Non-human primates (NHP) provide important animal models for studies on immune responses to infections and vaccines. When assessing cellular immunity in NHP, cytokines are almost exclusively analyzed utilizing cross-reactive anti-human antibodies. The functionality of antibodies has to be empirically established for each assay/application as well as NHP species. A rational approach was employed to identify monoclonal antibodies (mAb) cross-reactive with many NHP species. Panels of new and established mAbs against human Interferon (IFN)-? and Interleukin (IL)-2 were assessed for reactivity with eukaryotically expressed recombinant IFN-? and IL-2, respectively, from Old (rhesus, cynomolgus and pigtail macaques, African green monkey, sooty mangabey and baboon) and New World NHP (Ma's night monkey, squirrel monkey and common marmoset). Pan-reactive mAbs, recognizing cytokines from all NHP species, were further analyzed in capture assays and flow cytometry with NHP peripheral blood mononuclear cells (PBMC). Pan-reactive mAb pairs for IFN-? well as IL-2 were identified and used in ELISA to measure IFN-? and IL-2, respectively, in Old and New World NHP PBMC supernatants. The same mAb pairs displayed high functionality in ELISpot and FluoroSpot for the measurement of antigen-specific IFN-? and IL-2 responses using cynomolgus PBMC. Functionality of pan-reactive mAbs in flow cytometry was also verified with cynomolgus PBMC. The development of well-defined immunoassays functional with a panel of NHP species facilitates improved analyses of cellular immunity and enables inclusion in multiplex cytokine assays intended for a variety of NHP.
In this study, we sought out to study the anti-cancer effects of propranolol (Pro) in combination with tumor lysate vaccine on lymphocyte proliferation activities as well as on IL-2, IL-4, IL-10, IL-12, IL-17 and IFN-? cytokine concentration in the tumor microenvironment (TME). A tumor model was established in inbred Balb/C mice using transplantation of tumor to the flank of native mice. Tumor-bearing mice were immunized with lysate tumor cells (vaccine), a combination of Pro/Vaccine (Vac). Control groups consisted of tumor-bearing mice receiving only propranolol or PBS three times with one week interval via subcutaneous (s.c) injection. One week after the last immunization, tumor was removed, homogenate was prepared and the levels of IL-12, IL-17, 1L-2, IL-10, IL-4, IFN-? cytokine concentrations were evaluated by commercial ELISA kits. In addition, spleen cell suspension was used for the lymphocyte proliferation assay using the BrdU method. Results from this study indicated that Pro/Vac had the ability to significantly increase lymphocyte proliferation, and to suppress tumor growth. Administration of breast tumor lysates with propranolol increased the concentration of IL-12, IL-17, 1L-2 and IFN-? cytokines in tumor microenvironment. This study has proved the efficiency of propranolol as an adjuvant in combination with the tumor vaccine model on tumor suppression via cytokine pattern modulation in tumor microenvironment.
