Native/citrullinated ll37-specific t-cells help autoantibody production in systemic lupus erythematosus

ABSTRACT LL37 exerts a dual pathogenic role in psoriasis. Bound to self-DNA/RNA, LL37 licenses autoreactivity by stimulating plasmacytoid dendritic cells-(pDCs)-Type I interferon (IFN-I) and

acts as autoantigen for pathogenic Th17-cells. In systemic lupus erythematosus (SLE), LL37 also triggers IFN-I in pDCs and is target of pathogenic autoantibodies. However, whether LL37

activates T-cells in SLE and how the latter differ from psoriasis LL37-specific T-cells is unknown. Here we found that 45% SLE patients had circulating T-cells strongly responding to LL37,

which correlate with anti-LL37 antibodies/disease activity. In contrast to psoriatic Th17-cells, these LL37-specific SLE T-cells displayed a T-follicular helper-(TFH)-like phenotype, with

CXCR5/Bcl-6 and IL-21 expression, implicating a role in stimulation of pathogenic autoantibodies. Accordingly, SLE LL37-specific T-cells promoted B-cell secretion of pathogenic anti-LL37

antibodies _in vitro_. Importantly, we identified abundant citrullinated LL37 (cit-LL37) in SLE tissues (skin and kidney) and observed very pronounced reactivity of LL37-specific SLE T-cells

to cit-LL37, compared to native-LL37, which was much more occasional in psoriasis. Thus, in SLE, we identified LL37-specific T-cells with a distinct functional specialization and antigenic

specificity. This suggests that autoantigenic specificity is independent from the nature of the autoantigen, but rather relies on the disease-specific milieu driving T-cell subset

polarization and autoantigen modifications. SIMILAR CONTENT BEING VIEWED BY OTHERS REGULATORY T CELLS IN PSORIATIC ARTHRITIS: AN IL-17A-PRODUCING, FOXP3INTCD161 + RORΓT + ICOS + PHENOTYPE,

THAT ASSOCIATES WITH THE PRESENCE OF ADAMTSL5 AUTOANTIBODIES Article Open access 30 November 2022 DIRECT TISSUE-SENSING REPROGRAMS TLR4+ TFH-LIKE CELLS INFLAMMATORY PROFILE IN THE JOINTS OF

RHEUMATOID ARTHRITIS PATIENTS Article Open access 27 September 2021 TYPE I INTERFERON DRIVES T CELL CYTOTOXICITY BY UPREGULATION OF INTERFERON REGULATORY FACTOR 7 IN AUTOIMMUNE KIDNEY

DISEASES IN MICE Article Open access 20 May 2025 INTRODUCTION Systemic Lupus Erythematosus (SLE) is an autoimmune disease with a prevalence of about 20–50 cases per 100,000 in Northern

Europe and USA1, characterized by immune-complex formation and deposition, which result in inflammation and tissue damage. In SLE, altered clearance of dying cells determines persistent

exposure of autoantigens and activation of antigen-presenting cells (APCs), mainly _via_ Toll-like receptors (TLR), thus favoring adaptive immune response licensing1,2,3. SLE autoantibodies

are preferentially directed against nuclear antigens (ANA)1,2,3, but in a substantial subset of SLE patients, antibodies target cytoplasmic proteins in neutrophils4,5. Neutrophils are indeed

crucial players in SLE pathogenesis5,6. Pathways linked to neutrophil activation (apoptosis, neutrophil-extracellular-trap release, NET, extrusion of oxidized DNA by living cells) were

believed implicated in autoimmune B-cell activation5,6,7. Although life expectancy has greatly improved, SLE remains a devastating disease with a standardized mortality ratio in excess of

three8, thus novel therapeutic strategies and new therapeutic targets are necessary1,2. We have discovered new autoantibody specificities and key self-proteins of the group of antimicrobial

peptides (AMPs), which could drive pathogenic events in SLE9. LL37 is of particular interest, since it binds and protects self-nucleic acids from degradation, forming complexes that prolong

DNA/RNA half-life. These LL37-nucleic acid complexes act as danger-associated molecular patterns (DAMPs)9,10,11,12. Complexes formed by LL37 with DNA/RNA elicit the production of type I

interferon (IFN-I) and other pro-inflammatory cytokines by plasmacytoid, myeloid dendritic cells (pDCs, mDCs), and monocytes9,10,11,12. Anti-LL37 antibodies favor the up-take of LL37-DNA

complexes into DCs9, which enhances IFN-I production. Finally, LL37-DNA complexes can directly stimulate B-cells to produce antibodies, including anti-LL37 antibodies13. Since neutrophils

accumulate in SLE target organs, including skin and kidneys, and are important releasers of LL37, we anticipate that increased LL37 concentration and LL37 binding to self-DNA/RNA, coupled to

generation of anti-LL37/LL37/DNA/RNA immune-complexes, would concur to pathogenic events in SLE14,15,16. Notably, in psoriasis LL37 frequently acts as T-cell autoantigens17. We hypothesized

that LL37 is highly immunogenic for T-cells because its sequence contains multiple binding motifs for several HLA-class I/II alleles. However, it is presently unclear whether: (a) anti-LL37

antibodies correlate with disease activity; (2) LL37-DNA complexes and/or NET/NET-like structures are present in lupus-target tissues; (3) T-cells responding to LL37 exist and correlate

with, and/or participate to, SLE pathogenesis. The latter aspect is important in that T-cells with T-helper follicular (TFH) functions are necessary for isotype immunoglobulin (Ig)-switch

and somatic hypermutation, the processes that generate high affinity autoantibodies17,18,19,20. By analyzing distinct patient cohorts, here we demonstrate that high anti-LL37 antibody levels

specifically circulate in SLE and not in psoriasis or control chronic diseases, and correlate with disease activity. LL37 and citrullinated LL37 (cit-LL37) are present in SLE skin/kidney,

and SLE T-cells proliferate to both native-LL37 and cit-LL37 in up to 45% of SLE patients, with cit-LL37 being a more efficient T-cell antigen than native-LL37. SLE T-cell responses

significantly correlate with anti-LL37 antibodies and disease activity, suggesting that LL37-directed responses are markers of active/severe SLE. Notably, unlike autoreactive psoriasis

T-cells, SLE native-LL37/cit-LL37-specific T-cells often possess a T-follicular helper (TFH)-like phenotype, and drive the production of pathogenic anti-LL37/anti-DNA/RNA antibodies _in

vitro_. Thus, the efficient dual immune amplification role of LL37 on both innate and adaptive immunity, favoured by the LL37 adjuvant activity and immunogenicity, operates also in SLE.

However, the intrinsic properties of this antigen, although efficacious in both settings, lead to different outcomes, dictated by the disease-specific milieu. RESULTS LL37, CIT-LL37 AND

LL37-DNA COMPLEXES ARE PRESENT IN LUPUS TARGET ORGANS LL37 is produced by activated neutrophils and is abundantly released in neutrophil-extracellular-like (NET)-structures9. We previously

hypothesized that neutrophils release high amounts of LL37 and DNA in SLE tissues, favoring IFN-I production and the activation of inflammatory pathways, which would fuel autoimmunity9.

Thus, here we have searched for LL37 and LL37-DNA complexes in SLE-target tissues. Moreover, since peptidyl arginine deaminase (PAD) enzymes can become activated during inflammation21,22,23

and perform citrullination of arginine-rich proteins, we specifically searched for the presence of cit-LL37 in SLE/cutaneous lupus (CLE) tissues. To do so, we took advantage of specially

developed monoclonal antibodies directed to either native LL37 (Mab137) or cit-LL37 (Mab142), which lack cross-reactivity between the two LL37 forms and to unrelated control

proteins/peptides (in their native and citrullinated forms, Fig. 1A). We detected both native-LL37 and cit-LL37 in SLE and CLE target organs by immunohistochemistry (Fig. 1B,C) and laser

scanner confocal microscopy (LSM) (Fig. 1D,E). Quantification, of native LL37 and cit-LL37 showed that the two LL37 forms were mostly present in the skin (dermis) and the kidney sections in

comparable amounts (Fig. 1B,D, respectively) (see Suppl. Methods). Of note, healthy donor (HD) skin did not express significant amounts of either LL37 forms, as expected, as HD skin does not

usually have a neutrophil infiltrate (Fig. 1B,C, lower panels). In addition, we clearly detected LL37 complexed with DNA in the kidneys of patients with lupus nephritis (Fig. 1F). Within

four of seven kidney biopsies analyzed (57%), DNA appeared in the form of filaments bound to LL37 and/or cit-LL37 (Fig. 1F). LL37, and LL37-DNA complexes co-localized with, or were in the

proximity of, the interferon-induced GTP-binding protein Mx1 (Mx1), an IFN-I-activated gene product24, and immuno-globulin (IgG) deposition (Fig. 1G, Fig. S1). An estimation of the percent

of IgG staining which co-localizes with LL37 staining is shown in Fig. 1H (see also Suppl. Methods). Since complement deposition in the form of membrane-attack-complexes (MAC) can be

responsible for hypercitrullination of multiple substrates in other settings25,26, we assessed the simultaneous presence of cit-LL37 and complement C9 deposition, which indeed could

co-localize in SLE kidney (Fig. S2). These results suggest that LL37 forms complexes with self-DNA and autoantibodies in SLE-affected tissues, which can enable LL37 to act as an adjuvant and

exert local interferogenic activity, revealed by the expression of Mx1. The results also demonstrate that a consistent part of LL37 undergoes citrullination in SLE-affected tissues.

