br B Proliferation of gd T cells
(B) Proliferation of gd T17 Pam3CSK4 in the lungs from healthy SPF mice, tumor-bearing SPF mice, and tumor-bearing GF mice was assessed by flow cytometric analysis of Ki67 expression.
(C) The number and IL-17 expression of gd T cells in the lungs were analyzed 36 h after local delivery of LPS and PGN.
(D) Recombinant mouse IL-1b and IL-23 were administered to healthy mice through intratracheal instillation. The number of total gdT cells and the frequency IL-17A+ or Ki67+ gdT cells were analyzed 36 h post-dosing.
See also Figure S4.
most of them did not express RORgt (Figure 4A). Vg6+ gd T cells are long-lived, tissue-resident cells that colonize the lung during embryonic development and undergo self-renewal under ho-meostatic conditions (Haas et al., 2012). Consistent with this, the IL17-producing Vg6+Vd1+ T cells that expanded in tumor-bearing lungs were highly resistant to radiation and reconstitu-tion. Twenty weeks after transplantation of lethally irradiated KP-CD45.1 mice with the bone marrow from CD45.2 donors, gd T cells were readily detected in tumor-bearing lungs. How-ever, despite the robust reconstitution of Tbet+ gd T cells or the Vg4+ gd T cell compartment by the donor bone marrow, RORgt+ gd T cells were largely of the recipient origin in these chi-meras and donor-derived Vg6+Vd1+ T cells were virtually absent (Figure S4A).
The lung-resident origin of these gd T cells strongly indicated that tumor-associated gd T expansion was largely due to local proliferation rather than recruitment from the circulation. Indeed, we observed increased proliferation of gd T cells, particularly those expressing IL-17, in tumor-bearing lungs compared to healthy lungs, which was largely abrogated in GF mice (Fig-ure 4B). By contrast, no apparent changes in the proliferation of RORgt- gd T cells or Th17 cells were observed in GF mice (Fig-ure S4B). Moreover, associated with the increased bacterial load in the lung, IL-17 production from gd T cells in the tumor-bearing lungs was substantially enhanced as compared to that from gd T cells in healthy lungs (Figure S4C). Reducing the commensal mi-crobiota with the 4Abx cocktail dramatically decreased the abundance of gd T17 cells in the tumor-bearing lung, resulting
in lower IL-17A levels in the BALF or serum (Figure S4D). In the GF reconstitution experiment described above, we found that microbiome exposure robustly restored the gd T17 cell compart-ment in ex-GF mice (Figure S4E). Further supporting a model in which tumor-associated increase in the local bacterial burden in-duces proliferation and activation of tissue-resident gd T17 cells, local administration of TLR ligands (such as lipopolysaccharide, LPS, and peptidoglycan, PGN) triggered the expansion and IL-17 production of gd T cells in the lung (Figure 4C). In addition, direct stimulation of the lung gd T cells via intratracheal delivery of IL-1b and IL-23, the pro-inflammatory mediators produced by myeloid cells upon microbial exposure, potently induced their proliferation and activation (Figure 4D).
gd T cells in the tumor-bearing lungs from SPF mice showed minimal capacity for interferon (IFN)-g or tumor necrosis factor (TNF-a) production coupled with low expression of the transcrip-tion factor Tbet (Figure S5). In contrast to splenic gd T cells, gd T cells in the tumor-bearing lungs were largely positive for PLZF (promyelocytic leukemia zinc finger) (Figure 5A), which is a transcription factor essential for the development of tissue-resident IL17-producing Vg6+ gd T cells (Lu et al., 2015). In addi-tion, they displayed low surface expression of CD27 (Figure 5B), which is critical for the differentiation of IFN-g-producing gd T cells (Ribot et al., 2009). gd T cells in the tumor-bearing SPF lungs exhibited elevated surface expression of activation and/or maturity markers including CD44, CD69, and PD1 compared to those in the spleen (Figures 5C–5E). However, gd T cells from the tumor-bearing lungs of the GF mice displayed a different phenotype: they had higher expression of IFN-g, Tbet, and CD27, and significantly lower expression of PLZF as well as CD44, CD69, and PD1 (Figures 5A–5E and S5). These re-sults suggest commensal microbiota are critical for the differen-tiation and activation/maturation of tumor-associated gd T cells in the lung.