Altered function and differentiation of age-associated B cells contribute to the female bias in lupus mice

Differences in immune responses to viruses and autoimmune diseases such as systemic lupus erythematosus (SLE) can show sexual dimorphism. Age-associated B cells (ABC) are a population of CD11c+T-bet+ B cells critical for antiviral responses and autoimmune disorders. Absence of DEF6 and SWAP-70, two homologous guanine exchange factors, in double-knock-out (DKO) mice leads to a lupus-like syndrome in females marked by accumulation of ABCs. Here we demonstrate that DKO ABCs show sex-specific differences in cell number, upregulation of an ISG signature, and further differentiation. DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c− effector B cell populations with pathogenic and pro-inflammatory function as demonstrated by BCR sequencing and fate-mapping experiments. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs, resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function and differentiation of ABCs that accompanies TLR7-driven immunopathogenesis.

Treg and Oligoclonal Expansion of Terminal Effector CD8+ T Cell as Key Players in Multiple Myeloma

The classical paradigm of host-tumor interaction, i.e. elimination, equilibrium, and escape (EEE), is reflected in the clinical behavior of myeloma which progresses from the premalignant condition, Monoclonal Gammopathy of Unknown Significance (MGUS). Despite the role of other immune cells, CD4+ regulatory T cells (Treg) and cytotoxic CD8+ T cells have emerged as the dominant effectors of host control of the myeloma clone. Progression from MGUS to myeloma is associated with alterations in Tregs and terminal effector CD8+ T cells (TTE). These changes involve CD39 and CD69 expression, affecting the adenosine pathway and residency in the bone marrow (BM) microenvironment, together with oligoclonal expansion within CD8+ TTE cells. In this mini-review article, in the context of earlier data, we summarize our recent understanding of Treg involvement in the adenosine pathway, the significance of oligoclonal expansion within CD8+ TTE cells and BM-residency of CD8+ TTE cells in MGUS and newly diagnosed multiple myeloma patients.

Sex-specific differences in the function and differentiation of ABCs mark TLR7-driven immunopathogenesis

ABSTRACTSex differences characterize immune responses to viruses like SARS-CoV2 and autoimmune diseases like SLE. ABCs are an emerging population of CD11c+ T-bet+ B cells critical for antiviral responses and autoimmune disorders. DEF6 and SWAP70, are two homologous molecules whose combined absence in double-knock-out mice (DKOs) leads to a lupus syndrome in females marked by an accumulation of ABCs. Here we demonstrate that DKO ABCs exhibit sex-specific differences in their expansion, upregulation of an ISG signature, and further differentiation. BCR sequencing and fate mapping experiments reveal that DKO ABCs undergo oligoclonal expansion and differentiate into both CD11c+ and CD11c− effector populations with pathogenic and proinflammatory potential. Tlr7 duplication in DKO males overrides the sex-bias and further augments the dissemination and pathogenicity of ABCs resulting in severe pulmonary inflammation and early mortality. Thus, sexual dimorphism shapes the expansion, function, and differentiation of ABCs contributing to the sex-bias that accompanies TLR7-driven immunopathogenesis.

CD8dimCD3+ lymphocytes in fever patients might be biomarkers of active EBV infection and exclusion indicator of T-LGLL

Background: Massive monoclonal or oligoclonal expansion of CD8+ T cells is a notable feature of primary infections of the Epstein–Barr virus (EBV). However, the clinical significance of this expansion is not clear. Results: An increase in the CD8dimCD3+ lymphocyte subset in patients with active EBV infection was due to caspase-8-dependent apoptosis was found using flow cytometry in this study. The number of these cells was associated with the illness severity. Pan-T-cell antigen and receptor analyses were also compared in patients with active EBV infections and T-cell large granular lymphocytic leukemia to provide additional diagnostic information. Conclusion: The increase in CD8dimCD3+ cells could be a biomarker of active EBV infection and an exclusion indicator of T-cell large granular lymphocytic leukemia with flow cytometric analysis.