ANTI-NATIVE LL37 AND ANTI-CIT-LL37-SPECIFIC ANTIBODY REACTIVITY CORRELATES WITH SLEDAI AND DECLINES IN INACTIVE SLE We have shown previously that a consistent proportion of SLE patients

harbor circulating anti-LL37 antibodies9,13, as compared to HD. In this study, anti-LL37 antibody reactivity correlated with SLE severity9. A second study reported the presence of

circulating anti-LL37 antibodies in SLE but did not confirm a correlation with disease activity27. Thus, we assessed the presence of anti-LL37 antibodies in SLE cohorts (Table S1), and a

small group of CLE patients28 (Table S2). Sera from SSc, RA, UC and psoriasis patients, diseases in which LL37 is hyper-expressed in tissues, and HD, served as controls10,29,30,31,32,33. In

addition, we searched for anti-cit-LL37 antibodies. Twenty-one (53%) and eighteen (48%) of 40 SLE patients had detectable and significant antibody reactivity to native LL37 and cit-LL37,

respectively (Fig. 2A). Compared to HD, we observed a weaker and/or occasional reactivity to native LL37/cit-LL37 in control psoriasis psoriasis, RA, SSc and UC patients, which was

significantly weaker when compared to SLE reactivity. Most importantly, antibody responses to control reverse LL37 (REV) and cit-REV peptide were negative in SLE, which suggests that

circulating SLE antibodies truly recognized cit-LL37 and not merely citrulline (Fig. 2B). In contrast, reactivity to cit-REV was detected in a significant proportion of RA sera (14 of 70 RA,

20%), which is consistent with the reported capacity of RA patients to mount a broad antibody response to citrullinated proteins23 (Fig. 2B). Most importantly, anti-LL37 and anti-cit-LL37

antibodies correlated with disease activity only in SLE patients (using Systemic Lupus Erythematosus Disease Activity Index, SLEDAI, see Methods). We observed no correlation with disease

activity (see Methods), in control disease patients, [for psoriasis, psoriasis-activity index, PASI: Spearman’s r (a-LL37) = −0.31 N = 33, p = 0.31; r (a-cit-LL37) = −0.34, N = 33, P = 

0.32]; for RA [DAS28: Spearman’s r (a-LL37) = −0.06 N = 40, p = 0.9; r (a-cit-LL37) = 0.07, N = 40, P = 0.69]; for UC [Mayo endoscopic Score, Spearman’s r (a-LL37) = −0.05 N = 58, p = 1.1; r

(a-cit-LL37) = 0,002, N = 58, P = 1.0]; for SSc [Rodnan skin score: Spearman’s r (a-LL37) = −0.21 N = 33, P = 0.1; r (a-cit-LL37) = 0,0008, N = 33, P = 0.99]. We confirmed SLE correlations

data in a replication cohort (Replication cohort1, See Table S1, Fig. 2D). Anti-LL37 antibodies also correlated with serum IFN-α in two patients cohorts (discovery cohort: Spearman r = 0.46,

p = 0.0019, N = 38, for anti-LL37 abs, r = 0.3, p = 0.035, N = 3 for anti-cit-LL37 abs; replication cohort2: r = 0.42, p = 0.04, N = 18 for anti-LL37 abs, r = 0.5, p = 0.016, N = 18, N = 18

for anti-cit-LL37 abs). To observe how antibody reactivity behaved during the course of disease, we selected twenty-four patients with active disease (SLEDAI > 4, range 4-to-32; mean

SLEDAI = 12.6 ± 7.5), which were positive for anti-native LL37 and anti-cit-LL37 antibodies at first sampling. During the following 6-to-36 months, the SLEDAI decreased significantly in

twenty-one (88%) (Fig. 2E, left panel) but not in three of them (12%, Fig. 2F, left panel) in response to treatment (Plaquenil, prednisone, See also Table S1). In the LL37-responder

patients, anti-native LL37 and anti-cit-LL37 antibody reactivity declined in parallel with SLEDAI reduction (Fig. 2E, right panels). In contrast, in the three unresponsive patients (Fig. 2F,

right panels) anti-native LL37 and anti-cit-LL37 antibody reactivity increased. These results show that when a significant anti-LL37 humoral response is specifically present in SLE, this

correlates with disease activity and declines with it, suggesting a role of antibody reactivity to LL37 and cit-LL37 as disease markers and, possibly, involvement in disease pathogenesis.

T-CELLS RESPOND TO LL37 AND CIT-LL37 IN SLE AND THEIR RESPONSES CORRELATE WITH SLEDAI AND ANTI-LL37/ANTI-CIT-LL37 ANTIBODIES A previous _in silico_ analysis identified promiscuous T-cell

epitopes for multiple HLA-DR alleles in the LL37 sequence, explaining why LL37 is very likely to be immunogenic for T-cells, an assumption verified in psoriasis17. Thus, given the consistent

expression of LL37 in SLE tissues, and the LL37 ability to form LL37-DNA complexes _in vivo_, we tested whether LL37 behaves as a T-cell autoantigen also in SLE/CLE. SLE/CLE blood

mononuclear cells (PBMCs) were cultured with LL37 (Fig. S3, for gating strategy) or control unrelated AMPs human β-defensin-3 (HBD3) and α-defensins HNP1–3, or with tetanus toxoid (TT) and

phytohemagglutin (PHA), as positive controls. At day 6, we assessed BrdU-incorporation in CD3+CD4+ T-cells and considered T-cell responses positive those with stimulation index was ≥3 (see

Methods)17. Cumulative data in Fig. 3A show that CD3+CD4+ cells from 18 of 40 SLE (45%) (and from three of five CLE patients), proliferated in response to LL37, whereas responses to HBD3 or

HNP1–3 were not significant (Fig. 3A). No SSc or HD showed a significant response to LL37 and other AMPs, whereas ten of twenty-nine PSO patients (34%) responded in a significant manner, as

expected17. T-cells from two out of 15 (13%) RA patients proliferated to LL37. We observed proliferation to cit-LL37 in 11 of 25 (44%) SLE and 3 of 5 CLE patients but we observed no

responses in HD (Fig. 3B). T-cells from two of 15 RA (13%) and 3 of 21 PSO (14%) patients also responded to cit-LL37, suggesting that cit-LL37 can be occasionally immunogenic also in

psoriasis and RA. In these assays, the control reverse LL37 peptide (REV, Table S2) did not elicit T-cell responses, ensuring that T-cell activation by LL37 was sequence dependent and

independent of its DNA binding ability9,17. Indeed, the REV peptide has an inverse LL37 sequence (REV), but retains DNA-binding activity17. Moreover, we also observed T-cell proliferation

(Fig. S4A) to shorter (13–15mer) overlapping LL37-peptides (Table S2, Fig. S4A) and to cit-LL37, all completely devoid of adjuvant activity for DCs, as shown in Fig. S4B. When we plotted the

values of LL37/cit-LL37 induced T-cell proliferation against disease activity as assessed by SLEDAI, we found a positive correlation (Fig. 3C). We also correlated the same parameters to

“clinical” SLEDAI (without inclusion of data regarding presence/absence of anti-DNA antibodies, or complement reduction) and we found similar correlations (T-cell response to LL37 versus

clinical SLEDAI: r = 0.52, P = 0.0007, N = 40; T-cell responses to cit-LL37 versus clinical SLEDAI: r = 0.49, p = 0.02, N = 24). Interestingly, T-cell responses to both native LL37 and

cit-LL37 were more frequent in SLE patients with malar rash according to Chi-square test (z = 1.84, P = 0.033, N = 25, for response to LL37 and z = 1.7, P = 0.047, N = 25 for response to

cit-LL37). Moreover, the magnitude of the T-cell responses (as SI) was higher in SLE patients with malar rash than in patients with no malar rash (Mann-Whitney test: P = 0.039, N = 25, for

response to native LL37 and P = 0.017, N = 25, for response to cit-LL37) and in patients with renal involvement, as compared to patients without renal involvement (Mann-Whitney test: P = 

0.045, N = 30 for response to native LL37, and P = 0.034, N = 25, for response to cit-LL37). Of note, the magnitude of T-cell response to LL37 (but not to cit-LL37) was also higher in

patients with anti-DNA antibody positivity, than in patients with anti-DNA antibody negativity (Mann-Whitney test: P = 0.0078, N = 17). In psoriasis patients we confirmed a positive and

significant correlation between T-cell response to LL37 and PASI (Spearman’s r = 0.33, p = 0.04, N = 29)17, whereas T-cell responses to LL37/cit-LL37 in RA did not correlate with disease

activity (expressed as DAS28) (Spearman’s r = 0.07, p = 0.34, N = 38, for both responses). Most importantly, T-cell responses to native LL37/cit-LL37 correlated with the presence of

autoantibodies to both native LL37 and cit-LL37, in SLE (Fig. 3D). These data indicate that LL37 can act as an autoantigen for SLE CD4 T-cells in both its native and citrullinated form.

