Figure 7. The expression of three acute phase proteins after IL-6 neutralization. Mice were injected intraperitoneally with mouse anti-IL-6 Ab 3 and 24 h post cryo-thermal therapy and sacrificed on the 2nd day. Serum was collected for western blot analysis. Protein amount was evaluated and visualized with Ponceau S. Blots were evaluated with Quantity One 1-D. Results are expressed as relative pixel intensity normalized with control group. Data are shown as mean ± SD. ***p < 0.001 by student t test. n=3 with three technical replicates.

Figure 8. Time course verification of proteins associated with complement system and adaptive immunity using parallel reaction monitoring. The dash line represents the protein expression level in healthy mice. Data are shown as mean ± SD. **p < 0.01, ***p < 0.001 by two-way ANOVA with the Bonferroni correction. n=5~6 for each condition, except that on the 28th day, only 3 mice left in the control group.

Figure 9. The maturation of DCs and activation of CD4+CD26+ T cells. (A) The percentage of CD86+MHCII+ DCs, gated on CD11c+. Lymphocytes were isolated from spleens 1 day, 3 dayand 5 day post therapy and stained with the following Abs: anti-mouse-CD11c, anti-mouse- MHCII and anti-mouse-CD86. (B) The percentage of CD26+ T cells gated on CD3+CD4+. Lymphocytes were isolated from spleen on the 2nd day post therapy and stained with the following Abs: anti-mouse-CD3, anti-mouse-CD4 and anti-mouse-CD26. Data are shown as mean ± SD. **p < 0.01, ***p<0.001, by student t test, n=3.

In addition, CTSL was also observed up-regulated on the 5th day after therapy (Figure 8). CTSL plays important roles in antigen processing and maturation of antigen presenting cells (APCs) [52]. Such up-regulation suggested a possible maturation of APCs (like dendritic cells). Therefore, we performed flow cytometry to examine the maturation of dendritic cells in spleens of treated mice. As a result, matured DCs (CD11c+CD86+MHCII+) showed a significant increase on the 3rd day after therapy and was even higher on the 5th day, while it remained at a relative low level in the untreated mice (Figure 9A). Concomitant with the “acute” response, DPP4 (known as CD26), with a soluble form in circulating system [53], was also shown up-regulated on the 2^nd day after therapy and returned to the base level afterwards (Figure 8). CD26 is a maker of T cell activation which is highly expressed in the activated and antigen-reactive memory T cells. CD26high T cell could produce T-helper 1 (Th1) cytokines to elicit type-1 anti-tumor response (CD4+CD26+ T cell) or exert cytotoxic T lymphocyte activity (CD8+CD26+ T cell) [54-56]. The up-regulation of CD26 on the 2nd day suggested that more T memory cells were activated and proliferated upon “acute” response. To further confirm this suggestion, we performed flow cytometric analysis upon lymphocytes isolated from splenocytes 2 days post therapy. As a result, we found a strikingly increased percentage of CD4+CD26+ T cell (Figure 9B) as compared to that of untreated mice. Therefore, these results confirmed that more CD4+ memory T cells were activated and proliferated upon “acute” response. By this manner, these cells could potentially serve as Th1 cells to initiate and amplify the adaptive immunity against tumor.

Since we observed the maturation of DCs and proliferation of Th1 memory T cells, we next

examined the secretion of Th1 cytokines (IL-12 and IFN-γ) to further investigate whether protective Th1 adaptive immunity was activated under “acute” response. IL-12, naturally produced by dendritic cells, is important in directing the development of Th1 cells [57]. The source of IFN-γ is restricted to CD4+ Th1 cells, once antigen-specific immunity develops [58]. Hence, we examined their splenic expression on the 3rd day post therapy using RT-PCR. As shown in Figure 10, both IL-12 and IFN-γ exhibited the highest expression after the therapy, while in the untreated mice, their expression were inhibited compared to both healthy and treated mice. Therefore, the protective T helper type 1 immune response appeared to be activated after the therapy.