RNA sequencing dataset from "Van Dis et al., IFN-γ-independent control of M. tuberculosis requires CD4 T cell-derived GM-CSF and activation of HIF-1α. 2022"

The prevailing model of protective immunity to tuberculosis is that CD4 T cells produce the cytokine IFN-γ to activate bactericidal mechanisms in infected macrophages. Although IFN-γ-independent CD4 T cell based control of M. tuberculosis infection has been demonstrated in vivo it is unclear whether CD4 T cells are capable of directly activating macrophages to control infection in the absence of IFN-γ. We developed a co-culture model using CD4 T cells isolated from the lungs of infected mice and M. tuberculosis-infected murine bone marrow-derived macrophages (BMDMs) to investigate mechanisms of CD4 dependent control of infection. This dataset represents RNA sequencing data from M. tuberculosis-infected wild-type and Ifngr-/- murine bone marrow-derived macrophages (BMDMs) after 24 hours of lung CD4 T cell co-culture.

CD4 T cells induced differential regulation of 1825 genes in wild-type BMDMs and 1142 genes in Ifngr-/- BMDMs compared to untreated. Although wild-type and Ifngr-/- BMDMs infected with M. tuberculosis were transcriptionally very similar prior to activation, the transcriptome of these genotypes of macrophages diverged after CD4 T cell co-culture. This is likely due in part to the presence or absence of IFN-γ signaling as IFN-γ alone regulates the expression of >2500 genes during M. tuberculosis infection. Still, 769 genes were altered in an IFN-γ-independent manner, with >2-fold upregulation in both wild-type and Ifngr-/- BMDMs. We found no difference in macrophage polarization between untreated M. tuberculosis-infected wild-type and Ifngr-/- BMDMs, and no significant increase in the expression of genes associated with M2 macrophages after CD4 T cell co-culture in either genotype. However, there was significant upregulation of genes associated with M1 macrophages after CD4 T cell co-culture in both genotypes, with wild-type BMDMs slightly more polarized. Collectively, these results show comparable patterns of activation in wild-type and Ifngr-/- macrophage during CD4 T cell co-culture, indicating that CD4 T cells elicit significant polarizing, inflammatory and antimicrobial effects in M. tuberculosis-infected macrophages irrespective of IFN-γ signaling.

RNA-sequencing on M. tuberculosis-infected murine bone marrow-derived macrophages (BMDMs) after 24 hours of lung CD4 T cell co-culture. Four independent experiments were performed. For each experiment, wild-type or Ifngr-/- BMDMs were seeded in 24-well plates, infected in DMEM with 5% horse serum and 5% FBS by 4-hour phagocytosis at a multiplicity of infection of 1, and co-cultured with lung CD4 T cells isolated from the lungs of 7–12-week-old wild-type mice 21 – 28 d after ~500 CFU aerosol infection with M. tuberculosis Erdman using mouse CD4 (L3T4) MicroBeads from Miltenyi Biotec (130-117-043) according to the manufacturer’s protocol. At 24 hours postinfection, CD4 T cells were separated from BMDMs using CD4 (L3T4) Dynabeads from Invitrogen (11445D) according to the manufacturer’s protocol, and BMDMs were lysed in 1 mL TRIzol (Invitrogen). Total RNA was extracted using chloroform and 2 mL Heavy Phase Lock Gel tubes from QuantaBio (2302830), and the aqueous layer was further purified using RNeasy Mini spin columns from Qiagen (74104). RNA quality was determined using an Agilent 2100 Bioanalyzer and RNA concentration was determined using the Qubit Quantitation Platform at the Functional Genomics Laboratory of The California Institute for Quantitative Biosciences (University of California, Berkeley). RNA-seq libraries were prepared by the DNA Technologies and Expression Analysis Core (University of California, Davis) and differential gene expression was analyzed by the Genome Center and Bioinformatics Core Facility (University of California, Davis).