Eight patients, that at first sampling had T-cell proliferating to LL37, were available for longitudinal follow-up and were re-tested for T-cell reactivity to LL37/cit-LL37 (after up to 36

months of successful standard therapy, as above). Their T-cell responses decreased (and this decrease was statistically significant for proliferation to native LL37). Reduction in magnitude

of the responses coincided with a reduction in SLEDAI (Fig. 3E). Of interest, in the same patients, the antibody reactivity to native LL37 and cit-LL37 significantly declined in parallel

(Fig. 3F). These studies provide correlative evidence for the participation of autoreactive native-LL37/cit-LL37-specific T-cells to LL37 antibody production and disease activity. Moreover,

T-cell responses to native LL37 and cit-LL37 may behave as biomarkers in SLE, at least in the LL37-responder patients. LL37/CIT-LL37-SPECIFIC T-CELLS SUSTAIN THE PRODUCTION OF PATHOGENIC

ANTI-NATIVE LL37, AND ANTI-DNA/RNA ANTIBODIES We then assessed the helper T-cell ability for autoantibodies production of SLE LL37-responder T-cells34. As shown in Fig. 4A, when we cultured

SLE PBMCs for 7 days in the presence of LL37 or cit-LL37 or control antigens, anti-LL37 (left panel) and anti-cit-LL37 (right panel) antibodies became apparent in culture supernatants of

PBMCs stimulated with LL37 and cit-LL37, (but not in cultures stimulated with control peptides). Noteworthy, culture of PBMCs of psoriasis patients whose T-cells proliferate to LL37, did not

lead to significant anti-LL37/cit-LL37 antibody production, as for HD PBMCs, suggesting that psoriasis LL37-specific T-cells did not primarily work to favor autoantibody production _in

vivo_. Moreover, unrelated antibody specificities, such as anti-HNPs and anti-TT antibodies, did not increase in SLE cultures stimulated by native LL37 or cit-LL37 (Fig. S5), thus T-cell

help was specific for anti-LL37 antibody production. Interestingly, upon sequential stimulation with autologous PBMCs and native LL37, both an anti-DNA- and RNA-antibody reactivity became

also detectable in SLE cultures (Fig. 4B). Immortalization by Epstein Barr Virus (EBV, 33) of the SLE/CLE B-cells expanded by LL37 stimulation, generated B-LCLs, some of which produced

anti-native LL37/cit-LL37 antibodies and/or anti-DNA antibodies (Fig. 4C), and similar amounts of total IgG (positive supernatants 125 ±  21 pg/mL versus negative supernatants 145 ±  16 

pg/mL, p = 0.1, N = 4, Student’s t-test for unpaired samples). These anti-LL37 containing supernatants, but not anti-LL37-negative supernatants, induced extrusion of NET-like filaments

decorated by LL37 (Fig. 4D,E) by SLE neutrophils _in vitro_. Thus, cit-LL37-specific T-cells (and not only native-LL37 specific T-cells), sustain the production of anti-LL37 antibodies, with

consequent SLE neutrophils release of interferogenic LL37-DNA complexes. CIRCULATING SLE LL37/CIT-LL37-SPECIFIC T-CELLS CAN EXPRESS A TFH-LIKE PHENOTYPE While CD3+CD4+ T cells proliferated

in response to LL37 and cit-LL37 in both SLE and psoriasis, only in SLE a substantial humoral anti-LL37/cit-LL37 response developed9,17,34,35(Fig. 2), suggestive of a preferential expansion

of TFH in SLE. To verify this hypothesis, we assessed the expression of TFH markers in PBMCs cultured in the presence of LL37/cit-LL37 in SLE or CLE, and in control psoriasis patients. In

SLE (but not in psoriasis), we observed a significant up-regulation of CXCR5 (cumulative data in Fig. 5A; gating strategy in Fig. S6A–C) and IL-21 production (Fig. 5B) by LL37/cit-LL37

proliferating T-cells. Of note, secreted IL-21 levels correlated with the magnitude of LL37/cit-LL37-induced T-cell proliferation (for LL37: Spearman r = 0.64, p = 0.0001, N = 31; for

cit-LL37: r = 0.5, P = 0.0013, N = 24). To avoid bias due to prolong _in vitro_ culture, we assessed transcription factors expression at shorter time points (48 hours), in a limited number

of patients. While both SLE and psoriasis activated (CD38hi,36) T-cells could upregulate Ror-ɣt expression37, only CD38hi SLE T-cells, but not CD38hi psoriasis T-cells, could up-regulate

Bcl-6, upon stimulation with LL37/cit-LL37 (Fig. 5C) (see Fig. S6D for gating strategy). For the unambiguous identification of LL37/cit-LL37-specific T-cells, we cloned peripheral T-cells

from one SLE and one CLE patient. We obtained several T-cell clones for which we report the HLA-restriction and the fine epitope mapping in Fig. 5D. Some of the T-cell clones proliferated

exclusively after stimulation with native LL37, or exclusively to cit-LL37-derived epitopes. Other clones proliferated to both native LL37 and cit-LL37 (Fig. 5D), but did not cross-react to

the control peptide HA306–320, a promiscuously presented peptide derived from the hemoagglutinin protein of _Influenza_ A (in both its native and citrullinated form). Th17 or TFH-like

attributes were also clonally distributed. For instance, CLE6 clone 6 and SLE2 clone A3 produced the largest amounts of IL-21 (Fig. 5E) and expressed the TFH markers CXCR5, ICOS, PD1 and

Bcl-6, and low level of Ror-ɣt (Fig. 5F). CLE6 clone 1, was higher producer of IL-17 (Fig. 5E) and showed the highest expression level of CCR6 and Ror-ɣt, with modest Bcl-6 expression (Fig. 

5F). Altogether, these results show that in SLE, but not in psoriasis, at least part of the native-LL37 or cit-LL37-specific T-cells belong to the TFH- like subset, whereas Th17-types of

cells were present in both pathologies. CIT-LL37 HAS STRONGER AGONIST ACTIVITY COMPARED TO NATIVE-LL37 FOR T-CELL ACTIVATION The identification of T-cells that promiscuously recognize the

native and citrullinated forms of LL37 provided the tools to assess whether citrullination conferred advantages for T-cell activation. We used native or citrullinated peptides for which HLA

binding capacity were similar (Fig. S7) (see also Methods). Proliferative responses to native-LL37 and cit-LL37 were similar in magnitude (Fig. 6A) when we used both antigens in high dose.

However, T-cells responded better to cit-LL37 than the native counterpart at lower antigen doses. This happened in 12 LL37/cit-LL37 responders, while in three LL37-non-responders we observed

no proliferation (Fig. 6A). Consistent with these findings, proliferation of PBMCs was still significant only in response to cit-LL37 (although at the highest dose) when patients were in

remission (Fig. 6B). To substantiate these findings, we used peptide-MHC-tetramer staining to visualize the specificity, frequency and cross-reactivity of circulating SLE/CLE native

LL37/cit-LL37-specific T-cells after a seven-day culture (gating strategy in Fig. S8A). In three patients analyzed, the frequency of peptide-MHC-tetramer positive T-cells was higher upon

culture of PBMCs with cit-LL37 compared to native-LL37 (Fig. 6C, upper panels). This suggests that T-cells may have undergone preferential proliferative expansion when stimulated with

cit-LL37. In the same culture conditions, PBMCs of HLA-matched HD did not stain significantly with LL37/cit-LL37 peptide-MHC-tetramers (Fig. S8B,C). Of interest, we observed heterogeneous

responses among different patients (Fig. 6D–G). For instance, SLE2 and CLE6 had T-cells recognizing both native LL37 and cit-LL37, with a proportion of these cells exclusively recognizing

cit-LL37 epitopes, while SLE22 had T-cells recognizing only cit-LL37. Furthermore, at remission, we observed not staining for native-LL37 in T-cells from SLE2, SLE22 and CLE6 PBMCs, while we

still observed some staining upon stimulation with cit-LL37 (Fig. 6G, lower panel). Altogether, these data support the contention that cit-LL37, at least when presented in the context of

selected HLA alleles, can act as stronger TCR-agonist for SLE T-cells, compared to native LL37. Responder T-cells remain detectable during clinical remission. Thus, these results suggest

that the presence, at flares, of cit-LL37 in lupus target organs may enhance T-cell activation and would favor further damage and increased autoreactivity. DISCUSSION In this study, we

report that T-cells belonging to the TFH-like/Th17 subsets recognize the autoantigen LL37 in SLE and are likely to favor the emergence of autoantibodies to LL37 and DNA/RNA in _in vitro_

cultures and possibly _in vivo_. Consistent with this finding, we observed, by confocal microscopy, that LL37, DNA and IgG co-localize in SLE target organs, while statistical studies

indicate that the magnitude of anti-LL37 antibody and T-cell reactivity correlate with each other and with SLE disease activity, as captured by SLEDAI, and decrease in patients with inactive

disease. We provide evidence that the post-translational modification by citrullination of LL37 is readily detectable in SLE affected target organs (both skin and kidneys) and that,

compared to native-LL37, cit-LL37 may further enhance T-cell responses. Altogether, these data strongly support the contention that adaptive immune responses directed against the autoantigen

