Occasional complete responses to immune checkpoint inhibitor therapy demonstrate that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) can be immune-sensitive when appropriately targeted. Here, we analyzed AML/MDS patients (n=14) treated with the anti-TIM3 sabatolimab and the hypomethylating agent decitabine in a phase Ib clinical trial (NCT03066648) using single-cell RNA and T cell receptor (TCR) sequencing (n=6) and functional co-culture assays. Unlike T cell-restricted CTLA4 and PD1, TIM3 was broadly expressed across natural killer (NK)-cell, myeloid-cell, and T-cell populations. Therapy induced expansion of cytotoxic NK-cell subsets and enhanced type I interferon signaling. Fewer than 1% of bone marrow CD8+ T cells displayed a canonical exhaustion phenotype, and treatment preferably expanded small CD8+ T-cell clones in responders. Responders exhibited greater expansion of cytotoxic CD4+ T cells and B cells, as exemplified by a patient with pre-existing CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) achieving an outstanding complete response lasting 23 months. Over 20% of this patient's lymphocytes were T-LGLL cells expressing a TCR capable of recognizing autologous blasts. Overall, our results suggest that anti-TIM3 combined with decitabine engages a distinct mechanism of immune activation compared to anti-PD1 and anti-CTLA4, preferentially expanding NK-cell and CD4+ T-cell populations.

ABSTRACT:Cytomegalovirus (CMV) reactivation reshapes immune reconstitution and remains a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). To understand the impact of asymptomatic CMV viremia on immune reconstitution, we profiled longitudinal blood samples (n = 32) from donor-recipient pairs before and after HSCT with single-cell RNA and T-cell receptor (TCR) sequencing for 3 months after transplantation. To analyze the development of antigen-specific T-cell responses in detail, we sorted and TCR-sequenced T cells specific to the immunodominant CMV epitope pp65. After HSCT, an early increase in CD14+ monocytes was observed, followed by expansion of T and natural killer (NK) cells. Persisting patient-cell chimerism was observed in the CD4+ T-cell fraction. CMV-specific T cells and CMV-associated adaptive NK cells matured slower in grafts from seronegative donors compared to those from seropositive donors. The early maturation of adaptive NK cells was markedly more rapid in patients with CMV-seropositive donors, with enhanced recall function; however, adaptive NK cell cytotoxicity was higher in patients with CMV-seronegative donors. Large oligoclonal CD8+ T-cell expansions against the immunodominant CMV pp65NLV epitope comprised up to 63.6% of the CD8+ T-cell repertoire, showing terminal maturation with upregulation of canonical NK cell receptors. Our study presents, to our knowledge, the first comprehensive single-cell transcriptomic characterization of adaptive immune recovery after allogeneic HSCT, with a particular focus on CMV reactivation. Our findings highlight the impact of donor CMV serostatus on immune maturation in recipients with CMV reactivation, which may help predict the timing of immune reconstitution and inform strategies for management of posttransplant complications.

