B Lymphocytes Accumulate and Proliferate in Human Skin at Sites of Cutaneous Antigen Challenge

B cells play important roles in skin diseases (Egbuniwe et al., 2015Egbuniwe I.U. Karagiannis S.N. Nestle F.O. Lacy K.E. Revisiting the role of B cells in skin immune surveillance.Trends Immunol. 2015; 36: 102-111Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) and in cutaneous homeostasis (Geherin et al., 2016Geherin S.A. Gómez D. Glabman R.A. Ruthel G. Hamann A. Debes G.F. IL-10+ innate-like B cells are part of the skin immune system and require α4β1 integrin to migrate between the peritoneum and inflamed skin.J Immunol. 2016; 196: 2514-2525Crossref PubMed Scopus (31) Google Scholar, Geherin et al., 2012Geherin S.A. Fintushel S.R. Lee M.H. Wilson R.P. Patel R.T. Alt C. et al.The skin, a novel niche for recirculating B cells.J Immunol. 2012; 188: 6027-6035Crossref PubMed Scopus (63) Google Scholar; Nihal et al., 2000Nihal M. Mikkola D. Wood G.S. Detection of clonally restricted immunoglobulin heavy chain gene rearrangements in normal and lesional skin: analysis of the B cell component of the skin-associated lymphoid tissue and implications for the molecular diagnosis of cutaneous B cell lymphomas.J Mol Diagn. 2000; 2: 5-10Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Mature class-switched IgG+ B cells have been detected in normal human skin (Saul et al., 2016Saul L. Ilieva K.M. Bax H.J. Karagiannis P. Correa I. Rodriguez-Hernandez I. et al.IgG subclass switching and clonal expansion in cutaneous melanoma and normal skin.Sci Rep. 2016; 6: 29736Crossref PubMed Scopus (33) Google Scholar) featuring clonally restricted B-cell receptors, indicating narrow antigenic repertoires (Nihal et al., 2000Nihal M. Mikkola D. Wood G.S. Detection of clonally restricted immunoglobulin heavy chain gene rearrangements in normal and lesional skin: analysis of the B cell component of the skin-associated lymphoid tissue and implications for the molecular diagnosis of cutaneous B cell lymphomas.J Mol Diagn. 2000; 2: 5-10Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). However, the involvement of B cells during an antigenic stimulus in human skin remains unexplored. B cells are relatively scarce in normal human skin (Supplementary Figure S1), explaining why past studies have primarily focused on T cells, which constitute the major skin-resident lymphocyte population (Clark et al., 2006bClark R.A. Chong B.F. Mirchandani N. Yamanaka K.I. Murphy G.F. Dowgiert R.K. et al.A novel method for the isolation of skin resident T cells from normal and diseased human skin.J Invest Dermatol. 2006; 126: 1059-1070Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar; Jiang et al., 2012Jiang X. Clark R.A. Liu L. Wagers A.J. Fuhlbrigge R.C. Kupper T.S. Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity.Nature. 2012; 483: 227-231Crossref PubMed Scopus (605) Google Scholar; Sanchez Rodriguez et al., 2014Sanchez Rodriguez R. Pauli M.L. Neuhaus I.M. Yu S.S. Arron S.T. Harris H.W. et al.Memory regulatory T cells reside in human skin.J Clin Invest. 2014; 124: 1027-1036Crossref PubMed Scopus (224) Google Scholar). We investigated the dynamics of B-cell infiltration in the skin after local antigen challenge with varicella-zoster virus (VZV; provided by Prof A.N. Akbar, University College London, UK) and candida (Candin) antigens (Allermed Laboratories, San Diego, CA) by taking skin biopsies from and inducing suction blisters over the site of intradermal injection in human skin in vivo. The induction of skin suction blisters has been reproducibly employed in examining the cellular kinetics of skin-infiltrating immune subsets, including T cells in delayed-type hypersensitivity responses after intradermal injection of recall antigens (Vukmanovic-Stejic et al., 2013Vukmanovic-Stejic M. Sandhu D. Sobande T.O. Agius E. Lacy K.E. Riddell N. et al.Varicella zoster–specific CD4+Foxp3+ T cells accumulate after cutaneous antigen challenge in humans.J Immunol. 