CARD11 Mutation Induces Oligoclonal Expansion of T-Cells, and Accelerates ATL Development in Combination with HBZ

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma that develops in about 5% of human T-cell leukemia/lymphoma virus 1 (HTLV-1) carriers. In addition to viral oncogenes, namely tax and HTLV-1 bZIP factor (HBZ), gene mutations, highly enriched for T-cell receptor (TCR)-NF-kB signaling, should be involved in the development of ATL. Among gene mutations, mutation in CARD11, a cytoplasmic scaffolding protein required for TCR-induced NF-kB activation, was detected in 24% of ATL patients. Here we generated a mouse model for conditional expression of a human ATL-derived CARD11(E626K) gain-of-function mutant, and demonstrated CARD11 activation induced oligoclonal expansion of T-cell and infiltration to many organs. We also showed that expression of HBZ accelerated mutant CARD11-induced lymphoproliferative diseases. We introduced a human Card11(E626K) into the mouse genome at the ROSA26 locus. After crossing with CD4-Cre Tg, CARD11(E626K)CD4-Cre mice was obtained. In CD4+ cells from CARD11(E626K)CD4-Cre, the amount of cleaved BCL-10 and NF-kBp65 increased compared with those in WT CD4+cells, confirming the activation of NF-kB. About half of CARD11(E626K)CD4-Cre mice died on or after 6 months after birth. At 6 months, leukocytosis was observed in CARD11(E626K)CD4-Cre, and accordingly the number of CD4+ cells cells was about 1.43 times greater than those in WT mice. The most affected organ in CARD11(E626K)CD4-Cre mice was lung. Alveolar septum was thickened by infiltrated cells at 6 months, and worsened subsequently. CD3+ T-cell accumulated around capillary blood vessels, and had high proliferation indices (>50%), as assayed by Ki-67 staining. CARD11(E626K)CD4-Cre mice developed lymphadenopathy (4/8 mice (50%) at 6M and 4/6 mice (66.7%) at 12M). Normal lymph node (LN) architecture was barely preserved, and medullary sinus was expanded with CD3+ T-cell. Some of them were positive for FoxP3, and had moderate proliferation indices (25%-50%). Among CD4+ T-cell, the proportion of naive T-cell (Tn) decreased, and that of effector/memory T-cell (Tem) and regulatory T-cell (Treg) increased compared to WT LNs. The proportion of Treg in CD4+ T-cell from LN of 6M- and 12M-old CARD11(E626K)CD4-Cre mice was 25% and 40%, respectively, which values were much larger than the normal range as 10-15%. We next examined the clonality of CD4+ cells in spleen and swollen LNs from CARD11(E626K)CD4-Cre mice. The clonality of the TCR repertoires of 20 individual Vb gene famines from Vb1-20 was assessed by a PCR. The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 5 splenic CD4+ cells, and 1 of 2 LN CD4+ cells from CARD11(E626K)CD4-Cre mice. To assess the effect of HBZ constant expression on CARD11(E626K)CD4-Cre mice, we generated HBZ Tg, in which HBZ cDNA was expressed under the CD4 promoter. Similar to the previous report (by Satou et al.), our HBZ Tg showed increased number of Tem, destroyed architecture of lung such as thickened alveolar septum by infiltrated cells and decreased alveolar space by edema, and the lymphadenopathy after 12M (66.7%). We then crossed CARD11(E626K)CD4-Creand HBZ Tg, and obtained the compound mice. The compound mice caused more aggressive lymphoproliferative diseases compared with CARD11(E626K)CD4-Cre mice. Most of compound mice died within 8M. At 6M, architecture of lung, kidney, spleen, and LN was destroyed. In lung, alveolar space of lung was scarcely observed caused of T-cell invasion. Alveolar septum was thickened with infiltrated cells, and CD3+ cells accumulated around capillary blood vessel. Some of them were positive for FoxP3, and indicated moderate proliferation indices (25-50%). T-cell invasion was also observed in kidney. Lymphadenopathy was detected in 6 of 9 (66.7%) with completely destroyed architecture, increment of the proportion of Fox3+ cells, and moderate proliferation indices (25-50%). The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 4 splenic CD4+ cells, and 2 of 2 LNs CD4+ cells from compound mice. These results suggest that CARD11 mutant-induced NFkB activation and constant HBZ expression may have similar effects, such as T-cell infiltration into organs and LN adenopathy, and that the simultaneous occurrence of both may have additive effects. Disclosures Sugiyama: Chordia Therapeutics, Japan.: Current Employment. Morishita:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Shimoda:Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck & Co.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Celgene: Honoraria; Shire plc: Honoraria; Novartis: Honoraria, Research Funding; Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria.

