Nuclear antigen–reactive CD4+ T cells expand in active systemic lupus erythematosus, produce effector cytokines, and invade the kidneys

Abstract

Systemic lupus erythematosus is a systemic and chronic autoimmune disease characterized by loss of tolerance towards nuclear antigens with autoreactive CD4+ T cells implicated in disease pathogenesis. However, very little is known about their receptor specificity since the detection of human autoantigen specific CD4+ T cells has been extremely challenging. Here we present an analysis of CD4+ T cells reactive to nuclear antigens using two complementary methods: T cell libraries and antigen-reactive T cell enrichment. The frequencies of nuclear antigen specific CD4+ T cells correlated with disease severity. These autoreactive T cells produce effector cytokines such as interferon-γ, interleukin-17, and interleukin-10. Compared to blood, these cells were enriched in the urine of patients with active lupus nephritis, suggesting an infiltration of the inflamed kidneys. Thus, these previously unrecognized characteristics support a role for nuclear antigen-specific CD4+ T cells in systemic lupus erythematosus. Systemic lupus erythematosus is a systemic and chronic autoimmune disease characterized by loss of tolerance towards nuclear antigens with autoreactive CD4+ T cells implicated in disease pathogenesis. However, very little is known about their receptor specificity since the detection of human autoantigen specific CD4+ T cells has been extremely challenging. Here we present an analysis of CD4+ T cells reactive to nuclear antigens using two complementary methods: T cell libraries and antigen-reactive T cell enrichment. The frequencies of nuclear antigen specific CD4+ T cells correlated with disease severity. These autoreactive T cells produce effector cytokines such as interferon-γ, interleukin-17, and interleukin-10. Compared to blood, these cells were enriched in the urine of patients with active lupus nephritis, suggesting an infiltration of the inflamed kidneys. Thus, these previously unrecognized characteristics support a role for nuclear antigen-specific CD4+ T cells in systemic lupus erythematosus. Systemic lupus erythematosus (SLE) is a systemic and chronic autoimmune disease characterized by loss of tolerance toward nuclear antigens due to defective disposal of biological waste such as apoptotic material containing ribonucleoproteins and nucleosomes.1Casciola-Rosen L.A. Anhalt G. Rosen A. Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.J Exp Med. 1994; 179: 1317-1330Crossref PubMed Scopus (1505) Google Scholar Autoantibodies directed against nuclear antigens are characteristic of SLE and usually occur before overt disease.2Arbuckle M.R. McClain M.T. Rubertone M.V. et al.Development of autoantibodies before the clinical onset of systemic lupus erythematosus.N Engl J Med. 2003; 349: 1526-1533Crossref PubMed Scopus (1803) Google Scholar In addition, autoreactive CD4+ T cells are implicated in the pathogenesis of SLE by promoting autoantibody production by B cells and directly propagating organ damage in inflamed target organs.3Suarez-Fueyo A. Bradley S.J. Tsokos G.C. T cells in systemic lupus erythematosus.Curr Opin Immunol. 2016; 43: 32-38Crossref PubMed Scopus (75) Google Scholar Although the detection of nuclear antigen–reactive T cells has been reported previously, the evidence for their existence remains circumferential until now and it is presently unclear how they interplay in the pathogenesis.4Crow M.K. DelGiudice-Asch G. Zehetbauer J.B. et al.Autoantigen-specific T cell proliferation induced by the ribosomal P2 protein in patients with systemic lupus erythematosus.J Clin Invest. 1994; 94: 345-352Crossref PubMed Scopus (44) Google Scholar, 5Engler J.B. Undeutsch R. Kloke L. et al.Unmasking of autoreactive CD4 T cells by depletion of CD25 regulatory T cells in systemic lupus erythematosus.Ann Rheum Dis. 2011; 70: 2176-2183Crossref PubMed Scopus (15) Google Scholar, 6Greidinger E.L. Gazitt T. Jaimes K.F. Hoffman R.W. Human T cell clones specific for heterogeneous nuclear ribonucleoprotein A2 autoantigen from connective tissue disease patients assist in autoantibody production.Arthritis Rheum. 