Single-cell multiomics of CD4+ T cell clones in HIV-1 infected individuals

The clonal expansion of HIV-1-infected CD4+ T cells is a major barrier to cure. Using single-cell ECCITE-seq, we examined the transcriptional landscape, upstream immune regulators, HIV-1 RNA expression, and T cell clonal expansion dynamics of 215,458 CD4+ T cells (267 HIV-1 RNA+ cells and 68 expanded HIV-1 RNA+ T cell clones) from six HIV-1-infected individuals (during viremia and after suppressive antiretroviral therapy) and two uninfected individuals, in unstimulated conditions and after CMV and HIV-1 antigen stimulation. We found that despite antiretroviral therapy, antigen and TNF responses persisted and shaped T cell clonal expansion. HIV-1 resided in Th1 polarized, antigen-responding T cells expressing Bcl-2 family anti-apoptotic genes. HIV-1 RNA+ T cell clones were larger in clone size, established during viremia, persistent after viral suppression, and enriched in GZMB+ cytotoxic effector memory Th1 cells. Targeting HIV-1-infected cytotoxic CD4+ T cells and drivers of clonal expansion provides a new direction for HIV-1 eradication. Overall design: Study was completed in two arms, unstimulated and antigen stimulated CD4+ T cells. Paired PBMC samples from 6 individuals from the Sabes cohort during acute/recent viremia were used in both arms, 2 uninfected controls were recruited for comparison. For the unstimulated arm, CD4+ T cells were isolated from PBMC by negative selection and stained with a CITE-seq panel. For the stimulated arm, CD8+ T cells were depleted from PBMC. CD8- PBMC were pulsed with either CMV peptides or HIV peptides for 9 hours in the presence of T20 ART. Cells were then stained and antigen specific cells sorted on a FACS ARIA sorter for CD3+CD8-CD19-CD56- Acitvation inducible Marker (AIM, CD69+CD154+). Memory cells were also sorted as CD3+CD8-CD19-CD56-CD69-CD154-CD45RO+. Sorted cells were then hashed and stained with the CITE panel as previous. In either case, labeled cells were loaded into the 10X chromium controler for 5' immune profiling with feature barcoding. This resulted in libraries for the single cell transcriptome, surface proteome, and T cell receptor sequences. Libraries were sequenced with either the minimum requirements (R1 26, R2 90 for transcriptome and CITE-seq) on a HiSeq 4000 or in 150PE mode on a Novaseq 6000. In parallel, bulk TCR repetiore libraries were generated from remaining unstimulated CD4+ T cells using the NEBNext Human ImmuneProfiling kit and sequence on a MiSeq in 300PE mode.