Clonal expansion of CD4(+) cytotoxic T lymphocytes in patients with IgG4-related disease

Abstract

Background: IgG4-related disease (IgG4-RD) is a systemic condition of unknown cause characterized by highly fibrotic lesions with dense lymphoplasmacytic infiltrates. CD4(+) T cells constitute the major inflammatory cell population in IgG4-RD lesions.

Objective: We used an unbiased approach to characterize CD4(+) T-cell subsets in patients with IgG4-RD based on their clonal expansion and ability to infiltrate affected tissue sites.

Methods: We used flow cytometry to identify CD4(+) effector/memory T cells in a cohort of 101 patients with IgG4-RD. These expanded cells were characterized by means of gene expression analysis and flow cytometry. Next-generation sequencing of the T-cell receptor β chain gene was performed on CD4(+)SLAMF7(+) cytotoxic T lymphocytes (CTLs) and CD4(+)GATA3(+) TH2 cells in a subset of patients to identify their clonality. Tissue infiltration by specific T cells was examined by using quantitative multicolor imaging.

Results: CD4(+) effector/memory T cells with a cytolytic phenotype were expanded in patients with IgG4-RD. Next-generation sequencing revealed prominent clonal expansions of these CD4(+) CTLs but not CD4(+)GATA3(+) memory TH2 cells in patients with IgG4-RD. The dominant T cells infiltrating a range of inflamed IgG4-RD tissue sites were clonally expanded CD4(+) CTLs that expressed SLAMF7, granzyme A, IL-1β, and TGF-β1. Clinical remission induced by rituximab-mediated B-cell depletion was associated with a reduction in numbers of disease-associated CD4(+) CTLs.

Conclusions: IgG4-RD is prominently linked to clonally expanded IL-1β- and TGF-β1-secreting CD4(+) CTLs in both peripheral blood and inflammatory tissue lesions. These active, terminally differentiated, cytokine-secreting effector CD4(+) T cells are now linked to a human disease characterized by chronic inflammation and fibrosis.

Keywords: CD4(+) cytotoxic T cells; Fibrosis; IL-1β; IgG(4); IgG(4)-related disease; T(H)2 cells; rituximab.

Figure 1. Expansions of T_EM cells with a cytolytic phenotype in IgG4-RD

(A) CD4⁺CD62L^loCD27^lo T_EM cells in two non-atopic (P2 & P19) two atopic (P12 & P25) IgG4-RD patients and 2 representative healthy controls. (B) CD4⁺CD45RO⁺CD62L^loCD27^lo T_EM cells in peripheral blood of IgG4-RD subjects. Boxplot displays the 25^th to 75^th percentiles. P values are based on the Mann-Whitney test. (C) Heat map depicting differentially expressed immune-related genes in T_EM cells from four IgG4-RD patients compared to CD4⁺CD45RO⁺ T cells from four healthy controls. Upregulated genes representing modified Th1, cytolytic and myeloid signatures are highlighted.

Figure 2. Expanded T_EM cells in IgG4-RD subjects exhibit a functional cytolytic profile

(A & B) Key hits from the gene expression analysis from Fig. 1 were validated using flow cytometry in T_EM cells (A) and using TCR Vβ-specific antibodies (B). (C) Levels of ThPOK and Runx3 in CD4⁺SLAMF7⁺ CTLs from IgG4-RD subjects and CD4⁺CD45RO⁺ T cells in healthy controls. Error bars show SEM. P values are based on the Mann-Whitney test. (D) Granzyme B and CD107a staining on CD4⁺ CTLs from an IgG4-RD patient, before and after stimulation with anti-CD3 . (E) Cytotoxicity of in vitro expanded CD4⁺ CTLs derived from two subjects (P2 and P25) against an allogeneic EBV-transformed B cell target cell line, measured after 12 hours of co-culture with or without anti-CD3 (10 μg/mL) at varying CD4⁺ CTL: target ratios. The data are representative of two separate experiments done on 4 patients.

Figure 3. The TCR Vβ repertoire of expanded T_EM cells in IgG4-RD

(A) TCRβ repertoire of the expanded circulating T_EM subset in 4 IgG4-RD subjects (P31, P28, P27 and P25) and from total CD4+ T cells from four healthy controls, represented as bubble charts, where the size and color corresponds to the frequency of the observed Vβ-Jβ rearrangements. (B) TCRβ repertoire of expanded circulating CD4⁺GATA-3⁺ T_H2 cells in four IgG4-RD subjects with an atopic history (P3, P10, P39 & P54), contrasted with the expanded CD4⁺SLAMF7⁺ CTLs from the same individuals. See legend to Fig.3A for details.