Recent clinical trials in AML combining immune checkpoint inhibitors (anti-PD1, anti-CTLA4) with hypomethylating agents have yielded only modest response rates. However, there has been exceptional responders achieving durable complete responses in all these trials. TIM3 is a checkpoint molecule expressed both on immune and leukemic cells, making it an interesting target in AML. Here, we conducted a comprehensive immunomonitoring of a phase Ib trial (NCT03066648) evaluating decitabine in combination with anti-TIM3 antibody sabatolimab (MBG453). We studied paired bone marrow (BM) and peripheral blood samples from 11 unfit newly diagnosed (ND) or relapsed/refractory (R/R) AML patients and 1 MDS patient with single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-seq) and flow cytometry. We also performed co-culture assays of primary immune and leukemic cells with scRNA+TCRαβ-seq readout in a patient with a durable complete response. In scRNA+TCRαβ-seq data, HAVCR2 (encoding TIM3) is highly expressed in NK cells, myeloid cells, and unconventional T cells, with some expression in CD8+ T cells. This contrasts with PDCD1 (encoding PD1) and CTLA4, which are primarily expressed in CD8+ and CD4+ T cells, respectively, albeit at low levels in AML. At baseline, responders had higher proportions of CD4+ T cells and B lymphocytes, whereas non-responders had more cytotoxic CD8+ T cells, which was confirmed by flow cytometry. After starting anti-TIM3+HMA therapy, both groups showed increased numbers of CD8+ T cells and NK cells. Responders to anti-TIM3+HMA therapy generally had more mature (CD56dim and adaptive) NK cells, with key transcriptional changes in type I/II interferon (IFN) and NF-κB pathways. Functional co-culture assayswith primary blasts from a complete responder revealed three NK activation states: cell-contact activated, cytokine-secreting, and type I IFN-responsive. NK cells showed enhanced type I IFN responses after therapy correlating with clinical remission, but this response was reduced when NK cells were co-cultured with blasts from a relapse time point, suggesting potential immune evasion. Cytotoxic CD4+ T cells were more abundant and highly clonal in pre-treatment samples from responders. In an exceptional responder with concomitant CD4+ T-LGLL diagnosis, the dominant CD4+ T-LGLL clone comprised 21% of the TCR repertoire at baseline, persisted at remission, but fell to 7% at relapse. Transcriptomic analysis showed upregulation of IFNG and TNF post-therapy, with a subset peaking soon after treatment. To assess whether the T-LGLL clone targets patient's leukemic cells, we engineered Jurkat reporter cells with the TCRαβ from the CD4+ T-LGLL clone and performed co-culture assays with scRNA-seq readout. In comparison to mock transduced cells, T-LGLL reporter cells showed upregulation of type I IFN genes when co-cultured with patient's leukemia cells, especially in presence of antigen-presenting cells. In co-culture of patient's own T-LGLL cells, pre-therapy cells responded strongly to blasts from screening but lost reactivity to relapse blasts, while post-therapy T-LGLL cells retained activity to both. Exhausted CD8+ T cells were rare in AML BM (<0.5% pre-treatment), most CD8+ T cells exhibited effector or memory phenotypes. Functional co-culture assays showed that CD8+ T cells retained robust activation capacity, marked by TNF and IFNG expression, when co-cultured with autologous blasts. Anti-TIM3+HMA therapy preferentially expanded small CD8+ T cell clones in responders, whereas non-responders showed expansion of larger, more cytotoxic clones, some of which were recognized to target viral epitopes. ScRNA-seq analysis of myeloid cells revealed that responders had a higher proportion of differentiated myeloid cells, such as classical monocytes. In the exceptional responder, relapse blasts in co-culture upregulated immune evasion genes (e.g., CD274 encoding PDL1) and showed the highest HLA class I expression, indicating putative immune escape mechanisms from T and NK cells, respectively.Our study provides a comprehensive analysis of anti-TIM3 in combination with decitabine in AML/MDS. We demonstrate that TIM3 blockade modulates the immune landscape by activating mature and adaptive NK cells, promotes cytotoxic CD4+ T cells, and primes small CD8+ T cell clones for expansion. Our results suggest that cytotoxic CD4+ T-LGLL cells may boost responses to immune checkpoint therapy in AML.

