Allergen-specific immunotherapy modulates the balance of circulating Tfh and Tfr cells

Abstract

Follicular helper T cells (Tfh cells) are a CD4 T-cell subset specialized in providing help for the development and maintenance of B-cell responses.1Crotty S. Follicular helper CD4 T cells (TFH).Annu Rev Immunol. 2011; 29: 621-663Crossref PubMed Scopus (2026) Google Scholar Tfh cells are essential for germinal center formation and the development of effective humoral immunity.2Johnston R.J. Poholek A.C. DiToro D. Yusuf I. Eto D. Barnett B. et al.Bcl6 and Blimp-1 are reciprocal and antagonistic regulators of T follicular helper cell differentiation.Science. 2009; 325: 1006-1010Crossref PubMed Scopus (1142) Google Scholar In humans, circulating resting memory Tfh cells are minimally defined as CD3+CD4+CD45RO+CXCR5+.3Locci M. Havenar-Daughton C. Landais E. Wu J. Kroenke M.A. Arlehamn C.L. et al.Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses.Immunity. 2013; 39: 758-769Abstract Full Text Full Text PDF PubMed Scopus (641) Google Scholar Follicular regulatory T cells (Tfr), a T-cell subset believed to originate from thymic-derived FOXP3+ T-cell precursors, have been shown to regulate and suppress germinal center reactions.4Linterman M.A. Pierson W. Lee S.K. Kallies A. Kawamoto S. Rayner T.F. et al.Foxp3+ follicular regulatory T cells control the germinal center response.Nat Med. 2011; 17: 975-982Crossref PubMed Scopus (913) Google Scholar Several reports suggest that Tfh cells likely play a role in mediating allergic disease5Coquet J.M. Schuijs M.J. Smyth M.J. Deswarte K. Beyaert R. Braun H. et al.Interleukin-21-producing CD4(+) T cells promote type 2 immunity to house dust mites.Immunity. 2015; 43: 318-330Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 6Kobayashi T. Iijima K. Dent A.L. Kita H. Follicular helper T cells mediate IgE antibody response to airborne allergens.J Allergy Clin Immunol. 2017; 139: 300-313.e7Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar; however, there is a lack of human studies of Tfh cell biology in the context of allergy7Kemeny D.M. The role of the T follicular helper cells in allergic disease.Cell Mol Immunol. 2012; 9: 386-389Crossref PubMed Scopus (27) Google Scholar, 8Varricchi G. Harker J. Borriello F. Marone Hon G. Durham S.R. Shamji M.H. T follicular helper (Tfh) cells in normal immune responses and in allergic disorders.Allergy. 2016; 71: 1086-1094Crossref PubMed Scopus (73) Google Scholar and allergen-specific immunotherapy (AIT). Given the critical role of Tfh and Tfr cells in the regulation of IgE production in the context of allergic disease in murine models,6Kobayashi T. Iijima K. Dent A.L. Kita H. Follicular helper T cells mediate IgE antibody response to airborne allergens.J Allergy Clin Immunol. 2017; 139: 300-313.e7Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar we hypothesized that AIT modulates Tfh and/or Tfr cells. A total of 70 subjects were recruited for this study, including 25 timothy grass allergic patients, 32 patients who received subcutaneous shots of AIT and were in treatment maintenance at the time of blood draw, and 13 nonallergic healthy controls identified as having negative skin prick test results to a panel of 32 allergen extracts and no clinical history of allergy (see Table E1 in this article's Online Repository at www.jacionline.org). The effect of AIT on Tfh cell frequency was assessed by flow cytometry, quantifying the total peripheral memory Tfh cell population (defined as CD3+CD4+CD45RO+CXCR5+) (Fig 1, A). We found a significant reduction in Tfh cells in AIT-treated patients compared with untreated allergic donors (median 6.4% in allergics, 3.5% in AIT-treated patients; P = .008; Fig 1, B). Reduction in Tfh cells after AIT was also seen compared with nonallergic controls (median, 5.1%) but this difference did not reach statistical significance (P = .13; Fig 1, B). To verify our manual gating analysis results, we