Loss of tolerance to self-antigens could be accounted for by the loss of suppressive mechanisms and/or a gain of effector function. The CD4+CD25+Foxp3+ population contains both suppressive and effector cell types, and the relative balance between these subsets in health and disease remains unclear (17
). Here, we reveal functional differences within the CD4+CD25+ compartment in children with newly diagnosed type 1 diabetes. Two independent variables were identified: a stable increase in CD4+CD25+ proliferative capacity and cytokine production—IL-17 and TNF-α—and a transient loss of suppressive function during the first 9 months from diagnosis. Thus, recent onset type 1 diabetes is marked by increased effector function within the CD4+CD25+ compartment but without lasting disruption of Treg function.
Future studies on the functional heterogeneity of the CD4+CD25+Foxp3+ compartment will be aided by phenotypic definitions of regulatory and nonregulatory cells such as those based on CD45RA/CD25 (17
), CCR6 (24
), and HLA-DR/CD25 (18
). We did not see changes in the frequency of putative nonregulatory CD45RA−CD25+Foxp3lo
cells or HLA-DR− cells, including additional multiple phenotypic subset analyses. Because we detected no change in the absolute numbers of CD4+CD25hi
, we speculate that the functional makeup of that subset may change either in suppressive capabilities or in the ability to traffic to inflammatory sites. Phenotypic heterogeneity may be more evident in an islet-specific population and/or within the pancreas itself. It is interesting to note that changes in number or function of circulating CD25+ subsets in autoimmunity have mainly been seen during acute episodes or flares of autoimmune activity in systemic lupus erythematosus, multiple sclerosis, and rheumatoid arthritis. In type 1 diabetes onset, there may simply not be acute immune alterations, or these may predate clinical manifestations. Although our studies in type 1 diabetes did not reveal differences in individual subsets between diabetic and healthy control subjects, the enhanced degree of heterogeneity within the CD4+CD25hi
compartment (Supplementary Fig. 4
) was notable and appears to reflect the more complex nature of the phenotype of the CD45RA−Foxp3hi
activated Tregs. The functional significance of this phenotypic heterogeneity deserves further study.
Most striking were our observations that the CD4+CD25+ compartment from type 1 diabetic subjects had increased proliferative capacity that correlated with increases in select cytokines—IL-17 and TNF-α but not IFN-γ. Unlike healthy control subjects, this change in function was uncoupled from suppressive activity. IL-17 producers have been implicated in the pathology of a number of autoimmune diseases (40
), perhaps reflecting their relative resistance to Treg suppression (41
). We find that IL-17 and TNF-α from healthy control subject CD4+CD25− targets can be readily inhibited by type 1 diabetic CD4+CD25+ cells (), suggesting that IL-17/TNF-α producers within the CD4+CD25+ population in type 1 diabetes are particularly resistant to suppression. Thus, in type 1 diabetes, as disease progresses, there may emerge distinct effector populations, such as IL-17/TNF-α producers, that may be less effectively controlled by Tregs. Future studies determining the stability of Foxp3 expression, using measures of Foxp3 methylation, and its relationship to IL-17 production in the type 1 diabetic CD4+CD25+ population will be important in light of current discussions on functional plasticity (43
). IL-17 is the subject of intensifying study in type 1 diabetes (37
). Indeed, anti–IL-17 treatment of older NOD mice with established insulitis, but not young mice prior to insulitis, prevented diabetes (44
). It will be important to determine when these cytokine-producing cells emerge during type 1 diabetes development and also whether they are enriched for distinct islet specificities.
In most settings, the production of IL-17 and suppressive function are mutually exclusive (45
). The generation of induced Tregs and Th17 subsets is driven by shared factors, such as transforming growth factor-β, but regulated by distinct cofactors (46
). Alternatively, clonal analysis suggests that CD4+CD25+ cells can be functionally flexible, producing IL-17 or being suppressive, depending on the cytokine milieu (18
). The inflammatory cytokines IL-1β and IL-6 have been shown to potentiate IL-17 production (18
). It is interesting to note that IL-1β and IL-6 appear elevated in monocytes from type 1 diabetic subjects (48
), and we also found elevated IL-6 in cocultures with type 1 diabetic CD4+CD25+ cells and control targets and APCs (data not shown). Therefore, in type 1 diabetes, these IL-17 producers could originate from a distinct nonregulatory anti-islet effector subset or be more easily induced from a previously suppressive population. Our investigations uniquely identify that the elevated IL-17 and TNF-α production associated with type 1 diabetic CD4+CD25+ cells was not associated with a concomitant loss in suppression of CD4+CD25− target proliferation or cytokine production.