T cell large granular lymphocytic leukemia (T-LGLL) is a rare hematological disease of clonally expanded mature cytotoxic T cells. T-LGLL is thought to originate as a response to chronic (auto)antigen stimulus, as it often presents with autoimmune-mediated cytopenias and rheumatoid arthritis (RA). Approximately 40-50% of CD8+ T-LGLL patients have somatic STAT3 mutations in CD8+ T cells. However, previous analyses done in bulk have not been able to detangle whether STAT3 mutations initiate aberrant T cell expansion or accrue as subclonal events that give T cells a proliferative advantage independent of antigen drive. Here, we used single-cell whole genome sequencing (scWGS) to profile the life histories of CD8+ T cells in five T-LGLL patients. Understanding the underlying clonal structure of STAT3 mutations across T cell clones could pave the way for improved diagnostic, monitoring, and treatment options for T-LGLL patients. Methods We sequenced the whole genomes of 120 T cells flow-sorted from peripheral blood samples of 5 T-LGLL patients, leveraging primary-template directed amplification (ResolveDNA, Bioskryb Genomics). We genotyped DNA amplified from individually sorted T cells for STAT3 and selected 5-26 STAT3mt and 8-15 STAT3wt single T cells per patient for WGS with a target coverage of 15×. T-LGLL patients were aged 42-78, sampled either at diagnosis (n=3) or follow-up (n=2). All patients harbored at least one STAT3 variant: Y640F (n=4), N647I (n=1), D661Y (n=2) or D661V (n=1). RA preceded T-LGLL diagnosis in 3 patients. 4 patients received treatment for T-LGLL (mean 2.6 lines of treatment). Results STAT3 mt LGL CD8+ T cells harbored an elevated single-nucleotide variant (SNV) burden compared to STAT3wt CD8+ T cells in 2/5 patients and an elevated insertion-deletion burden in 2/5 patients (p<0.05, Wilcoxon). Overall mutation load increased with increasing patient age. Mutational signature analysis revealed that the clock-like mutational processes SBS1, SBS5, and SBSblood accounted for most SNVs in both STAT3mt and STAT3wt T cells. We used shared and unique somatic variants across sequenced CD8+ T cells to reconstruct molecular phylogenies for each patient. We complemented our phylogenetic trees by extracting the T cell receptor (TCR) of each sequenced cell. Each patient had a unique phylogenetic tree structure, but several general patterns emerged. First, we observed few shared mutations between T cells that belonged to different TCR clones, consistent with T cells developing from a polyclonal population of hematopoietic stem and progenitor cells. Second, the CD8+ T cell repertoires of LGLL patients were strikingly clonal. Despite our small sampling of T cells from each patient, we captured at least two expanded TCR clones from each patient. Third, compared with TCR Vβ antibody staining of CD8+ T cells from the same samples, STAT3 variants did not necessarily arise in the dominant Vβ clone. In patients with one STAT3 variant in one expanded TCR clone (n=3), the acquisition of STAT3 could be unambiguously timed after TCR rearrangement, implying that STAT3 variants arise as subclonal events during or after clonal expansion. The STAT3 mutation was timed to occur years or even decades before T-LGLL diagnosis and predated RA in the patients in whom the comorbidity was diagnosed. Additionally, a patient with four different STAT3 variants displayed recurrent hits of the gene in numerous different TCR clones, demonstrating remarkably strong selection for STAT3 mutations. Finally, a patient with an unusual double-positive CD4+CD8+ T-LGLL phenotype had multiple different clones with the same STAT3 variant. In this patient, the acquisition of the STAT3 variant was timed before TCR rearrangement, early in T cell development. Discussion We capitalized on the record resolution of scWGS to elucidate the clonal evolution of T-LGLL. Our study highlights the contribution of somatic mutations to functional shifts in healthy T cells, even when not instigating malignant transformation into an aggressive hematological cancer. Our results uncover extensive heterogeneity in the phylogenetic histories of CD8+ T cells in different T-LGLL patients. This suggests that T-LGLL may be a more complex disease than previously thought, shaped by patient-specific selection pressures. Our findings hint at different models of STAT3 mutation acquisition in T-LGLL, which may reflect distinct clinical entities to be explored in larger cohorts.

Clonal hematopoiesis (CH) becomes more prevalent with aging and may influence inflammatory diseases by altering immune function. While CH of indeterminate potential (CHIP) promotes inflammation in nonmalignant conditions, its relationship with rheumatoid arthritis (RA) remains unknown. We analyzed CHIP mutations in RA using two population-level cohorts and patients with newly diagnosed RA. CHIP was associated with prevalent RA in 10,089 FINRISK study participants with whole-exome sequencing (OR, 2.06; P = 0.029) and in the FinnGen cohort (n = 520,210; OR, 1.49; P < 0.001) using single-nucleotide polymorphism array-based CHIP annotation. In FinnGen, CHIP was also associated with inferior overall survival in participants with RA (P = 0.013). In newly diagnosed RA (n = 573), DNMT3A-mutated seropositive patients had increased inflammatory markers and disease activity compared with patients without CHIP. In contrast, TET2 mutations were enriched in seronegative RA (P = 0.009). Our findings provide further evidence for the context-dependent association between CHIP and inflammation, with potential therapeutic implications.

