Passive transfer of flt-3L-derived dendritic cells delays diabetes development in NOD mice and associates with early production of interleukin (IL)-4 and IL-10 in the spleen of recipient mice

Abstract

CD11c+/CD11b+dendritic cells (DC) with high levels of major histocompatibility complex (MHC) class II and co-stimulatory molecules have been derived from spleen cells cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) + flt-3L + interleukin (IL)-6 (flt-3L-DC). Investigating in vivo the function of DC in non-obese diabetic mice (NOD), we showed that a single injection of this in vitro-derived subset of DC prevents the development of diabetes into prediabetic female mice. In contrast, DC derived from bone marrow cells cultured with GM-CSF + IL-4 [bone marrow (BM)-DC] induced no protection. Moreover, protection against diabetes following injection of flt-3L-DC was associated with IL-4 and IL-10 production in the spleen and the pancreatic lymph nodes of recipient mice, indicating that this DC population is able to polarize the immune response towards a Th2 pathway. As we shown previously, NOD BM-DC exhibit an enhanced capacity to produce IL-12p70 in response to lipopolysaccharide (LPS) and anti-CD40 stimulation compared to BM-DC from control mice. In contrast, NOD flt-3L-DC, as their control mouse counterpart, produced no IL-12p70 to these stimuli. Our findings show that a subset of DC, characterized by a mature phenotype and the absence of IL-12p70 production can be derived from NOD mouse spleen favouring IL-4 and IL-10 regulatory responses and protection from diabetes development.

Fig. 1

Phenotype of NOD flt-3L-DC. Bulk culture of flt-3L-DC (a) and BM-DC (b) or culture of B LPS from 8-week-old-NOD female mice were double-stained with anti-CD11c (a,b) or anti-B220 (c) antibodies and the indicated antibodies and analysed by flow cytometry. Histograms represent I-A, CD80, CD86, CD40, CD11b, Gr-1, B220, K^d and CD8α expression among CD11c⁺ gated cells (a,b) or I-A, CD80, CD86, CD40 expression among the B220⁺ gated cells.

Fig. 2

Transfer of flt-3L-DC protects NOD mice against diabetes development. Five-week-old NOD female mice received a single i.v. injection of 5 × 10⁵ purified (purity > 95%) BM-DC (P = 0·2387, n = 8) or flt-3L-DC (P = 0·0015, n = 7). Splenocytes from 4-week-old NOD females were used as negative control (n = 8). Kaplan–Meier actuarial graph survival and log-rank test were used for statistical analysis of diabetes development. Data shown are representative of four independent experiments (seven to 15 mice/group).

Fig. 3

Ex-vivo autologous MLR from DC-treated mice. Cells from individual DC (BM-DC and flt-3L-DC)-treated or control mice (three mice/group) were isolated from the spleen and the pancreatic LN on day 3 post-injection and cultured (3 × 10⁵ cells/0·2 ml) for 3 days in RPMI 5% FCS. Cell proliferation was measured by [³H]-Tdr incorporation during the last 16 h of culture. Data represent cpm/culture × 10⁻³ (mean ± s.d.).

Fig. 4

Transient increase of autologous MLR following flt-3L-DC injection. Cells from individual flt-3L-DC-treated or control mice (three mice/group) were isolated on day 3 (a) or day 7 (b,c) post-injection and cultured (3 × 10⁵ cells/0·2 ml) for 3 days in RPMI 5% FCS. On day 7, cells were also cultured in the presence of anti-CD3 antibody (1 µg/ml) (c). Cell proliferation was measured by [³H]-Tdr incorporation during the last 16 h of culture. Data represent cpm/culture (mean ± s.d.).