Our study reveals a novel functional dysregulation in the CD4+CD25+ compartment in children with type 1 diabetes. The type 1 diabetic CD4+CD25+ population not only contains cells with the ability to suppress proliferation and cytokine production but also contains cells with heightened capacity to produce specific proinflammatory cytokines IL-17 and TNF-α. This prospective longitudinal study reveals a temporal change from diagnosis in Treg control of CD4+ T–cell proliferation. Children had normal regulatory function at diabetes onset, which declined between 2 weeks and 6 months and returned to normal levels by 9 months (). This timeframe parallels the clinical remission in type 1 diabetes, which is a period that follows diagnosis by a few weeks and is denoted by a decline in HbA1c
that lasts through 9–12 months (49
) (). The underlying mechanism of remission remains unknown. A loss of regulatory function during remission appears counterintuitive to an improvement in β-cell function if these changes in immunity are indeed causally linked to the metabolic changes (33
). There are a number of ways in which this apparent discrepancy could be explained and tested experimentally. Changes in the peripheral blood may reflect a temporary but beneficial mobilization of Tregs to the pancreas, as speculated by others (39
), possibly in response to the alleviation of metabolic stress after insulin treatment. Although we were unable to find differences in surface marker expression, the loss of regulatory function in the CD4+CD25+ compartment could still reflect a change in the effector/regulatory balance in favor of effector T cells. This might be explained by a change in antigen presenting function in the less metabolically stressed pancreas that could change the sequestration of effectors in the tissue. Alternatively, although the kinetics of metabolic and immune changes appear synchronous, they may not be synchronous in their downstream effects. Thus, the transient decline in Treg function during remission may precede, or predict, the exit from the honeymoon period. Such a scenario would fit with models of diabetes as a relapsing/remitting disease (33
) and might suggest that other waves of decline and gain in circulating Treg function may have occurred prior to clinical disease onset. Natural history studies are currently focusing on immune function prior to the final loss of β-cell function that precipitates the disease and will be essential to our understanding of disease development.
On the other hand, the metabolic and immune changes may well be distinct. Indeed, our analysis highlights that the entry into clinical remission is characteristically homogeneous within our patient group (Supplementary Fig. 2
), whereas the changes in regulatory function are variable. Some individuals exhibit no Treg defects, whereas others exhibit transient defects or have a stable Treg defect during the remission period (Supplementary Fig. 6
). Extended analysis past 12 months might reveal a linkage between these functional differences and the timing of exit from remission. However, studies of metabolic function and immunity remain challenging, as tight metabolic control is a necessary clinical goal in the management of type 1 diabetes. This necessity underscores the challenge of combining independent strategies for controlling metabolic function and anti-islet immunity (34
). Regardless, the remission period needs to be better understood from both metabolic and immune function standpoints. Despite limitations in our ability to discern this interplay, our demonstration of immune changes from diabetes onset in children with type 1 diabetes provides a platform for future immune intervention.
Understanding the developmental and functional relationship between the Tregs and nonregulatory T cells within the CD4+CD25+ compartment in type 1 diabetes, as in other autoimmune states, will inform decisions and provide insight into therapeutic strategies. Immune intervention trials for type 1 diabetes are performed, to date, in this early window of time after diagnosis. The identification of enhanced IL-17 expression within the CD4+CD25+ population could add a wrinkle to Treg-based therapy because these effector cells could be preferentially expanded in cultures of type 1 diabetic CD4+CD25+ T cells and may also be relatively resistant to Treg control. In a similar manner, the identification of some patients with transient decreases in Treg activity in a defined window 3–6 months postdiagnosis could alter the efficacy of certain immune interventions in these particular individuals. In conclusion, our results provide new insight into the basal immune activity and kinetic changes within the CD4+CD25+ compartment of children during the first year of type 1 diabetes.