Immune aplastic anemia (AA) is a life-threatening bone marrow failure disorder driven by an autoimmune T cell attack against hematopoietic stem and progenitor cells (HSPCs). However, the exact autoantigen targets and role of other immune cells in the pathogenesis of AA are unknown. Here, we analyzed a cohort of 218 patients with AA using single-cell RNA and T cell receptor (TCR) αβ sequencing, TCRβ sequencing, flow cytometry, and plasma cytokine profiling. We identified natural killer (NK) cells and CD8+ terminally differentiated effector T (TEMRA) cells expressing NK receptors with AA-associated TCRβ motifs as the most dysregulated immune cell populations in AA bone marrow. Functional coculture experiments using primary HSPCs and immune cells showed that NK cells cannot kill HSPCs alone but may sensitize HSPCs to CD8+ T cell-mediated killing through production of interferons. Furthermore, HSPCs induced activation of T cell clones with CD8+ TEMRA NK-like phenotype in coculture. Our results reveal a convergent phenotype of innate and adaptive immune cells that may drive AA.

Somatic mutations in T cells can cause cancer but also have implications for immunological diseases and cell therapies. The mutation spectrum in nonmalignant T cells is unclear. Here, we examined somatic mutations in CD4+ and CD8+ T cells from 90 patients with hematological and immunological disorders and used T cell receptor (TCR) and single-cell sequencing to link mutations with T cell expansions and phenotypes. CD8+ cells had a higher mutation burden than CD4+ cells. Notably, the biggest variant allele frequency (VAF) of non-synonymous variants was higher than synonymous variants in CD8+ T cells, indicating non-random occurrence. The non-synonymous VAF in CD8+ T cells strongly correlated with the TCR frequency, but not age. We identified mutations in pathways essential for T cell function and often affected lymphoid neoplasia. Single-cell sequencing revealed cytotoxic TEMRA phenotypes of mutated T cells. Our findings suggest that somatic mutations contribute to CD8+ T cell expansions without malignant transformation.

Primer sequences of the primers utilized in Sanger sequencing.

