population of
CD4+CD25+Foxp3+ T cells, but two cases showed increased population of CD4+CD25+Foxp3+ T cells. In 3
of 8 cases Foxp3 expression at the cellular (protein) and mRNA level were inconsistent.
Conclusion: Repeated DC vaccination may commonly induce Treg and helper T cells at the cellular level.
However, there are a few discrepancies of Treg expression at cellular and mRNA level.
� 2008 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Contents lists available at ScienceDirect
Journal of Dermatological Science
journa l homepage: www. int l .e lsev ierhea l th .com/ journa ls / jods
1. Introduction
The incidence and mortality of malignant melanoma isincreasing among Asians and Caucasians [1]. Surgical treatmentis effective for early-stage melanoma, but new strategies arerequired for patients with advanced melanoma that is highlyresistant to conventional chemotherapy and radiotherapy. Immu-notherapy with autologous dendritic cell (DC) vaccination is anattractive strategy [2]. DCs are differentiated from peripheral bloodmonocytes and administered to patients after pulsing with tumorantigens with the goal of inducing an active immune response [2].This is a safe procedure that can lead to induction of an anti-tumoreffect due to proliferation and activation of melanoma-specificcytotoxic T cells [3,4]. However, potential disadvantages of DCvaccination include induction of tolerance caused by cytokines
* Corresponding author. Tel.: +81 75 251 5586; fax: +81 75 251 5586.
E-mail address: [email protected] (N. Nakai).
0923-1811/$30.00 � 2008 Japanese Society for Investigative Dermatology. Published b
doi:10.1016/j.jdermsci.2008.11.007
produced by regulatory T cells (Treg) [5], loss of MHC class Iexpression [3,6], and mutations in or downregulation of tumorantigens [4]. This has led to the suggestion that cancerimmunoediting has limited clinical efficacy.
We showed positive delayed type hypersensitivity (DTH)responses and induction of vaccine peptide-specific IFN-g-produ-cing T cells to melanoma antigen in DC vaccination in our previousreport [3]. While we have also shown histopathologically that DCvaccination causes loss of MHC class I expression and down-regulation of tumor antigens in metastases [3], and an increase ofTreg as well as DC, CD4+, CD8+, and IFN-g+ cells in DTH site [7].Furthermore, we found no statistically significant increase insurvival in patients treated with DC vaccination compared withthose receiving conventional therapy [8].
In recent years Banerjee et al. [9] reported that cytokine-matured DC led to rapid enhancement of Treg in vitro and in vitroin myeloma patients. This fact and our previous result [7] suggestthat DC vaccination may have the possibility to induce tolerance oftumor as well as antigen-specific anti-tumor immunity.
y Elsevier Ireland Ltd. All rights reserved.
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N. Nakai et al. / Journal of Dermatological Science 54 (2009) 31–3732
In the current study, we examined changes in populations ofCD4+CD25+ and CD4+CD25+Foxp3+ T cells in peripheral bloodmononuclear cells (PBMCs) obtained pre- and post-DC vaccinationfrom 9 patients with advanced melanoma. The change in the Foxp3mRNA level in CD4+ T cells from pre- to post-DC vaccination wasalso investigated.
2. Patients and methods
2.1. Patients
Nine patients with stage III or IV melanoma were judged to beeligible for the study. All were patients that participated inprevious study [3]: patient 1, 2, 3, 4, 5, 6, 7, 8, and 9 werecorresponded with patient 5, 3, 7, 4, 6, 16, 8, 2, and 13, respectively.Six of 9 were also patients that participated in previous study [7]:patient 1, 2, 3, 5, 6, and 8 were corresponded with patient 3, 2, 5, 4,7, and 1, respectively. Entry criteria included age over 18 years old,expression of HLA-A2 or HLA-A24 on PBMCs, and anticipatedsurvival of greater than 3 months. Tumor lysate was used inpatients from whom we were able to excise the tumor or patientswho did not exhibit HLA-A2 or HLA-A24. The characteristics of thepatients are listed in Table 1. The study protocol was approved bythe Institutional Ethical Review Board of the Graduate School ofMedical Science, Kyoto Prefectural University of Medicine.Informed consent was obtained from the patients before entryinto the study.