2013; 190: 977-986Crossref PubMed Google Scholar); innate lymphoid cells after challenge with house dust mite (Salimi et al., 2013Salimi M. Barlow J.L. Saunders S.P. Xue L. Gutowska-Owsiak D. Wang X. et al.A role for IL-25 and IL-33–driven type-2 innate lymphoid cells in atopic dermatitis.J Exp Med. 2013; 210: 2939-2950Crossref PubMed Scopus (634) Google Scholar); and different leukocyte subsets (including B cells) during acute inflammation (Akbar et al., 2013Akbar A.N. Reed J.R. Lacy K.E. Jackson S.E. Vukmanovic-Stejic M. Rustin M.H. Investigation of the cutaneous response to recall antigen in humans in vivo.Clin Exp Immunol. 2013; 173: 163-172Crossref PubMed Scopus (26) Google Scholar; Jenner et al., 2014Jenner W. Motwani M. Veighey K. Newson J. Audzevich T. Nicolaou A. et al.Characterisation of leukocytes in a human skin blister model of acute inflammation and resolution.PLoS One. 2014; 9: e89375Crossref PubMed Scopus (24) Google Scholar). Flow cytometric analysis (FACSCanto II or LSRFortessa Cell Analyser - Becton Dickinson, Franklin Lakes, NJ) showed CD19+CD20+ B cells in low numbers (mean percentage of total lymphocyte population) in fluid from skin suction blisters induced without antigen challenge (mean = 0.05%, n = 3) (Figure 1a and b). This, in addition to immunofluorescence studies on normal skin (Supplementary Figure S1), confirmed that B cells were scarce under homeostatic conditions in unperturbed skin. After intradermal challenge with VZV antigen, CD20+ B cells (percentage of total live lymphocytes) were detected on day 3 (mean = 0.26%, n = 3) and day 7 (mean = 5.07%, n = 3) blisters (Figure 1c). Blister fluid obtained from sites injected with sterile saline (Advanz Pharma, London, UK) contained lower percentages of B cells on both day 3 and day 7 compared with VZV blisters (Figure 1c). A trend toward lower absolute numbers of cells extracted from saline blister fluid (n = 5, mean = 5.1 cells per ml) than that from VZV blister fluid (n = 5, mean = 48.1 cells per ml) was seen, although this did not reach statistical significance.Figure 1Characterization of skin-resident B cells with and without VZV antigen challenge. Suction blisters were induced (without VZV antigen challenge) on the skin of healthy individuals (n = 3), and BF was collected within 24 hours after blister formation for flow cytometric (FACS) analysis. Matched donor PBMCs were also analyzed by FACS. (a) Lymphocytes were identified on the basis of SSC and FSC profiles in peripheral blood and skin suction blisters. (b) Proportions of CD19+CD20+ B cells and CD3+ T cells were evaluated from the total lymphocyte population within donor PBMCs (n = 3; percentage mean ± SEM; B cells = 3.6 ± 0.3; T cells = 62 ± 19) and unchallenged BF (n = 3; percentage mean ± SEM; B cells = 0.05 ± 0.01; T cells = 53 ± 16). Representative FACS plot shown for an individual donor. (c) Donors received intradermal injections of VZV antigen or sterile saline, and blisters were raised over injection sites. Blister aspirates and matched donor PBMCs collected either 3 d (n = 3) or 7 d (n = 3) after injection were analyzed by FACS for B (CD20+) and T (CD3+) cells. Blisters on d 0 (n = 2) were induced without previous VZV challenge. Representative FACS plot shown for an individual donor. (d) Quantification of B and T cells within d 3 (n = 3) and d 7 (n = 3) VZV and saline blister aspirates compared with those within d 0 (n = 2) blisters. (e) d 3 blister aspirates and PBMCs analyzed by FACS for expression of CLA (a skin-homing marker) on CD20+ B cells and CD3+ T cells. Plots represent an individual donor. (f) Quantification of CLA+ B and T cells within d 3 (n = 3) and d 7 (n = 3) VZV blister aspirates and matched donor PBMCs. (g–h) Phenotypic analysis and quantification of mature (CD20+CD22+) and memory (CD20+CD27+) B cells within