Text S1 - Phosphoantigen/IL2 Expansion and Differentiation of Vγ2Vδ2 T Cells Increase Resistance to Tuberculosis in Nonhuman Primates

Fig. S1. Picostim/IL2 treatment, while expanding Vγ2Vδ2 T cells, could confer immune resistance to TB lesions in lungs after pulmonary Mtb infection. Shown are dditional digital photos of cut sections of lung lobes from other macaques(a total of 27). Please see Fig. 2 legend in Text for detailed description of photos. Fig. S2. Shown are additional histopathology photos for other macaques from Picostim/IL-2-treated and control groups. Please see Fig. 3b legend in Text for detailed description of photos. Original magnification ×100 for all photos. Fig. S3a. Phosphoantigen-expanded Vγ2Vδ2 T cells in pulmonary compartments possess the capacity to de novo produce anti-Mtb cytokines IFNγ, perforin and granulysin without phosphoantigen HMBPP stimulation in vitro. Shown are representative flow cytometry histograms (left) of IFNγ-producing(top panels) and perforin-producing(lower panels) Vγ2Vδ2 T effector cells gated on CD3 and graph data (right) of numbers of Vγ2Vδ2 T effector cells in BALF collected overtime from Picostim/IL-2-treated and control groups. Effector cells were measured by ICS without HMBPP stimulation. See Fig. 4a legend in Text for detailed description. Fig. S3b. Additional representative in situ confocal microscopic images (63× NA) of Vγ2Vδ2 T effector cells producing perforin and granulysin in lung tissue sections from other macaques. See Fig. 4b legend in Text for detailed description. Fig. S3c. Immunohistochemistry analysis of Vγ2 T cells in lung parenchyma and granuloma tissues. Note that more Vγ2 T cells were detected in “tiny”, small and large granulomas tissues in Picostim/IL2-treated macaques than those in control IL2 alone- and saline/BSA-treated macaques. Magnifications were indicated. Immunohistochemistry analysis of Vγ2 T cells was essentially the same as previously described. Fig. S3d. Vγ2Vδ2 T effector cells that expanded and differentiated in vivo at day 14 after Picostim/IL-2 treatment could recognize Mtb-infected autologous macrophages, leading to inhibition of intracellular Mtb growth, and such inhibition could be reduced by antibodies against granulysin/perforin. Macaque PBMC frozen down at day 14 after Picostim/IL-2 treatment were cultured for 7 days in presence of HMBPP/IL2, and used to purify Vγ2Vδ2 T cells as described in Methods. Vγ2Vδ2 T cells were incubated for 4 days with autologous Mtb-infected monocytes(prepared using day 56 PBMC) at E∶T ratio of 10 in the presence of anti-perforin/granulysin Abs(see clones ID in Methods, 10 µg/ml for each) or IgG isotype control (10 ug/ml) as described in Methods. The cultured cells were lysed, and CFU counts in lysate were determined as described in Methods. N = 3. Fig. S4. Shown are SDS-PAGE and Western blot data for analysis of recombinant macaque perforin and granulaysin proteins purified from E-coli expression system [29]. See Fig. 5 legend in Text for details. Fig. S5. Picostim/IL-2 treated macaques exhibited greater numbers of IFNγ-producing CD4+ T cells (top) and CD8+ T cells(bottom) in BALF than saline/BSA-treated or IL-2-treated macaques. Effector cells were measured by direct ICS without antigen stimulation in vitro. See Fig. 6 legend in Text for details. (PDF)