2.2. Preparation of monocyte-derived melanoma antigen-pulsed DCs
for vaccination
Preparation of monocyte-derived melanoma antigen-pulsedDCs and the vaccination protocol have been described previously[3]. In short, monocytes were isolated from peripheral blood, anddifferentiated to DCs with IL-4 and GM-CSF for 7 days followed bymaturation with TNF-a and Poly(I:C) for 3 days. Mature DCs werecoincubated with a cocktail of melanoma peptides at 50 mg/ml for6 h. Immature DCs were cultured for 24 h in medium containing100 mg/ml of autologous tumor lysate on day 7, and then in amedium containing 1000 U/ml of TNF-a and 20 mg/ml of Poly(I:C)for an additional 3 days to induce terminal differentiation intomature DCs. Patients were vaccinated in the groin with 10 � 106
cells per injection in 10 intradermal injections as one course oftreatment every week or every other week.
2.3. Evaluation of clinical outcome
Clinical outcome was evaluated based on analysis of wholebody CT images before and after one course of treatment (10vaccinations) [3,8].
2.4. Delayed type hypersensitivity response
Delayed type hypersensitivity was examined as describedpreviously [3,7]. Briefly, patients received intradermal injections of200 ml of PBS with 10 mg of MAGE-1, 2, 3, tyrosinase and MART-1respectively, or 20 mg of tumor lysate at separate sites on theforearm. Forty-eight hours later, DTH was assessed by measuringthe area of erythema and induration using two-dimensionalmeasurements. DTH was scored positive if the area of erythemaand induration was greater than 10 mm.
2.5. Enzyme-linked immunosorbent spot assay
Enzyme-linked immunosorbent spot (ELISPOT) assay for thedetection of antigen-specific interferon (IFN)-g-producing T cells
Table 2ADTH to the tumor lysate and individual melanoma peptides.
Patient
no.
HLA-A24-peptides HLA-A02-peptides Tumor
lysateMAGE-1 MAGE-2 MAGE-3 Tyrosinase MAGE-3 MART-1
1 + + + +
2 + + + + + +
3 + + + �4 +
5 + + + + +
6 + + � � �7 � �8 + + + + + + +
9 +
Blanks indicate the peptides or tumor lysate without application for patients.
N. Nakai et al. / Journal of Dermatological Science 54 (2009) 31–37 33
was performed, as described previously [3]. Briefly, PBMCs(5 � 105 cells/well) were added to plates precoated with 10 mg/ml of a primary anti-IFN-g monoclonal antibody (R&D Systems,Minneapolis, MN, USA) in the presence or absence of 10 mg/ml ofpeptide antigens and incubated for 16 h. The antigens were HLA-A-24 and HLA-A-2 restricted melanoma peptides and tumor lysateused in the DC vaccine. PBMCs with no peptide were used as anegative control and PBMCs with 50 mol/l of PMA was used as apositive control. A positive response was arbitrarily defined as atleast a two-fold increase in peptide-specific IFN-g ELISPOTs and atleast 10 spots/5 � 105 PBMCs at any time point post-DC vaccina-tion.
2.6. Purification of CD4+ T cells from PBMCs
CD4+ T cells were captured with antibodies coupled to MACSbeads according to the manufacturer’s instructions (CD4 Microbe-ads, Miltenyi Biotec, Bergisch Gladbach, Germany). In short, PBMCsfrom 9 patients and 5 healthy individuals, respectively, wereisolated by density gradient centrifugation using Ficoll-PaqueTMand resuspended in phosphate-buffered saline (PBS) (pH 7.2)containing 0.5% bovine serum albumin (BSA) and 2 mM EDTA (107
cells in 80 ml). After addition of 20 ml of CD4 microbeads per 107
PBMCs, the PBMCs were incubated for 15 min at 4 8C, washed with1 ml of buffer per 107 cells, and applied to an MS column andseparated magnetically. After removal of the unlabeled cellfraction, the magnetically labeled cells were flushed out by firmlypushing the plunger into the column. The purity of CD4+ T cells wasconsistently higher than 95% in all experiments, as determined bystaining with anti-CD4 antibody and flow cytometry.
