CD8+ TEMRA Memory T cells Associate with Asthma Symptom Duration and Escape Proliferation Arrest in Severe but Not Mild Asthma

Aberrant immune response is a hallmark of asthma, with 5-10% of patients suffering from severe disease exhibiting poor response to standard treatment. A better understanding of the immune responses contributing to disease heterogeneity is critical for improving asthma management. Here we show a significant association of airway CD8+ effector memory T cells re-expressing CD45RA (TEMRAs), but not CD8+ CD45RO+ or tissue resident memory (TRM) T cells, with asthma duration in patients with severe asthma (SA) but not mild to moderate asthma (MMA). Higher frequencies of IFN-g+ CD8+ TEMRAs compared to IFN-g+ CD45RO+ T cells were detected in SA airways, and the TEMRAs from SA but not MMA patients proliferated ex vivo, although both expressed cellular senescence-associated biomarkers. Prompted by the transcriptomic profile of SA CD8+ TEMRAs and proliferative response to IL-15, airway IL15 expression measured higher in SA compared to MMA patients. Our findings add a new dimension to understanding asthma heterogeneity identifying IL-15 as a potential target for treatment. Cryopreserved cell suspensions were thawed as above and resuspended in FACS buffer but instead of stimulation were incubated with optimally diluted labeled monoclonal antibodies for 30 min at 0oC. Cells were washed and then sorted on an Aurora CS cell sorter using SpectroFlo software. Up to 100,000 T cells from three populations (CD8+ naïve, CD8+ CD45RO+ memory, and CD8+ TEMRAs) were sorted directly in to 1 mL of RLT buffer (Qiagen). Libraries were generated with the Takara SMART-Seq Stranded kit using Takara SMARTer RNA Unique Dual Index A and B Kits according to the manufacturer’s instructions. RNA was normalized to 5 ng/uL with water for a total volume of 7 uL of input RNA. RNA fragmentation was carried out for 6 minutes. 10 cycles were used for PCR1, followed by ribosomal RNA depletion using scZapR. No samples were pooled prior to PCR2 where 12 cycles were completed. Library quantification and assessment was done using a Qubit FLEX fluorometer and an Agilent TapeStation 4150/Fragment Analyzer 5300. Libraries were normalized and pooled to 2nM by calculating the concentration based off the fragment size (base pairs) and the concentration (ng/uL) of the libraries. Sequencing was performed on an Illumina NextSeq 2000, using a P3 200 flow cell. The pooled library was loaded at 750 pM. Sequencing was carried out 2x101 bp, with a target of 40 million reads per sample. A custom run chemistry was used to incorporate 3 dark cycles at the start of R2 that mask the 3 bp of the Takara adapter present in the read. Sequencing data was demultiplexed by the on-board Illumina DRAGEN FASTQ Generation software (v3.10.12).