Background Impaired immune cell reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) poses risks for both disease recurrence and treatment-related complications, such as cytomegalovirus (CMV) reactivation. CMV strongly shapes the immune system, but the development of anti-CMV T cells and CMV-associated adaptive NK cells in vivo is still largely uncharacterized. Understanding these dynamics post-allo-HSCT may have clinical implications, since NK cells mediate a graft-versus-leukemia effect, particularly in myeloid malignancies, and adaptive NK cells have recently been associated with favorable immunotherapy responses in both hematological and solid cancers. Here, with longitudinal sample collection pre and post-allo-HSCT, we aimed to determine how patient and donor-derived NK and T cells synergistically contribute to immune system regeneration, how clonotypes develop and persist, and whether donor/recipient CMV serostatus and CMV reactivation post-allo-HSCT influence NK and T cell phenotypes. Methods We sequenced 32 longitudinal CD45+ sorted peripheral blood samples with single-cell RNA and T cell receptor sequencing (scRNA+TCRαβ-seq) from 5 allo-HSCT donor-recipient pairs (pre-transplant sample from donor and recipient and 3-4 follow-up samples per patient over 3 months) and 6 healthy controls (Figure). All recipients were CMV seropositive, while 2 and 3 donors were seropositive and negative, respectively. CMV reactivation occurred in 4 patients during follow-up, on average 32 days post-allo-HSCT. We screened 32 common viral epitopes from post-transplant samples (n=3, 5 samples) with scRNA+TCRαβ+pMHC-seq (Immudex dCODE Dextramers). We then profiled T cells specific to CMV pp65 NLV-multimer with TCRβ-seq (allo-HSCT donor-recipient pairs n=3, 13 samples, healthy n=4), and traced CMV-recognizing TCRs in the scRNA+TCRαβ-seq data. Results We analyzed a total of 197,887 immune cells, including 37,123 NK cells and 59,852 CD8+ T cells. After allo-HSCT, an early increase in classical CD14+ monocytes was observed, followed by expansion of NK and T cell populations. At the time of CMV viremia, NK cell and CD8+ T effector memory/terminally differentiated effector memory (CD8+ T EM/EMRA) populations increased regardless of donor CMV serology. CD56 bright, CD56 dim and adaptive NK cells were present in all patients post-transplant, with patient-specific heterogeneity in phenotype and temporal dynamics. The proportion of adaptive NK cells out of CD45+ cells was higher post-viremia in 2 patients with CMV+ donors (18.9% and 7.4%), compared to 2 patients with CMV- donors (3.1% and 1.7%). Surprisingly, the expansion of adaptive NK cells (3.8%) was also noted in a patient with a CMV- donor without CMV reactivation post-allo-HSCT. After viremia, adaptive NK cells upregulated IFNG production, KLRC2/NKG2C and KLRC3/NKG2E and downregulated KLRC1/NKG2A. CD8+ T cell clonality markedly increased after CMV viremia in all patients with CMV reactivation, as measured by proportion of large and hyperexpanded clones in the scTCRαβ-seq data. Hyperexpanded clones consisted mostly of CD8+ T EM/EMRA phenotypes and were present only in samples collected during or after viremia. With scRNA+TCRαβ+pMHC-seq, pp65 NLV was discovered to be the immunodominant CMV epitope. Characterizing pp65 NLV-specific TCRs in the scRNA+TCRαβ data, CMV-specific CD8+ T cells belonged to highly clonal T EM/EMRA clusters. Further, a subset enriched to a T EMRA phenotype expressing NK cell receptors, including KIR3DL2, KIR2DL3, KLRC2/NKG2C KLRC3/NKG2E, and FCGR3A/CD16, and the exhaustion markers TIGIT, TOX, and CD160 at a higher level than other CD8+ T cells. The donor-derived CMV-specific TCR repertoire evolved in recipients after viremia. For example, in patient 2 we noted an 18.5-fold expansion of a donor-derived CMV-specific clone during viremia that was not originally the most dominant anti-CMV T cell clone in the donor. Conclusions We delineated NK cell and antigen-specific T cell dynamics in the unique setting of allo-HSCT and CMV reactivation, highlighting the manyfold expansion of donor-derived T cell clones and the expansion and evolution in the transcriptional profile of adaptive NK cells during CMV viremia. Our study sheds light on the immunobiology of these two cell types with implications in transplant outcome and translational relevance as cancer immunotherapy.

Figures describing the following; length distribution of somatic insertions and deletions in the exome sequencing data, coverage of mononucleotide microsatellite sites in both exome and MiSeq sequencing data, mutation frequencies at mononucleotide microsatellite sites in the exome sequencing data, number of somatic deletions and insertions in the exome sequencing data, model p-values, expected -log10(p) values, mutation frequency, significance and normalized allelic fraction (NAF) of the top genes, overlap between validation sets 1,2, and 3, and mapped base pairs in the MiSeq sequencing data as well as the supplementary methods and extended literature evaluation of the candidate genes.

Mutation significance and sequencing information on the MiSeq sequenced microsatellites.

The median coverage at different genes in the MiSeq data. frequency and significance in exome and MiSeq data (per microsatellite and per gene) as well as the NAFs in the MiSeq data.

<p>Clinical and sequencing related information about the 24 exome and 93 MiSeq sequenced tumors.</p>

<p>Summary of all the genes included in the MiSeq validation (Sets A and B); mutation frequency and significance in exome and MiSeq data (per microsatellite and per gene) as well as the NAFs in the MiSeq data. repeats. microsatellite classes</p>

Additional information about the mutations in sets A, B and C.