CD8+ T cell-mediated endotheliopathy is a targetable mechanism of neuro-inflammation in Susac syndrome

Neuroinflammation is often associated with blood-brain-barrier dysfunction, which contributes to neurological tissue damage. Here, we reveal the pathophysiology of Susac syndrome (SuS), an enigmatic neuroinflammatory disease with central nervous system (CNS) endotheliopathy. By investigating immune cells from the blood, cerebrospinal fluid, and CNS of SuS patients, we demonstrate oligoclonal expansion of terminally differentiated activated cytotoxic CD8+ T cells (CTLs). Neuropathological data derived from both SuS patients and a newly-developed transgenic mouse model recapitulating the disease indicate that CTLs adhere to CNS microvessels in distinct areas and polarize granzyme B, which most likely results in the observed endothelial cell injury and microhemorrhages. Blocking T-cell adhesion by anti-α4 integrin-intervention ameliorates the disease in the preclinical model. Similarly, disease severity decreases in four SuS patients treated with natalizumab along with other therapy. Our study identifies CD8+ T-cell-mediated endotheliopathy as a key disease mechanism in SuS and highlights therapeutic opportunities.

Deconstructing the Clonal Advantage and Clonal Stability of 5q- Candidate Genes in Del(5q) MDS on a Single Cell Level

Hematopoietic Stem/Progenitor cells (HSPCs) with 5q haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) acquire a clonal advantage in the bone marrow and out-compete normal hematopoiesis. A critical, yet unsolved question remains: how does genetic haploinsufficiency in del(5q) cells contribute to the clonal advantage of HSPCs? We investigated the role of haploinsufficiency for three candidate genes in the common deleted region on chromosome 5 (Csnk1a1, Egr1 and Apc) in direct competition with each other and wild-type (wt) cells on a single cell level by employing a novel lentiviral genetic barcoding strategy. We introduced genotype and cell-specific barcodes into HSPCs from murine models haploinsufficient for Csnk1a1, Egr1 or Apc. Barcoded HSPCs were sort-purified, genotypes mixed and subsequently competitively transplanted into lethally irradiated mice and re-transplanted after 16 weeks in a secondary transplant. The barcoded progeny was reliably recovered from peripheral blood and relative contribution of the barcoded clones to differentiated blood lineages was followed over 32 weeks. Despite heterogeneity in clonal evolution among the mice, all haploinsufficient clones had the potential to outcompete wt clones (3 of 5 mice in the primary transplant, 3 out of 4 mice in the secondary transplant). Csnk1a1 haploinsufficient clones showed the largest clonal abundance and clonal persistence. Expansion of oligoclonal Csnk1a1 haploinsufficient HSPCs was further enhanced in the secondary transplant in all mice. Egr1 haploinsufficient clones showed potential for prominent oligoclonal expansion in one mouse, but decreased in abundance in all secondary transplants. Apc haploinsufficient clones showed persistence but not expansion in 3 out of 5 mice. These results were validated by conventional competitive transplants, which demonstrated that Csnk1a1 and Egr1 haploinsufficient cells achieved the highest advantage over wt hematopoiesis in the primary transplant and more enhanced in the secondary transplant. Since Csnk1a1 regulates β-catenin protein stability, we hypothesized that the clonal expansion of Csnk1a1 haploinsufficient HSPCs is dependent on β-catenin levels. We performed a second genetic barcoding competitive transplant, comparing Csnk1a1-/+ HSPC directly to double haploinsufficient Csnk1a1-/+/Ctnnb1-/+ (β-catenin encoding) HSPCs. We included additional double haploinsufficient mutants Csnk1a1-/+/Apc-/+ and Csnk1a1-/+/Egr1-/+. Results showed pronounced expansion of Csnk1a1-/+ clones, while Csnk1a1-/+/Ctnnb1-/+ clones were outcompeted over time, suggesting that the advantage of Csnk1a1-/+ clones is β-catenin dependent. Csnk1a1-/+/Egr1-/+ and Csnk1a1-/+/Apc-/+ clones were less advantageous than Csnk1a1-/+ clones. To further investigate the mechanism of clonal fitness in Csnk1a1-/+ haploinsufficient HSPCs, we performed droplet based single cell RNA sequencing of Csnk1a1-/+ and wt Lin-Sca1+cKit+ (LSK) HSPCs. Csnk1a1 -/+ LSK were characterized by a higher fraction of cells expressing cell cycle genes compared to wt cells. In line, transcriptional alterations in the most primitive HSCs suggest that the clonal advantage is conveyed by canonical Wnt signaling activating downstream targets such E2F proteins. Csnk1a1-/+ haploinsufficient multipotent progenitors and myeloid/lymphoid primed progenitors expressed marked upregulation of metabolic pathways, mitochondrial respiration, cell cycle and differentiation, ubiquitination/proteasome system and deregulation of ribosome biogenesis. In conclusion, we demonstrate using a novel genetic barcoding approach in a competitive transplant setting that Csnk1a1-/+ haploinsufficient HSPCs have the potential for oligoclonal expansion and clonal persistence. Wnt/β-catenin signaling plays a central role in the clonal expansion. Interestingly, in Csnk1a1 haploinsufficiency the HSC state is preserved and the increased proliferation and metabolic activation are hallmark features of differentiating progenitor cells at MPP stage, increasing with cell cycle activation, thus ensuring clonal stability and preventing HSC exhaustion over time. Disclosures Brümmendorf: University Hospital of the RWTH Aachen: Employment; Janssen: Consultancy; Pfizer: Consultancy, Research Funding; Merck: Consultancy; Novartis: Consultancy, Research Funding; Ariad: Consultancy. Ebert:Celgene: Research Funding; Deerfield: Research Funding; Broad Institute: Other: Contributor to a patent filing on this technology that is held by the Broad Institute..

