Signaling Lymphocytic Activation Molecule Family Member 7 Engagement Restores Defective Effector CD8+ T Cell Function in Systemic Lupus Erythematosus

Abstract

Objective: Effector CD8+ T cell function is impaired in systemic lupus erythematosus (SLE) and is associated with a compromised ability to fight infections. Signaling lymphocytic activation molecule family member 7 (SLAMF7) engagement has been shown to enhance natural killer cell degranulation. This study was undertaken to characterize the expression and function of SLAMF7 on CD8+ T cell subsets isolated from the peripheral blood of SLE patients and healthy subjects.

Methods: CD8+ T cell subset distribution, SLAMF7 expression, and expression of cytolytic enzymes (perforin, granzyme A [GzmA], and GzmB) on cells isolated from SLE patients and healthy controls were analyzed by flow cytometry. CD107a expression and interferon-γ (IFNγ) production in response to viral antigenic stimulation in the presence or absence of an anti-SLAMF7 antibody were assessed by flow cytometry. Antiviral cytotoxic activity in response to SLAMF7 engagement was determined using a flow cytometry-based assay.

Results: The distribution of CD8+ T cell subsets was altered in the peripheral blood of SLE patients, with a decreased effector cell subpopulation. Memory CD8+ T cells from SLE patients displayed decreased amounts of SLAMF7, a surface receptor that characterizes effector CD8+ T cells. Ligation of SLAMF7 increased CD8+ T cell degranulation capacity and the percentage of IFNγ-producing cells in response to antigen challenge in SLE patients and healthy controls. Moreover, SLAMF7 engagement promoted cytotoxic lysis of target cells in response to stimulation with viral antigens.

Conclusion: CD8+ T cell activation in response to viral antigens is defective in SLE patients. Activation of SLAMF7 through a specific monoclonal antibody restores CD8+ T cell antiviral effector function to normal levels and thus represents a potential therapeutic option in SLE.

Figure 1. Skewed distribution of CD8+ T cell differentiated subsets in peripheral blood from SLE patients

(A) PBMC isolated from SLE patients were stained for CD8+ T cells differentiated subsets by examining the expression of CCR7 and CD45RA. (B) Distribution of CD8+ T cells differentiated subsets in SLE patients compared to healthy controls. Frequency of (C) naïve CD8+ T cells (D) CM, (E) EM and (F) TDEM CD8+ T cells in three cohorts: inactive SLE (SLEDAI<4), active SLE (SLEDAI≥4) and healthy controls (CON). Naive (CCR7+CD45RA+), CM: Central Memory (CCR7+CD45RA−), EM: Effector Memory (CCR7−CD45RA−), TDEM: Terminally Differentiated Effector Memory (CCR7−CD45RA+). DN: double negative (CD3+CD4−CD8−) (mean ± SEM; SLE n=45, controls n=41).

Figure 2. SLAMF7 expression is reduced on CD8+ T cells isolated from SLE patients compared to healthy controls

SLAMF7 expression was assessed by flow cytometry on T cells isolated from peripheral blood. (A) Frequency (%) of SLAMF7 expression on CD4+, CD8+ and double negative (DN) T cells isolated from SLE patients and controls. Representative plots, cumulative data and (B) correlation with disease activity: inactive SLE (SLEDAI<4), active SLE (SLEDAI≥4) and healthy controls (CON). (C) Frequency of SLAMF7 expression on central memory (CM), effector memory (EM) and terminally differentiated effector memory (TDEM) CD8+ T cells. (D) Assessment of SLAMF7 expression by CM, EM and TDEM CD8+ T differentiated subset in three cohorts: inactive SLE (SLEDAI<4), active SLE (SLEDAI≥4) and healthy controls (CON) (mean ± SEM; SLE n=16 to 27, controls n=13 to 22).

Figure 3. Expression of perforin, GzmA and GzmB is restricted to the SLAMF7+ CD8+ T cells population

Frequencies of CD8+ T cells expressing perforin, GzmA and GzmB was assessed by flow cytometry. (A) Representative flow cytometric profile of SLAMF7 vs perforin, GzmA and GzmB. Numbers indicate percentages. (B) Cumulative data from from SLE patients and healthy controls (mean ± SEM; SLE n=18, controls n=15).

Figure 4. SLAMF7 engagement restores effector function of SLE CD8+ T cells in response to antigenic stimulation

PBMC from SLE and controls were stimulated with CEF (CMV-EBV-Flu peptide mix) for 6 hours in presence of anti-SLAMF7 mAb or an isotype control (ISO). CD107a expression and IFNg production were assessed by flow cytometry at the end of the stimulation. SEB (Staphyloccal Enterotoxin B) was used as positive control. Numbers indicate percentages. (A) Representative flow plots and (B) cumulative data (mean ± SEM; SLE n=8, controls n=8).

Figure 5. SLAMF7 ligation enhances cytotoxic activity of healthy and SLE CD8+ T cells in response to viral antigens

CEF stimulated PBMC were enriched for CD8+ T cells. CD8+ T cells were treated with anti-SLAMF7 mAb or an isotype control (ISO), and co-cultured for 6h with autologous CEF pulsed CD4+ T cells and un-pulsed CD4+ T cells. CEF specific killing of target CD4+ T cells was expressed as Aqua positive cells (dead cells marker) in response to different ratios of CD8+ T cells. Un-pulsed CD4+ T cells were used as control to determine target cell death background. (A) Representative dot plots showing CD4+ T cells killing (%) in response to SLAMF7 or ISO treated CD8+ T cells. Cumulative healthy healthy controls (n=4) and SLE (n=5) data are depicted in (B) and (C). Results are expressed as [Aqua+ pulsed CD4+ T cells] – [Aqua+ un-pulsed CD4+ T cells] (mean ± SEM).