<p>Frequency of mutations observed at different mononucleotide repeat sites.</p>

Background: Immune aplastic anemia (AA) is a bone marrow failure (BMF) disorder driven by an autoimmune response against the hematopoietic stem and progenitor cells (HSPCs), yet the mechanisms of breaking self-tolerance and sustaining inflammation are incompletely understood. Aims: Characterize and functionally validate the immune cell phenotypes and T cell receptor (TCR) targets in AA and associate them with clinical variables. Methods: With scRNA+TCRαβ-seq, we profiled AA (n=13, N=17) from bone marrow (BM) and peripheral blood (PB) and compared these to healthy controls (HC) (n=35) and hematological malignancies (n=36). With TCRβ-seq, we profiled AA (n=144, N=248) from BM and PB and compared these to other BMFs (n=137), other autoimmune disorders (n=45), hematological malignancies (n=39), and HC (n=826, N=866). With multiplexed cytokine platform, we profiled AA (n=78, N=88) and HC (n=20). Findings were validated in co-cultures of HPSCs with immune cells with scRNA+TCRαβ-seq readout (AA n=7, HC n=4; N=57). The presence of clonal hematopoiesis, somatic loss of HLA alleles, and somatic mutations in the T cells were assessed in a subset of patients. Results: Compared to HC, the most statistically significant finding in the AA BM was the expansion of NK cell cluster. The increased proportion of NK cells was validated in a larger flow cytometry cohort (AA n=49, HC n=26) and was associated with more severe disease. NK cells had mostly terminally differentiated or adaptive NK cell phenotype with upregulation of cytotoxic markers and lineage-defining KLRC2/NKG2C and LAG3. Patients with the highest NK cell infiltration had also distinct cytokine profiles, with upregulated IL2/4/13/20 and CCL28. Compared to HC, patients with AA had also significantly more cytotoxic CD8+ T cells. The most expanded CD8+ T cell populations were terminally differentiated, highly cytotoxic CD8+ TEMRA cells, with upregulation of exhaustion markers (LAG3, TIGIT), but with retained stimulation capacity in vitro. Interestingly, these clonally expanded T cells also upregulated NK cell receptors, such as FCGR3A/CD16 and KLRC1/2/3. The expansion of these NK-like CD8+ TEMRA cells was validated in an additional scRNA-seq cohort (AA n=8, HC n=2) and was more pronounced in severe AA than in non-severe AA. The TCR-seq analysis showed oligoclonal expansions in patients with AA. With a sophisticated in-house TCR-seq analysis pipeline, we found 200 TCR-motifs that were associated with AA, and not found in HC, MDS, or other associated disorders or viral infections. These motifs were successfully validated in two large AA and HC TCR-seq cohorts and were associated with clinical variables such as loss of HLA class I alleles. T cells with AA-associated TCR motifs had NK-like CD8+ TEMRA phenotype, implying NK receptor expression in T cells could be a result of chronic antigen stimulation. Finally, the function of NK cells, NK-like CD8+ TEMRA cells, and clones with AA-associated TCR-motifs was assessed with autologous immune cell and HPSC co-cultures with scRNA+TCRαβ-seq readout and results will be presented at the conference. Summary/Conclusion: Our results show convergent shift of both innate and adaptive immune cells, where NK cells gain T cell-like memory properties and T cells gain NK-like TCR independent killing abilities to maintain autoinflammation in AA. Keywords: Autoimmunity, Aplastic anemia, T cell, Natural killer

Inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of genetic disorders characterized by insufficient blood cell production and increased risk of transformation to myeloid malignancies. While genetically diverse, IBMFS are collectively defined by a cell-intrinsic hematopoietic stem cell (HSC) fitness defect that impairs HSC self-renewal and hematopoietic differentiation. In IBMFS, HSCs frequently acquire mutations that improve cell fitness, a phenomenon known as somatic compensation. Somatic compensation can occur via distinct genetic processes such as loss of the germline mutation or somatic alterations in pathways affected by the disease-causing gene. While the clinical implications of somatic compensation in IBMFS remain to be fully discovered, understanding these mutational processes can help understand disease pathophysiology and may inform future diagnostic and therapeutic approaches. In this review, we highlight current understanding about somatic compensation in IBMFS.

