Dynamic changes in cellular infiltrates with repeated cutaneous vaccination: a histologic and immunophenotypic analysis

Jochen T Schaefer, James W Patterson, Donna H Deacon, Mark E Smolkin, Gina R Petroni, Emily M Jackson, Craig L Slingluff Jr, Jochen T Schaefer, James W Patterson, Donna H Deacon, Mark E Smolkin, Gina R Petroni, Emily M Jackson, Craig L Slingluff Jr

Abstract

Background: Melanoma vaccines have not been optimized. Adjuvants are added to activate dendritic cells (DCs) and to induce a favourable immunologic milieu, however, little is known about their cellular and molecular effects in human skin. We hypothesized that a vaccine in incomplete Freund's adjuvant (IFA) would increase dermal Th1 and Tc1-lymphocytes and mature DCs, but that repeated vaccination may increase regulatory cells.

Methods: During and after 6 weekly immunizations with a multipeptide vaccine, immunization sites were biopsied at weeks 0, 1, 3, 7, or 12. In 36 participants, we enumerated DCs and lymphocyte subsets by immunohistochemistry and characterized their location within skin compartments.

Results: Mature DCs aggregated with lymphocytes around superficial vessels, however, immature DCs were randomly distributed. Over time, there was no change in mature DCs. Increases in T and B-cells were noted. Th2 cells outnumbered Th1 lymphocytes after 1 vaccine 6.6:1. Eosinophils and FoxP3+ cells accumulated, especially after 3 vaccinations, the former cell population most abundantly in deeper layers.

Conclusions: A multipeptide/IFA vaccine may induce a Th2-dominant microenvironment, which is reversed with repeat vaccination. However, repeat vaccination may increase FoxP3+T-cells and eosinophils. These data suggest multiple opportunities to optimize vaccine regimens and potential endpoints for monitoring the effects of new adjuvants.

Trial registration: ClinicalTrials.gov Identifier: NCT00705640.