LL37 may participate to events relevant to SLE pathogenesis. Moreover, T/B-cell responses to LL37 are markers of active SLE. Indeed, by exploring distinct SLE cohorts, we found that

anti-LL37 antibodies not only correlate with disease activity (SLEDAI) but also with circulating type I IFN serum levels. These data confirm and extend our previous report9, in contrast to

observations by others that did not find a correlation between anti-LL37 antibodies and disease activity27. We ascribe this discrepancy to the larger proportion of untreated patients with

severe disease and nephritis, coupled to a short disease duration, in our cohorts. By using high-resolution confocal microscopy, we observed the presence of DNA filaments decorated with LL37

in kidney biopsies from patients with lupus nephritis, highly suggestive of LL37-DNA complexes formation _in vivo_. In the kidney, we could also demonstrate the co-localization of LL37 with

IgG, suggesting the formation/deposition of LL37/DNA-immune complexes _in vivo_, where they might act by inhibiting NET-like structures degradation (38). The concomitant presence of Mx1, an

IFN-α induced protein23 co-localizing with LL37 and IgG, provides supplemental evidence for a role of LL37-DNA complexes and anti-LL37 antibodies in IFN-I production _in vivo_9,38. Of note,

anti-LL37 antibodies likely aggravate tissue inflammation by enhancing the uptake of LL37-DNA complexes into pDCs, an ability demonstrated _in vitro_9. The role and mechanism of NET

formation in SLE is currently under debate6,7,39,40,41. Experimental models of SLE, although with several limitations, tend to exclude a pathogenic role for NETosis in SLE42,43, apparently

in accordance with the finding that NETosis-defective individuals (with impaired NADPH activity) are more prone to lupus-like disease than HD39,40,41,42,43. On the other hand, NET-like

structures form in both a NADPH-dependent, or independent manner, and NET-like cell death due to autoantibodies does not seem to rely on NADPH activity40,44,45,46,47. In our experiments, we

observe that patient-derived anti-LL37 antibodies generated _in vitro_ stimulate neutrophil extrusion of DNA filaments decorated by LL37. Thus, while we did not clarify in detail the

mechanism that triggers the LL37-DNA complexes release, it is out of doubt that autoantibodies are actively responsible for NET-like structures formation _in vitro_. Of interest, the

presence of consistent amounts of cit-LL37 in both skin and kidney, demonstrated for the first time in SLE tissues, makes also plausible the occurrence of the “leukotoxic

hypercitrullination” (LTH) described by Andrade’s group in RA25,26. In LTH, neutrophils undergo a NET-like cell death upon membrane attack complexes (MAC) formation. Clear documentation of

complement activation, consumption and deposition in tissues exists for SLE48,49,50. Thus, it is likely that LTH could explain the observed co-localization of C9 and cit-LL37 in SLE-affected

kidneys. These data are anyway consistent with work showing that citrullination occurs in lupus kidneys (as consequence of inducible nitric oxide synthase –iNOS- activation51). The main

evidence that LL37 is a T-cell autoantigen in SLE reinforces the role of LL37 and anti-LL37 antibodies in SLE pathogenesis9,13. Interestingly though, T-cells also recognize LL37 as an

autoantigen in psoriasis17, but autoantibodies to LL37 are infrequent in psoriasis35. In this respect, it is interesting that in both psoriasis and SLE the LL37-specific Th17-cells do exist

and express Ror-ɣt but only in SLE, and not in psoriasis, they can express the TFH-markers Bcl-6 and high IL-21, at polyclonal and clonal levels. In addition, despite LL37 is over-expressed

in RA synovia, UC gut and SSc skin, the magnitude of autoreactivity to LL37 in RA, UC and SSc is lower than in SLE17,27,29,30,31,32,33,34. Not only are T-cells specific for LL37 (and

cit-LL37) more frequent in SLE than in RA and SSc, but correlation studies strongly suggest that these cells are instrumental for production of high affinity anti-LL37 antibodies exclusively

in SLE, and functional experiments show that they do not help autoantibody production in psoriasis. The reasons why LL37 is a more potent target of both antibodies and T-cells in SLE than

in RA and SSc, and why TFH-like cells are more easily apparent in SLE than in psoriasis are unclear. It is possible that LL37/LL37-DNA, and likely LL37-RNA-complexes11 reached the highest

levels in SLE target organs most likely due to defective removal, rather than to enhanced release, which favors higher anti-LL37 immunity. Indeed, SLE has a recognized association with

insufficient clearance of dead cells, and impaired nuclease activity38,44,52,53,54. In an old study, lower serum DNase1 levels associated with active SLE, the lowest DNAse I activity

detected in patients with active renal involvement52. Persistence of LL37-DNA complexes can chronically activate DNA-binding receptors, and further fuel inflammation with production of the

main actor in SLE, IFN-I. A non-mutually exclusive possibility is that the site where LL37 release and citrullination occur is determinant (the kidney). An argument to support this it is the

high percentage of patients with severe SLE, accompanied by renal manifestations, who show high T-cell reactivity in our study. Indeed, impairment of NET-like structures degradation can

correlate with lupus nephritis38. However, the magnitude of T-cell response to native/cit-LL37 also correlated with malar rash, suggesting skin involvement to be important for response to

LL37. Our study finds that T-cell responses to LL37 correlate with disease activity and anti-LL37-antibodies irrespective of whether T-cells are specific for native LL37 or cit-LL37. This

raises the question as to whether LL37 citrullination is or is not involved in loss of T-cell tolerance in SLE. Since SLE T-cells can often cross-recognize native LL37/cit-LL37,

citrullination may appear as a null event in the priming/activation of LL37 specific T-cells. Notably, though, in our experiments relatively low doses of cit-LL37 (more efficiently than

native LL37), induced T-cell proliferation (as seen in BrdU-primary proliferation assays) and T-cell expansion, (as evidenced by increased percentage of peptide-MHC-tetramer staining). In

addition, cit-LL37, more effectively than native LL37, recalled the residual LL37-specific T-cell responses persisting at low frequency at remission. Furthermore, in several patients,

T-cells appeared exclusively activated by cit-LL37 and these T-cells helped anyway the production of antibodies to native LL37 _in vitro_. This is reminiscent of an _in vivo_ animal model of

autoimmunity described by Mamula _et al_., where a modified version of a self-antigen stimulated T-helper cells which drove antibodies against the original unmodified self-antigen55. Thus,

it is possible that SLE LL37-specific T-cells, at least in a SLE sub-population, are primarily specific for cit-LL37, as cit-LL37 acts as a stronger agonist for these cells. Notably, recent

works have linked a specific SLE-associated polymorphism (A20) with enhanced protein citrullination56, PAD4 polymorphisms with renal involvement57, and SLE-specific citrullinated antigens as

antibody targets58, supporting a role for this post-translational modification in SLE pathogenesis. Cit-LL37-specific T-cells may thus cross-recognize native LL37 at a given threshold

concentration, which may reach high levels during neutrophil activation. Our results could support this, since functional experiments and peptide-MHC-binding assays show that citrullinated

LL37 epitopes have sufficient likelihood to be presented to T-cells by a wide array of MHC-II alleles, including DR15, DR1, DRB5, DR10, DR4, some of which (DR15, DRB5) are over-represented

in SLE59,60. In conclusion, both native LL37 and cit-LL37 are likely players in SLE, the former mainly for its adjuvant activity9,10, the latter for its increased T-cell immunogenicity. Less

clear, and worth of further investigation, is the contribution of cit-LL37 to the immunogenicity of LL37 in psoriasis, a disease characterized by decreased citrullinated proteins in

hyperproliferative epidermis, but also by neutrophil infiltrate61. Also, we are aware that the role of citrullination in autoreactivity in SLE could be restricted to the LL37-directed

response, as additional post-translational modifications are reasonably considered dominant in SLE62. Of interest for the rheumatology field, the correlation between T-cell responses and

antibody levels, and of both responses with disease activity (SLEDAI), renders LL37-directed responses interesting biomarkers, at least in patients that respond to LL37. It remain to

systematically address whether a native/cit-LL37-directed autoreactivity marks nephritis, or skin rash, or both, and whether targeting of the dual effects of LL37 in the activation of both

innate and adaptive immune responses9,10,11,17 is a novel avenue for therapy in SLE (as in psoriasis). For instance, we are currently investigating whether polyanionic compounds or

