Introduction
Type 1 diabetes is a multifactorial autoimmune disorder triggered by islet antigen-specific CD4
+ and CD8
+ T cell-mediated destruction of insulin-producing cells in the pancreas. Although the presence of various autoantibodies can predict the onset of type 1 diabetes, it remains unclear if the corresponding autoreactive B cells play a determinative role in the underlying immunopathology. Clinical studies have shown that B cell depletion with rituximab temporarily slows the decline of C-peptide levels in the blood of individuals with type 1 diabetes [
1]. Moreover, B cells are present in the pancreatic islets at the time of diagnosis [
2] and persist in situ throughout the course of disease [
3].
A previous study identified transitional CD27
−IgD
+IgM
− B cell expansions in individuals with type 1 diabetes and healthy carriers of the
PTPN22 genetic variant 1858T, which predisposes to a variety of autoimmune disorders [
4]. In contrast, no disease-specific alterations in the B cell compartment were detected in another study, designed to quantify the expression levels of CD19, CD24, CD27, CD38, IgD and IgM in individuals with type 1 diabetes and age- and sex-matched healthy donors [
5]. Equivalent results were obtained in a comprehensive analysis of children with newly diagnosed type 1 diabetes compared with healthy control individuals [
6]. However, increased frequencies of marginal zone CD19
+CD21
+CD23
− B cells and decreased frequencies of regulatory CD1d
+CD5
+CD19
+ and follicular CD19
+CD21
−CD23
+ B cells have been reported in Chinese individuals with type 1 diabetes [
7]. Similarly, decreased frequencies of CD40
+ and interleukin (IL)-10
+ B cells were detected in another cohort of individuals with type 1 diabetes relative to healthy donors [
8]. In addition, high-affinity insulin-binding naive B cells are lost from the anergic compartment in individuals with newly diagnosed type 1 diabetes, but return in individuals with long-standing type 1 diabetes [
9]. Thus, whilst differences are present in those with type 1 diabetes compared with healthy individuals, a consistent disease-relevant phenotype in the circulating B cell pool has not been delineated.
To inform this ongoing debate, we conducted an extensive flow cytometric analysis of B cell subsets in individuals with type 1 diabetes and age- and sex-matched healthy donors.
Discussion
The generation of memory B cells from naive precursors is critical for the induction and maintenance of protective antibody responses to infectious agents [
17]. Substantial phenotypic heterogeneity exists among memory B cells, and in various autoimmune conditions, such as rheumatoid arthritis and systemic lupus erythematosus, altered subset profiles correlate with disease activity [
18]. However, few such associations have been described in individuals with type 1 diabetes.
In this study, we examined the phenotypic characteristics of naive and memory B cells in adults with type 1 diabetes and age- and sex-matched healthy donors. Our data showed that CXCR3 expression was reduced on memory B cells in individuals with long-standing diabetes. These changes were associated with raised serum concentrations of BAFF and the CXCR3 ligands CXCL10 and CXCL11. In line with previous studies, we also found that CXCR3 expression was reduced on CD3
+ T cells in individuals with long-standing diabetes [
19,
20].
Although the changes in CXCR3 expression were only significant in one of the newly diagnosed cohorts, they showed the same downward trend that we observed in the long-standing diabetes cohorts, along with the same significant increases in serum levels of CXCL10 and CXCL11. We therefore suggest that the lack of statistical significance in one of the newly diagnosed cohorts was due to a small sample size, rather than a change in CXCR3 expression limited to individuals with long-standing diabetes.
CXCR3 is constitutively expressed or readily upregulated on a substantial proportion of memory B cells [
21,
22]. In contrast to individuals with rheumatoid arthritis or systemic lupus erythematosus [
23], we found that expression levels of CD24 and CXCR3 on switched CD21
+CD24
+CD27
+CD38
intCD95
+IgD
− memory B cells were reduced in individuals with long-standing diabetes. These cells are analogous to a highly activated population of CD21
+CD24
+CD27
+CD38
+CD95
+CXCR3
+IgD
− B cells, in which decreased expression levels of CD21 and increased expression levels of CD95 and CXCR3 correlate with activation status [
18]. Moreover, they are clearly distinct from regulatory CD27
−CD24
hiCD38
hi B cells, which are defective in immune-deficient and other autoimmune conditions [
5,
24,
25]. It is also notable that we did not detect increased expression levels of CD95 on switched memory B cells, as described previously in individuals with rheumatoid arthritis [
26]. Instead, we found that CD95 expression was reduced on switched CD27
+IgD
− memory B cells in individuals with long-standing diabetes, consistent with the findings of a recent study by Hanley and colleagues, who also reported lower frequencies of CD24
++CD38
++ B cells in people with type 1 diabetes [
27]. This latter phenotype is indicative of regulatory B cells [
28], but does not match the profiles we identified in areas 2 and 6 of the SPADE tree, where reduced expression of CD24 was apparent in newly diagnosed individuals and those with long-standing diabetes. However, it has been reported that people with asthma have decreased percentages of CD24
+CD27
+ B cells, which are required for the induction of IL10
+ T cells [
29]. Phenotypically, these cells would be represented in area 2 of the B cell SPADE tree, although the physiological function of the decrease in CD24 expression that we observed is currently unknown.
