We next conducted a cellular analysis to evaluate the degree of lymphocyte activation in both groups of disease-free mice (Fig. 3). Consistent with the adjuvanticity

of LPS, treated mice displayed overall increased lymphocyte number and activity in the spleen (Sp) and, more notably, in the lymph nodes draining the pancreas (pLN) (Fig. 3A). Gross subdivision of lineages as CD8 or CD4 T cells, B cells or Dendritic cells did not reveal preferential expansion (not shown and Fig. S2). Splenic B lymphocytes showed an activated phenotype as indicated by elevated MHC Class II expression (Fig. S2). Moreover, seric IgG and IgM titres were readily increased (not shown). Despite the systemic effect of long-term LPS treatment, effector CD4 T cells were not significantly affected as measured by the frequency of CD69+ or CD44high cells in spleen and pLN. Noteworthy, CD4+CD69+CD44hi cells, previously

described as primed cells enriched in diabetogenic PCI-32765 purchase effectors [10], were found in the spleen and pLN of both groups of mice (Figs. 3B and S3). Similarly, while T lymphocyte differentiation into IFN-γ-producing helper cells is essential for diabetes establishment in NOD mice [50], LPS-treated and healthy controls displayed similar frequency of CD4+IFN-γ+ cells in spleen and pLN (Figs. 3C and S3). Infiltration of CD4+IFN-γ+ selleck inhibitor cells was also detected in pancreas of healthy and LPS-protected animals (Fig. S3). Taken together these analyses indicate that LPS treatment while preventing

disease development did not induce immune paralysis, nor impaired Th1 differentiation, an effector class essential for diabetes establishment in NOD mice [50]. We conclude that LPS-induced protection in NOD mice, similarly to spontaneous protection, operated by a mechanism that did not impede pancreatic islet infiltration and effector CD4+ cell activation. We Sinomenine next assessed whether Treg undergo phenotypic modifications in vivo upon LPS treatment by analysing the frequency and numbers (Figs. 4, 5 and S4–S6) of specific CD4 cell subsets. Historically, Treg were first defined as CD4+CD25+ T cells [51]. Yet, activated conventional T cells also express CD25 in a transient manner upon activation. LPS treatment in NOD mice did not increase the frequency and number of CD4 cells expressing CD25 (Fig. 4A). While CD4+CD25+ cells expressing high levels of l-selectin have been shown to be particularly potent in preventing diabetes occurrence [18], the frequencies of CD62LhiCD4+CD25+ splenocytes in each experimental group were not significantly different (data not shown). CD103-expressing CD4+CD25+ cells display increased regulatory function in vivo [52, 53] and CD103 expression is likely a molecular signature of pancreatic Treg [10]. Strikingly, LPS-protected mice presented an increased frequency and number of CD103+CD4+CD25+ cells, both in the spleen and in pLN (Figs. 4B and S4), when compared with healthy controls.