MICa/b-dependent activation of natural killer cells by CD64+ inflammatory type 2 dendritic cells contributes to autoimmunity

Abstract

Primary Sjögren's syndrome (pSS) is an inflammatory autoimmune disorder largely mediated by type I and II interferon (IFN). The potential contribution of innate immune cells, such as natural killer (NK) cells and dendritic cells (DC), to the pSS pathology remains understudied. Here, we identified an enriched CD16⁺ CD56hi NK cell subset associated with higher cytotoxic function, as well as elevated proportions of inflammatory CD64⁺ conventional dendritic cell (cDC2) subtype that expresses increased levels of MICa/b, the ligand for the activating receptor NKG2D, in pSS individuals. Circulating cDC2 from pSS patients efficiently induced activation of cytotoxic NK cells ex vivo and were found in proximity to CD56⁺ NK cells in salivary glands (SG) from pSS patients. Interestingly, transcriptional activation of IFN signatures associated with the RIG-I/DDX60 pathway, IFN I receptor, and its target genes regulate the expression of NKG2D ligands on cDC2 from pSS patients. Finally, increased proportions of CD64hi RAE-1⁺ cDC2 and NKG2D⁺ CD11b⁺ CD27⁺ NK cells were present in vivo in the SG after poly I:C injection. Our study provides novel insight into the contribution and interplay of NK and cDC2 in pSS pathology and identifies new potential therapy targets.

Keywords: RIG-I; Sjögren's syndrome; dendritic cells; interferon; natural killer cells.

Figure 1. Proportions and phenotypical characteristics of natural killer cell subsets from pSS patients

1. A

   Flow cytometry dot plots showing analysis of NK cell subsets present in Lineage (CD3, CD19, CD14) negative, HLADR negative lymphocytes, and defined by CD16 vs. CD56 expression in the PB of a representative HD (left) and pSS (right) individuals. Gating defining CD56^hi CD16⁺ NK cells (III) is highlighted in red.

2. B

   Summarized proportions of CD56^hi CD16⁺ circulating NK cells in 56 healthy donors (HD; gray) and total 48 primary Sjögren's syndrome (pSS; blue) patients or stratified according to the absence (No HQ, n = 25 biological replicates) or the presence of hydroxychloroquine (HQ; n = 17 biological replicates) or other (OT; n = 6 biological replicates) treatments (purple).

3. C

   Analysis of expression of IFNγ and CD107a on NK cells. Representative flow cytometry dot plot is included in the top. Proportions of total CD107a⁺ (bottom, left) and IFNγ⁺ CD107a⁺ (bottom, right) cells within the CD56^hi CD16⁺ NK cell subset from n = 16 HD (gray) and n = 19 total or n = 8 HQ and n = 11 No‐HQ pSS patients (blue).

4. D

   Functional characteristics of NK cells from pSS patients. Summary of proportions of dead target K562‐GFP cells cultured for 16 h in the absence (light gray) or the presence of isolated circulating NK cells from HD (n = 10; biological replicates) or pSS (n = 12 biological replicates) patients.

5. E–G

   Histological immunofluorescence analysis of merged expression of CD56 (white) and Granzyme B (red) on low (left) and highly (right) infiltrated glandular areas (E) or with CD19 (red) and IL‐17 (green) (G) from the section of SG tissue from a representative pSS patient. Cell nuclei were stained with DAPI (blue). Cells co‐expressing CD56 and Granzyme B (E) or expressing CD19 (G) are highlighted with red arrows. In panel (G), IL17⁺ and CD56⁺ are also highlighted in green and white arrows, respectively. Original magnification 40×. (F): Image J quantification of proportions of area of CD56⁺ NK cells co‐expressing Granzyme B within the total the mentioned total high and low infiltrated areas from n = 7 tested pSS patients. Data are normalized to the number of total DAPI⁺ cells detected on each area considered as 100%.

Data information: (B–D) Data are represented as box and whiskers plots with maximum and minimum range and a median value central band. Statistical significance was calculated with a two‐tailed Mann Whitney test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.

Figure 2. Frequencies, phenotypic and functional characteristics of myeloid cell subsets from pSS patients

1. A

   Box and whiskers plots showing proportions from live lymphocytes of non‐classical (NC; right) CD16⁺ CD14^lo Mo (left), cDC2 (middle), and cDC1 (right) in the PB from HD (gray; n = 56 biological replicates) and total pSS patients (blue; n = 48: biological replicates) or stratified according to the absence (No HQ, n = 25) or the presence of hydroxychloroquine (HQ; n = 17) or other (OT; n = 6) treatments (purple).

