| Part 1 Study on immunopathogenesis of mycobacterial infectionObjective To investigate the immune responses of lymphocyte popμlations during mycobacterial infection in a non-human primate model. Methods Systemic BCG infection introduced by intraveneous challenge. Whole blood was collected from the infected macaques over time. Lymphocytes in pμlmonary alveoli were obtained from bronchial alveoli lavage (BAL) fluid and BAL was performed on selected time points. The whole blood staining, cell staining with BAL fluid, enzyme-linked immunospot assay (ELISPOT), intracellμlar cytokine staining (ICS) and BCG CFU counting in the peripheral blood were employed to detect the numbers and function of T lymphocytes in the macaque model during mycobacterial infection. ResμIts The percentage of CD3+CD4+ and CD3+CD8+ cells and their subsets in CD3+ T cells in both peripheral blood and pμlmonary compartment kept similar during mycobacteral infection. But the PPD-specific CD3+CD4+ and CD3+CD8+ cells increased significantly, which lasted for six weeks. Importantly, the systemic infection resμlted in predominant increases in numbers and effector memory subset of Vγ2Vδ2＋T cells and peaked on 3-4 weeks after infection. The response of Vγ2Vδ2＋T cells to the stimμlation of mycobacterial specific antigen, HMBPP, increased dramatically, which lasted up to 10 weeks. Meanwhile, the BCG CFU in the peripheral blood decreased to undetectable from week 3 after infection.Conclusion CD3+CD4+, CD3+CD8+ and Vγ2Vδ2+ T cells may all contribute to the immune protection against the mycobacterial infection. Vγ2Vδ2+ T cells which expanded and activated significantly during BCG infection probably play a key role in the protective immune response against the infection. Part 2 Study on immune pharma-kinetics of HMBPPObjective To investigate the immune pharma-kinetics of HMBPP (4-hydroxy-3-methyl-but-2-enyl-pyrophosphate) using a non-human primate model. Methods Peripheral blood lymphocytes (PBL) were isolated from the blood of untreated macaques and stimμlated with HMBPP in vitro to test the proliferation of Vγ2Vδ2+ T cells. Then the macaques were divided into 4 groups: control, HMBPP-treated, IL-2-treated and HMBPP＋IL-2 cotreated. The animals were treated with saline intramuscμlarly only, or HMBPP intramuscμlarly and/or IL-2 subcutaneously, respectively. Whole blood and BAL fluid were collected from the macaques over time The whole blood staining, cell staining with BAL fluid, ELISPOT and ICS were employed to detect the responses of T lymphocytes in the treated macaque model. ResμIts Both HMBPP in vitro stimμlation and HMBPP＋IL-2 co-treatment in vivo induced the expansion of Vγ2Vδ2＋T cells in both peripheral blood and pμlmonary compartment. Importantly, HMBPP-specific Vγ2Vδ2＋T cells increased dramatically in HMBPP+IL-2 group, but there were no changes of PPD-specific CD3+CD4+ and CD3+CD8+ cells in this group. Conclusion HMBPP and IL-2 co-treatment coμld induce the expansion and activation of Vγ2Vδ2＋T cells in vivo both in peripheral blood and pμlmonary compartment. Therefore, Vγ2Vδ2+ T cells can be artificially manipμlated in vivo using a macaque model.Keywords HMBPP, Vγ2Vδ2+ T cells, non-human primate modelPart 3 Study on immunotherapy of mycobacterial infectionObjective To investigate the immunotherapeutic effects on mycobacterial infection in a non-human primate model. Methods The macaques were divided into 4 groups: control, Picostim treated, IL-2 treated and Picostim+IL-2 cotreated. All animals were infected with BCG by intraveneous injection and treated with saline only, or Picostim and/or IL-2, respectively. Whole blood was collected from the infected macaques over time. Lymphocytes in pμlmonary alveoli were obtained from BAL fluid and BAL was performed on selected time points. The whole blood staining, cell staining with BAL fluid, ELISPOT, ICS and BCG CFU counting in the peripheral blood were employed to detect the numbers and function of T lymphocytes to evaluate the therapeutic effects during infection and treatments. ResμIts Compared with control group, the percentage of CD3+CD4+ and CD3+CD8+ cells and their subsets in CD3+ T cells in both peripheral blood and pμlmonary compartment kept similar during infection and treatment in the other 3 groups. And the PPD-specific CD3+CD4+ and CD3+CD8+ cells were similiar among 4 groups. Strikingly, Vγ2Vδ2+ T cells expanded in Picostim+IL-2 cotreated group from day 4 in both peripheral blood and pμlmonary compartment and lasted for more than 6 weeks after infection and cotreatment. Compared with control group, the expanstion of Vγ2Vδ2＋T cells appeared and peaked earlier and lasted longer in Picostim+IL-2 cotreated group and its effector memory subset was predominant. Most importantly, both IFN-γand perforin producing HMBPP-specific Vγ2Vδ2+ T cells increased dramatically and peaked much earlier in Picostim+IL-2 cotreated group than those in control group. Meanwhile, the BCG CFU counting in the peripheral blood of cotreated group was lower that other groups. Conclusion Picostim+IL-2 cotreatment coμld affect both the phenotype and function of Vγ2Vδ2+ T cells significantly which makes the antigen specific responses enhanced dramatically. It probably contributes to the control of the pathogen from the begaining of infection so that to allivate the symptoms of infection and improve the prognosis.
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