LL37-specific aptamers reduce the immunogenicity of LL37, in addition to the concomitant block of its adjuvant activity in psoriasis9. METHODS STUDY DESIGN SLE/CLE blood (20 mL), kidney and

skin biopsies, and blood from SLE, SSc, RA and UC patients were obtained in Rome, Italy, Policlinico Umberto I, Department of Internal Medicine and Medical Specialties - Rheumatology Unit,

from University Hospital (CHUV), Department of Dermatology and Immunology, Lausanne, from the Swiss SLE cohort study (SSCS) and from Sandro Pertini Hospital, Rome. Sera and PBMCs from PSO

patients were from University of Tor Vergata, Rome and Humanitas Hospital, Milan, Italy. Sera and skin from HD, matched for age and sex with SLE/CLE patients, were from blood Centers,

Policlinico Umberto I, Italy and Geneva University Hospital, Switzerland. Disease activity in SLE patients was assessed by SLEDAI 200063. For cutaneous lupus (CLE), we used CLASI64. For

psoriasis (PSO) patients we used PASI (17). RA were diagnosed according to 2010 ACR/EULAR classification criteria65. UC disease activity was assessed by endoscopic Mayo 58 and UC patients

with clinical and endoscopic activity (Mayo Score Full ≥3) were evaluated. Blood samples were collected at time of endoscopy66. SSc patients satisfied the ACR/EULAR 2013 classification

criteria67. When possible, SLE/CLE patients did not take medications in the last three months before first blood sampling. Alternatively, we enrolled SLE/CLE patients during washout, and

before entering a new protocol treatment. To corroborate data of the discovery cohort in different conditions, the replication cohorts (See Table S1) also included patients that were treated

(exclusion criteria: patients treated with biologics). For longitudinal studies, the available patients who gave informed consent, were re-tested after taking medications, usually

hydroxychloroquine and/or prednisone (see Table S1). Observation time between 6-to-36 months from first sampling (during a stable remission, after reaching an inactive disease state, SLEDAI 

< 4). HLA-Class II typing (HLA-DR) was performed in eight of the twenty-one SLE/CLE patients tested, in the reference center for HLA typing: Geneva University Hospital, Switzerland, and

“Centro Transfusionale”, Rome, Italy. The assessed HLA-haplotypes are: SLE2: DRB1*1501, DRB1*1601; CLE6: DRB1*0101, DRB1*1601; SLE1: DRB1*0301, DRB1*0401; SLE4: DRB1*0401, DRB1*1101; SLE17:

DRB1*1101, DRB1 16*01. SLE22: DRB1*0301, DRB1*1501; SLE5: DRB1*1001, nd; SLE8: DRB1*0401, DRB1*0701. All samples were obtained upon approval by Ethic Committees of University La Sapienza,

Tor Vergata, Sandro Pertini Hospital in Rome, and Humanitas Hospital, Milan, Italy, and of CHUV and Swiss Ethics, Switzerland. All blood and tissue donors gave informed consent, according to

Helsinki declaration. ANTIGENS We list the antigenic peptides used in this study in Table S3. LL37 and HBD3 were from Innovagen. LL37 was also from Proteogenix (France). REV and scramble

LL37 (SCR) peptides from AnaSpec Inc. Overlapping LL37-peptides (Supplementary Table 117), as well as the citrullinated LL37 (citrullinated at all five arginin positions) were made by Anawa,

Trading SA (Zurich, CH) or Citomatik (Italy). Byotinilated native LL37 and cit-LL37 were also from Anawa. HNP1–3 was from Hycult Biotechnology. Citrullinated SCR was synthesized by Biomatik

(Italy). Citrullinated and non-citrullinated vimentin and enolase were synthesized by Genscript as described68. Citrullinated and non-citrullinated vinculin and HA peptides synthesized by

Fmoc as described69. GENERATION OF ANTI-LL37/CIT-LL37 ANTIBODIES Antibodies Mab137 and Mab142, specific for native LL37 (Mab137) and cit-LL37 (Mab142) were produced in the Antibody Facility

of University of Geneva, CMU, CH (http://www.unige.ch/antibodies) as described70. For confocal detection a secondary anti-mouse antibody conjugated with AlexaFluor-647 or AlexaFluor-488 was

used (Abcam). Isotype controls were from Hycult. ISOLATION OF BLOOD PDCS Buffy coats of HD were from Centro Trasfusionale, Policlinico Umberto I, Rome, IT. After separation of mononuclear

cells by Ficoll centrifugation, pDCs were purified as described9,10, by using the Diamond Plasmacytoid Dendritic Cell Isolation Kit (Miltenyi Biotec) to obtain 99% purity. ELISA FOR

AUTOANTIBODY DETECTION IN SERA AND CULTURE SUPERNATANTS Anti-native/cit-LL37, other antigen antibodies (anti-DNA and anti-RNA), and total anti-human IgG were measured by ELISA as described9.

Briefly, 96-well flat-bottom plates (Non-Binding surface polystyrene, Corning, USA) are coated with 2 μg/mL of native LL37/cit-LL37 or control proteins or human DNA or human RNA9 or

anti-human IgG antibody (Sigma) in carbonate buffer (0.1 M NaHCHO3, pH 9) for 2 hours (or over night) and washed four times with PBS 0.1% Tween-20. This washing buffer was used for washing

at all steps. The blocking buffer containing 2% bovine serum albumin (BSA, Sigma) in PBS was used for at least 1 hour (or over night) to saturate unspecific binding sites. After washing,

sera were diluted at various concentrations (usually 1:100 or 1:200) in PBS 2% BSA (cell culture supernatants were diluted 1:2, 1:4 or 1:10) followed by 1 hour incubation with a horseradish

peroxidase–conjugated (HRP) goat anti-human IgG (Sigma-Aldrich), diluted 1:10,000 in PBS. The color was developed for 5 minutes with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate

(Sigma-Aldrich). The reaction was stopped by adding 50 μl of 2 N H2SO4, and absorbance determined at 450 nm with a reference wavelength of 540 nm. Anti-dsDNA in sera of lupus patients were

detected by the standard clinical test Kallestad _Crithidia luciliae_ Complete Kits for detection and semi-quantitation of autoantibodies to native DNA (nDNA) antigens by indirect

fluorescence antibody (IFA) procedure. ELISA for detection of anti-tetanus antibodies (anti-TT) was from Abnova (Taiwan). PURIFICATION OF NEUTROPHILS, NET-INDUCTION AND QUANTIFICATION

Neutrophils were isolated from peripheral blood of SLE patients with Polymorphoprep (Euroclone). Yield, measured by flow cytometry for CD15 expression, was 95 to 97% (the remaining cells

were eosinophils and monocytes expressing low levels of CD15)9. Purified neutrophils were seeded at 5 × 105 cells in 200 μL of complete medium and stimulated with anti-LL37 (clone 8A8.1,

IgG2b, 10 μg/mL) as in our previous study9, or with IgG control antibodies or LL37-positive and LL37-negative supernatants (diluted 1:4 in medium). The release of NET-DNA was measured in

cell-free supernatant collected after 3 hours of activation following addition of the PicoGreen using the PicoGreen assay kit. Samples were excited at 480 nm and read at 530 nm with a

fluorimeter. PRODUCTION OF RECOMBINANT HLA CLASS II MOLECULES Human recombinant HLADRA1*01:01, HLA-DRB1*01:01, and HLA-DRB1*15:01 were expressed in _E. coli_ and purified in a series of

liquid chromatography based steps71. PEPTIDE-MHC CLASS II BINDING ASSAYS We performed peptide-MHC binding assays by two different protocols by ImmunAware, Denmark and Benaroya Institute

(USA). In the first protocol, binding was measured using a Luminescent Oxygen Channelling Immunoassay (LOCI), marketed by Perkin Elmer as AlphaScreen72. This is a nonradioactive bead-based

homogenous proximity assay, where the donor beads containing a photosensitizer compound upon excitation with light at a 680 nm wavelength, converts ambient oxygen to energy-rich, short-lived

singlet oxygen; that is transferred to a acceptor beads in close proximity only. The acceptor beads can respond to singlet oxygen with a luminescence/ fluorescence cascade leading to an

amplified signal in the 520–620 nm range. As one tag binding to the donor beads: a biotin group engineered unto the HLA β chain. As the other tag binding to the acceptor beads:

conformation-dependent HLA class II specific antibody (L243). Thus, only when a fully folded complex is present will the donor and acceptor beads come in sufficient close proximity and give

a measurable signal. Biotinylated recombinant HLA class II molecules were diluted from an 8 M urea buffer into a folding buffer (PBS, 20% glycerol, Protease inhibitor mix) with peptide

concentrations ranging from 0.13 nM to 10,000 nM. The analysed peptides were tested in 5-to-8 five-fold concentration titrations spanning from 20 μM to 0.3 nM. The reaction mixtures were

incubated for 48 h at 18 °C to allow for peptide-HLA complex formation reaching steady state. After incubation, 15 μL of the folding mixture was transferred to a 384-well Optiplate