Figures

Figure 1
Figure 1
Mel48 Protocol schema. All patients were vaccinated 6 times at the primary vaccination site, on weeks 0, 1, 2, 4, 5, and 6. At the replicate vaccination sites, the number of vaccines given depended on when the vaccination site was biopsied, as shown schematically here. V = vaccination, vertical black bar = vaccination site biopsy.
Figure 2
Figure 2
Lymphohistiocytic infiltrate increasing over time. H&E stained histologic sections of replicate vaccination site, representative for each time point (A: no vaccine; B: 1 week after 1st vaccine; C: 1 week after 3rd vaccine; D: 1 week after 6th vaccine; E: 6 weeks after 6th vaccine). Top panel: The three compartments: superficial papillary dermis; middle panel: deep dermis, lower panel: subcutis. Note the significant increase of the inflammatory infiltrate between the first (B) and third (C) vaccination in all compartments. Bar = 100 μm.
Figure 3
Figure 3
Pools of eosinohilis in the mid and deep layers following the third vaccine. (a) Numerous eosinophils are present in the subcutis. Bar = 200 μm. (b) High-power view. Note the distinctive cytologic detail, including the bilobed nucleus in a round cell with numerous, red cytoplasmic granules. Bar = 20 μm (c) Focal areas of fat necrosis (empty spaces of various sizes) are present. Bar = 200 μm (d) Note sites of vaccine deposits (large, "empty" spaces walled off by macrophages. Bar = 100 μm.
Figure 4
Figure 4
Perivascular T-and B-cell infiltrate. (a) Prominent infiltrate of inflammatory cells composed of lymphocytes and macrophages. (b) CD3+ T-cells (brown chromagen) cluster around blood vessel. (c) CD20+ B-cells (brown chromagen) group peripheral to the T-cell zone. Bar = 100 μm in a-c. (d) Double-staining for CD20+ B-cells (brown membranous stain) and CD8 (purple membranous stain). Counter-staining with hematoxylin marks nuclei blue. Note the group of B-cells located distant from blood vessel and next to the perivascular zone. The latter is composed of purple T-cells (we show the CD8+ population here) Bar = 50 μm.
Figure 5
Figure 5
Boxplots by time and layer of all 36 study patients: T cells, B cells, and dendritic cells. This figure illustrates T cell (CD3), B cell (CD20), immature (CD1a) and mature (CD83) dendritic cells in each of the three evaluated skin compartments (S = superficial, M = mid and D = deep) over time (A = without vaccine; B = 1 week after first vaccine; C = 1 week after third vaccine; D = 1 week after sixth vaccine; E = 6 weeks after last vaccine). The inner box of the boxplot represents the 25th and 75th percentiles, while the whiskers indicate the range. To facilitate data display, the square roots of values were used with the y-axis labelled on the regular scale.
Figure 6
Figure 6
Boxplots by time and layer of the "adjuvant and peptide group": T cells, B cells, and dendritic cells. This figure illustrates T cell (CD3), B cell (CD20), immature (CD1a) and mature (CD83) dendritic cells in each of the three evaluated skin compartments (S = superficial, M = mid and D = deep) over time (A = without vaccine; B = 1 week after first vaccine; C = 1 week after third vaccine; D = 1 week after sixth vaccine; E = 6 weeks after last vaccine). The inner box of the boxplot represents the 25th and 75th percentiles, while the whiskers indicate the range. To facilitate data display, the square roots of values were used with the y-axis labelled on the regular scale.
Figure 7
Figure 7
Boxplots by time and layer of all 36 study patients: Th1, Th2, and Foxp3 (Figure 7 demonstrates all 36 study patients. Figure 8 only shows the "adjuvant and peptide group"). This figure demonstrates Th1 lymphocytes (Tbet+) and three negative regulators: Th2 lymphocytes (GATA3+), eosinophils and regulatory T-cells (FoxP3+) in each of the three evaluated skin compartments (S = superficial, M = mid and D = deep) over time (A = without vaccine; B = 1 week after first vaccine; C = 1 week after third vaccine; D = 1 week after sixth vaccine; E = 6 weeks after last vaccine). The inner box of the boxplot represents the 25th and 75th percentiles, while the whiskers indicate the range. To facilitate data display, the square roots of values were used with the y-axis labelled on the regular scale.
Figure 8
Figure 8
Boxplots by time and layer of the "adjuvant and peptide group": Th1, Th2, and Foxp3.This figure demonstrates Th1 lymphocytes (Tbet+) and three negative regulators: Th2 lymphocytes (GATA3+), eosinophils and regulatory T-cells (FoxP3+) in each of the three evaluated skin compartments (S = superficial, M = mid and D = deep) over time (A = without vaccine; B = 1 week after first vaccine; C = 1 week after third vaccine; D = 1 week after sixth vaccine; E = 6 weeks after last vaccine). The inner box of the boxplot represents the 25th and 75th percentiles, while the whiskers indicate the range. To facilitate data display, the square roots of values were used with the y-axis labelled on the regular scale.