2.7. Real-time RT-PCR
RNA was extracted from the CD4+ T cells and reverse-transcribed using an OmniscriptTM RT Kit (Qiagen, Hilden,Germany). Briefly, 2 ml of 10 x RT buffer, 2 ml of dNTP Mix, 2 mlof Oligo-dT primer, 1 ml of RNase inhibitor, 1 ml of Omniscriptreverse transcriptase, RNase-free water, and the extracted RNAsolution were placed in a tube in a final volume of 20 ml for eachsample. The tube was mixed thoroughly and incubated for 60 minat 37 8C. Matching primers and dye probes for Foxp3(Hs00203958_m1) and b-actin (Hs99999903_m1) were purchasedfrom Applied Biosystems (Foster City, CA, USA). Samples of 12.5 mlof Taqman1 universal master mix, 1.25 ml of primers, 8.75 ml ofdistilled water, and 2.5 ml of sample cDNA were placed in tubesand real-time PCR was performed using the 7300 Real-Time PCRSystem (Applied Biosystems), followed by quantification of RNAlevels by RQ software (Applied Biosystems).
2.8. Flow cytometry
CD4, CD25 and Foxp3 expression levels were determined with ahuman Treg FlowTM Kit (BioLegend, San Diego, CA) using theconjugated Abs CD4-PE-Cy5, CD25-PE, FOXP3-Alexa Fluor1 488, ormouse isotype control IgG1-Alexa Fluor1 488. Cell staining wasperformed according to the manufacturer’s instructions. In short,1 � 106 PBMCs were resuspended in staining buffer (PBS contain-ing 3% fetal bovine serum) in a tube. After centrifugation, 20 ml ofCD4 PE-Cy5/CD25 PE cocktail was added to each tube and thePBMCs were incubated at room temperature in the dark for 20 min.After washing once with 1 ml of cell staining buffer, 1 ml of 1�FOXP3 Fix/Perm solution (Biolegend) was added to each tube. Aftervortexing, the PBMCs were incubated at room temperature in thedark for 20 min, washed with cell staining buffer and then with1 ml of 1� FOXP3 Perm buffer (Biolegend), and incubated with1 ml of 1� FOXP3 Perm buffer in the dark for 15 min. After spinning
down and removal of supernatant, the PBMCs were resuspendedwith 100 ml of 1� FOXP3 Perm buffer and then combined with 5 mlof Alexa Fluor1 488 anti-human FOXP3 antibody or 5 ml of AlexaFluor1 488 mouse IgG1, k isotype control and incubated at roomtemperature in the dark for 30 min. The PBMCs were then washedwith cell staining buffer and resuspended in 0.5 ml of cell stainingbuffer. Populations of CD4+CD25+ T cells and CD4+CD25+Foxp3+ Tcells were determined using a FACSCalibur flow cytometer (BDBiosciences) and BD CellQuest software.
2.9. Statistical analysis
Comparisons were performed by Student’s t-test with asignificance level of P < 0.05.
3. Results
3.1. Outcomes
Nine patients with advanced malignant melanoma wereenrolled in the study: 2 with stage IIIB disease and 7 with stageIV disease (Table 1). All patients received at least one completecourse of treatment. After completion of one course, two cases(patient 3 and 4) showed stable disease, five (patients 1, 5, 6, 7 and9) showed progression of disease, and two (patients 2 and 8) thatwas performed as an adjuvant treatment after removing thelymph node metastasis showed no evidence of disease from thetime of study entry to the completion of one vaccination course(Table 1). The 2 stage III patients survived for more than 140weeks. The median survival period was 99.3 weeks for 3 stage IVpatients (patients 4, 5, and 6) with metastases in one organ withpositive responses in DTH or ELISPOT assays or accumulation ofIFN-g+, CD4+, and CD8+ T cells at DTH sites (Table 1) [7]. Themedian survival period was 27.5 weeks for 2 stage IV patients(patients 7 and 9) with metastases in one organ and negativeresponses in both DTH and ELISPOT assays or with a positive DTHresponse only (Table 1). A median survival period of 26.5 weekswas observed for 2 stage IV patients (patients 1 and 3) withmetastases in two different organs despite positive responses inDTH and ELISPOT assays and confirmation of IFN-g+, CD4+, andCD8+ T cells at DTH sites (Table 1) [7]. Positive DTH responses to atleast one antigen were seen in 8 of 9 patients (Table 2A). Whileinduction of vaccine peptide-specific IFN-g producing T cells to atleast one antigen by ELISPOT assay was seen 6 of 9 patients(Table 2B).