also analyzed the data using a FLOCK-based automated gating strategy, which produced similar results (see Fig E1 in this article's Online Repository at www.jacionline.org). Given that the reduction in Tfh cells in patients treated with AIT is most prominent in the CXCR5hi subset, we characterized gene expression in CXCR5hi versus CXCR5lo cells in 5 allergic donors with a predominant CXCR5hi Tfh population (Fig 1, A, red box) and 5 AIT-treated donors with a predominantly CXCR5lo Tfh population (Fig 1, A, blue box). CXCR5hi and CXCR5lo cells were sorted and high-quality RNA (RNA Integrity Number [RIN] > 7.5) was obtained for subsequent transcriptomic analysis. As expected, transcription of CXCR5 was significantly reduced in CXCR5lo cells compared with CXCR5hi cells (Fig 1, C). Paired comparisons of CXCR5hi versus CXCR5lo samples identified 26 genes with significantly different expression levels (multiple hypothesis adjusted P value <.05; Fig 2, D). Examination of the 26 differentially expressed genes revealed that several had known immunological functions, including regulatory activity (FOXP3, CCR8, LAG3, CD200, LRRC32, and CD70), inflammation (CCL5, LGALS3, and ENC1), cytotoxicity (GZMA), and proliferation (MKI67). Unsupervised hierarchical clustering of samples based on the expression pattern of the 26 genes resulted in separation of CXCR5hi from CXCR5lo cells independent of the AIT status of the donor from which the samples were derived (Fig 2, E). Thus, the differences observed are intrinsic properties of CXCR5hi versus CXCR5lo cells and are independent of AIT treatment status. To determine how the identified differences in mRNA levels between CXCR5hi and CXCR5lo cell populations impacted protein expression, we performed fluorescence-activated cell sorting analyses for proteins from 7 out of the 26 differentially expressed genes, based on antibody availability (Fig 2, A). In addition, we included antibodies against Helios because transcription of this gene was borderline significant in our analysis (P adjusted = .16; see Table E2 in this article's Online Repository at www.jacionline.org) and Helios has been shown to be important for regulatory T-cell function.9Kim H.J. Barnitz R.A. Kreslavsky T. Brown F.D. Moffett H. Lemieux M.E. et al.Stable inhibitory activity of regulatory T cells requires the transcription factor Helios.Science. 2015; 350: 334-339Crossref PubMed Scopus (252) Google Scholar Significantly higher expression in CXCR5lo versus CXCR5hi cells was detected for FOXP3, Helios, and Granzyme A. Comparing the frequency of Helios and Granzyme A expression in CXCR5loFOXP3+ versus CXCR5loFOXP3− cells revealed an enrichment of FOXP3+Helios+ cells, which was significantly higher than FOXP3+Granzyme A+ cells (median, 7.0 and 0.5, respectively, P < .0001) (Fig 2, B), suggesting that Helios and FOXP3 are frequently coexpressed on a single-cell level, whereas Granzyme A is expressed by a different subpopulation within the CXCR5lo cells. Given the upregulation of Foxp3 and Helios in CXCR5lo cells, we analyzed the median fluorescence intensity of CXCR5 in classical Tfh (CXCR5+FOXP3−Helios−) versus Tfr (CXCR5+FOXP3+Helios+) cells (Fig 2, C). A pronounced difference in CXCR5 expression (P > .0001) was observed in the Tfr subset compared with Tfh cells, indicating that Tfr cells fall within the CXCR5lo fraction of Tfh cells in our gating strategy. Thus, the increase in CXCR5lo cells observed in AIT-treated patients may be associated with a relative increase in Tfr cells. To assess the functionality of the stimulated Tfr cells, we performed intracellular cytokine staining which revealed that IL-10 production is more than 6-fold higher in Tfr cells compared with Tfh cells, a fold difference higher than any other cytokine measured (see Fig E2 in this article's Online Repository at www.jacionline.org). Finally, we wanted to determine whether the observed changes in CXCR5+ cells in the AIT cohort are associated