Serum concentrations of CXCL10 are known to be elevated in individuals with recent-onset type 1 diabetes [
30‐
32], although there has been one report of decreased CXCL10 levels in children with type 1 diabetes [
33] (reviewed in [
34]). In a longitudinal study of children enrolled at diagnosis of type 1 diabetes, significantly higher CXCL10 levels were found in these participants compared with healthy control individuals [
30]. Serum CXCL10 concentrations remained elevated at 1 month post diagnosis and then declined over the next 9-–30 months, persisting at levels above those detected in healthy control individuals [
30]. Another study demonstrated that CXCL10 levels increase over time in at-risk individuals, peaking as they become seropositive for islet autoantibodies, irrespective of further progression to clinical type 1 diabetes [
35]. In line with these earlier reports, we found increased serum concentrations of CXCL10 in adults with newly diagnosed type 1 diabetes and slightly lower levels in adults with long-standing type 1 diabetes (but elevated compared with healthy control individuals). We further demonstrated increased serum levels of CXCL11 in adults with type 1 diabetes, irrespective of time from diagnosis. Decreased expression levels of CXCR3 on memory B cells in individuals with type 1 diabetes may therefore reflect ligand-induced receptor internalisation and/or downregulation [
36]. However, it has also been shown that reduced expression of CXCR3 can lead to increased ligand concentrations, as the receptor has a scavenger function [
37]. An alternative possibility is that memory CXCR3
hi B cells traffic to the pancreas or pancreatic lymph nodes in individuals with type 1 diabetes, as shown previously for memory T cells [
38,
39]. A similar phenomenon may explain why circulating switched memory B cells in individuals with type 1 diabetes also express lower levels of the adhesion molecule CD24.
CXCR3 is widely expressed in the CD3
+ compartment, especially among T helper type 1-polarised CD4
+ and effector CD8
+ T cells [
40]. In line with previous studies [
19,
20], we found reduced expression levels of CXCR3 on circulating CD3
+ T cells in individuals with type 1 diabetes. These findings are compatible with disease-associated trafficking of CD3
+CXCR3
hi T cells to the pancreas [
19,
20,
33]. In addition, we observed a simultaneous decrease in CD38 expression on a proportion of these CD3
+ T cells. High levels of CD38 have been described on a subpopulation of peripheral CD4
+CD25
+CD127
dim regulatory T cells, which are sensitive to therapeutic intervention with the CD38-specific monoclonal antibody daratumumab in individuals with multiple myeloma [
41]. It has also been reported that CD8
+CXCR3
+ T cells exert regulatory functions in humans [
42]. Alternatively, the CD27
+CD38
+CD95
+CXCR3
+ T cell population found in area 4 may represent a stem cell-like memory (T
SCM) subset [
43]. It is notable in this regard that autoantibodies specific for CD38 have been detected in individuals with type 1 diabetes [
44].
In addition to CXCL10 and CXCL11, our analysis of serum chemokines and cytokines revealed elevated concentrations of BAFF in individuals with long-standing diabetes. This cytokine plays a key role in the development of diabetes in NOD mice [
45,
46], and previous studies have reported increased serum levels of BAFF in individuals with autoimmune thyroid diseases [
47] and rheumatoid arthritis [
48]. Decreased expression of the BAFF receptor has also been reported on B cells in children with type 1 diabetes [
15]. Intriguingly, BAFF has been shown to enhance the chemotaxis of primary human B cells in response to a variety of chemokines [
16]. A disease-relevant synergy may therefore exist in the setting of type 1 diabetes among raised levels of BAFF, CXCL10 and CXCL11.
Future studies will focus on investigating the ability of B cells from individuals with type 1 diabetes to migrate in response to physiological levels of CXCL10 and CXCL11, in the presence and absence of BAFF. It will be important to ascertain whether abnormal blood glucose and insulin levels in individuals with type 1 diabetes contribute to the loss of CXCR3 on memory B cells by studying the effects of ex-vivo culture and by correlating HbA
1c values with chemokine and chemokine receptor expression levels. It is notable in this context that glycosylation of the extracellular domains may increase signalling via CXCR3 [
49]. A detailed study of postmortem pancreatic histological samples, available from the nPOD collection, may allow the identification of CXCR3
++ B cells that trafficked to the pancreas during life, and were thus lost from the periphery. Similarly, the source of raised CXCR3 ligands in the serum remains unknown. Although there is some evidence that the inflamed pancreas can produce CXCR3 ligands in individuals with type 1 diabetes [
39], it is also possible that these chemokines are produced in response to elevated glucose levels in the periphery, an effect that could be determined by longitudinal measurements of chemokine levels in conjunction with measurements of HbA
1c.
In conclusion, we have identified a mechanistically cohesive immune profile associated with type 1 diabetes. The key abnormalities suggest long-term disruption of a chemokine ligand/receptor system that controls B cell migration. On this basis, we propose that related parameters may find utility as cellular and/or soluble biomarkers to monitor and/or predict the development of islet-specific autoantibody responses.