d 0 (n = 2) donor PBMCs and blister aspirates (non-VZV), compared with those within d 3 (n = 3) and d 7 (n = 3) donor PBMCs and VZV aspirates. Representative flow cytometry plots are shown. Kruskal‒Wallis test with Dunn’s multiple comparisons post-test was used for analysis. Error bars represent mean ± SEM. (i) Live lymphocytes were identified within peripheral blood and BF on the basis of SSC and FSC profiles and by staining with a live/dead (viability) marker, followed by identification of CD3–CD27+ lymphocytes. (j) Identification of plasma cell (CD38hiCD138+) and plasmablast (CD38intCD138+) subsets from within CD3–CD27+ B cells in peripheral blood and BF. FACS plots are shown for one individual each evaluated on d 3 and d 7. (k) Total IgG antibody titres within d 7 BF aspirates from saline- (control, n = 4), VZV- (n = 10), and CAN- (n = 4) challenged skin and from donor sera (n = 13) were analyzed using the ImmunoCAP and Total IgG ELISA assays. Each symbol on the plot represents one individual. ∗∗∗∗P < 0.0001. One-way ANOVA with Tukey’s multiple comparison post-test was used for analysis. (l) Reactivity of d 7 BF (CAN and VZV) and HV serum IgG to VZV antigen were tested using a modified indirect ELISA assay. Specific signal (optical density) was measured at 1:10, 1:100, 1:1,000, and 1:10,000 dilutions (left hand graphs). Antibody reactivity was then determined as absorbance at 1:100 dilution, relative to a positive control antibody (16 ng/ml anti-VZVgE) for each sample tested (right-hand graph). Error bars represent mean ± SEM. BF, blister fluid; CAN, candida; CLA, cutaneous lymphocyte antigen; conc, concentration; d, day; FSC, forward scatter; FSC-A, forward scatter area; HV, healthy volunteer; K, thousand; SSC, side scatter; SSC-A, side scatter area; VZV, varicella-zoster virus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Characterization of skin-resident B cells with and without VZV antigen challenge. Suction blisters were induced (without VZV antigen challenge) on the skin of healthy individuals (n = 3), and BF was collected within 24 hours after blister formation for flow cytometric (FACS) analysis. Matched donor PBMCs were also analyzed by FACS. (a) Lymphocytes were identified on the basis of SSC and FSC profiles in peripheral blood and skin suction blisters. (b) Proportions of CD19+CD20+ B cells and CD3+ T cells were evaluated from the total lymphocyte population within donor PBMCs (n = 3; percentage mean ± SEM; B cells = 3.6 ± 0.3; T cells = 62 ± 19) and unchallenged BF (n = 3; percentage mean ± SEM; B cells = 0.05 ± 0.01; T cells = 53 ± 16). Representative FACS plot shown for an individual donor. (c) Donors received intradermal injections of VZV antigen or sterile saline, and blisters were raised over injection sites. Blister aspirates and matched donor PBMCs collected either 3 d (n = 3) or 7 d (n = 3) after injection were analyzed by FACS for B (CD20+) and T (CD3+) cells. Blisters on d 0 (n = 2) were induced without previous VZV challenge. Representative FACS plot shown for an individual donor. (d) Quantification of B and T cells within d 3 (n = 3) and d 7 (n = 3) VZV and saline blister aspirates compared with those within d 0 (n = 2) blisters. (e) d 3 blister aspirates and PBMCs analyzed by FACS for expression of CLA (a skin-homing marker) on CD20+ B cells and CD3+ T cells. Plots represent an individual donor. (f) Quantification of CLA+ B and T cells within d 3 (n = 3) and d 7 (n = 3) VZV blister aspirates and matched donor PBMCs. (g–h) Phenotypic analysis and quantification of mature (CD20+CD22+) and memory (CD20+CD27+) B cells within d 0 (n = 2) donor PBMCs and blister aspirates (non-VZV), compared with those within d 3 (n = 3) and d 7 (n = 3) donor PBMCs and VZV aspirates. Representative flow cytometry plots are shown. Kruskal‒Wallis test with Dunn’s multiple comparisons post-test was used for analysis. Error bars represent