IgG subclass switching and clonal expansion in cutaneous melanoma and normal skin

Louise Saul,Victoria Sanz-Moreno,Carl Hobbs,Debra H Josephs,Ciaran Healy,Frank O Nestle,Isabella Tosi,Anthony Cheung,Katie E Lacy,Isabel Correa,Sara Lombardi,Sophia N Karagiannis,Silvia Crescioli,Heather J Bax,Federica Villanova,Kristina M Ilieva,Isioma U Egbuniwe,Panagiotis Karagiannis,Mark Harries,James Spicer ,Irene Rodríguez-Hernández ,Jenny Geh ,David Fear

doi:10.1038/srep29736

Abstract

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.

Highlights

Medicine, King’s College London & NIHR Biomedical Research Centre at Guy’s and St
We found that a small proportion of circulating CD45+CD3-CD14-CD19+CD22+B cells in healthy volunteers (n = 24) and patients with melanoma (n = 49) express the skin-homing Cutaneous Leucocyte-associated Antigen (CLA) (Fig. 1a, Figure S1a)
Cutaneous B cell infiltrates from non-malignant skin and melanoma lesion samples were confirmed by flow cytometric analyses of CD45+CD19+CD22+B cells (Fig. 1d, for matched normal skin and melanoma lesion B cells and peripheral blood B cells from a single donor, representative of n = 4; Figure S2 for further examples of CD45+CD19+B cells from normal skin and melanoma lesion samples)

Summary

Introduction

Medicine, King’s College London & NIHR Biomedical Research Centre at Guy’s and St. Thomas’s Hospitals and King’s College London, King’s College London, London SE1 9RT, United Kingdom. 2Division of Cancer Studies, Faculty of Life Sciences and Medicine, King’s College London, 3rd Floor Bermondsey Wing, Guy’s Hospital, Great Maze Pond, London SE1 9RT, United Kingdom. 3Breast Cancer Research Unit, Division of Cancer Studies, Faculty of Life Sciences and Medicine, King’s College London, 3rd Floor Bermondsey Wing, Guy’s Hospital, London, United Kingdom. 4Tumour Plasticity Laboratory, Randall Division of Cell and Molecular Biophysics, New Hunt’s House, Guy’s Campus, King’s College London, London SE1 1UL, United Kingdom. 5Skin Tumor Unit, St. Class switch recombination (CSR) and SHM, involving the enzyme Activation-induced cytidine Deaminase (AID) can occur both in lymph node germinal centers and in tissues (e.g. lung, nasal mucosa) in response to antigenic challenge. This provides an enriched antibody repertoire with reactivity and affinity against encountered antigens and of different isotypes, conferring the potential to generate antibodies with a variety of Fc-mediated immune effector functions[1,2,3]. Clonal expansion of IgG-expressing clones against tumor-associated antigens has been reported to correspond to clinical tumor regression[10] Taken together, these studies support potential functions for mature humoral responses in normal and inflamed cutaneous sites. We describe evidence for the presence of cutaneous mature B cells, distinct IgG subclass distribution profiles, clonal expansion, somatic hypermutation in the IgG heavy chain variable regions, and predicted antigen binding site characteristics of the mature humoral response repertoire in cutaneous malignant melanoma lesions and in normal skin

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