Figure 4. Expansion of CD4⁺SLAMF7⁺ CTLs in tissue lesions of IgG4-RD subjects

(A) Expansion of CD4⁺SLAMF7⁺ CTLs in 101 IgG4-RD subjects. (B) CD4⁺CD62L^loCD27^loSLAMF7⁺ CTLs in the aortic wall of a subject with IgG4-related aortitis (P51) and the involved nasal septum of a subject with IgG4-RD (P43). (C) Immunofluorescence staining of CD4+SLAMF7+ CTLs and CD4+GATA-3+ Th2 cells in the affected tissues of IgG4-RD subjects (lymph node biopsy from P11 and salivary gland biopsy from P25). CD4 (red), DAPI (blue), GATA-3 (magenta) and SLAMF7 (green) staining are shown. (D) Quantification of CD4+SLAMF7+, CD4+GATA-3+ and CD4+SLAMF7-GATA-3- cells in tissue biopsies from 10 IgG4-RD patients (atopic patients are marked with asterisks).

Figure 5. Disease associated CD4⁺ CTLs secrete IFNγ, IL-1β and TGF-β1 upon in vitro restimulation

(A) Intracellular staining for IFN-γ and IL-4 in restimulated CD4⁺ CTLs from seven IgG4-RD patients. The empty histogram (red) depicts an unstimulated control. (B & C) ELISPOT detection of the frequency of IL-1β producers among re-stimulated CD4⁺CD45RO⁺ T cells from seven IgG4-RD subjects compared to healthy donors (error bars show SEM, unpaired t-test) (A) and CD4⁺ CTLs from four IgG4-RD subjects compared to naïve CD4⁺CD45RA⁺ T cells from the same individuals (p< 0.05, paired t-test) (B). (D) Western Blot detection of IL-1β from culture supernatants of in vitro expanded T cells, maintained in IL-2 for two weeks (10 ng/mL). Supernatants from CD4⁺CD45RO⁺ T cells from a healthy donor and CD4⁺CD45RO⁺SLAMF7⁺ or CD4⁺CD45RO⁺SLAMF7⁻ T cells from an IgG4-RD subject were used without any stimulation (US), with 5 μg/mL LPS or 3μg/mL anti-CD3. LPS stimulated PBMCs from a healthy donor were used as a positive control. (E) Detection of TGF-β1 production among naïve CD4⁺ cells and CD4⁺SLAMF7⁺ CTLs from four IgG4-RD subjects by ELISA, with or without in vitro re-stimulation (with 3 μg/mL anti-CD3). Error bars show SEM. P values are based on the Mann-Whitney test.

Figure 6. Expansion of IL-1β and TGF-β1 secreting CD4⁺ CTLs in IgG4-RD lesions

(A) Immunofluorescence staining of IL-1β-producing CD4⁺ SLAMF7⁺ cells in the tissues of 2 IgG4-RD subjects (lymph node biopsy from P11 and salivary gland biopsy from P25) and a healthy control subject (labial salivary gland biopsy). CD4 (red), SLAMF7 (green) DAPI (blue) and IL-1β (white). (B) Immunofluorescence staining of TGF-β1-producing CD4⁺ SLAMF7⁺ cells in the tissues of 2 IgG4-DS and 2 Sjögren’s syndrome subjects (submandibular salivary gland biopsies). CD4 (red), Granzyme A (green) DAPI (blue) and IL-1β (cyan) staining are shown.

Figure 7. Rituximab-mediated depletion of CD4⁺ CTLs

(A) Decline in CD4⁺ SLAMF7⁺ CTLs (n = 8 subjects) over 6–14 months following rituximab infusion shown as a change in cell counts and proportion. (B) Effect of rituximab on CD4⁺ CD45RA⁺ naïve T cell counts in the peripheral blood of IgG4-RD patients. (C & D) Effect of rituximab on CD4⁺GATA-3⁺ T_H2 cells and CD4⁺CD25⁺Foxp3⁺ cells in the peripheral blood of IgG4-RD patients 90–120 days after rituximab therapy. (E) Decline in the number and proportion of an expanded CD4⁺ CTL clone tracked using a TCR-Vβ specific antibody following rituximab therapy in an IgG4-RD subject (P25). (F) Decline in circulating CD4⁺ CTL numbers at day 70–95 following rituximab therapy (normalized to the pretreatment levels) is plotted against the IgG4-RD Responder Index, a clinical measure of disease activity.