Oligoclonal Expansion and T-Cells Subpopulations in Patients with Aplastic Anemia at Different Stages of the Disease

Background. The main mechanism of the bone marrow (BM) failure in idiopathic aplastic anemia (AA) has an immunomediated character. Researching the T-cell clone's effect in the AA pathogenesis is very relevant at the present time. Oligoclonal expansion of T cells is frequent in AA and the identification of immunodominant T-cell clones can correlate with the disease activity and may possibly serve as response predictor to immunosuppressive therapy (IST). The aim. To identify T-cells subpopulations, expression of PD-1 and PD-L1 on T-cells and TCR-Vβ repertoires by flow cytometry in different groups of AA patients. Methods. Thirty AA patients (pts) with median age of 30.5 (19-71), m/f ratio 1:1,3 were divided in 3 groups: pts with newly diagnosed (ND) AA (n=13), pts with overall response to IST (OR) (n=10), non-response pts (NR) for 2 and more lines of IST (n=7). Flow cytometry was performed with BD FACS Canto II. We used commercial kit (IOTest® Beta Mark TCR Vb Repertoire) for evaluation of TCR-Vβ repertoire in the bone marrow (BM) of these patients. We performed analysis of BM samples from healthy donors as a control group (n=8). Due to low amount of donor samples the maximal value each of the 24 subclones (for CD4+ (T-helpers - Th) and CD8+ cells (T-cytotoxic cells - TCL)) was accepted as threshold. We concluded the presence of clonal expansion if TCR subclone exceeded this threshold. We identified different T-cell subpopulations in all 3 groups of AA and healthy donors by flow cytometry: double positive T-cells (CD3+CD4+CD8+), double negative T-cells (CD3+CD4- CD8-), Th (CD3+CD4+), TCL (CD3+CD8+), NK-T-cells (CD3+CD56+) out of CD3+ cells. Among Th and TCL cells was determined naive T-cells (CD28+CD95-), effector T-cells (CD28-CD95+), memory T-cells (CD28+CD95+), regulatory T-cells (CD4+CD127-CD25high) and subpopulations Th and TCL co-expressed PD-1 and PD-L1. Multiple comparisons were assessed by ANOVA or Kruskal Wallis test by GraphPad Prism software. Results. In our study all 30 AA patients had an immunodominant TCR-Vβ clones among Th and/or TCL cells. We identified the most common clonotypes in comparison with healthy donors - Vβ1, Vβ2, Vβ3 among the Th cells and Vβ3, Vβ9, Vβ13.1 among the TCL cells. In ND group Vβ1 was highly expanded in 5 (38.5%), Vβ3 - in 7 (53.8%) pts among Th, and Vβ3 - in 3 (23.1%) and Vβ9 - in 4 (30.8%) out of 13 pts among TCL. In OR group Vβ2 expansion was in 4 (40%) and Vβ3 - in 5 (50%) pts among Th; Vβ3 in 6 (60%) and Vβ9 in 6 (60%) out of 10 pts among TCL. In NR group the most frequent was Vβ13.1 clone in TCL - in 3 (42.9%) out of 7 pts. In NR group in overall clonal expansion was less frequent than in ND and OR groups. We also analyzed the previously mentioned subpopulations of T-cells in patients with AA in three groups (ND, OR, NR) compared to healthy donors (table 1). We obtained significant differences in the count of naive Th and TCL cells, memory T-cells in all three groups of AA patients compared to donors: proportion of naive Th and TCL cells was significantly higher and proportion of memory Th cells was lower in the donor group than in AA pts. The percent of TCL effectors was higher in ND AA pts compare to donors. We also found that cell count of activated Th (CD4+CD25+) was higher in the group of refractory pts. In OR pts proportion of PD-1-positive Th was higher than in donors. In NR pts Th and TCL co-expressed with PD-L1 were lower compare to donors (table 1). Conclusions. In our study we found immunodominant clonotypes in different AA pts and depletion of the pool of naive T cells. Dynamic observation of changes in the most common clonotypes in AA pts during treatment will provide suitable therapy tactics (allogenic bone marrow transplantation or IST). Disclosures No relevant conflicts of interest to declare.