(PerkinElmer) followed by addition of 15 μL of a solution containing L243 acceptor beads and streptavidin donor beads (final concentration of beads was 5 μL/mL). The plates were incubated

over night at 18 °C, and then measured in enVisionTM, Perkin Elmer. Assay signals measured in OD450 were were plotted against the offered peptide concentrations, and analyzed by non-linear

regression using GraphPad Prism. The peptide concentration resulting in half saturation, the half maximal effective concentration (EC50), was estimated by fitting the experimental data to

the equation: Y = Bmax*X/(KD + X), where Y is the OD450 measurement of complexes formed and X is the concentration of ligand (peptide) offered. The EC50 approximates the KD as long as the

receptor concentration used is less than the KD thus avoiding ligand depletion. For the second binding protocol72,73, we used purified recombinant DRB1*0401, DRB1*1601 or DRB5*0101 and

DRB1*1001 proteins for _in vitro_ peptide-binding assays. Increasing concentrations of each non-biotinylated test peptide was incubated in competition with a previously determined fixed

concentration of biotinylated reference peptide (1 µM of NFIRMVISNPAAT for DRB1*0401 and DRB1*1001, 1.5 µM of NFIRMVISNPAAT for DRB1*1601, and 0.8 µM PKYVKQNTLKLAT for DRB5*0101) and

captured in microtiter plate wells coated with 10 μg/mL of L243 (anti-HLA-DR) antibody. After washing, residual biotinylated reference peptide was labelled using europium conjugated

streptavidin (Perkin Elmer) and quantified using a Victor3 D time resolved fluorimeter (Perkin Elmer). We also calculated the IC50 binding values from the resulting curves, as the peptide

concentration needed for 50% inhibition of reference peptide binding. REFERENCES * Tsokos, G. C. Systemic lupus erythematosus. _N. Engl. J. Med._ 365, 2110–2121 (2011). Article  PubMed  CAS

  Google Scholar  * Tan, E. M. Autoantibodies, autoimmune disease, and the birth of immune diagnostics. _J. Clin. Invest._ 122, 3835–3836 (2012). Article  PubMed  PubMed Central  CAS  Google

Scholar  * Thoeger, Z., Sharabi, A. & Mozes, E. Novel approaches to the development of targeted therapeutic agents for systemic lupus erythematosus. _J. Autoimmun._ 54, 60–71 (2014).

Article  CAS  Google Scholar  * Fumeaux, D., de Seigneux, S., Chizzolini, C. & Martin, P. Y. Renal disease in ANCA-associated vasculitis. _Rev. Med. Suisse._ 10, 493–497 (2014). PubMed 

Google Scholar  * Pieterse, E. & van der Vlag, J. Breaking immunological tolerance in systemic lupus erythematosus. _Front. Immunol._ 5, 164–171 (2014). Article  PubMed  PubMed Central 

CAS  Google Scholar  * Lood, C. _et al_. Neutrophil extracellular traps enriched in oxidized mitochondrial DNA are interferogenic and contribute to lupus-like disease. _Nat. Medicine._ 22,

146–153 (2017). Article  CAS  Google Scholar  * Caielli, S. _et al_. Oxidized mitochondrial nucleoids released by neutrophils drive type I interferon production in human lupus. _J. Exp.

Med._ 213, 697–713 (2016). Article  PubMed  PubMed Central  CAS  Google Scholar  * Yurkovich, M., Vostretsova, K., Chen, W. & Aviña-Zubieta, J. A. Overall and cause-specific mortality in

patients with systemic lupus erythematosus: a meta-analysis of observational studies. _Arthritis Care Res (Hoboken)._ 66, 608–616 (2014). Article  PubMed  Google Scholar  * Lande, R. _et

al_. Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus. _Sci. Transl. Med._ 3, 73ra19–38 (2011). Article  PubMed 

PubMed Central  Google Scholar  * Lande, R. _et al_. Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. _Nature._ 449, 564–569 (2007). Article  ADS  PubMed  CAS

  Google Scholar  * Ganguly, D. _et al_. Self-RNA-antimicrobial peptide complexes activate human dendritic cells though TLR7 and TLR8. _J. Exp. Med._ 206, 1983–1994 (2009). Article  PubMed 

PubMed Central  CAS  Google Scholar  * Chamilos, G. _et al_. Cytosolic sensing of extracellular self-DNA transported into monocytes by the antimicrobial peptide LL37. _Blood._ 120, 3699–3707

(2012). Article  PubMed  PubMed Central  CAS  Google Scholar  * Gestermann, N. _et al_. Netting neutrophils activate autoreactive B cells in Lupus. _J. Immunol._ 200, 3364–3371 (2018).

Article  PubMed  CAS  Google Scholar  * Kreuter, A. _et al_. Expression of antimicrobial peptides in different subtypes of cutaneous lupus erythematosus. _J. Am. Acad. Dermatol._ 65, 125–133

(2011). Article  PubMed  CAS  Google Scholar  * Sun, C. L., Zhang, F. Z., Li, P. & Bi, L. Q. LL-37 expression in the skin in systemic lupus erythematosus. _Lupus._ 20, 904–911 (2011).

Article  PubMed  CAS  Google Scholar  * Almaani, S., Meara, A. & Rovin, B. H. Update on Lupus Nephritis. _CJASN._ 12, 825–835 (2017). Article  PubMed  Google Scholar  * Lande, R. _et

al_. The antimicrobial peptide LL37 is a T-cell autoantigen in psoriasis. _Nat. Commun._ 5, 5621–5635 (2014). Article  ADS  PubMed  CAS  Google Scholar  * Cannons, J. L., Lu, K. T. &

Schwartzberg, P. L. T follicular helper cell diversity and plasticity. _Trends Immunol._ 34, 200–207 (2013). Article  PubMed  PubMed Central  CAS  Google Scholar  * Park, J. J. _et al_.

Insights into the role of follicular helper T cells in autoimmunity. _Immune Netw._ 14, 21–29 (2014). Article  PubMed  PubMed Central  Google Scholar  * Kilsgård, O. _et al_.

Peptidylarginine deiminases present in the airways during tobacco smoking and inflammation can citrullinate the host defense peptide LL-37, resulting in altered activities. _Am. J. Respir.

Cell Mol. Biol._ 46, 240–248 (2012). Article  PubMed  CAS  Google Scholar  * Dwivedi, N. & Radic, M. Citrullination of autoantigens implicates NETosis in the induction of autoimmunity.

_Ann. Rheum. Dis._ 73, 483–491 (2014). Article  PubMed  CAS  Google Scholar  * Zhou, Y. _et al_. Spontaneous secretion of the citrullination enzyme PAD2 and cell surface exposure of PAD4 by

neutrophils. _Front Immunol._ 8, 1200–1215 (2017). Article  PubMed  PubMed Central  CAS  Google Scholar  * Vejlstrup, A., Møller, A. M., Nielsen, C. H. & Damgaard, D. Release of active

peptidylarginine deiminase into the circulation during acute inflammation induced by coronary artery bypass surgery. _J. Inflamm. Res._ 12, 137–144 (2019). Article  PubMed  PubMed Central 

CAS  Google Scholar  * Airò, P. _et al_. Upregulation of myxovirus-resistance protein A: a possible marker of type I interferon induction in systemic sclerosis. _J. Rheumatol._ 35, 2192–2200

(2008). Article  PubMed  CAS  Google Scholar  * Romero, V. _et al_. Immune-mediated pore-forming pathways induce cellular hypercitrullination and generate citrullinated autoantigens in

rheumatoid arthritis. _Sci. Transl. Med._ 30, 209ra150 (2013). Google Scholar  * Konig, M. F. & Andrade, F. A. Critical reappraisal of neutrophil extracellular traps and NETosis mimics

based on differential requirements for protein citrullination. _Front. Immunol._ 7, 461–478 (2016). PubMed  PubMed Central  Google Scholar  * Kienhöfer, D. _et al_. No evidence of pathogenic

involvement of cathelicidins in patient cohorts and mouse models of lupus and arthritis. _PLoS One._ 9, e115474 (2014). Article  ADS  PubMed  PubMed Central  CAS  Google Scholar  * Kuhn, A.

& Landmann, A. The classification and diagnosis of cutaneous lupus erythematosus. _J. Autoimmun._ 48-49, 14–19 (2014). Article  PubMed  CAS  Google Scholar  * Harder, J. & Schröder,

J. M. Psoriatic scales: a promising source for the isolation of human skin-derived antimicrobial proteins. _J. Leukoc. Biol._ 77, 476–486 (2005). Article  PubMed  CAS  Google Scholar  *

Kim, H. J. _et al_. LL-37 suppresses sodium nitroprusside-induced apoptosis of systemic sclerosis dermal fibroblasts. _Exp. Dermatol._ 20, 843–845 (2011). Article  PubMed  CAS  Google

Scholar  * Hoffmann, M. H. _et al_. Cathelicidins LL-37 and rCRAMP are associated with pathogenic events of arthritis in humans and rats. _Ann. Rheum. Dis._ 72, 1239–1248 (2013). Article 

PubMed  CAS  Google Scholar  * Takahashi, T. _et al_. A potential contribution of antimicrobial peptide LL-37 to tissue fibrosis and vasculopathy in systemic sclerosis. _Br. J. Dermatol._

175, 1195–1203 (2016). Article  PubMed  CAS  Google Scholar  * Schauber, J. _et al_. Heterogeneous expression of human cathelicidin hCAP18/LL-37 in inflammatory bowel diseases. _Eur. J.