References

    1. Puzanov I, Nathanson KL, Chapman PB, Xu X, Sosman JA, McArthur GA, Ribas A, Kim KB, Grippo JF, Flaherty KT. PLX4032, a highly selective V600EBRAF kinase inhibitor: Clinical correlation of activity with pharmacokinetic and pharmacodynamic parameters in a phase I trial. J Clin Oncol (Meeting Abstracts) 2009;27:9021.
    1. Rosenberg SA, Yang JC, White DE, Steinberg SM. Durability of complete responses in patients with metastatic cancer treated with high-dose interleukin-2: identification of the antigens mediating response. Ann Surg. 1998;228:307–319. doi: 10.1097/00000658-199809000-00004.
    1. Powell DJ Jr, Dudley ME, Hogan KA, Wunderlich JR, Rosenberg SA. Adoptive transfer of vaccine-induced peripheral blood mononuclear cells to patients with metastatic melanoma following lymphodepletion. J Immunol. 2006;177:6527–6539.
    1. Hunder NN, Wallen H, Cao J, Hendricks DW, Reilly JZ, Rodmyre R, Jungbluth A, Gnjatic S, Thompson JA, Yee C. Treatment of metastatic melanoma with autologous CD4+ T cells against NY-ESO-1. N Engl J Med. 2008;358:2698–2703. doi: 10.1056/NEJMoa0800251.
    1. Wallack MK, Sivanandham M, Balch CM, Urist MM, Bland KI, Murray D, Robinson WA, Flaherty L, Richards JM, Bartolucci AA, Rosen L. Surgical adjuvant active specific immunotherapy for patients with stage III melanoma: the final analysis of data from a phase III, randomized, double-blind, multicenter vaccinia melanoma oncolysate trial. J Am Coll Surg. 1998;187:69–77. doi: 10.1016/S1072-7515(98)00097-0. discussion 77-69.
    1. Sondak VK, Liu PY, Tuthill RJ, Kempf RA, Unger JM, Sosman JA, Thompson JA, Weiss GR, Redman BG, Jakowatz JG. et al.Adjuvant immunotherapy of resected, intermediate-thickness, node-negative melanoma with an allogeneic tumor vaccine: overall results of a randomized trial of the Southwest Oncology Group. J Clin Oncol. 2002;20:2058–2066. doi: 10.1200/JCO.2002.08.071.
    1. Kirkwood JM, Ibrahim JG, Sosman JA, Sondak VK, Agarwala SS, Ernstoff MS, Rao U. High-dose interferon alfa-2b significantly prolongs relapse-free and overall survival compared with the GM2-KLH/QS-21 vaccine in patients with resected stage IIB-III melanoma: results of intergroup trial E1694/S9512/C509801. J Clin Oncol. 2001;19:2370–2380.
    1. Schadendorf D, Nestle FO, Broecker EB, Enk A, Grabbe S, Ugurel S, Edler L, Schuler G. Dacarbacine (DTIC) versus vaccination with autologous peptide-pulsed dendritic cells (DC) as first-line treatment of patients with metastatic melanoma: Results of a prospective-randomized phase III study. J Clin Oncol (Meeting Abstracts) 2004;22:7508.
    1. Morton DL, Mozzillo N, Thompson JF, Kelley MC, Faries M, Wagner J, Schneebaum S, Schuchter L, Gammon G, Elashoff R, Group MCT. An international, randomized, phase III trial of bacillus Calmette-Guerin (BCG) plus allogeneic melanoma vaccine (MCV) or placebo after complete resection of melanoma metastatic to regional or distant sites. J Clin Oncol (Meeting Abstracts) 2007;25:8508.
    1. Schwartzentruber DJ, Lawson D, Richards J, Conry RM, Miller D, Triesman J, Gailani F, Riley LB, Vena D, Hwu P. A phase III multi-institutional randomized study of immunization with the gp100: 209-217(210M) peptide followed by high-dose IL-2 compared with high-dose IL-2 alone in patients with metastatic melanoma. J Clin Oncol (Meeting Abstracts) 2009;27:CRA9011.
