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Letters to the Editor
Somatic STAT3 mutations in CD8+ T cells of healthy blood donors carrying human T-cell leukemia virus type 2
Chronic viral antigen stimulation may underlie CD8+ T-cell expansion and T-cell large granular lymphocyte leukemia (T-LGLL).1 In T-LGLL, CD8+ T-cell expansions are associated with somatic mutations that solidify clon- al dominance, such as in the case of activating STAT3 mutations which are found in 40% of patients.2 However, whether chronic exposure to viral antigens are associated with somatic mutations in expanding CD8+ T cells among individuals without clinically detectable lymphoproliferations is currently not known.
Human T-cell leukemia virus type 2 (HTLV-2) prefer- entially targets CD8+ T cells, causing strong expansion of the infected CD8+ T-cell clones.3 Whereas HTLV-1 is the causative agent of adult T-cell leukemia/ lym- phomas,4 the etiologic role of HTLV-2 in lymphoprolifer- ative diseases is less clear. In 1992 an incidental case of LGL leukemia with HTLV-2 seropositivity was described.5 Later, Thomas et al.6 reported anti-HTLV antibody positivity in 44% of T-LGLL patients. While some cross-reactivity cannot be excluded, 7.5% (4 of 53) of T-LGLL patients tested positive for HTLV-2 by both western blotting and polymerase chain reaction (PCR),6 suggesting that HTLV-2 may participate in T-LGLL pathogenesis in a minority of cases. In this study, we examined whether CD8+ T cells from healthy, asympto- matic blood donors with chronic HTLV-2 infection har- bor somatic mutations in STAT3 or other immune-asso- ciated genes, potentially identifying invidiuals at risk of subsequent lymphoproliferative diseases.
This study was conducted with samples from the HTLV Outcomes Study (HOST) which includes samples from subjects recruited from five major US blood dona- tion centers.7 HTLV status was analyzed with an ezyme- linked immunosorbant (ELISA) assay, followed by west- ern blot confirmation and HTLV-1 versus HTLV-2 typing by either real-time PCR or a type specific ELISA.7 At each visit, cohort participants were interviewed in detail for symptoms, followed by a physical and neurological examination. Informed consent was obtained from all participants. The study and sample collection were approved by the University of California San Francisco committee on human research and other Institutional Review Boards. We obtained frozen peripheral blood mononuclear cells (PBMC) of 30 HTLV-2 infected and 35 HTLV-2 uninfected blood donors from University of California San Francisco and Vitalant Research Institute (CA, USA). The PBMC samples collected between 2000 and 2008 were randomly selected from HOST.
We separated CD4+ and CD8+ T cells from PBMC samples of HTLV-2 positive (n=30) and negative (n=35) healthy blood donors. The presence of STAT3 mutations in the sorted fractions was analyzed by ultra-deep tar- geted amplicon sequencing, covering hotspot regions of STAT3 gene (median coverage =7,472, sensitivity =0.5% variant allele frequency [VAF]). The sequencing was per- formed with Illumina Miseq System (Online Supplementary Figure S1A), and variant calling was per- formed as previously reported.8 Somatic nonsynony- mous STAT3 mutations were discovered in CD8+ T cells from four of 30 (13.3%) HTLV-2 positive subjects, whereas no STAT3 mutations were discovered in HTLV- 2 negative subjects using deep amplicon sequencing of STAT3 (Figure 1A; Fisher’s exact test P=0.04). Furthermore, no STAT3 mutations were discovered in
CD4+ T cells, indicating that the mutations were specific for the CD8+ T-cell subset. Among the four STAT3 mutations detected, three were missense STAT3 muta- tions (Y640F, N647I and D661Y) and one was a non- frameshift insertion (Y657_K658insY), with VAF of 11.9%, 0.5%, 4.9%, and 1.2%, respectively (Figure 1B). All the mutations identified in CD8+ T cells were locat- ed in the SH2 domain of STAT3 and have been previous- ly reported in T-LGLL (Online Supplementary Figure S1A).2 The proportion of differentiated, putatively cytotoxic CD57+, CD16+ CD8+ T cells were higher in STAT3 mutated compared to STAT3 unmuted HTLV-2 positive individuals (Figure 1C). In addition, higher level of the cytotoxic marker perforin was noted in CD8+ T cells of STAT3 mutated, HTLV-2 positive individuals (Online Supplementary Figure S1B and C). TCRb deep sequencing9 of sorted CD8+ T cells revealed higher clonality index in the STAT3 mutated compared to STAT3 unmuted indi- viduals (Figure 1D). The VAF of the STAT3 Y640F muta- tion was consistent with clonal event in the largest TCRBV03-01 clone; the other three STAT3 mutations with smaller VAF likely occurred as subclonal events or in the smaller CD8+ T-cell clones (Figure 1E).
The age distribution was similar between HTLV-2 neg- ative subjects (median age 52 year), HTLV-2 positive subjects without STAT3 mutations (median age 53 years), and HTLV-2 positive subjects with STAT3 muta- tions (median age 58.5 years) (P-value between HTLV-2 positive subjects with and without STAT3 mutations, P=0.50; Mann–Whitney U test) (Figure 1F). No statistical difference in viral load was detected between STAT3 mutated (median =0.149) and unmuted HTLV-2 positive subjects (median =0.0005) (P=0.5; Mann–Whitney U test) (Figure 1G). No serial HTLV-2 viral load measure- ments were available; however, HTLV-2 viral load has been reported to be stable over time.10 There was no dif- ference in total white blood cell and lymphocyte counts between STAT3 mutated and unmuted cases (Figure 1H and I).
In order to characterize a larger spectrum of somatic variants in genes linked to immune regulation, we ana- lyzed CD8+ T cells from 28 HTLV-2 positive subjects using a custom next generation sequencing panel cover- ing the coding regions of 2,533 immune-related genes.11 Samples were sequenced with Illumina HiSeq or NovaSeq 6000 system (Online Supplementary Figure S2), and somatic variant calling followed a previously described approach.11 Variants were filtered using popu- lation based filtering, MuTect2 filters and against CD4+ and CD8+ panels of normals from 21 healthy controls (Online Supplementary Figure S2). Variants only found in CD8+ T cells, using sorted CD4+ T cells as matched nor- mals, were evaluated further. Sequencing coverages are presented in the Online Supplementary Figure S2. Two STAT3 mutations (Y640F and D661Y) were detected in two of four STAT3 mutant cases. N647I and Y657_K658insY variants did not pass filtering due to lower sequencing coverage compared to amplicon sequencing; their presence was confirmed with visual inspection of the sequencing data using Integrative Genomics Viewer.12 In addition to STAT3 mutations, we identified a total of 66 coding somatic variants in 61 genes in CD8+ T cells (Figure 2A). Nineteen (68%) of the subjects had at least one variant, and the median number of variants was one per subject.
Eight subjects (29%) harbored variants in the genes previously discovered in LGLL (STAT3, KMT2D, TYRO3, DIDO1, BCL11B, CACNB2, KRAS, LRBA and FANCA),13 and five subjects (18%) harbored genes involved in JAK-
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haematologica | 2022; 107(2)