2004; 50: 2216-2222Crossref PubMed Scopus (32) Google Scholar, 7Kattah N.H. Newell E.W. Jarrell J.A. et al.Tetramers reveal IL-17-secreting CD4+ T cells that are specific for U1-70 in lupus and mixed connective tissue disease.Proc Natl Acad Sci U S A. 2015; 112: 3044-3049Crossref PubMed Scopus (18) Google Scholar, 8Lu L. Kaliyaperumal A. Boumpas D.T. Datta S.K. Major peptide autoepitopes for nucleosome-specific T cells of human lupus.J Clin Invest. 1999; 104: 345-355Crossref PubMed Scopus (155) Google Scholar, 9Okubo M. Yamamoto K. Kato T. et al.Detection and epitope analysis of autoantigen-reactive T cells to the U1-small nuclear ribonucleoprotein A protein in autoimmune disease patients.J Immunol. 1993; 151: 1108-1115PubMed Google Scholar Understanding how these cells participate in autoimmunity will be critical to the design of effective treatment. The detection of autoantigen-specific T cells is hindered by their extraordinarily low frequencies in circulating blood.10Taniguchi R.T. DeVoss J.J. Moon J.J. et al.Detection of an autoreactive T-cell population within the polyclonal repertoire that undergoes distinct autoimmune regulator (Aire)-mediated selection.Proc Natl Acad Sci U S A. 2012; 109: 7847-7852Crossref PubMed Scopus (81) Google Scholar Previous studies of human naive CD4+ T-cell repertoires have used tetramer-based technology to effectively provide precise information on T-cell responses to specific antigen epitopes; however, this method requires prior knowledge of the human leukocyte antigen types of the donor.11Kwok W.W. Tan V. Gillette L. et al.Frequency of epitope-specific naive CD4+ T cells correlates with immunodominance in the human memory repertoire.J Immunol. 2012; 188: 2537-2544Crossref PubMed Scopus (78) Google Scholar, 12Moon J.J. Chu H.H. Pepper M. et al.Naive CD4+ T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude.Immunity. 2007; 27: 203-213Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar, 13Su L.F. Kidd B.A. Han A. et al.Virus-specific CD4+ memory-phenotype T cells are abundant in unexposed adults.Immunity. 2013; 38: 373-383Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar Alternative techniques to simultaneously detect and enumerate rare T-cell populations with reactivity to different antigens without prior knowledge of human leukocyte antigen types were developed by using libraries of amplified polyclonal T cells14Geiger R. Duhen T. Lanzavecchia A. Sallusto F. Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells.J Exp Med. 2009; 206: 1525-1534Crossref PubMed Scopus (169) Google Scholar and by enrichment of CD154-expressing T cells after stimulation with an antigen termed ARTE (antigen-reactive T-cell enrichment).15Bacher P. Schink C. Teutschbein J. et al.Antigen-reactive T cell enrichment for direct, high-resolution analysis of the human naive and memory Th cell repertoire.J Immunol. 2013; 190: 3967-3976Crossref PubMed Scopus (105) Google Scholar CD4+ T cells mainly exert their function through secretion of cytokines upon antigenic activation16Zhu J. Paul W.E. Peripheral CD4+ T-cell differentiation regulated by networks of cytokines and transcription factors.Immunol Rev. 2010; 238: 247-262Crossref PubMed Scopus (379) Google Scholar and by propagating tissue inflammation such as in lupus nephritis (LN) that represents one of the most serious complications of SLE.17Brentjens J.R. Sepulveda M. Baliah T. et al.Interstitial immune complex nephritis in patients with systemic lupus erythematosus.Kidney Int. 1975; 7: 342-350Abstract Full Text PDF PubMed Scopus (108) Google Scholar,18Suarez-Fueyo A. Bradley S.J. Klatzmann D. Tsokos G.C. T cells and autoimmune kidney disease.Nat Rev Nephrol. 2017; 13: 329-343Crossref PubMed Scopus (59) Google Scholar Analysis of the T-cell repertoire present in renal biopsies from patients with LN revealed oligoclonality of kidney-infiltrating CD4+ T cells, indicating accumulation of antigen-specific T cells in inflamed kidneys.19Murata H. Matsumura R. Koyama A. et al.T cell receptor repertoire of T cells in the kidneys of patients with lupus nephritis.Arthritis Rheum. 2002; 46: 2141-2147Crossref PubMed Scopus (49) Google Scholar Our previous study revealed urinary T cells in patients with active LN as a precise biomarker that resemble the phenotype of intrarenal cells.20Enghard P. Humrich J.Y. Rudolph B. et al.CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients.Arthritis Rheum. 