    1. Brinckerhoff LH, Kalashnikov VV, Thompson LW, Yamshchikov GV, Pierce RA, Galavotti HS, Engelhard VH, Slingluff CL Jr. Terminal modifications inhibit proteolytic degradation of an immunogenic MART-1(27-35) peptide: implications for peptide vaccines. Int J Cancer. 1999;83:326–334. doi: 10.1002/(SICI)1097-0215(19991029)83:3<326::AID-IJC7>;2-X.
    1. Davis ID, Chen W, Jackson H, Parente P, Shackleton M, Hopkins W, Chen Q, Dimopoulos N, Luke T, Murphy R. et al.Recombinant NY-ESO-1 protein with ISCOMATRIX adjuvant induces broad integrated antibody and CD4(+) and CD8(+) T cell responses in humans. Proc Natl Acad Sci USA. 2004;101:10697–10702. doi: 10.1073/pnas.0403572101.
    1. Celis E. Overlapping human leukocyte antigen class I/II binding peptide vaccine for the treatment of patients with stage IV melanoma: evidence of systemic immune dysfunction. Cancer. 2007;110:203–214. doi: 10.1002/cncr.22744.
    1. Spaner DE, Astsaturov I, Vogel T, Petrella T, Elias I, Burdett-Radoux S, Verma S, Iscoe N, Hamilton P, Berinstein NL. Enhanced viral and tumor immunity with intranodal injection of canary pox viruses expressing the melanoma antigen, gp100. Cancer. 2006;106:890–899. doi: 10.1002/cncr.21669.
    1. Yamshchikov GV, Barnd DL, Eastham S, Galavotti H, Patterson JW, Deacon DH, Teates D, Neese P, Grosh WW, Petroni G. et al.Evaluation of peptide vaccine immunogenicity in draining lymph nodes and peripheral blood of melanoma patients. Int J Cancer. 2001;92:703–711. doi: 10.1002/1097-0215(20010601)92:5<703::AID-IJC1250>;2-5.
    1. Slingluff CL Jr, Yamshchikov GV, Hogan KT, Hibbitts SC, Petroni GR, Bissonette EA, Patterson JW, Neese PY, Grosh WW, Chianese-Bullock KA. et al.Evaluation of the sentinel immunized node for immune monitoring of cancer vaccines. Ann Surg Oncol. 2008;15:3538–3549. doi: 10.1245/s10434-008-0046-4.
    1. Slingluff CL Jr, Petroni GR, Yamshchikov GV, Hibbitts S, Grosh WW, Chianese-Bullock KA, Bissonette EA, Barnd DL, Deacon DH, Patterson JW. et al.Immunologic and clinical outcomes of vaccination with a multiepitope melanoma peptide vaccine plus low-dose interleukin-2 administered either concurrently or on a delayed schedule. J Clin Oncol. 2004;22:4474–4485. doi: 10.1200/JCO.2004.10.212.
    1. Chianese-Bullock KA, Pressley J, Garbee C, Hibbitts S, Murphy C, Yamshchikov G, Petroni GR, Bissonette EA, Neese PY, Grosh WW. et al.MAGE-A1-, MAGE-A10-, and gp100-derived peptides are immunogenic when combined with granulocyte-macrophage colony-stimulating factor and montanide ISA-51 adjuvant and administered as part of a multipeptide vaccine for melanoma. J Immunol. 2005;174:3080–3086.
    1. Slingluff CL Jr, Petroni GR, Chianese-Bullock KA, Smolkin ME, Hibbitts S, Murphy C, Johansen N, Grosh WW, Yamshchikov GV, Neese PY. et al.Immunologic and clinical outcomes of a randomized phase II trial of two multipeptide vaccines for melanoma in the adjuvant setting. Clin Cancer Res. 2007;13:6386–6395. doi: 10.1158/1078-0432.CCR-07-0486.
    1. Diggle P, Liang K-Y, Zeger SL. Analysis of longitudinal data. Oxford New York: Clarendon Press; Oxford University Press; 1994.
    1. Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med. 1998;4:328–332. doi: 10.1038/nm0398-328.
    1. Nakai N, Asai J, Ueda E, Takenaka H, Katoh N, Kishimoto S. Vaccination of Japanese patients with advanced melanoma with peptide, tumor lysate or both peptide and tumor lysate-pulsed mature, monocyte-derived dendritic cells. J Dermatol. 2006;33:462–472. doi: 10.1111/j.1346-8138.2006.00110.x.