3.2. CD4+CD25+Foxp3+ T cells and CD4+CD25+ T cells in PBMCs in
melanoma patients and healthy individuals
The frequencies of CD4+CD25+Foxp3+ T cells in melanomapatients before vaccination were slight higher than healthy
Table 2BIFN-g ELISPOTs to the tumor lysate and individual melanoma peptides.
Patient
no.
HLA-A24-peptides HLA-A02-peptides Tumor
lysateMAGE-1 MAGE-2 MAGE-3 Tyrosinase MAGE-3 MART-1
1 + + ND ND
2 + + + + � ND
3 + + + +
4 +
5 + + + + +
6 � � � � �7 � �8 + + + + + + ND
9 �
ND, not done. Blanks indicate the peptides or tumor lysate without application for
patients.
N. Nakai et al. / Journal of Dermatological Science 54 (2009) 31–3734
individuals (patients, 6.4 � 2.3%; healthy individuals, 4.4 � 1.1%),however, the statistical significance was not confirmed (P = 0.1)(Fig. 1). While the frequencies of CD4+CD25+ T cells between the twowere also no statistical significance (patients, 13.9 � 6.9%; healthyindividuals, 13.2 � 2.0%) (Fig. 1).
3.3. Pre- and post-vaccination blood analysis
Of 8 evaluable cases, 5 (patients 2, 3, 4, 5, and 6) showedcomparatively favorable conditions without remarkable elevationof CRP and LDH in blood tests before and after the post-treatmentsample collection point and 3 (patients 1, 7, and 8) showed poorresults in blood tests after the post-treatment sample collectionpoint (Table 3). Patient 7 showed severe liver tumor proliferationwith ascites after 12 DC vaccinations, with severe elevation of CRPand LDH and decreases in red blood cell count, platelet count,hemoglobin, total protein, and albumin. Patient 8 also showedsevere elevation of CRP, LDH and g-GTP due to rapid progression ofmetastases in lung, liver, biliary tract, pharynx, and cervical lymphnodes after 71 vaccinations. Patient 1 showed progressive diseasewith severe elevation of CRP and LDH and decreases in red bloodcell count and hemoglobin due to enlargement of the primarytumor and metastatic lung tumors.
3.4. CD4+CD25+ and CD4+CD25+Foxp3+ T cells in PBMCs pre- and
post-vaccination
Of the 9 cases, 7 (patients 1, 2, 3, 4, 6, 7, and 9) showed increasesin the populations of CD4+CD25+Foxp3+ T cells in PBMCs after thepost-treatment sample collection point (Fig. 2, Table 4). While 8(patients 1, 2, 3, 4, 5, 7, 8, and 9) showed increase in thepopulations of CD4+CD25+ T cells in PBMCs after the post-treatment sample collection point. The increase or decrease inCD4+CD25+ T cells corresponded to the change inCD4+CD25+Foxp3+ T cells from before to after treatment in 6patients (patients 1, 2, 3, 4, 7, and 9). However, two (patients 5 and8) showed increase in CD4+CD25+ T cells populations (Fig. 2,
Fig. 1. The frequencies of CD4+CD25+Foxp3+ T cells and CD4+CD25+ T cells in PBMCs in he
CD4+CD25+Foxp3+ T cells and CD4+CD25+ T cells in CD4+ T cells from PBMCs. healthy
Table 4) and decrease in CD4+CD25+Foxp3+ T cells population.While one (patient 6) showed slight decrease in CD4+CD25+ T cellspopulations and slight increase in CD4+CD25+Foxp3+ T cellspopulation. Eight of 9 patients (patients 1, 2, 3, 4, 5, 7, 8, and 9)showed significant change of T cells population after the post-treatment sample collection point (Fig. 2, Table 4), whereas 1 case(patient 6) showed subtle change.