mean ± SEM. (i) Live lymphocytes were identified within peripheral blood and BF on the basis of SSC and FSC profiles and by staining with a live/dead (viability) marker, followed by identification of CD3–CD27+ lymphocytes. (j) Identification of plasma cell (CD38hiCD138+) and plasmablast (CD38intCD138+) subsets from within CD3–CD27+ B cells in peripheral blood and BF. FACS plots are shown for one individual each evaluated on d 3 and d 7. (k) Total IgG antibody titres within d 7 BF aspirates from saline- (control, n = 4), VZV- (n = 10), and CAN- (n = 4) challenged skin and from donor sera (n = 13) were analyzed using the ImmunoCAP and Total IgG ELISA assays. Each symbol on the plot represents one individual. ∗∗∗∗P < 0.0001. One-way ANOVA with Tukey’s multiple comparison post-test was used for analysis. (l) Reactivity of d 7 BF (CAN and VZV) and HV serum IgG to VZV antigen were tested using a modified indirect ELISA assay. Specific signal (optical density) was measured at 1:10, 1:100, 1:1,000, and 1:10,000 dilutions (left hand graphs). Antibody reactivity was then determined as absorbance at 1:100 dilution, relative to a positive control antibody (16 ng/ml anti-VZVgE) for each sample tested (right-hand graph). Error bars represent mean ± SEM. BF, blister fluid; CAN, candida; CLA, cutaneous lymphocyte antigen; conc, concentration; d, day; FSC, forward scatter; FSC-A, forward scatter area; HV, healthy volunteer; K, thousand; SSC, side scatter; SSC-A, side scatter area; VZV, varicella-zoster virus.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Markers of skin-homing B cells remain undefined, although cutaneous lymphocyte antigen (CLA) is expressed on circulating B cells after percutaneous immunization with tetanus toxoid toxin (Kantele et al., 2003Kantele A. Savilahti E. Tiimonen H. Iikkanen K. Autio S. Kantele J.M. Cutaneous lymphocyte antigen expression on human effector B cells depends on the site and on the nature of antigen encounter.Eur J Immunol. 2003; 33: 3275-3283Crossref PubMed Scopus (31) Google Scholar). We examined circulating CD20+ B cells by flow cytometry for expression of classical T-cell skin-homing markers CLA, CCR4, and CCR10 (Clark et al., 2006aClark R.A. Chong B. Mirchandani N. Brinster N.K. Yamanaka K-i Dowgiert R.K. et al.The vast majority of CLA+ T cells are resident in normal skin.J Immunol. 2006; 176: 4431-4439Crossref PubMed Scopus (550) Google Scholar; McCully et al., 2012McCully M.L. Ladell K. S. B. skin expression in human T 2012; PubMed Scopus Google Scholar). The percentages of B cells were than those of B cells between and cells) (Supplementary Figure B cells were identified within VZV blister aspirates on day 3 after challenge (mean = n = 3) and within cutaneous blisters on day 7 after VZV challenge (mean = n = T cells were mean = day mean = n = 3) (Figure and as (Clark et al., 2006aClark R.A. Chong B. Mirchandani N. Brinster N.K. Yamanaka K-i Dowgiert R.K. et al.The vast majority of CLA+ T cells are resident in normal skin.J Immunol. 2006; 176: 4431-4439Crossref PubMed Scopus (550) Google Scholar). We investigated antibody from cutaneous B cells. Flow cytometric of mature and memory B cells within suction blister aspirates on 3 and 7 after VZV challenge (Figure and circulating plasma cells expression of expression of and with or without expression et al., A. B. B. C. A. et human B and plasma cells. in and of circulating normal and plasma PubMed Scopus Google Scholar). We detected of in CD3–CD27+ circulating plasma cells in blood not in blister of on day 3 and one on day after VZV injection (Figure and B cells were also identified within the blister fluid and peripheral blood 3 and day Figure to have et al., The of plasma Immunol. 