Somatic mutations accumulate in cells during aging, and the mutation rate and spectrum vary between tissues. Mutation accumulation can be associated with inflammation: for example, myeloid driver gene mutations in hematopoietic stem cells (clonal hematopoiesis, CH), has been linked with atherosclerosis. Somatic mutations can also accumulate in T cells, where they are connected with immune dysregulation in large granular lymphocytic leukemia, characterized by autoimmunity and STAT3 mutations in T cells. However, the mutation spectrum in T cells in other hematological disorders is not yet fully discovered. We hypothesized that T cell lineage specific somatic mutations are associated with T cell clonality in various diseases involving the hematopoietic system. We systematically characterized the spectrum of somatic mutations in CD4+ and CD8+ T cells in altogether 207 samples from 95 patients with chronic graft-versus-host disease (cGVHD, number of patients: 40), myelodysplastic syndrome (MDS, n = 15), immune thrombocytopenia (n = 2), aplastic anemia (AA, n = 21, previously published), and immunodeficiency (n = 17, previously published). Age at sampling was similar between disease cohorts. DNA was extracted from bead separated CD4+ and CD8+ cells from peripheral blood or bone marrow samples. We applied a custom-made targeted NGS panel, consisting of 2533 immune-related genes. Variant calling was performed using Mutect2. In variant filtering, we used 42 healthy T cell and skin samples as panel of normals. To explore the association between somatic mutations and clonal T cell expansions, we performed TCRb deep sequencing from 148 matched patient samples. With mean sequencing depth of 382x, we identified 888 non-synonymous somatic variants in total. 784 (86%) of the variants were only found in either CD4+ or CD8+ fraction. 52 variants were shared between individual patient's CD4+ and CD8+ T cells, suggesting that they were acquired prior to T cell lineage differentiation. On the average, we observed 3.8 fraction-specific variants per sample. Mean variant allele frequency (VAF) was 4.5% and 12.7% for fraction-specific and shared variants, respectively. Variant types included missense (91%), nonsense (5.1%), splice-site (1.7%), frameshift (1.3%), and non-stop (0.2%) mutations. The most common mutational signatures were age-associated Signature 1 and defective DNA mismatch repair associated Signature 15. Immunodeficiency patients had lower number of fraction-specific variants compared to AA, MDS and cGVHD patients (p = 0.027). In all cohorts, CD8+ T cells had acquired more variants than CD4+ T cells (p = 0.00012; mean [median] for CD8: 4.4 [4] and CD4: 3.2 [2]). The highest fraction-specific VAF in each sample was associated with the largest T cell clone size in CD8+ but not in CD4+ samples (R = 0.59, p = 3.1e-09 and p = ns, respectively). In CD8+ T cells, age was associated with number of fraction-specific variants (R = 0.38, p = 1.1e-4) but not with the highest CD8-specific VAF (p = ns). Among the variants shared between individual patients' CD4+ and CD8+ T cells, we found mutations in DNMT3A (n = 3) and TET2 (n = 2) and ASXL1 (n = 1), the most recurrently mutated CH genes. In addition to shared mutations, in 12% of patients had fraction-specific myeloid driver gene mutations (e.g. DNMT3A, TET2, KRAS, NRAS) in CD4+ or CD8+ T cells. To identify pathways enriched for putative driver mutations, we performed analyses of fraction-specific T cell variants with OncodriveFM. The most significantly mutated gene ontology pathways included T cell co-stimulation, leukocyte migration, Ras protein signal transduction, and positive regulation of MAP kinase activity. In conclusion, T cell fraction-specific somatic mutations are common in patients with various hematological diseases, and they are enriched in pathways important for T cell function. CD8+ T cells accumulate more mutations than CD4+ T cells. In CD8+ T cells, the number of somatic mutations was associated with age, reflecting stochastic accumulation of mutations. Based on mutational signatures, somatic mutations in T cells are mainly result of endogenous mutational processes. In addition, mutational clone size CD8+ T cells was significantly associated with the largest TCR clone, suggesting that mutations have accumulated in expanded CD8+ T cell clones. Further studies are warranted to determine the functional effects of somatic mutations in T cells.