3.5. Foxp3 mRNA levels in CD4+ T cells pre- and post-vaccination
The Foxp3 mRNA level was determined in 8 of 9 cases, of which5 (patients 1, 2, 3, 4, and 5) showed increased Foxp3 mRNA afterthe post-treatment sample collection point (P < 0.05,<0.0005, and<0.005 for patients 1, 2 and 5, respectively) (Fig. 3, Table 4). Four ofthese cases (patients 1, 2, 3, and 4) also had increased populationsof CD4+CD25+Foxp3+ T cells in PBMCs after treatment. However,the Foxp3 mRNA level in fifth case (patient 5) increased despitedecreased CD4+CD25+Foxp3+ T cells in PBMCs. Three cases(patients 6, 7 and 8) showed decreased Foxp3 mRNA after thepost-treatment sample collection point (P < 0.05, <0.005 and<0.0005 for patients 6, 7 and 8, respectively) (Fig. 3, Table 4).Among the 3 cases one (patient 8) also had decreasedCD4+CD25+Foxp3+ T cells, but two (patient 6 and 7) had increasedCD4+CD25+Foxp3+ T cells in PBMCs after the post-treatmentsample collection point.
4. Discussion
Our results showed that CD4+CD25+ T cells in PBMCs increasedin 8 of 9 patients after the post-treatment sample collection point,which indicated that activated helper T cells and Treg wereinduced by DC vaccination. CD4+CD25+ T cells include both Tregand activated T cells [10], and in some studies CD4+CD25high T cellshave been regarded as Treg cells and gated on top of the 4%CD4+CD25+ population in flow cytometry [10]. To address thisissue, we performed triple staining for CD4, CD25 and Foxp3 todetermine the Treg levels in PBMCs after DC vaccination. Thetranscription factor Foxp3 is considered to be a master control geneof naturally occurring regulatory T cells derived from the thymus[11]. CD4+ Treg cells have previously been identified as a smallsubset (5–6%) of the overall CD4+ T cell population [12], and ourstudy showed that CD4+CD25+Foxp3+ T cells increased in 7 of 9patients and were present at levels of 3.2–10.8% (average, 6.4%)before DC vaccination and 2.0–15.6% (average, 8.7%) after DCvaccination. While healthy individuals also showed the frequen-cies of 3.1–5.7% (average, 4.4%). According to these results ourexperimental technique and the method of evaluation havereliability. Some previous reports [13–15] mentioned thatpopulation of Treg in advanced malignant diseases were highthan healthy person. However, no remarkable difference wasdetected in our results (P = 0.1), suggesting that Treg maypreferentially move to and accumulate in tumors and ascites thanin blood [16].
althy individuals and melanoma patients before vaccination. Percent values showed
control, n = 5; patients, n = 9.
Table 3Results of blood tests before and after vaccination in 9 patients.
RBC WBC PLT Hb GOT GPT LDH g-GTP TP Alb BUN Cre CRP
(�104/ml) (�103/ml) (�104/ml) (g/dl) (IU/l) (IU/l) (IU/l) (IU/l) (g/dl) (g/dl) (mg/dl) (mg/dl) (mg/dl)
Patient 1 Pre (0) 219 # 4.8 25.7 7.4 # 20 7 630 " 34 7.0 3.8 # 26.6 " 1.30 " 0.20
Post (10) 265 # 6.5 20.4 8.9 # 41 " 31 1185 " 41 8.3 4.0 37.3 " 1.14 " 16.80 "
Patient 2 Pre (0) 527 " 5.3 22.7 16.7 " 42 " 56 " 165 46 8.0 4.7 12.1 0.78 0.70 "Post (63) 480 7.6 " 21.0 15.3 " 36 " 55 " 162 36 7.3 4.1 10.6 0.87 0.37 "
Patient 3 Pre (0) 350 # 3.3 # 12.6 # 11.1 # 17 12 182 18 6.9 3.3 # 14.3 0.66 0.02
Post (10) 337 # 3.8 15.5 # 11.8 18 12 214 16 7.2 ND 16.2 0.58 0.01
Patient 4 Pre (0) 340 # 3.5 38.1 " 11.8 41 " 15 313 " 19 7.4 4.6 11.0 0.55 0.00
Post (63) 372 3.8 27.8 12.1 31 25 185 22 7.3 4.6 15.1 0.79 0.00
Patient 5 Pre (0) 337 # 3.6 19.2 10.7 # 17 12 171 16 7.2 4.3 18.0 0.67 0.00
Post (15) 359 # 3.7 20.4 11.2 # 18 13 189 17 7.1 4.4 14.5 0.60 0.02
Patient 6 Pre (0) 370 4.0 26.5 10.9 # 23 7 165 18 7.0 3.4 # 13.8 0.53 0.30
Post (20) 265 # 4.2 24.8 8.2 # 14 4 # 196 18 7.1 3.9 17.4 0.84 0.50 "
Patient 7 Pre (0) 293 # 5.5 17.4 10.3 # 16 12 204 45 8.0 4.1 11.7 0.49 7.30 "Post (12) 202 # 4.3 2.8 # 6.4 # 23 9 515 " 23 6.1 # 2.4 # 20.3 " 0.99 18.00 "
Patient 8 Pre (0) 378 3.3 # 13.5 # 12.9 20 26 205 66 " 7.3 4.5 16.0 0.72 0.20
Post (71) 395 7.5 " 31.8 11.5 # 21 22 1363 " 455 " 7.2 3.4 # 13.5 0.68 6.42 "
Patient 9 Pre (0) 344 # 6.1 14.4 # 11.8 43 " 41 " 237 12 6.4 3.6# 23.5 " 0.95 0.10
Post (10) ND ND ND ND ND ND ND ND ND ND ND ND ND
Arabic numerals in parentheses indicate the total number of DC vaccinations at the time of collection of PBMCs. ND, not done.
