CD8⁺ T cells and pDCs play a dominant role to promote allograft long-term survival.

<p>The purified CD4⁺ T cells (5×10⁶), CD8⁺ T cells (5×10⁶), pDCs (5×10⁵) and total splenotytes (5×10⁶) originated from recipients with long-term allograft survival were then adoptively transferred into naive recipients respectively. One day after the adoptive transfer, the mice were transplanted with cardiac allograft. The survival time of cardiac allografts was monitored. The data shown are representative of three independent experiments (*P<0.05).</p

Flt3L combined with Rapa promotes the production of CD4⁺CD25⁺Foxp3⁺ and CD8⁺CD25⁺Foxp3⁺ T cells.

<p>Spleen cells isolated from mice treated with Flt3L/Rapa and other control mice at the time of rejection or at study endpoint (POD 100) were analyzed to determine the proportion of CD4⁺ CD25⁺ Foxp3⁺ and CD8⁺ CD25⁺ Foxp3⁺ T cells. (A, B) The expression of Foxp3 by CD4⁺ CD25⁺ and CD8⁺ CD25⁺ T cells was analyzed by flow cytometry after intracellular staining. The bar graph was a summary of percentages of CD4⁺ CD25⁺ Foxp3⁺ T cells and CD8⁺ CD25⁺ Foxp3⁺ T cells in the recipients. The data shown are representative of three independent experiments that yielded comparable results (* <i>P</i><0.05).</p

Flt3L combined with low-dose of Rapa prolongs cardiac allograft survival and attenuates infiltrates in the graft.

<p>(A) Heart allograft survival. The cardiac allograft survival time in recipients treated with Flt3L (10 µg/day, i.v.) and low-dose of Rapa (2 mg/kg/day; p.o, POD0-15) was significant longer than those of the untreated and monotherapy groups (n = 10) (* <i>P</i><0.05, combination therapy group vs. other groups). (B) Histology of cardiac allografts. Grafts were harvested at the time of rejection or at study endpoint (POD 100) and evaluated by H&E staining of paraffin sections (original magnification, ×200). The results are representative of three independently performed experiments.</p

Flt3L/Rapa therapy favors anergic induction in alloreactive T cells.

<p>(A) Splenic T cells originated from cardiac allograft recipient mice at the time of rejection or at study endpoint (POD 100) were used as responder cells for MLR assay with donor-derived DCs (DC: T ratio of 1∶5). Bar graph was the summary of proliferation index (PI) of T cells isolated from different recipients. (B) The culture supernatants were harvested and cytokine IL-10 levels were quantification by ELISA. The data shown are representative of three independent experiments (*<i>P</i><0.05).</p

Treatment with Flt3L/Rapa enhances the autophagy in the cardiac allograft.

<p>(A, B) The cytoplasmic extracts of cardiac allograft tissue were subjected to Western blotting for evaluating the expression level of Beclin-1 and LC3B, Immunohistochemistry was conducted for examining the expression of Beclin-1 by the cardiac allograft of each group (original magnification, ×200). (C) Heart allograft survival. The cardiac allograft survival time in recipients treated with Flt3L/Rapa and 3-MA (24 mg/kg, i.p, 5, 10, 15 days) was significant shorter than that of the recipients treated with Flt3L/Rapa (* <i>P</i><0.05). (D) The percentage of pDC, CD8a⁺ DC, CD4⁺ CD25⁺ Foxp3⁺ and CD8⁺ CD25⁺ Foxp3⁺ T cells was assessed by FACS analysis (* <i>P</i><0.05, combination therapy group vs. other groups at endpoint). All data presented here are representative of three separate experiments with consistent results.</p

DCs from recipients treated with Flt3L/Rapa display a tolerogenic phenotype.

<p>Spleen cells isolated from mice treated with Flt3L/Rapa and other control mice at the time of rejection or at study endpoint (POD 100) were stained with PE-cy7-labeled anti-mouse CD11c mAb, Per-cy5.5-labeled anti-mouse B220 mAb, APC-cy7-labeled anti-mouse CD8a mAb, FITC-labeled anti-mouse CCR9 mAb, APC-labeled anti-mouse CD80 mAb and APC-labeled anti-mouse CD40 mAb. CD11c⁺ and CD11c⁺ B220⁺ gated DCs were analyzed by flow cytometry for expression of the various DC markers. (A) The bar graph was a summary of percentages of pDC and CD8a⁺ DC in the recipients. (B) CD11c⁺-gated DCs were analyzed for expression of costimulatory markers CD80 and CD40 to assess their maturation state. (C) The percentage of CCR9⁺ cell of gated pDC subsets was assessed by FACS analysis. The results are representative of three independently performed experiments (* P<0.05).</p

Correlation of PCT with CRE and Uric acid.

<p>Spearman correlation test was performed to analyze the correlation between serum PCT and CRE, uric acid levels in GA patients (n = 51). PCT was not correlated neither with CRE (r = 0.07, <i>p</i> = 0.63) nor Uric acid (r = 0.15, <i>p</i> = 0.30).</p

PCT is highly expressed in GA in comparison to RA, AS and Healthy control.

<p>(A) The comparison of serum PCT in GA (n = 51), RA (n = 37), AS (n = 41) and healthy control (n = 33) were determined by Mann–Whitney U-test. In GA patients PCT was significantly higher level observed than in RA (p = 0.002), AS (p = 0.0007) and healthy control (p<0.0001), whilst no significant differences in between RA and AS were observed. (B) The GA patients were divided into GA (tophi) (n = 29) and GA (none tophi) (n = 22) which indicates GA patients with tophi or not respectively. GA Serum PCT level was compared among these groups.</p

Correlation between PCT and ASDAS, CRP, ESR and WBC.

<p>Correlation of the serum levels of PCT with ASDAS, CRP, ESR and WBC was evaluated by Spearman correlation test in AS patients (n = 41). PCT was significantly positively correlated with CRP (P = 0.001). No correlation between PCT and ASDAS, ESR and WBC were detected.</p

Positive correlation of serum PCT with VAS, CRP, ESR, and WBC in GA patients.

<p>The determination of linear relationships between PCT and CRP, ESR, VAS and WBC in GA patients (n = 51) was performed by Spearman correlation coefficient. PCT is significantly positively correlated with VAS (r = 0.39; <i>p</i> = 0.004), CRP (r = 0.52; <i>p</i>< 0.0001), ESR (r = 0.28; <i>p</i> = 0.045). There was no association between PCT and WBC.</p