2009; 60: 199-206Crossref PubMed Scopus (100) Google Scholar,21Enghard P. Rieder C. Kopetschke K. et al.Urinary CD4 T cells identify SLE patients with proliferative lupus nephritis and can be used to monitor treatment response.Ann Rheum Dis. 2014; 73: 277-283Crossref PubMed Scopus (46) Google Scholar Although these cells are enriched for CXC chemokine receptor 3 and CC chemokine receptor 5 expressing T cells indicative of a T helper cell 1 (Th1) cell population and contain CD154+ T cells suggestive of recent antigen encounter,22Klocke J. Kopetschke K. Griessbach A.S. et al.Mapping urinary chemokines in human lupus nephritis: potentially redundant pathways recruit CD4+ and CD8+ T cells and macrophages.Eur J Immunol. 2017; 47: 180-192Crossref PubMed Scopus (16) Google Scholar,23Kopetschke K. Klocke J. Griessbach A.S. et al.The cellular signature of urinary immune cells in lupus nephritis: new insights into potential biomarkers.Arthritis Res Ther. 2015; 17: 94Crossref PubMed Scopus (38) Google Scholar their antigen specificity toward nuclear antigens remains unclear. In this study, we present an analysis of CD4+ T cells reactive to nuclear antigens in a total of 17 healthy individuals, 12 subjects with inactive SLE, and 20 subjects with active SLE by using T-cell libraries and ARTE techniques to analyze the frequencies of autoreactive CD4+ T cells in peripheral blood and urine of patients with LN as well as the cytokine production. Given the high to detect rare antigen-specific T cells, libraries of amplified CD4+ T cells were and for reactivity against nuclear T-cell libraries from 12 patients with SLE and healthy were with the nuclear antigens and in the of cells by the of cell proliferation in response to antigenic stimulation the amplified T-cell to the a used as a proliferation and the of the stimulation of cells to to the B as the In to nuclear we as an autoantigen patients with 5 healthy The detection of antigen-specific cells by a of with the antigen and the of antigen-specific T-cell and by using M. D. et to CD4+ T cells specific for antigens to Med. PubMed Scopus Google Scholar R. Duhen T. Lanzavecchia A. Sallusto F. Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells.J Exp Med. 2009; 206: 1525-1534Crossref PubMed Scopus (169) Google Scholar the frequencies of antigen-specific T cells were the frequencies of nuclear antigen–reactive CD4+ T cells were in patients with active SLE with healthy subjects and patients with inactive SLE The total of autoreactive CD4+ T cells in a of to cells 1 cells, with the frequencies in patients with active SLE antigen–reactive CD4+ T cells were in patients with active These cells were in 5 of patients with inactive SLE, 3 of healthy circulating autoreactive T patients with active SLE a of autoreactive T-cell with reactive T cells against 3 nuclear In the target in patients with inactive SLE and healthy circulating nuclear antigen–reactive T cells are for SLE, in active SLE, their is and their target structures are autoreactive T cells a role in propagating autoimmune we the of autoreactive T cells with disease by the SLE disease the frequencies of CD4+ T cells reactive to and correlated with In the frequency of T cells reactive to a 3 a due to T-cell in active T-cell libraries a of high to detect very rare cells within a low of However, this on of cells, the cell and a to the reactive we with the ARTE The ARTE requires of cells, is in patients with SLE present with and of this and to the of reactive cells, we 5 nuclear antigens and used this to peripheral blood cells before enrichment of CD154-expressing cells patients with healthy a of the surface and T cells reactive to nuclear antigens be within a population Although of and in individuals, patients with active SLE frequencies of T cells with nuclear antigens to stimulation healthy and patients with inactive SLE frequencies and cells patients with inactive by the the frequencies of nuclear antigen–reactive T cells were in patients with active disease in healthy and patients with inactive disease and The frequency of nuclear antigen–reactive T cells correlated with disease suggesting their potential role in SLE pathogenesis CD4+ T cells reactive to a antigen frequencies the the of a toward frequencies of antigen-reactive T cells in patients with active using T-cell and ARTE of autoantigen-specific CD4+ T cells in patients with active techniques are as by stimulation and T-cell responses stimulation the ARTE revealed frequencies of nuclear antigen–reactive CD4+ T cells the T-cell T-cell and ARTE techniques the detection of autoreactive T cells in healthy individuals, a for the of human of human Clin Invest. 