N. Nakai et al. / Journal of Dermatological Science 54 (2009) 31–37 35
While reduced induction of CD4+CD25+Foxp3+ T cells after thepost-treatment sample collection point was only found in 2patients (Table 4). These results suggested that activation of helperT cells and induction of Treg might commonly occur as serialevents in DC vaccination with a few exceptions.
Foxp3 mRNA levels after the post-treatment sample collectionpoint increased in 5 of 8 cases, with a corresponding increase inCD4+CD25+Foxp3+ T cells in 4 of these 5 cases (Table 4). Thissuggested that Treg increased at the mRNA and cellular levels inthe 4 cases and that the risk of immunotolerance specific formelanoma antigen might begin to increase gradually, as Yamazakiet al. [17,18] mentioned that Treg can proliferate in culture and in
Fig. 2. The frequencies of CD4+CD25+ T cells and CD4+CD25+Foxp3+ T cells in PBMCs befo
Foxp3+ in CD4+ T cells from PBMCs.
vivo when stimulated by antigen-loaded mature DC. Further, Tregproliferation also occurs in the presence of IL-2 [10]. In fact IL-2positive cells were detected in DTH site in our previous report [7],which may support the possibility of the melanoma antigen-specific immunotolerance in our study. The fifth case (patient 5)with increased Foxp3 mRNA after the post-treatment samplecollection point had a decreased population of CD4+CD25+Foxp3+ Tcells in the same sample. This patient showed a stable result inperipheral blood test (Table 3), positive DTH response tomelanoma antigens, and induction of vaccine peptide-specificIFN-g-producing T cells by ELISPOT assay (Tables 2A and 2B) at thepost-treatment sample collection point, however, the patient died
re and after vaccination in 9 patients. Flow cytometry diagrams showed CD25+ and
Table 4Summary of changes in the populations of CD4+CD25+ T cells and CD4+CD25+Foxp3+ T cells, and the Foxp3 mRNA level in peripheral CD4+ T cells before and after DC
vaccination in 9 patients.
Patient no. CD4+CD25+Foxp3+ T cells (%) in
CD4+ T cells
CD4+CD25+ T cells (%) in CD4+ T
cells
Foxp3 mRNA level in CD4+
T cells after vaccine compared
with that of before vaccinePre Post Pre Post
1 8.1 12.5 (10) 19.3 44.3 (10) 1.5 � 0.3 (10)
2 4.7 7.7 (63) 14.0 20.7 (63) 2.0 � 0.2 (63)
3 8.1 13.8 (10) 9.8 23.8 (10) 1.8 � 0.3 (10)
4 3.2 5.8 (63) 5.7 8.3 (63) 1.2 � 0.2 (63)
5 5.5 2.0 (15) 14.0 23.7 (15) 3.0 � 0.3 (15)
6 5.6 6.4 (20) 8.1 7.4 (20) 0.7 � 0.2 (20)
7 6.5 15.6 (12) 21.3 28.1 (12) 0.5 � 0.1 (12)
8 10.8 4.9 (71) 25.3 40.6 (71) 0.5 � 0.0 (71)
9 5.1 9.6 (10) 7.2 15.9 (10) ND
Arabic numerals in parentheses indicate the total number of DC vaccinations at the time of collection of PBMCs. ND, not done.
