Proliferation-competent Tcf1+ CD8 T-cells in dysfunctional populations are CD4 T-cell help independent

Effector CD8 T-cell responses in chronic infection and cancer are maintained by proliferation competent progenitors, which share features with the progenitors of memory T-cells in acutely resolved infections. Combining classical assessment of population dynamics with single cell RNA-sequencing, we discovered a switch in the need for CD4 help between related populations in acute and chronic infection. While in acute infection memory T-cells but not effector T-cells are CD4 help dependent, we found the opposite in chronic infection. Thus, progenitor cells in chronic infection acquire a level of autonomous function that is not seen among their counterparts in acute infection. Vice versa, the dependece of the effector cells highlight why a functional antigen-specific CD4 T-cell compartment is critical in therapeutic settings. Single cell splenocyte suspensions were obtained by mashing total spleens through a 100 μm nylon cell strainer (BD Falcon) and red blood cells were lysed with a hypotonic ACK buffer. Transgenic CD8+ T-cells were isolated using the mouse CD8+ T-cell enrichment kit (Miltenyi Biotech, Bergisch-Gladbach, Germany). 2x10e3 CD45.1+ P14 TCRαβ were transferred into naïve CD45.2+ C57BL/6 mice. 5x10e6 pfu wild-type LCMV c13 was diluted in PBS and injected intravenously. In vivo depletion of CD4 lymphocytes was achieved by intraperitoneal injection of 300 µg anti-mouse CD4 antibody (clone GK1.5; Bio X Cell, USA) diluted in PBS and injected on day -2, +1, and thereafter on each fifth day after infection. At day 40 post infection, splenocytes were enriched for CD45.1 + P14 T-cells using anti-CD45.1 biotin/ anti-biotin conjugated microbeads and magnetic MACS cell separation (Miltenyi Biotech, Bergisch-Gladbach, Germany). Single-cells were sorted on BD FACS Fusion (100 micron nozzle, standard operation settings, single-cell purity, index sorting). Individual cells meeting the gating strategy were sorted directly in lysis buffer into individual wells of a low-binding PCR plate. Immediately after sorting, each plate was spun down, snap-freezed on dry ice and stored at -80oC until use. The single-cell libraries were generated using the previously described SCRB-seq protocol (doi: https://doi.org/10.1101/003236), with some modifications. After RNA purification and reverse transcription, the generated single-cell cDNA was amplified for 22 cycles. The barcoded single-cell amplicons were purified with the use of (0.6x) Agencourt AMPure XP beads. 1 ng of the resulting amplified cDNA were used for library preparation with the Illumina Nextera XT DNA Library reagents (FC-131-1024, Illumina). After PCR amplification of the fragmented libraries, the samples were purified with (0.6x) Agencourt AMPure XP beads and eluted in 10 µl of molecular grade water. The quality of the resulting library was assessed with the use of Agilent High Sensitivity DNA Kit (5067-4626, Agilent). The library quantification was performed based on the Illumina recommendations (SY-930-1010, Illumina) with the use of KAPA SYBR FAST qPCR Master Mix (KK4600, Kapa Biosystems). The samples were sequenced on Illumina HiSeq 2500 system at the following conditions – high output run mode, paired-end, 16 bp read 1, 49 bp read 2, single-indexed sequencing resulting in 1 million reads per single-cell. A total of 1728 single-cells were sequenced – 864 ctrl and 864 w/o CD4 single-cells. The cells per each condition were derived from three experimental animals (288 single-cells from an animal).