with an induction of the Tfr phenotype (FOXP3+Helios+), and whether they can be induced directly by T cell receptor (TCR) stimulation, which is associated with IL-2 production. Purified Tfh cells (CD45RO+CXCR5+) from 8 donors were cultured for 5 days in medium alone or in the presence of TCR stimulation (anti-CD3/38 beads) with or without anti–IL-2. Flow cytometric analysis revealed a significant reduction in CXCR5 expression in the presence of anti-CD3/28 stimulation (median, 77.7%) compared with culture in medium alone (median, 94.9%), which was largely rescued in the presence of anti–IL-2 (median, 86.4%) (Fig 2, D). Tfr cells (CXCR5+FOXP3+Helios+) were significantly increased in the presence of anti-CD3/28 stimulation (median, 8.9%) compared with medium alone (median, 1.5%). Addition of anti–IL-2 partially reversed Tfr induction (median, 5.0%) after TCR stimulation (Fig 2, E). On the basis of these data, we speculate that TCR stimulation and/or the presence of IL-2 during in vitro culture may have agonistic effects on the development/survival of Tfr cells over CXCR5hi Tfh cells. In the context of AIT, we hypothesize that repeated administration of allergen extracts elicits IL-2 production from allergen-specific T-cell responses, which globally impairs CXCR5 expression in memory Tfh cells and induces/retains Tfr-cell populations. This could be an important mechanism contributing to the induction of tolerance during AIT. Patients were recruited following Institutional Review Board approval (La Jolla Institute for Allergy and Immunology, La Jolla, Calif) (Federal Wide Assurance no. 00000032). All patients enrolled in this study provided written consent. For the allergic cohort, timothy grass (TG)-allergic donors with skin prick test wheal of 3 mm or more in diameter to TG and a clinical history consistent with seasonal grass pollen allergy were recruited (n = 25). The second cohort consisted of TG-allergic subjects who receive subcutaneous shots of AIT for a minimum of 6 months and were in treatment maintenance at the time of the blood draw (n = 32). Allergen extracts used were from ALK America (Port Washington, NY). Select allergens were from Greer, Lenoir, NC (primarily molds) and Hollister-Stier, Spokane, Wash (AP dog). The patients received 1 shot per month. Because of concerns about increased risk for anaphylaxis after a large volume blood draw, patients were recalled on another day outside the shot day for blood sample collection. Blood was drawn within a month of the last allergy shot. Nonallergic, healthy control donors were identified as having negative skin prick test results to a panel of 32 allergen extracts and no clinical history of allergy (n = 13). A complete summary of all donors recruited for this study is provided in Table E1. To assess Tfh cell frequency, PBMCs from all 3 donor cohorts were thawed, washed, and stained with an antibody cocktail for CD4 (eBioscience, San Diego, Calif, clone RPA-T4), CD3 (BD, Franklin Lakes, NJ, clone UCHT1), CD45RO (eBioscience, clone UCHL1), CXCR5 (BD, clone RF8B2), and CD19 (BD, San Diego, Calif; clone HIB19), CD14 (BD, clone M5E2), CD8 (BD, clone RPA-T8), and live/dead aqua fixable viability dye for exclusion. Cells were stained for 20 minutes at room temperature, washed, and analyzed by flow cytometry. Frequency and CXCR5 median fluorescence intensity of Tfh cells was determined by gating on the CD4+CD3+CD45RO+CXCR5+ population. Expression of markers of interest identified by RNA-Seq analysis was assessed by surface staining PBMCs ex vivo with the panel described above, except CXCR5 (Biolegend, San Diego, Calif; clone J252D4), which performed better during fixation, in addition to CCR8 (R&D Systems, Minneapolis, Minn; clone 191704) (panel 1) and CD200 (Biolegend, clone OX-104) (panel 3). Subsequently, cells were treated with FOXP3 Fixation/Permeabilization Kit (eBioscience) and intracellular staining was performed for FOXP3 (ebioscience, clone 