N. Nakai et al. / Journal of Dermatological Science 54 (2009) 31–3736
after about 1 year due to steady disease progression. Abe et al. [19]also described that a discrepancy of Foxp3 expression in the mRNAand its protein level in PBMC occurred in adult T cell leukemia. Onepossible explanation for this discrepancy in patient 5 mightsuggest the probable disease progression in the near future; that is,nuclei of CD4+ T cells might have started to upregulate transcrip-tional activity of Foxp3 mRNA.
Three cases showed decreased Foxp3 mRNA levels after thepost-treatment sample collection point, of which one (patient 7)had decreased Foxp3 mRNA despite a remarkable increase inCD4+CD25+Foxp3+ T cells (Table 4). This patient already hadextremely severe progressive disease based on physical status andblood tests (Table 3). This inconsistency of Foxp3 expression mightpossibly suggest that transcription of Foxp3 in CD4+ T cells alreadyhave started to decrease due to the serious physical status despiteincreased Foxp3 at the cellular level, as Xu et al. [20] provided thatexpression levels of Treg in chronic hepatitis B and chronic severehepatitis B, respectively were different. One patient (patient 8)showed a decreased Foxp3 expression at both mRNA and cellular
Fig. 3. Changes in the Foxp3 mRNA level in peripheral CD4+ T cells before and after DC vac
vaccinations at the time of collection of PBMCs. +P < 0.05 vs. pre-vaccination; ++P < 0.0
level with increased population of CD4+CD25+ T cells after thepost-treatment sample collection point. These results, the induc-tion of positive DTH responses (Table 2A), and induction of vaccinepeptide-specific IFN-g producing T cells (Table 2B) suggested thateffective anti-tumor immunity for melanoma might be inducedand contributed to his survival for 175 weeks. As for patient 6Foxp3 mRNA level decreased slightly, but we could not interpretthe result with cellular level because of subtle changes inCD4+CD25+ and CD4+CD25+Foxp3+ T cells between pre- andpost-vaccination samples.
There might be two possibilities that the development of Tregoccurred by natural tumor progression [21] or by DC vaccination[17,18]. Though a control study could not be performed because ofthe nature of clinical research with human subjects, however, weregarded the data of pre-vaccination as control data without DCvaccination because enrolled patients in the present study wereadvanced melanoma without any vaccination. Actually, the totalnumbers of vaccination varied from 11 to 93 times. However, theanalyses were performed consistently in continuous DC vaccina-
cination in 8 patients. Arabic numerals in each graph indicate the total number of DC
05 vs. pre-vaccination; +++P < 0.0005 vs. pre-vaccination.
N. Nakai et al. / Journal of Dermatological Science 54 (2009) 31–37 37
tion, further, the results in Treg population and its mRNA level hadcertain variations after the post-treatment sample collection point.Therefore, we do not think our results as simple influence of thechanges in disease progression [21]. Banerjee et al. [9] alsodescribed that Foxp3 regulatory T cells expanded by humandendritic cells in vivo in 3 of 3 myeloma patients. Fundamentally,our results supported their result and if their trial had beenperformed in many patients and also be analyzed by mRNA levelsome discrepancies like our results might have appeared.
We have found no statistical significant change in survival inpatients treated with DC vaccine [8], which suggests that theclinical efficacy may be a result of interactions among the tumorand host-immunity involving the tumor microenvironment,effector cells, and Treg [4,22,23]. Elimination of Treg andstrengthening of antigen-specific anti-tumor immunity is impor-tant for improving the clinical efficacy of DC vaccination [24–27],and transient deletion or suppression of Treg based on avoidance ofautoimmune disease and combination therapy with stronginduction of innate immunity may be appropriate strategies[12,28].
In conclusion, activated helper T cells and Treg commonlyincrease at cellular level by DC vaccination, however, there are alsoa few discrepancies in Treg expression at cellular and mRNA level,therefore, the investigation of the discrepancy may be futureproblem and strengthening of antigen-specific anti-tumor immu-nity and suppressing of immunological regulatory functions arerequired to improve the efficacy of DC vaccination.
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