2015; PubMed Scopus Google Scholar). and IgG are in response to VZV with IgG 9: PubMed Google Scholar). We detected IgG and Total IgG and at mean titres within VZV (n = mean = and candida (n = mean = blister than within saline (n = mean = although statistical was not (Figure IgG titres within VZV blisters were than and titres (Supplementary Figure VZV and saline aspirates lower titres for antibody than We also tested the of healthy serum (n = 19) and day 7 blister fluid = and candida = to IgG by were in blister fluid after both VZV and candida antigen challenge in addition to healthy serum with in signal the (Figure measured at 1:100 and relative to the positive control antibody that is that antibody in the skin in response to antigen challenge. studies on skin identified CD20+ B cells within 24 hours after VZV and candida skin challenge (Figure We the of CD3+ and CD20+ immune cells within (Supplementary Figure and mean CD20+ cell within the to within skin (mean ± analysis B-cell numbers from ± n = to day (n = 5, VZV = ± n = candida = ± day 3 (n = 5, VZV = ± n = candida = ± and day 7 (n = VZV = ± n = candida = ± per (Figure We also B-cell within VZV- and skin on day 7 (Figure and B cells accumulate and within cutaneous sites of antigen challenge. from skin biopsies and 7 after intradermal challenge with (a) candida and (b) VZV antigens were for CD3+ T cells CD20+ B cells and lymphocytes within were with = skin biopsies were analyzed per per Representative are shown. Quantification of CD3+ T and CD20+ B cells within candida and VZV in a and 0 biopsies were from healthy skin without previous VZV or candida antigen challenge. were within to and the mean of cells was per Error bars represent mean ± SEM. Quantification of CD3+ T and CD20+ B cells within candida and VZV in a and Error bars represent mean ± SEM. were analyzed with ANOVA with Tukey’s multiple comparison Representative immunofluorescence staining of cells, by B cells within day 7 skin VZV, = < < < ∗∗∗∗P < 0.0001. VZV, varicella-zoster virus.View Large Image Figure ViewerDownload Hi-res image Download (PPT) this the and of B cells to cutaneous immune responses in healthy human skin in a human in challenge with recall antigens in mature B-cell and a B-cell component at sites of cutaneous antigen challenge. A model in which from the to cutaneous sites of antigen the expression of skin-homing markers as CLA and However, the of B cells cells not in during responses in the skin. studies are to the that control the of B cells to cutaneous sites and in human studies were by the of on the Cutaneous was obtained from in this of the for the are in the and within the is from which have J. N. N. E. The of We with healthy provided tissue for this We the at and and the at College for We also Rustin and Vukmanovic-Stejic College London, London, for on and of varicella-zoster virus and candida skin as as College for with skin suction blisters. The was by the for at and and College The are for and the to the and of the The from the and the and the at College and the and and and for in of for and and - The expressed are those of the and not those of the the for or the of human studies were by the of on the Cutaneous was obtained from in this < mean = were for varicella-zoster virus (VZV; provided by Prof A.N. Akbar, University College London, UK) or candida skin (Allermed Laboratories, San Diego, CA) on the basis of previous or A total of of VZV or skin test antigen was the of the which was confirmed by at the injection a of sterile saline (Advanz Pharma, London, UK) was also injected a on the biopsies of were from sites of antigen 24 3 and 7 after challenge for immunofluorescence analysis. suction blisters were induced over sites of antigen challenge as 0 control biopsies were also from not to previous VZV or candida antigen challenge. Skin suction blisters were induced at as (Akbar et al., 2013Akbar A.N. Reed J.R. Lacy K.E. Jackson S.E. Vukmanovic-Stejic M. Rustin M.H. Investigation of the cutaneous response to recall antigen in humans in vivo.Clin Exp Immunol. 