2. B

   Representative confocal microscopy (40× magnification) showing immunofluorescence analysis of CD1c (white), HLADR (green), and CD56 (red) expression on an SG tissue section from a representative pSS patient. Cell nuclei were stained with DAPI (blue). Cells co‐expressing CD1c and HLADR markers with close proximity to CD56 cells are highlighted with a yellow arrow.

3. C

   Proportions of MICa/b⁺ and ULBP1⁺ cells from circulating cDC2 (left plots), cDC1 (middle plots), and CD14⁺ Mo (right plots) in 46 HD (gray) and total n = 42, n = 15 HQ, n = 22 No HQ, and n = 5 OT pSS patients (blue and purple).

4. D

   Representative confocal microscopy (40× magnification) showing immunofluorescence analysis of CD1c (white), HLADR (green), and MICab (red) expression on a representative SG tissue section from a pSS patient from a total of n = 4 individuals tested. Cell nuclei were stained with DAPI (blue). Cells co‐expressing CD1c, HLADR, and MICAB are highlighted with an orange arrow.

5. E

   Quantification of proportions of CD1c⁺ HLA‐DR⁺ cDC2 detected in SMSG from n = 4 pSS patients that either co‐express or not MICa/b.

6. F

   Proportions of the CD56^hi CD16⁺ NK cell subset proportions (left) after 16 h culture of sorted autologous CD56⁺ NK cell in media alone or in the presence of sorted autologous circulating Mo, cDC2, and cDC1 from n = 8 pSS patients at ratio 1:2 (myeloid cell:NK) and expression of CD107a (right) on CD56⁺ NK cells in these functional assays is shown.

Data information: (A, C, E, F): Data are represented as box and whiskers plots with maximum and minimum range and a median value central band. In panel (E), MICa/b⁺ and MICa/b⁻ cells from the same donor are paired. Statistical significance was calculated with a two‐tailed Mann Whitney (A, C) or a Wilcoxon matched pairs (E, F) tests. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.

Figure 3. Expression of CD64 and differential transcriptional signatures in circulating Mo, cDC2 and cDC1 from pSS patients

1. Mean of fluorescence intensity of CD64 on cDC2 (left), cDC1, non‐classical (NC) CD16⁺ CD14^lo Mo, and Transitional (T) CD16^hi CD14⁺⁺ in the PB from HD (gray; n = 48) and pSS patients (blue; n = 34 total patients, n = 13 HQ, and n = 21 No‐HQ as biological replicates). Statistical significance was calculated using a two‐tailed Mann Whitney test. *P < 0.05; **P < 0.01.

2. Significance of selected upregulated (positive z‐score > 1; red), downregulated (negative z‐score < −1; blue) canonical pathways predicted by Ingenuity Pathway Analysis (IPA) (full analysis shown in Appendix Table S2) from significant differentially expressed genes (DEG P < 0.05 after FDR correction, log2FC > 1.5 and < −1.5) in Mo, cDC2, and cDC1 from n = 4 pSS vs. n = 4 HD. Pathways that did not have a Z‐score or did not reach these mentioned value cut offs were labeled in gray.

3. Chordplot representing overlap of selected IPA‐predicted canonical pathways associated with the indicated innate immune pathways (right area, in different colors) and the DEG included within each of them in cDC2 from pSS patients. Changes and levels in transcriptional expression of each DEG are highlighted in red (upregulation) and blue (downregulation).

4. Venn's diagram of overlapping significant DEG included in selected IFN, PKR, and IRF canonical pathways in PB cDC2 from pSS patients. FDR‐corrected P values of differential transcriptional expression of indicated DEG are highlighted; P < 0.05; **P < 0.01; ***P < 0.001.

5. Heatmaps representing log2‐FC in transcription of selected Interferon Stimulated genes (ISG) or transcripts associated with the IFN pathway on each cell subset from the PB from pSS vs. HD (red, upregulated; blue, downregulated). Yellow dots size is proportional to statistical significance of DEG expression (P < 0.05 FDR corrected values).

6. Heatmap representing enrichment of selected DEG associated with IFN, RIG‐I, and IFN receptor pathways using Gene Set enrichment Analysis (GSEA). Overlap of each DEG with the indicated sets is highlighted in blue. FDR‐corrected p values for enrichment of each gene set are also included.

7. qPCR validation of the indicated transcript from sorted circulating cDC2 from n = 7 pSS patients (blue) vs. n = 6 HD (gray). Statistical significance was calculated using a two‐tailed U Mann Whitney test. **P < 0.01.

Data information: (A, G): Data are represented as box and whiskers plots with maximum and minimum range and a median value central band. Source data are available online for this figure.