    1. de Vries IJ, Bernsen MR, Lesterhuis WJ, Scharenborg NM, Strijk SP, Gerritsen MJ, Ruiter DJ, Figdor CG, Punt CJ, Adema GJ. Immunomonitoring tumor-specific T cells in delayed-type hypersensitivity skin biopsies after dendritic cell vaccination correlates with clinical outcome. J Clin Oncol. 2005;23:5779–5787. doi: 10.1200/JCO.2005.06.478.
    1. Bedrosian I, Mick R, Xu S, Nisenbaum H, Faries M, Zhang P, Cohen PA, Koski G, Czerniecki BJ. Intranodal administration of peptide-pulsed mature dendritic cell vaccines results in superior CD8+ T-cell function in melanoma patients. J Clin Oncol. 2003;21:3826–3835. doi: 10.1200/JCO.2003.04.042.
    1. Nakai N, Katoh N, Germeraad WT, Kishida T, Ueda E, Takenaka H, Mazda O, Kishimoto S. Immunohistological analysis of peptide-induced delayed-type hypersensitivity in advanced melanoma patients treated with melanoma antigen-pulsed mature monocyte-derived dendritic cell vaccination. J Dermatol Sci. 2009;53:40–47. doi: 10.1016/j.jdermsci.2008.07.013.
    1. Steinman RM, Banchereau J. Taking dendritic cells into medicine. Nature. 2007;449:419–426. doi: 10.1038/nature06175.
    1. Dhodapkar MV, Dhodapkar KM, Palucka AK. Interactions of tumor cells with dendritic cells: balancing immunity and tolerance. Cell Death Differ. 2008;15:39–50. doi: 10.1038/sj.cdd.4402247.
    1. Wells JW, Cowled CJ, Farzaneh F, Noble A. Combined triggering of dendritic cell receptors results in synergistic activation and potent cytotoxic immunity. J Immunol. 2008;181:3422–3431.
    1. Finotto S, Glimcher L. T cell directives for transcriptional regulation in asthma. Springer Semin Immunopathol. 2004;25:281–294. doi: 10.1007/s00281-003-0143-1.
    1. Slingluff CL Jr, Yamshchikov G, Neese P, Galavotti H, Eastham S, Engelhard VH, Kittlesen D, Deacon D, Hibbitts S, Grosh WW. et al.Phase I trial of a melanoma vaccine with gp100(280-288) peptide and tetanus helper peptide in adjuvant: immunologic and clinical outcomes. Clin Cancer Res. 2001;7:3012–3024.
    1. Ahmadzadeh M, Felipe-Silva A, Heemskerk B, Powell DJ Jr, Wunderlich JR, Merino MJ, Rosenberg SA. FOXP3 expression accurately defines the population of intratumoral regulatory T cells that selectively accumulate in metastatic melanoma lesions. Blood. 2008;112:4953–4960. doi: 10.1182/blood-2008-06-163048.
    1. Roncador G, Brown PJ, Maestre L, Hue S, Martinez-Torrecuadrada JL, Ling KL, Pratap S, Toms C, Fox BC, Cerundolo V. et al.Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. Eur J Immunol. 2005;35:1681–1691. doi: 10.1002/eji.200526189.
    1. Walker MR, Kasprowicz DJ, Gersuk VH, Benard A, Van Landeghen M, Buckner JH, Ziegler SF. Induction of FoxP3 and acquisition of T regulatory activity by stimulated human CD4+CD25- T cells. J Clin Invest. 2003;112:1437–1443.
    1. Morgan ME, van Bilsen JH, Bakker AM, Heemskerk B, Schilham MW, Hartgers FC, Elferink BG, van der Zanden L, de Vries RR, Huizinga TW. et al.Expression of FOXP3 mRNA is not confined to CD4+CD25+ T regulatory cells in humans. Hum Immunol. 2005;66:13–20. doi: 10.1016/j.humimm.2004.05.016.
    1. Walker MR, Carson BD, Nepom GT, Ziegler SF, Buckner JH. De novo generation of antigen-specific CD4+CD25+ regulatory T cells from human CD4+CD25- cells. Proc Natl Acad Sci USA. 2005;102:4103–4108. doi: 10.1073/pnas.0407691102.
    1. Klemke CD, Fritzsching B, Franz B, Kleinmann EV, Oberle N, Poenitz N, Sykora J, Banham AH, Roncador G, Kuhn A. et al.Paucity of FOXP3+ cells in skin and peripheral blood distinguishes Sezary syndrome from other cutaneous T-cell lymphomas. Leukemia. 2006;20:1123–1129. doi: 10.1038/sj.leu.2404182.