Gastroenterol. Hepatol._ 18, 615–621 (2006). Article  PubMed  CAS  Google Scholar  * Zhang, X. _et al_. Circulating CXCR5+CD4+helper T cells in systemic lupus erythematosus patients share

phenotypic properties with germinal center follicular helper T cells and promote antibody production. _Lupus._ 24, 909–917 (2015). Article  PubMed  CAS  Google Scholar  * Frasca, L. _et al_.

Anti-LL37 antibodies are present in Psoriatic. _Arthritis (PsA) patients: new biomarkers in PsA. Front. Immunol._ 9, 1936–1952 (2018). PubMed  Google Scholar  * Quarona, V. _et al_. CD38

and CD157: a long journey from activation markers to multifunctional molecules. _Cytometry B Clin. Cytom._ 84, 207–217 (2013). Article  PubMed  CAS  Google Scholar  * Gao, W., Wu, Y., Tian,

Y. & Ni, B. Yin-Yang regulation of RORγt Protein Complex in Th17 differentiation. _Int. Rev. Immunol._ 34, 295–304 (2015). Article  PubMed  CAS  Google Scholar  * Hakkim, A. _et al_.

Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. _Proc.Natl. Acad. Sci.USA_ 107, 9813–9818 (2010). Article  ADS  PubMed  PubMed Central  Google

Scholar  * Thieblemont, N., Wright, H. L., Edwards, S. W. & Witko-Sarsat, V. Human neutrophils in auto-immunity. _Semin. Immunol._ 28, 159–173 (2016). Article  PubMed  CAS  Google

Scholar  * Boeltz, S. _et al_. NET or not to NET: current opinions and state of the science regarding the formation of neutrophil extracellular traps. _Cell Death Differ._ 26, 395–408

(2019). Article  PubMed  PubMed Central  Google Scholar  * Singel, K. L. & Segal, B. H. NOX2-dependent regulation of inflammation. _Clin. Sci. (Lond)._ 130, 479–490 (2016). Article 

PubMed  PubMed Central  CAS  Google Scholar  * Campbell, A. M., Kashgarian, M. & Shlomchik, M. J. NADPH oxidase inhibits the pathogenesis of systemic lupus erythematosus. _Sci. Transl.

Med._ 4, 157ra141 (2012). Article  PubMed  PubMed Central  Google Scholar  * Kienhöfer, D. _et al_. Experimental lupus is aggravated in mouse strains with impaired induction of neutrophil

extracellular traps. _JCI Insight._ 2, 92920 (2017). Article  PubMed  Google Scholar  * Mistry, P. & Kaplan, M. J. Cell death in the pathogenesis of systemic lupus erythematosus and

lupus nephritis. _Clin.Immunol._ 185, 159–173 (2017). Article  CAS  Google Scholar  * Kraaij, T. _et al_. A novel method for high-throughput detection and quantification of neutrophil

extracellular traps reveals ROS-independent NET release with immune complexes. _Autoimm. Rev._ 15, 577–584 (2016). Article  CAS  Google Scholar  * Kenny, E. F. _et al_. Diverse stimuli

engage different neutrophil extracellular trap pathways. _Elife._ 6, e24437 (2017). Article  PubMed  PubMed Central  Google Scholar  * Pieterse, E. _et al_. Cleaved N-terminal histone tails

distinguish between NADPH oxidase (NOX)-dependent and NOX-independent pathways of neutrophil extracellular trap formation. _Ann Rheum. Dis._ 77, 1790–1798 (2018). Article  PubMed  CAS 

Google Scholar  * Ziskind, M. & Koffler, D. Cutaneous localization of the membrane attack complex in discoid and systemic lupus erythematosus. _N. Engl. J. Med._ 306, 264–270 (1982).

Article  PubMed  Google Scholar  * Botto, M. _et al_. Complement in human diseases: Lessons from complement deficiencies. _Mol. Immunol._ 46, 2774–2783 (2009). Article  PubMed  CAS  Google

Scholar  * Wang, S., Wu, M., Chiriboga, L., Zeck, B. & Belmont, H. B. Membrane attack complex (mac) deposition in lupus nephritis is associated with hypertension and poor clinical

response to treatment. _Semin. Arthritis Rheum._ 48, 256–262 (2018). Article  PubMed  Google Scholar  * Bollain-y-Goytia, J. J. _et al_. Widespread expression of inducible NOS and citrulline

in lupus nephritis tissues. _Inflamm. Res._ 58, 61–66 (2009). Article  PubMed  CAS  Google Scholar  * Chitrabamrung, S., Rubin, R. L. & Tan, E. M. Serum deoxyribonuclease I and clinical

activity in systemic lupus erythematosus. _Rheumatol. Int._ 1, 55–60 (1981). Article  PubMed  CAS  Google Scholar  * Borros, A. Systemic lupus erythematosus and DNA degradation and

elimination defects front. _Immunol._ 10, 1697–1707 (2019). Google Scholar  * Kenny, E. F. _et al_. mice spontaneously develop a systemic lupus erythematosus-like disease. _Eur. J. Immunol._

49, 590–599 (2019). Article  PubMed  CAS  Google Scholar  * Mamula, M. J. _et al_. Isoaspartyl post-translational modification triggers autoimmune responses to self-proteins. _J. Biol.

Chem._ 274, 22321–22227 (1999). Article  PubMed  CAS  Google Scholar  * Odqvist, L. _et al_. Genetic variations in A20 DUB domain provide a genetic link to citrullination and neutrophil

extracellular traps in systemic lupus erythematosus. _Ann. Rheum. Dis._ 78, 1363–1370 (2019). Article  PubMed  CAS  Google Scholar  * Massarenti, N. _et al_. Peptidylarginine deiminase-4

Gene Polymorphisms Are Associated With Systemic Lupus Erythematosus and Lupus Nephritis. _Scand. J. Rheumatol._ 48, 133–140 (2019). Article  PubMed  CAS  Google Scholar  * Wang, Y. Novel

biomarkers containing citrullinated peptides for diagnosis of systemic lupus erythematosus using protein microarrays. _Clin. Exp. Rheumatol._ 6, 929–936 (2019). Google Scholar  * Louthrenoo,

W. _et al_. The genetic contribution of HLA-DRB5*01:01 to systemic lupus erythematosus in Thailand. _Int. J. Immunogenet._ 40, 126–130 (2013). Article  PubMed  CAS  Google Scholar  * Niu,

Z., Zhang, P. & Tong, T. Value of HLA-DR genotype in systemic lupus erythematosus and lupus nephritis: a meta-analysis. Int. _J. Rheum. Dis._ 18, 17–28 (2015). Article  CAS  Google

Scholar  * Ishida-Yamamoto, A. _et al_. Decreased deiminated Keratin K1 in psoriatic hyperproliferative epidermis. _J. Invest. Dermatol._ 114, 701–705 (2000). Article  PubMed  CAS  Google

Scholar  * Barrera-Vargas, A. _et al_. Differential ubiquitination in NETs regulates macrophage responses in systemic lupus erythematosus. _Ann. Rheum. Dis._ 77, 944–950 (2018). PubMed  CAS

  Google Scholar  * Gladman, D. D., Ibañez, D. & Urowitz, M. B. Systemic lupus erythematosus disease activity index 2000. _J. Rheumatol._ 29, 288–291 (2002). PubMed  Google Scholar  *

Albrecht, J. & Werth, V. P. Clinical outcome measures for cutaneous lupus erythematosus. _Lupus._ 19, 1137–1143 (2010). Article  PubMed  PubMed Central  CAS  Google Scholar  * Aletaha,

D. _et al_. Rheumatoid arthritis classification criteria: an American College of Rheumatology/European League Against Rheumatism collaborative initiative. _Arthritis Rheum._ 62, 2569–2581

(2010). Article  PubMed  Google Scholar  * Rutgeerts, P. _et al_. Infliximab for induction and maintenance therapy for ulcerative colitis. _N. Engl. J. Med._ 353, 2462–2476 (2005). Article 

PubMed  CAS  Google Scholar  * LeRoy, E. C. _et al_. Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. _J. Rheumatol._ 15, 202–205 (1988). PubMed  CAS  Google

Scholar  * Brentville, V. A. _et al_. Citrullinated vimentin presented on MHC-II in tumor cells is a target for CD4+ T-cell-mediated antitumor immunity. _Cancer Res._ 76, 548–560 (2016).

Article  PubMed  CAS  Google Scholar  * van Heemst, J. _et al_. Crossreactivity to vinculin and microbes provides a molecular basis for HLA-based protection against rheumatoid arthritis.

