Original Study| Volume 23, ISSUE 3, e95-e102, April 2023

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Immunomodulatory Role of Diospyros peregrina Fruit Preparation in Breast Cancer by Utilizing Macrophage Mediated Antigen Presentation and T Helper Cell (Th) Differentiation

Published:January 05, 2023DOI:



      Diospyros peregrina is dioecious plant native to India and belonging to the family of Ebenaceae, is largely utilized in treatment of various ailments. Little has been known about the antitumor activity of Diospyros peregrina with only 1 previous study on Ehrlich Ascites Carcinoma in mice. Therefore, it prompted us to extensively explore the immunomodulatory effect in various cancer forms. The focal point of this study revolves around breast cancer, which is the second most common cancer in the world. In view of the increasing demands for noninvasive treatments, natural plant-based agents open up promising applications in cancer immunotherapy


      CD4+ lymphocytes were isolated from the peripheral blood mononuclear cells (PBMCs) of breast cancer patients and normal donor blood samples using magnetic-activated cell sorting (MACS) and cultured separately. Utilizing the plastic surface adherence property, the macrophages were isolated from CD4 negative lymphocytes of both breast cancer patients and normal donors. For the presentation of tumor antigens invitro, macrophages were pulsed with breast tumor associated antigen (BTAA) in presence or absence of Diospyros peregrina fruit preparation (DFP). Differentially pulsed and irradiated macrophages were co-cultured with autologous and allogenic lymphocytes. Supernatants hence collected from CD4+ lymphocytes were utilized for cytokine profiling using ELISA and proliferation was assessed by MTT assay. Cytotoxic T lymphocytes (CTLs) generated from CD4 negative lymphocytes culture (2 × 105) was incubated with MCF-7 (2 × 104) to check cytotoxicity using LDH release assay. CD4+ lymphocytes were treated in presence or absence of DFP, were analyzed using immunoblotting and RT-qPCR, to check DFP mediated T helper (Th) cell differentiation through investigation of signatory cytokines and transcription factors.


      It was found that DFP elevated the proliferation of CD4+ T lymphocytes (Th) in response to BTAA. DFP also helped in presenting BTAA pulsed macrophages directing in the cytotoxic T-lymphocyte mediated immune response. Results indicated that DFP preferentially highlighted Th1 commitment with type-1 specific cytokines IFN-g and IL-12 and was indifferent in Th2 manifestation. DFP was not only involved in the upregulation of Tbet mounted type-1 mediated immune response and activation of STAT1 but also it downregulated STAT6 and GATA3, the functional activators and regulators of type-2 immune response. Moreover, it was observed that DFP inhibited the tumor-promoting environment modulated through Tregs by downregulating Foxp3 and STAT5. Further, it was detected that DFP directs Th1 bias and results in attainment of better suppression of breast tumor


      The results collectively pointed out that DFP favored cell-mediated immune response from BTAA antigen presentation on macrophages and also helping in the robust proliferation of an entire spectrum of T helper lymphocytes which furthermore strengthen the underlying immune responses, hence, fencing the body, of the progression of breast cancer.


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