Background::One of the significant challenges for cell therapies, such as chimeric antigen receptor (CAR)-T cell therapy, is the poor infiltration of immune cells into tumor tissues. CAR-monocytes/macrophages (CAR-M) are promising therapies because of their enrichment in the tumor microenvironment. Thus, we constructed a novel CAR-M to facilitate the infiltration of T cells and other immune cells.Methods::The suicide gene inducible caspase-9 ( iCasp9) and anti-erb-b2 receptor tyrosine kinase 2 (HER2) CAR elements were transfected into THP1 (an immortalized human monocyte cell line) by lentivirus. The suicide efficiency and specific anti-tumor efficacy were assessed using flow cytometry, inCucyte, and tumor-bearing BALB/c-nude mouse models. The activation of related signaling pathways in CAR-THP1 activation was explored by transcriptome sequencing. Finally, the synergistic therapeutic efficacy of CAR-THP1 combined with RAK cell treatment was demonstrated in tumor-bearing NOD.CB17-Prkdc scid Il2rg tm1/Bcgen mouse models. Results::We developed a novel CAR-THP1, which incorporated iCasp9, CD3ζ, and CD147 intracellular segments, based on the first-generation HER2-CAR backbone. By constructing and comparing a series of CARs with different permutations, CAR-CD3ζ-CD147-iCasp9-THP1 was selected as the optimal combination. CAR-CD3ζ-CD147-iCasp9-THP1 initiated suicide quickly and efficiently under the control of iCasp9 gene, which enabled us to achieve controlled proliferation of CAR-THP1. CAR-THP1 also exhibited robust specific anti-tumor efficacy independently of T cells in vitro and in vivo. Through transcriptional sequencing, we found that CAR-THP1 tended to differentiate into the M1 phenotype and bridged innate and adaptive immunity. A combination of CAR-THP1 and Retronectin actived killer cells (RAKs) showed better therapeutic efficiency, as the metalloproteinases (MMPs) secreted by CAR-THP1 facilitated the degradation of the dense tumor matrix. This further assisted intratumoral infiltration of T cells and augmented the anti-tumor immune response. Conclusion::CAR-THP1 might be effective against HER2-positive tumor cells and has great potential for combination therapy with other immune cells.

Objective To evaluate the function of interleukin-35 (IL-35)-producing regulatory T cells (IL-35-Treg) in regulating intestinal inflammatory immune response. Methods The percentages and characteristics of IL-35-Treg in the intestinal lamina propria of transgenic mice expressing IL-35 were ana-lyzed by flow cytometry. A mouse model of inflammatory bowel disease ( IBD) was established by giving 1. 5％ DSS in drinking water. Influences of IL-35-Treg depletion on mouse weight, pathological injury and the secretion of IFN-γ were analyzed. Results IL-35-Treg were enriched in the intestinal lamina propria, and mainly derived from thymic Treg (tTreg). Intestinal IL-35-Treg expressed high levels of programmed death 1 (PD-1). Depletion of IL-35-Treg in mice with DSS-induced IBD resulted in an aggravation through up-regulating the expression of IFN-γ. Conclusion IL-35-Treg might play an important role in the regula-tion of intestinal inflammatory immune response.

Objective: To investigate the expression of granulocyte-colony stimulating factor receptor (G-CSFR) in a mouse model of colitis-associated cancer (CAC), and the roles of G-CSFR positive immune cells in the development of CAC. Methods: The C57BL/6 mouse model of CAC was established by azoxymethane and dextran sulphate sodium. Three different stages in the development of CAC, including inflammation (AD1), mild dysplasia (AD2) and adenocarcinoma (AD3) were simulated. Colon tissue was digested into single cell suspension and the expressions of G-CSF and G-CSFR were analyzed by real-time PCR and fluorescence activated cell sorter (FACS). The expressions of G-CSFR on T cell, macrophage and neutrophil were analyzed by FACS. Results: The establishment of mouse model can effectively simulate the disease progression of CAC. The results of real-time PCR detection showed that the expression level of G-CSF mRNA in AD1, AD2 and AD3 groups were 1.2, 7.3 and 18.0-fold changes of the control group, respectively. The differences between AD2, AD3 and control groups were statistically significant (P<0.05). G-CSFR mRNA levels in AD1, AD2 and AD3 groups were 1.5, 2.2 and 4.5-fold changes of the control group, respectively. The difference between AD3 and control groups was statistically significant (P<0.05). FACS showed that the percentages of CD45⁺ G-CSFR⁺ cells in colorectal tissues of the control group, AD1, AD2 and AD3 groups were (21.84±1.77)%, (41.48±4.15)%, (44.84±8.54)% and (57.76±1.95)%, respectively.The percentages of CD45⁺ G-CSFR⁺ cells in AD2 and AD3 groups were significantly higher than that of control group (P<0.05). The percentages of CD45⁺ G-CSFR⁺ macrophage in the colorectal tissues of the control group, AD1, AD2 and AD3 groups were (21.54±5.88)%, (47.14±5.25)%, (42.49±7.80)% and (29.25±8.24)%, respectively. The percentages of CD45⁺ G-CSFR⁺ T cells in these groups were (30.04±6.87)%, (29.65±8.08)%, (33.75±7.37)% and (33.32±9.85)%, respectively. The percentages of CD45⁺ G-CSFR⁺ granulocyte were (2.39±2.10)%, (4.05±1.56)%, (3.62±2.67)% and (2.26±0.85)%, respectively (P<0.05). The percentages of G-CSFR⁺ macrophage and G-CSFR⁺ T cells were significantly higher than that of G-CSFR⁺ granulocyte (P<0.05). The differences between AD1 and control group, AD2 and control group, AD1 and AD2 group, AD2 and AD3 group were statistically significant (P<0.05). Conclusions The expression of G-CSFR is significantly up-regulated in the development of CAC. The enrichment of G-CSFR⁺ macrophages in the colon tissue suggests G-CSFR⁺ macrophages participate in the development of CAC.