2015; PubMed Scopus Google Scholar However, their different to the it healthy subjects have circulating nuclear antigen–reactive CD4+ T the of autoreactive T cells in healthy we T-cell clones from CD4+ T-cell from healthy after stimulation of with nuclear antigens and a antigen from A of autoantigen-specific T-cell clones to their antigens after antigen as by the of with of specific 5 of of 3 of 5 of of 5 of and the antigen response for that nuclear antigen–reactive T cells in healthy are autoreactive CD4+ T cells Thus, we the existence of nuclear antigen–reactive T cells in healthy subjects within the of the detection the cytokine production of nuclear antigen–reactive CD4+ T CD154-expressing cells were for effector cytokines such as and after stimulation with a of nuclear antigens of and T cells were in patients with active SLE the frequency of autoreactive T cells from the frequency of and autoreactive T cells were in patients with active SLE in healthy and patients with inactive SLE the patients with active SLE frequencies of and autoreactive T cells healthy as well as of and autoreactive T cells patients with inactive SLE In addition, the frequency of and of and nuclear antigen–reactive T cells correlated with disease However, the frequency of cells with disease by the frequency of antigen-reactive CD4+ T cells were mainly of the In as well as were F. Lanzavecchia A. of CD4+ memory T for J Immunol. 2009; PubMed Scopus Google Scholar Compared to autoantibody the frequencies of CD4+ T cells with the of and with the of and nuclear antigen–reactive CD4+ T cells and autoantibodies for CD4+ T cells toward autoantibodies The population of nuclear antigen–reactive cells is to the renal they their K. M. K. et of immune response in proliferative lupus nephritis.Arthritis Rheum. PubMed Scopus Google Scholar the T-cell receptor repertoire of CD4+ T cells from peripheral blood with from urine of 5 patients with SLE and active we an repertoires of cells The frequency of the 20 most abundant T-cell clones in urine to a diversity these cells reactivity to a of nuclear we libraries of urinary CD4+ T cells from 3 patients with SLE and active LN CD4+ T cells were and active cells by the to upon stimulation with with of by a proliferation after polyclonal and of these with an stimulation of were for of urinary CD4+ T cells specific to the nuclear In to urinary CD4+ T cells, the frequency of nuclear antigen–reactive CD4+ T cells in peripheral blood a of the frequencies within the that in active the frequencies of urinary nuclear antigen–reactive CD4+ T cells were of peripheral this we urinary cells from active LN with and mixed the cells with to a of antigen-reactive cells in 1 using the ARTE The frequency of T cells in and of urinary and peripheral blood cells used to nuclear antigen–reactive T cells are enriched in urine as for the kidney by the stimulation Although the urinary T-cell and ARTE in their stimulation to the nuclear antigens T-cell analysis of in peripheral and urinary CD4+ T cells from total an nuclear stimulation in urine These that nuclear antigen–reactive CD4+ T cells and in the inflamed kidneys. Thus, tissue inflammation in SLE be an antigen-specific and directly by the autoreactive T A of tolerance against nuclear antigens is the of SLE, and B cells, autoreactive T cells are to a role in pathogenesis. However, as the detection of CD4+ T cells a in autoimmunity to little is known about nuclear antigen–reactive CD4+ T cells in for the detection and of antigen-specific CD4+ T cells have been The T-cell prior polyclonal of T cells to the detection of rare antigen-specific T-cell R. Duhen T. Lanzavecchia A. Sallusto F. Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells.J Exp Med. 2009; 206: 1525-1534Crossref PubMed Scopus (169) Google Scholar high and with of this we were to reactive T cells against 5 nuclear antigens in patients with active The of the detection that loss of clones the be and the for analysis of the is by the of usually the analysis of to the population with frequencies Our using the T-cell revealed that the frequency of circulating autoreactive CD4+ T cells for a autoantigen in patients with a disease cells in a cells, a frequency of the detection of autoantigen-specific CD4+ T cells using is et of CD4+ T cells that are reactive to antigens before on known as the ARTE P. Schink C. Teutschbein J. et al.Antigen-reactive T cell enrichment for direct, high-resolution analysis of the human naive and memory Th cell repertoire.J Immunol. 2013; 190: 3967-3976Crossref PubMed Scopus (105) Google Scholar this for the detection of nuclear CD4+ T cells, we were to and of nuclear antigen–reactive CD4+ T cells in active the frequency of T cells reactive with and C. healthy and patients with SLE with disease the of autoreactive T cells is a of in active SLE of the of antigen-specific T have the existence of autoreactive T cells in SLE, using cytokine of without to a population of these M.K. DelGiudice-Asch G. Zehetbauer J.B. et al.Autoantigen-specific T cell proliferation induced by the ribosomal P2 protein in patients with systemic lupus erythematosus.J Clin Invest. 1994; 94: 345-352Crossref PubMed Scopus (44) Google Scholar, 5Engler J.B. Undeutsch R. Kloke L. et al.Unmasking of autoreactive CD4 T cells by depletion of CD25 regulatory T cells in systemic lupus erythematosus.Ann Rheum Dis. 2011; 70: 2176-2183Crossref PubMed Scopus (15) Google Scholar, 6Greidinger E.L. Gazitt T. Jaimes K.F. Hoffman R.W. Human T cell clones specific for heterogeneous nuclear ribonucleoprotein A2 autoantigen from connective tissue disease patients assist in autoantibody production.Arthritis Rheum. 2004; 50: 2216-2222Crossref PubMed Scopus (32) Google L. Kaliyaperumal A. Boumpas D.T. Datta S.K. Major peptide autoepitopes for nucleosome-specific T cells of human lupus.J Clin Invest. 1999; 104: 345-355Crossref PubMed Scopus (155) Google Scholar The evidence on the frequency of nuclear T cells for reactivity against the a characteristic autoantigen target in mixed connective tissue disease and in a of patients with nuclear CD4+ T cells were using and and were reported to occur a frequency of to T cells and to N.H. Newell E.W. Jarrell J.A. et al.Tetramers reveal IL-17-secreting CD4+ T cells that are specific for U1-70 in lupus and mixed connective tissue disease.Proc Natl Acad Sci U S A. 2015; 112: 3044-3049Crossref PubMed Scopus (18) Google M. Yamamoto K. Kato T. et al.Detection and epitope analysis of autoantigen-reactive T cells to the U1-small nuclear ribonucleoprotein A protein in autoimmune disease patients.J Immunol. 1993; 151: 1108-1115PubMed Google Scholar is of the as the frequency of autoreactive cells in In we the reactivity against characteristic nuclear patients with active LN frequencies of antigen–reactive CD4+ T cells, patients in their target and the reactivity against a of antigens to patients with inactive The biological of this is presently CD4+ T cell with clinical different be redundant for the disease pathogenesis. to autoimmune we were to the existence of autoreactive CD4+ T cells in healthy A. T cells in 1 Clin Invest. 2017; PubMed Scopus Google A. M. et al.Frequency of circulating autoreactive T cells to in Immunol. 2012; PubMed Scopus Google Scholar frequencies to patients with active These cells were by the of cells with without antigen with such techniques as T-cell libraries ARTE However, by cell we were to that the autoantigen-specific CD4+ T specific to the of tolerance and frequencies of autoreactive T cells remains that of autoreactive clones a in the pathogenesis of nuclear antigen–reactive CD4+ T cells mainly to a and cytokine with the with autoantibody production that the role of nuclear antigen–reactive CD4+ T cells be of B cell has been to be in the pathogenesis of C. B. M. is for the of autoimmune in Immunol. Google C. B. M. receptor autoantibody production and in Immunol. Google Scholar cells have been to be into the inflamed renal tissue in P. Humrich J.Y. Rudolph B. et al.CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients.Arthritis Rheum. 