236A/E7), Helios (eBioscience, clone 22F6), and Ki-67 (Biolegend, clone Ki-67) (panel 1); FOXP3 and RANTES (Biolegend, clone VL1) (panel 2); and FOXP3 and Galectin-3 (Biolegend, clone M3/38) and Granzyme A (Biolgend, clone CB9) (panel 3). All data acquisition was performed using a BD LSR II flow cytometer and data were analyzed using FlowJo software (TreeStar, Ashland, Ore). All data acquisition was performed blinded. PBMCs from 5 allergic and 5 AIT-treated patients were stained with the antibody cocktail for CD4, CD3, CD45RO, CXCR5, and CD19, CD14, CD8, and live/dead aqua for exclusion as described above. Tfh cells were gated as CD4+CD3+CD45RO+CXCR5+ and directly sorted into 750 μL of Trizol LS (Invitrogen, Carlsbad, Calif). Data acquisition and cell sorting were performed using a FACSAria II flow cytometer (BD, 2 out of 5 AIT-treated donors had insufficient number of CXCR5hi cells for RNA-Seq analysis). One of the AIT CXCR5 samples was excluded from analysis because of low-quality mRNA (RIN < 7.5). RNA-Seq analysis was performed in CXCR5lo samples collected from 5 allergic and 4 AIT-derived samples and CXCR5hi samples from 5 allergic and 3 AIT-derived samples, leaving 2 CXCR5lo samples and 1 CXCR5hi sample without corresponding match. Total RNA was purified as described previouslyE1Seumois G. Zapardiel-Gonzalo J. White B. Singh D. Schulten V. Dillon M. et al.Transcriptional profiling of Th2 cells identifies pathogenic features associated with asthma.J Immunol. 2016; 197: 655-664Crossref PubMed Scopus (53) Google Scholar and is described in detail in the Online Repository. RNA sequence analysis was performed as described previouslyE1Seumois G. Zapardiel-Gonzalo J. White B. Singh D. Schulten V. Dillon M. et al.Transcriptional profiling of Th2 cells identifies pathogenic features associated with asthma.J Immunol. 2016; 197: 655-664Crossref PubMed Scopus (53) Google Scholar and is described in detail in the Online Repository. CD4+ T cells were isolated from previously frozen PBMCs by negative selection using CD4+ T-cell isolation kit II per the manufacturer's instructions (Miltenyi, San Diego, Calif). The isolated CD4 cell population was stained as described above. Tfh cells (CD4+CD3+CD45RO+CXCR5+) were isolated using a BD FACSAria II cell sorter. After sorting, cells were plated in a round-bottom 96-well plate at 2 × 105/well in 200-μL serum-free AIM-V medium (Life Technologies) with human rIL-7 (4 ng/mL). Tfh cells were incubated in medium alone, with TCR stimulation (Dynabeads, human Tactivator CD3/28, Life Technologies) or with human rIL-2 (125 μg/mL), all in the presence or absence of anti-human IL-2 (50 μg/mL). After 5 days of incubation, cells were harvested, washed, and surface stained with antibodies for CD4 (eBioscience, clone RPA-T4), CD3 (BD, clone UCHT1), CD45RO (eBioscience, clone UCHL1), and CXCR5 (Biolegend, clone J252D4). For exclusion, stains for CD19 (BD, clone HIB19), CD14 (BD, clone M5E2), and CD8 (BD, clone RPA-T8) were also performed. Intracellular stains for FOXP3 and Helios were performed as described above. A 1- or 2-tailed Mann-Whitney U test was used for statistical analysis as indicated in the figure legends. For paired sample comparison, Wilcoxon signed rank test was used. Differences with a P value of less than .05 were considered significant.Fig E2Cytokine production after PMA/ionomycin stimulation in Tfr cells ex vivo. Intracellular cytokine staining for IL-10, IFN-γ, IL-4, and IL-13 after 6 hours of stimulation with PMA/ionomycin was performed in Tfr cells from 4 healthy donors. Percentage of cytokine-producing cells out of the total CD4 subset is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Demographic and clinical information of donors recruited for this studyDonorCohortSexBirth yearRaceDonation dateWheal size∗Wheal size of skin prick test with TG.AIT report†Self-report on whether patient felt that AIT helped to improve allergic symptoms.D00013NonallergicM1979White2008-07-150NAD00014NonallergicF1982White2008-07-150NAD00030NonallergicF1954White2008-08-130NAD00032NonallergicM1982White2008-10-130NAD00034NonallergicF1971White2008-10-160NAD00036NonallergicM1987White2008-10-210NAD00038NonallergicF1980White2008-10-220NAD00049NonallergicF1957Hispanic2008-11-210NAD00055NonallergicF1948White2009-01-050NAD00082NonallergicM1976Black2009-12-170NAD00086NonallergicF1974White2010-03-090NAD00091NonallergicM1971Hispanic2010-04-220NAD00118NonallergicF1951White2008-12-120NAD00008AllergicM1980White2008-07-0811NAD00052AllergicF1968White2008-12-1210NAD00056AllergicM1967Hispanic2009-01-085NAD00073AllergicF1965Black2009-10-2612NAD00084AllergicM1979White2009-01-2017NAD00090AllergicM1967Hispanic2009-01-0810NAD00117AllergicF1958White2011-01-2436NAD00122AllergicF1972White2011-03-0120NAD00137AllergicF1985White2012-05-226NAD00139AllergicF1961White2012-06-0748NAD00140AllergicF1977White2012-06-1210NAD00141AllergicM1991White2012-06-1414NAD00145AllergicF1961White2012-07-058NAD00149AllergicF1992White2012-08-015NAD00156AllergicF1960Black2012-08-2217NAD00158AllergicM1982White2012-09-0431NAD00159AllergicF1976Black2012-09-0410NAD00160AllergicF1964Black2012-09-1131NAD00161AllergicF1981Hispanic2012-09-1117NAD00163AllergicM1984Black2012-09-1822NAD00166AllergicM1982White2012-09-2128NAD00167AllergicM1949White2012-09-2113NAD00168AllergicM1969White2012-09-2421NAD00170AllergicM1966White2012-09-2637NAD00172AllergicF1953White2012-09-2612NAD00031AITF1969Hispanic2008-08-257YesD00063AITM1970Asian/Pacific Islander2009-03-2515YesD00072AITF1954White2009-10-148YesD00080AITM1973White2009-12-0225YesD00087AITM1973White2010-03-2318YesD00106AITF1971White2010-09-169YesD00101AITM1964White2009-11-1217YesD00109AITF1980Black2010-09-2710YesD00111AITM1963White2010-09-2911YesD00114AITF1961White2010-10-2111UncertainD00126AITM1969White2011-03-1417YesD00127AITM1973White2010-03-2314YesD00129AITF1990White2012-01-2317UncertainD00130AITM1973White2012-01-2415YesD00132AITM1969White2012-02-1411YesD00133AITM1973White2012-02-1411YesD00134AITF1954White2012-02-1615YesD00135AITM1962White2012-03-1513YesD00136AITF1946White2012-03-1610YesD00142AITF1984White2012-06-285NoD00143AITF1960White2012-06-297YesD00144AITM1978White2012-07-029YesD00146AITM1982Black2012-07-097YesD00147AITM1981White2012-07-256YesD00148AITF1967Black2012-07-2510NoD00150AITF1966White2012-08-026YesD00151AITF1961White2012-08-029NoD00152AITF1974White2012-08-0310YesD00153AITM1967White2012-08-035YesD00155AITM1985Black2012-08-149YesD00157AITF1987White2012-08-2710YesD00162AITF1956White2012-09-147YesF, Female; M, male; NA, not applicable/available.∗ Wheal size of skin prick test with TG.† Self-report on whether patient felt that AIT helped to improve allergic symptoms. Open table in a new tab Table E2A list of genes significantly differentially expressed by the CXCR5lo vs CXCR5hi subsetS. no.GeneProteinFold changeP adjustedMedian CXCR5hiMedian CXCR5lo1CCR8CCR8>100.0080102LRRC32Garp33.3.0012543ZBTB32Zinc finger and BTB domain-containing protein 3211.6.0462194ENC1CD9311.5.00591075HPGDHydroxyprostaglandin dehydrogenase 15-(NAD)9.7.0285496SEMA5ASemaphorin 5A8.6.0005417CHN1Chimerin 18.3.009282348FOXP3Fox p 37.5.00012919LAG3Lag36.0.000137710GZMAGranzyme A6.0.0004426411CCDC141Coiled-coil domain containing 1415.5.004169012MKI67Ki675.5.01863513RTKN2Rhotekin 25.3.0083619114CLECL1C-type lectin like 14.5.000135915NKG7Natural killer cell granule protein 74.0.0235221016LGALS3Galectin 34.0.0047530217CD70CD703.6.02031118CCL5RANTES3.6.000350125419LOC541471ND3.2.0043912320ZEB2Zinc finger E-box-binding homeobox 22.9.0413710721CD200CD2002.9.0014914422MLF1IPCentromere protein U2.8.028267423CST7Cystatin-F2.6.00824262124LYARLYAR2.5.00814836625SMC4SMC-41.9.01823344126FLJ22184ND0.0.0288027IKZF2Helios11.9.160910428CXCR5CXCR50.4—2321968IKZF2 and CXCR5 shown for reference.ND, Not determined. Open table in a new tab F, Female; M, male; NA, not applicable/available. IKZF2 and CXCR5 shown for reference. ND, Not determined.