_Nat. Commun._ 6, 6681–6691 (2015). Article  ADS  PubMed  CAS  Google Scholar  * Blanc, C., Zufferey, M. & Cosson, P. Use of _in vivo_ biotinylated GST fusion proteins to select

recombinant antibodies. _ALTEX._ 31, 37–42 (2014). Article  PubMed  Google Scholar  * Justesen, S., Harndahl, M., Lamberth, K., Nielsen, L. L. & Buus, S. Functional recombinant MHC class

II molecules and high-throughput peptide-binding assays. _Immunome Res._ 5, 2–21 (2009). Article  PubMed  PubMed Central  CAS  Google Scholar  * Ettinger, R. A. & Kwok, W. W. A peptide

binding motif for HLA-DQA1*0102/DQB1*0602, the class II MHC molecule associated with dominant protection in insulin-dependent diabetes mellitus. _J. Immunol._ 160, 2365–2373 (1998). PubMed 

CAS  Google Scholar  * Scholz, E. M. _et al_. Human leukocyte antigen (HLA)-DRB1*15:01 and HLA-DRB5*01:01 present complementary peptide repertoires. _Front. Immunol._ 8, 984–995 (2017).

Article  PubMed  PubMed Central  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank the Antibody Facilities, Centre Médical Universitaire, Geneva, Switzerland for producing

the anti-native LL37 and anti-cit-LL37 monoclonal antibodies. We acknowledge ImmunAware, Copenhagen (Denmark) for producing peptide-MHC-tetramers and performing HLA-binding assays. We thank

the Benaroya Research Institute Tetramer Core for providing HLA-DR proteins, A.S.B Kampstra and Prof. REM Toes from Leiden University Medical Centre Netherlands, for providing some

citrullinated peptides. Funding: This work was supported by the following grants: Carlos and Elsie de Reuter Foundation, Centre Médical Universitaire, Geneva, Switzerland to L.F.; “Ernst et

Lucie Schmidheiny” Foundation, Geneva, Switzerland to L.F; “Fondazione Roma” (2015–2017), Rome, Italy, to AC, RL, LF; “Ricerca Finalizzata” (CO-2013-02356463), Ministry of Health, Italy;

“Fond National Suisse”, Switzerland to M.G.; Swiss National Found to CC (grant 310030–159999) and National Psoriasis Fundation, USA (2019–2021) to LF. AUTHOR INFORMATION Author notes * B.

Cerbelli Present address: Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University, Latina, Italy AUTHORS AND AFFILIATIONS * Istituto Superiore di Sanità, National

Centre for pre-clinical and clinical drug research and evaluation, Pharmacological research and experimental therapy Unit, 00166, Rome, Italy R. Lande, R. Palazzo, A. Butera, M. Boirivant, 

S. E. Auteri & L. Frasca * University Hospital CHUV, Dept. of Dermatology, 1011, Lausanne, Switzerland R. Lande, N. Gestermann, L. Feldmeyer, I. Surbeck, J. Di Lucca, F. Stuber, M.

Gilliet & L. Frasca * Translational tumor immunology group, Department of fundamental oncology, University of Lausanne, 1066, Epalinges, Switzerland C. Jandus & P. Romero * Istituto

Superiore di Sanità, National AIDS Center, 00166, Rome, Italy M. Falchi * Istituto Superiore di Sanità, Confocal Microscopy Unit, Core Facilities, Rome, Italy F. Spadaro * Division of

Rheumatology, Internal medicine and medical specialties, University Sapienza, Rome, Italy V. Riccieri, F. R. Spinelli, S. E. Auteri, C. Alessandri, F. Conti & G. Valesini * Department of

Translational Research, Benaroya Research Institute, Seattle, WA, USA E. A. James & S. Horstman * Department of Systems Medicine University of Tor Vergata, Dermatology Unit, Rome, Italy

E. Botti, B. Marinari & L. Bianchi * “Sandro Pertini” Hospital, IBD, GE Unit, Rome, Italy R. Pica * Department of Radiological, oncological and anatomo-pathological sciences, Sapienza

University, Rome, Italy B. Cerbelli * Department of Pathology, Sapienza University, Rome, Italy K. Giannakakis * Academic Department of Clinical Oncology, University of Nottingham,

Nottingham, UK I. Daniels & L. G. Durrant * Skin pathology laboratory, Dermatology Unit, IRCCS Humanitas clinical and research center, Rozzano, Milan, Italy A. Costanzo * Department of

Immunology and Allergy, University Hospital and Medical School, Geneva, Switzerland C. Chizzolini & L. Frasca Authors * R. Lande View author publications You can also search for this

author inPubMed Google Scholar * R. Palazzo View author publications You can also search for this author inPubMed Google Scholar * N. Gestermann View author publications You can also search

for this author inPubMed Google Scholar * C. Jandus View author publications You can also search for this author inPubMed Google Scholar * M. Falchi View author publications You can also

search for this author inPubMed Google Scholar * F. Spadaro View author publications You can also search for this author inPubMed Google Scholar * V. Riccieri View author publications You

can also search for this author inPubMed Google Scholar * E. A. James View author publications You can also search for this author inPubMed Google Scholar * A. Butera View author

publications You can also search for this author inPubMed Google Scholar * M. Boirivant View author publications You can also search for this author inPubMed Google Scholar * L. Feldmeyer

View author publications You can also search for this author inPubMed Google Scholar * I. Surbeck View author publications You can also search for this author inPubMed Google Scholar * J. Di

Lucca View author publications You can also search for this author inPubMed Google Scholar * F. Stuber View author publications You can also search for this author inPubMed Google Scholar *

F. R. Spinelli View author publications You can also search for this author inPubMed Google Scholar * E. Botti View author publications You can also search for this author inPubMed Google

Scholar * B. Marinari View author publications You can also search for this author inPubMed Google Scholar * L. Bianchi View author publications You can also search for this author inPubMed 

Google Scholar * R. Pica View author publications You can also search for this author inPubMed Google Scholar * B. Cerbelli View author publications You can also search for this author

inPubMed Google Scholar * K. Giannakakis View author publications You can also search for this author inPubMed Google Scholar * S. E. Auteri View author publications You can also search for

this author inPubMed Google Scholar * I. Daniels View author publications You can also search for this author inPubMed Google Scholar * L. G. Durrant View author publications You can also

search for this author inPubMed Google Scholar * S. Horstman View author publications You can also search for this author inPubMed Google Scholar * A. Costanzo View author publications You

can also search for this author inPubMed Google Scholar * P. Romero View author publications You can also search for this author inPubMed Google Scholar * C. Alessandri View author

publications You can also search for this author inPubMed Google Scholar * F. Conti View author publications You can also search for this author inPubMed Google Scholar * G. Valesini View

author publications You can also search for this author inPubMed Google Scholar * M. Gilliet View author publications You can also search for this author inPubMed Google Scholar * C.

Chizzolini View author publications You can also search for this author inPubMed Google Scholar * L. Frasca View author publications You can also search for this author inPubMed Google

Scholar CONTRIBUTIONS R.L. designed and performed part of the immunological experiments, provided expertise for pDCs/neutrophil experiments, analyzed data and contributed to writing the

manuscript. RPa performed most experiments, analyzed data, and performed statistical analyses. N.G. performed ELISA assays and analysed plasma cells. C.J. was involved in experiments

concerning T-cell cloning and peptide-MHC-tetramer staining, and contributed to drafting the manuscript; M.F. and F.S. acquired and elaborated confocal images of renal biopsies; V.R.

provided clinical expertise, and blood of SLE and SSc patients and controls, and clinical data, including levels of anti-DNA antibodies in serum samples. EAJ supervised binding assays to MHC

and production of peptide-MHC-tetramers, provided antigen-presentation expertise and contributed to drafting the manuscript. A.B. and M.B. analyzed data regarding control UC patients and

performed statistical analysis. L.F., J.D.L. and F.S. provided SLE and CLE samples (including skin biopsies) and patients’ data. I.S. performed immunohistochemistry. F.R.S. provided S.L.E.

samples/data and clinical expertise; E.B., B.M. and L.B. provided psoriasis patients’ samples and data, and clinical expertise. RPi provided UC patients’ samples and data, and clinical

expertise. B.C. and K.G. provided SLE kidney biopsies and patients’ data. S.E.A. participated in ELISA experiments and organized patients’ data of a replication cohort; I.D. and L.D.

provided expertise in selection of control antigenic peptides and their MHC-binding abilities. S.H. performed peptide-binding assays to MHC. AC provided psoriasis samples and data. P.R.

provided expertise in T-cell cloning and contributed to drafting the manuscript. C.A., F.C., G.V., provided SLE and RA patients/data and clinical expertise in SLE, RA and on citrullination.

M.G. provided clinical samples of SLE., CLE and data, and contributed to writing the manuscript. C.C. provided clinical expertise in SLE and SSc and clinical samples and data, and

contributed to the writing of the manuscript. L.F. conceived and directed the research, designed and performed experiments, performed statistical analyses and wrote the manuscript with input

from other co-authors. CORRESPONDING AUTHOR Correspondence to L. Frasca. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION.

RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and

reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes

were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If

material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain

permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE Lande, R., Palazzo, R., Gestermann, N. _et al._ Native/citrullinated LL37-specific T-cells help autoantibody production in Systemic Lupus Erythematosus. _Sci Rep_ 10, 5851 (2020).

https://doi.org/10.1038/s41598-020-62480-3 Download citation * Received: 09 January 2020 * Accepted: 12 March 2020 * Published: 03 April 2020 * DOI:

https://doi.org/10.1038/s41598-020-62480-3 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative