Aberrant expression of PD-1 on B cells and its association with the clinical parameters of systemic lupus erythematosus

Background: Programmed death 1 (PD-1) is an immunoregulatory receptor that inhibits T cell activation and proliferation upon binding to its cognate ligand (PD-L1). However, the role of the PD-1/PD-L1 axis in B cell function, especially in inflammatory and autoimmune disorders, is less clear. The aim of this study was to analyze the PD-1 expression patterns on multiple B cell subpopulations isolated from systemic lupus erythematosus (SLE) patients, and determine their clinical relevance. Results: The frequency of B cells increased significantly in patients with active SLE compared with healthy controls and patients with inactive SLE. In particular, the frequencies of the IgD CD27 and IgD CD27high (plasmablast cells) subpopulations were significantly higher in the patients compared to healthy individuals. Interestingly, the patients with active SLE harbored an increased proportion of the PD-1+ B cells, which correlated significantly with the disease severity (SLEDAI scores), incidence of lupus nephritis, and the circulating levels of autoantibodies and complement factors. Furthermore, the primary PD-1+ B cells isolated from the peripheral blood of SLE patients proliferated faster and secreted more anti-dsDNA antibodies and immunoglobulins in vitro compared to the PD-1+/- B cells from healthy controls. Conclusions: PD-1 is overexpressed on all B cell subpopulations of SLE patients and associated with disease progression.

The activation of T cells is primarily regulated by the programmed death 1 (PD-1) receptor and its ligands PD-L1 and PD-L2, which form an immune checkpoint that is essential for maintaining tolerance to self-antigens 6-8 and preventing autoimmune disorders 9-11 . Furthermore, the PD-1/PD-L axis is often disrupted in animal models simulating human autoimmune diseases [12][13][14] , and directly affects immune activation and homeostasis 15-17 . Interestingly, blocking either PD-1 or PD-L1 in a murine model of lupus-like nephritis significantly alleviated tissue inflammation and other symptoms by inhibiting the autoreactive T cells and concomitantly increasing the proportion of the immunosuppressive CD8+ subset [17][18][19] . In addition, anti-PD-L1 immunoglobulin improved the survival of these mice by delaying proteinuria onset 20 . However, the exact pathological relevance of the PD-1/PD-L1 axis in human SLE remains to be elucidated.
Studies show that antigen-primed SLE patients that are recalcitrant to immunosuppressive therapy harbor an expanded IgD -CD27 + class-switched memory B cell population 2, 21 , which can be attributed to aberrant B-cell receptor (BCR) editing and somatic hypermutation in the peripheral memory B cells 22 . These abnormal memory B cells significantly increase the risk of autoimmune responses on account of their lower antigendependent activation thresholds, as well as antigen-independent activation through the Bcell-activating factor, Toll-like receptor agonists or cytokines 23 . The IgD -CD27memory Bcell subset is also enriched in the SLE patients 24 , and correlates with increased disease severity and renal involvement 25 . Interestingly, IgD -CD27 -B cells harboring mutated BCRs have been detected in the peripheral blood and lymphoid tissues of healthy donors as well 26,27 . However, the functional relationship between the PD-1/PD-L1 axis and memory B cell activity in SLE is not clear. To this end, we analyzed the expression patterns of PD-1 on different B cell populations in SLE patients, and determined their correlation with clinical indices.

Patients
Seventy-four Asian-origin SLE patients diagnosed as per the 1997 American College of Rheumatology revised criteria 28 , and 54 matched healthy controls were enrolled at the Department of Rheumatology of the First Affiliated Hospital of Bengbu Medical College, China. The medical records of all participants were screened for age, gender, blood cell counts, 24-h urinary protein secretion, circulating levels of anti-dsDNA, anti-nucleosome, anti-Smith (anti-Sm), anti-Sjogren syndrome A (anti-SSA) and anti-Sjogren syndrome B (anti-SSB) antibodies, complement component 3 (C3) and C4, IgG, IgM and IgA, and the erythrocyte sedimentation rates (ESR). The disease activity was scored according to the SLE Disease Activity Index (SLEDAI) and the patients were classified into the inactive (SLEDAI <10) and active (SLEDAI ≥10) groups.

Enzyme linked immunosorbent assay (ELISA)
The levels of anti-dsDNA antibody and IgG in the supernatants (see above) were analyzed on days 1, 3, 5 and 7 of culture by ELISA (Biorbyt, San Francisco, CA, USA).

Statistical analysis
All data were presented as mean ± standard deviation, and compared by one-way analysis of variance or two-tailed Student t test as appropriate. The correlation between two variables was analyzed by Spearman or Pearson correlation coefficient. P values < 0.05 were considered statistically significant. All data were analyzed using SPSS 16.0 (IBM, Armonk, NY, USA).