Figure 4. Induction of MICa/b in response to poly I:C and dependency on RIG‐I, DDX60, IFIT1, and IFNAR in primary human cDC2

1. A–C

   Proportions of MICa/b⁺ cells in cDC2 from HD and pSS in PBMC (A) or preisolated cells (C) after 16 h poly I:C (PI:C) stimulation in cDC2 isolated from the blood of HD (biological replicates n = 24, A and n = 10, C) and pSS patients (biological replicates n = 24 A; n = 11 C). (B): Proportions of MICa/b⁺ cells on gated CD64⁺ and CD64⁻ subpopulations included in CD11c⁺ CD14⁻ cDC from PBMC of n = 16 pSS patients.

2. D

   Spearman correlations between proportions of CD86⁺ and MICa/b⁺ cells on cDC2 from HD and pSS after 16 h culture in media alone (left) or in the presence of poly I:C (right). P and R values of all combined cohorts or each individual study group are shown.

3. E

   Proportions of CD107a⁺ NK cells after culture in media alone or with autologous poly I:C stimulated cDC2 in the presence of either anti‐MICa/b or an isotypic control Abs (n = 10 biological replicates from pSS patients).

4. F–H

   Surface expression of MICa/b on isolated primary cDC2 from HD biological nucleofected with either DDX60 (n = 6 biological replicates) or RIG‐I (n = 9 biological replicates) (F) or IFIT1 (H) specific siRNAs or control scramble (SC; biological replicates n = 9 for F and n = 6 for H) siRNAs after 16 h in the presence of media or poly I:C (PI:C). (G): Fold change in RAE‐I expression in cDC2 from the spleen of wild type (WT) vs. Tyk2 knock out (KO) mice exposed to poly I:C compared to cells cultured in media.

Data information: (A, B, C, E, F, G, H): Data are represented as box and whiskers plots with maximum and minimum range and a median value central band. In panel (A and C), data from the same donor in media or poly I:C treatments have been paired in (A) and (C) panels. Statistical significance (A–H) was calculated using two‐tailed Wilcoxon matched pairs signed rank (A, C, E, F, G, H) and Mann Whitney tests (A, G) to compare changes under different conditions or between independent experimental groups, respectively (*P < 0.05; **P < 0.01; ****P < 0.0001). Source data are available online for this figure.

Figure 5. In vivo recruitment of activated NK cells and cDC2 to the SMSG after injection of mice with poly I:C

1. A

   Analysis of salivary gland function measured as saliva weight/body weight after Pilocarpine injection in mice treated with PBS (blue; biological replicates n = 8; n = 7; n = 6; n = 5) or poly I:C (red; biological replicates n = 10; n = 10; n = 10; n = 4) at 1, 2, 3, 8 weeks post injection, respectively.

2. B

   Absolute numbers of hematopoietic CD45.2 cells detected on homogenized submandibular salivary gland (SMSG) from mice injected with PBS (blue; biological replicates n = 19; n = 9; n = 8) and poly I:C (red; biological replicates n = 22, n = 10, n = 10; n = 10).

3. C, D

   Image of histological analysis of immune cell infiltrates and Pax5⁺ cells identifying B cells in SG from a representative mouse injected with poly I:C (C, left). Scale bar: 100 μm. Quantification of area of total immune infiltrates (C, right) and number of Pax5⁺ cells (D) detected in the SMSG from mice‐treated PBS (blue; biological replicates n = 5 and n = 6 for 3 and 8 weeks, respectively) or poly I:C (red; biological replicates, n = 5 and n = 6 for 3 and 8 weeks, respectively) is shown.

4. E–G

   Frequencies of CD11b⁺CD27⁺ NK1.1⁺ CD3⁻ NK cells (E) and proportions of NKG2D⁺ (F) and CD107a⁻ IFNγ⁺ (G) cells in this population in the SMSG of mice after 1 and 3 weeks after injection with PBS (blue; biological replicates n = 19 in E, F and n = 4 in G for week 1; n = 10 in E, F for week 3 and for week 2 in G) or poly I:C (red; biological replicates n = 19 in E, F and n = 5 in G for week 1; n = 10 in E–G for week 3).

5. H–J

   Proportions of CD11b⁺ CD11c^hi cDC2 (H), expression of CD64 (I) and RAE‐I (J) in this population infiltrated in the SMSG from mice after 1 and 3 weeks after injection with PBS (blue; biological replicates n = 19 in H, I and n = 9 in J for week 1; n = 8 in I and n = 10 in I, J for 3 weeks) and poly I:C (red; biological replicates n = 19 in H, I and n = 9 in J for week 1; n = 10 in H–J for week 3). Data from three independent experiments are included with biological replicates.

Data information: (A–J) Data are represented as box and whiskers plots with maximum and minimum range and a median value central band Statistical significance was calculated with a two‐tailed Mann Whitney test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.