2009; 60: 199-206Crossref PubMed Scopus (100) Google B. M. et is in in human J 2004; Full Text Full Text PDF PubMed Scopus Google Scholar nuclear antigen–reactive T cells to the they their T cells have been previously to disease and the phenotype of intrarenal cells in P. Humrich J.Y. Rudolph B. et al.CXCR3+CD4+ T cells are enriched in inflamed kidneys and urine and provide a new biomarker for acute nephritis flares in systemic lupus erythematosus patients.Arthritis Rheum. 2009; 60: 199-206Crossref PubMed Scopus (100) Google D. et in The immune cell in kidneys of lupus nephritis Immunol. PubMed Scopus Google we used urinary T cells as for renal T T cells revealed a is in with in kidney H. Matsumura R. Koyama A. et al.T cell receptor repertoire of T cells in the kidneys of patients with lupus nephritis.Arthritis Rheum. 2002; 46: 2141-2147Crossref PubMed Scopus (49) Google SLE nephritis is with an of intrarenal T J Dis. Full Text Full Text PDF PubMed Scopus Google Scholar indicating enrichment of T-cell clones in the inflamed kidney urinary T cells, we were to detect nuclear antigen–reactive T cells and their frequency enriched to T it that nuclear antigen–reactive cells directly participate in the of organ In we that nuclear antigen–reactive CD4+ T cells are in active they are mainly T cells and inflamed target such as the are in were from 17 healthy individuals, 12 patients with inactive SLE and 20 patients with active SLE In addition, urine were from 12 patients with active SLE and subjects their on the of the by the and and and urinary cells were using a on the surface were on cells in different of the and to and cells, the the cell cells were with for and with the antigens were by using a and were on and the by using were analyzed using used the antigens to CD4+ T cells in the T-cell of human protein of human protein of human protein of human protein by of human protein by 1 of 1 of and of human protein The to amplified peripheral and urinary T-cell libraries and as previously R. Duhen T. Lanzavecchia A. Sallusto F. Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells.J Exp Med. 2009; 206: 1525-1534Crossref PubMed Scopus (169) Google Scholar peripheral CD4+ T cells to urinary CD4+ T cells were using CD4 to The of CD4+ T-cell using on the of the and to for peripheral blood and urinary In cells were by cells from after depletion with and 1 to of of amplified CD4+ T cells were into on the of antigens to be stimulation with the cells were for the of cells were and into the T-cell in a of 1 cell to CD4+ T CD4+ T-cell libraries for a and a with were in the CD4+ T cells were with the antigen for and proliferation using a T-cell to the antigens by heterogeneous a of the proliferation as The as 5 of and the of in with as a with an stimulation of for peripheral blood for urinary were it cell of the frequency of antigen-specific CD4+ T cells using of to and 1 cells, as R. Duhen T. Lanzavecchia A. Sallusto F. Human naive and memory CD4+ T cell repertoires specific for naturally processed antigens analyzed using libraries of amplified T cells.J Exp Med. 2009; 206: 1525-1534Crossref PubMed Scopus (169) Google Scholar The of is on the that 1 CD4+ T cell is present in a of T-cell libraries the after antigen-specific the that the P. A. analysis of rare antigen-specific T A. 2013; PubMed Scopus Google Scholar cells were for with antigens and with and to cells were to CD154-expressing of cells with a cell clones were from antigen-reactive cells, were enriched by the ARTE CD154-expressing cells were with a and in 1 of and were cells for the and cells were in of in the of were for 3 to until clones were before cells were and into the T-cell in a of 1 cell to CD4+ T were with 1 of and clones as a from peripheral CD4+ T cells and urinary cells using the The amplified the as M. J. L. et repertoire analysis by complex of T J 2013; 13: PubMed Scopus Google Scholar were processed using L. M. J. et and to 2015; PubMed Scopus Google Scholar and were clustered and were with for U and were used in the with and for the of critical it is on the of were with the and were using by the of to provide of the the of cells and disease were with the and frequencies were the frequencies of nuclear antigen–reactive CD4+ T cells, the frequencies without were as A to it and are in and the and and for with and cell for these studies by from the within the to and by from and of and of to with

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