The B cell subpopulations are skewed in SLE
As shown in Table 1, the SLE patients and controls did not differ significantly in terms of age and gender, and the patients displayed the clinicopathological features of SLE. The relative proportion of CD19 + B cells was significantly higher in the SLE patients compared to controls (P<0.05), and slightly higher among those with active as opposed to inactive disease (Fig 1A-B). Furthermore, the SLE patients also harbored significantly expanded CD19 + IgD -CD27 -(double negative or DN) and CD19 + IgD -CD27 high plasmablast cell (PC) populations compared to the healthy controls (Fig 1C-D). Interestingly, while the overall high B cell frequency did not affect the clinical symptoms or circulating autoantibody levels in the patients (data not shown), it correlated positively with the SLEDAI score and 24-h urinary protein levels, and negatively with C3 levels (Fig 1E). In contrast, a positive correlation was seen between the frequency of PCs and the IgM and C3 levels.
Furthermore, the CD27 + class-switched memory (SM) and CD27non-switched memory (NSM) B cells respectively correlated with higher SLEDAI cores and 24h urinary protein levels. Both populations showed a significant positive correlation with IgG levels and a negative correlation with that of IgM. The naïve B cells on the other hand were negatively associated with both SLEDAI and IgG levels ( Table 2). Patients exhibiting the malar rash and positive for anti-histones and anti-SSA52 antibodies showed an increased proportion of both SM and naïve B cells whereas the presence of only anti-SSB and anti-SSA52 antibodies correlated with an increase in the NSM population (Table 3). The other B cell subsets however did not show any significant association with the clinical and biochemical indices of SLE (Table 3).

B cells of SLE patients express PD-1 and correlate with the clinical progression
The frequency of the PD-1 + B cells was significantly higher in the SLE patients compared to the healthy controls, as well as in the patients with active as opposed to inactive disease ( Fig. 2A-B). Furthermore, PD-1 was overexpressed on all B cell subpopulations in SLE patients ( Fig. 2C-D). The expanded PD-1 + B cell population in the patients was associated with increased SLEDAI scores, as well as higher 24-h urinary protein levels. In addition, the serum levels of IgG and IgM respectively correlated positively and negatively with these cells (Fig. 2E). The frequency of PD-1 + B cells was significantly higher in patients positive for the anti-dsDNA (P = 0.040), anti-histone (P = 0.025) and anti-SSA52 (P = 0.048) antibodies, and those presenting lupus nephritis (P <0.0001) and oral ulcers (P = 0.05). In contrast, no significant association was observed between PD-1 + B cells and the hematological manifestations of SLE, arthritis or serositis (Table 4). We also analyzed the clinical significance of the distinct B-cell subsets expressing PD-1 (Table 5), and found that the PD-1 + PCs correlated positively associated with SLEDAI scores, 24-h urinary protein secretion and IgG levels, PD-1 + SM B cells with SLEDAI scores and 24-h urinary protein levels, and the PD-1 + NSM and naive B cells with only IgG levels ( Fig. 2F). Based on these findings, we surmised that the PD-1-expressing B cells are the effectors of SLE progression.

PD-1 + B cells from SLE patients secrete large amounts of autoantibodies
To validate the above hypothesis, we isolated primary PD-1 + and PD-1 -B cells from the SLE patients and controls, and cultured them in vitro in the presence of CpG DNA. As shown in Fig. 3A-B, the PD-1 + B cells from SLE patients were highly responsive to CpG DNA stimulation and showed markedly higher proliferation rates compared to the PD-1 + B cells from healthy controls, as well as the PD-1 -B cells isolated from SLE patients or controls. In Although the above findings clearly implicate PD-1 signaling in B cell survival and function, its potential role in SLE is largely unknown. We detected a substantial CD19 + PD-1 + B cell population in the SLE patients, which correlated significantly with disease severity, inflammation and high levels of circulating autoantibodies. In vitro expansion of these cells was also associated with increased proliferation and secretion of IgG and anti-dsDNA antibodies. Contradictory to a previous study that reported an inhibitory effect of PD-1 on B cell activation 45 , our findings indicate that an aberrant PD-1-expressing B cell subset is the likely autoimmune effector in SLE. It is possible that the abnormal activation of these auto-reactive B cells is due to certain SLE-related pathological factors rather than PD-1, wherein the latter is merely a marker of this population and not functionally relevant. Furthermore, PD-1 might be upregulated on the B cells following their activation and in fact exert an inhibitory effect via negative feedback. A previous study showed elevated PD-1 on the IgM + IgD + CD27 + memory B cells as opposed to the naïve and SM populations 43 . Although all B-cell subpopulations in the SLE patients of our cohort overexpressed PD-1, only some of these subsets were associated with autoantibody production and clinical parameters. The mechanism underlying PD-1 overexpression in the autoreactive B cells, the functional importance of specific PD-1 + B cell subsets in SLE, and the potential interactions between the PD-1 + B cells and T cells remain to be elucidated.

Conclusions
To summarize, the B cell phenotypes and PD-1 expression pattern are skewed in SLE patients, and the expanded CD19 + PD-1 + population is primarily associated with the pathological changes in SLE.

Consent for publication
The consent to publish has been acquired from each patient at the beginning of study.

Availability of data and material
The data are owned by Changhao Xie. All data are available from the corresponding author on reasonable request.

Competing interests
The authors declare no financial interests.