    1. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M. et al.Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med. 2004;10:942–949. doi: 10.1038/nm1093.
    1. Poulter LW, Seymour GJ, Duke O, Janossy G, Panayi G. Immunohistological analysis of delayed-type hypersensitivity in man. Cell Immunol. 1982;74:358–369. doi: 10.1016/0008-8749(82)90036-3.
    1. Takemura S, Braun A, Crowson C, Kurtin PJ, Cofield RH, O'Fallon WM, Goronzy JJ, Weyand CM. Lymphoid neogenesis in rheumatoid synovitis. J Immunol. 2001;167:1072–1080.
    1. Ayyoub M, Monsarrat B, Mazarguil H, Gairin JE. Analysis of the degradation mechanisms of MHC class I-presented tumor antigenic peptides by high performance liquid chromatography/electrospray ionization mass spectrometry: application to the design of peptidase-resistant analogs. Rapid Commun Mass Spectrom. 1998;12:557–564. doi: 10.1002/(SICI)1097-0231(19980515)12:9<557::AID-RCM199>;2-D.
    1. Melief CJ, van der Burg SH. Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines. Nat Rev Cancer. 2008;8:351–360. doi: 10.1038/nrc2373.
    1. Speetjens FM, Kuppen PJ, Welters MJ, Essahsah F, Voet van den Brink AM, Lantrua MG, Valentijn AR, Oostendorp J, Fathers LM, Nijman HW. et al.Induction of p53-specific immunity by a p53 synthetic long peptide vaccine in patients treated for metastatic colorectal cancer. Clin Cancer Res. 2009;15:1086–1095. doi: 10.1158/1078-0432.CCR-08-2227.
    1. Leffers N, Lambeck AJ, Gooden MJ, Hoogeboom BN, Wolf R, Hamming IE, Hepkema BG, Willemse PH, Molmans BH, Hollema H. et al.Immunization with a P53 synthetic long peptide vaccine induces P53-specific immune responses in ovarian cancer patients, a phase II trial. Int J Cancer. 2009;125:2104–2113. doi: 10.1002/ijc.24597.
    1. Glenn GM, Taylor DN, Li X, Frankel S, Montemarano A, Alving CR. Transcutaneous immunization: a human vaccine delivery strategy using a patch. Nat Med. 2000;6:1403–1406. doi: 10.1038/82225.
    1. Shi Z, Curiel DT, Tang DC. DNA-based non-invasive vaccination onto the skin. Vaccine. 1999;17:2136–2141. doi: 10.1016/S0264-410X(98)00488-5.
    1. Guebre-Xabier M, Hammond SA, Ellingsworth LR, Glenn GM. Immunostimulant patch enhances immune responses to influenza virus vaccine in aged mice. J Virol. 2004;78:7610–7618. doi: 10.1128/JVI.78.14.7610-7618.2004.
    1. Vogt A, Mahe B, Costagliola D, Bonduelle O, Hadam S, Schaefer G, Schaefer H, Katlama C, Sterry W, Autran B. et al.Transcutaneous anti-influenza vaccination promotes both CD4 and CD8 T cell immune responses in humans. J Immunol. 2008;180:1482–1489.
    1. Kenney RT, Frech SA, Muenz LR, Villar CP, Glenn GM. Dose sparing with intradermal injection of influenza vaccine. N Engl J Med. 2004;351:2295–2301. doi: 10.1056/NEJMoa043540.
    1. Rosenberg SA, Sherry RM, Morton KE, Scharfman WJ, Yang JC, Topalian SL, Royal RE, Kammula U, Restifo NP, Hughes MS. et al.Tumor progression can occur despite the induction of very high levels of self/tumor antigen-specific CD8+ T cells in patients with melanoma. J Immunol. 2005;175:6169–6176.
    1. Slingluff CL Jr, Petroni GR, Olson W, Czarkowski A, Grosh WW, Smolkin M, Chianese-Bullock KA, Neese PY, Deacon DH, Nail C. et al.Helper T-cell responses and clinical activity of a melanoma vaccine with multiple peptides from MAGE and melanocytic differentiation antigens. J Clin Oncol. 2008;26:4973–4980. doi: 10.1200/JCO.2008.17.3161.

Source: PubMed

Sponsoren und Mitarbeiter

Krankheiten

Drogeninterventionen

3
Abonnieren