2013; 173: 163-172Crossref PubMed Scopus Google Scholar). was for hours by of a suction over sites of VZV and candida or saline intradermal injection either on day 3 or day 7 after the challenge. Suction was a blister was after which the was to Blisters were with after blister fluid was and analyzed by flow cytometry and lymphocyte were by from skin of at and London, Lymphocytes were also from suction blister aspirates and healthy donor peripheral blood were with the San Diego, Franklin Lakes, and CLA or one were in as were on a II or LSRFortessa Cell Analyser using tissue were from skin were with serum and with and were also were with IgG or IgG Cell were with were and using either a and or for for or and for or as in cells) were to the of B (CD20+) and T (CD3+) lymphocytes as as to of lymphocyte infiltration were per per from which were of CD20+ CD3+ and cells. were then for each lymphocyte cells were in were to a was between the and the cell were Total antibody titres in blister fluid and serum were by College London, using the ImmunoCAP which for of of within human serum or plasma of antibody also is by the of a with total with of in each to total serum an antibody is to the to which serum on the addition of the test are then after the are to the After a a is which to the antibody a that is to the of the antibody in this contained within the Total IgG antibody titres in VZV- and skin blister fluid and healthy sera were also measured by ELISA were with per of IgG antibody were and after were for an using of per and IgG were in ELISA and per were in to ELISA A total of per of IgG antibody was and was using The were analyzed using the analysis Total IgG titres were using the under a and were for the of of with VZV- and skin blister fluid and healthy sera a cell ELISA assay. ELISA were with per of VZV at 1:100 with in of a were and after were for an using of per were in ELISA and per were in to ELISA A total of per of IgG antibody was and was using The were analyzed using the analysis Antibody to VZV antigen was as or relative the reactivity of to VZV was evaluated as the mean of blister fluid or or donor sera from at 1:100 dilution, relative to the mean of the positive control antibody (16 statistical analysis was using San Diego, were for of using the and of sample (n were to statistical analysis was for with n Figure of circulating B cells skin-homing CLA, CCR4, and CCR10 in healthy blood lymphocytes obtained from healthy were for flow cytometric analysis (FACSCanto II or LSRFortessa Cell Analyser - Becton Dickinson, Franklin Lakes, with for and the skin-homing markers CLA, CCR4, and (b) The percentages of circulating (left and were determined by on (a) the FSC on the SSC (left followed by the CD20+ FSC Flow cytometry plot is shown for one donor. (c) plots the percentages of circulating (n = (n = and (n = B cells. Each symbol on the plot represents one individual. Error bars represent mean ± SEM. were using Kruskal‒Wallis test with Dunn’s multiple comparison CLA, cutaneous lymphocyte antigen; FSC, forward scatter; FSC-A, forward scatter area; K, thousand; not SSC, side scatter; SSC-A, side scatter Large Image Figure ViewerDownload Hi-res image Download Figure of antibody titres in VZV and saline blister fluid compared with those in total donor Total and antibody titres were analyzed within matched BF aspirates from saline control (n = 4) and VZV (n = challenge sites as as from donor sera (n = on day 7 after VZV or saline using an Each symbol on the plot represents one individual. were analyzed with Kruskal‒Wallis test with Dunn’s multiple comparison BF, blister fluid; not VZV, varicella-zoster virus.View Large Image Figure ViewerDownload Hi-res image Download Figure of day 3 VZV skin tissue the of CD3+ and CD20+ immune cells at sites in Representative immunofluorescence of and CD20+ B cells are with a = Large Image Figure ViewerDownload Hi-res image Download (PPT)