Oligoclonal Expansion of Effector Memory CD8+CD57+ T Cells May Sustain Bone Marrow Destruction in Aplastic Anemia

The character of oligoclonal expansion of CD8+CD28- lymphocytes in aplastic anemia (AA), described by Risitano et al. (Blood, 2002 and Lancet, 2004), strongly suggests an antigen driving mechanism of T cell activation leading to destruction of hematopoietic stem and progenitor cells. In this study, we focused on a subset of CD8+CD28- T lymphocytes termed effector memory cells because of their high antigen-affinity and ability to be rapid activated after antigen stimulation. We investigated the frequency and oligoclonal expansion of effector CD28-CD57+ memory cells in CD4+ and CD8+ T subsets and the T cell receptor (TCR) Vβ repertoire by flow cytometry, and next-generation sequencing (NGS). Peripheral blood mononuclear cells from 20 AA patients and 14 healthy controls were evaluated for Vβ usage by flow cytometry. At the time of sampling, none of the patients had yet received immunosuppressive therapy. CD28+CD57- and CD28-CD57+ cells in CD4+ and CD8+ populations were assessed using the IOTest Beta Mark and a LSRII Fortessa cytometer for acquisition. The mean + 3SD of each Vβ group in controls was used as a cut-off to determine Vβ skewing in patients. When the frequencies of CD28+ and CD57+ cells in CD4+ and CD8+ subsets were compared, no differences were found between patients and controls (p=0.6125). Polyclonal expansion was described in CD8+CD28+, CD4+CD28+ and CD4+CD57+ T cells in 60%, 45% and 70% of patients, respectively. Using the mean of CD8+CD57+ cell frequency in controls as a cut-off, 12 of 20 patients displayed expansion of effector memory CD8+ cells and 11 of them (55% of all patients) showed expansion in 1 to 6 Vβ families with 1 to 4 immunodominant clones, while only 4 of 14 (29%) healthy controls displayed expansion of two Vβ families in expanded CD8+CD57+ cells without polyclonal expansion of CD8+CD28+ cells. In four cases (20% of all cases) of the remaining 8 patients whose cells did not display expanded CD8+CD57+ cells, there was expansion of 1 to 3 Vβ families with 1 dominant clone. These findings suggest activation of the immune response, polyclonal expansion of effector CD28+ compartments, and oligoclonal activation of effector memory CD8+ cells, regardless of frequency in peripheral blood. The frequency of CD8+CD57+ cells modestly correlated with the mean Vβ expansion in each patient (r2=0.5831, p<0.001). Also, we analyzed VDJ combinations and complementary region 3 (CDR3) sequences in CD4+ and CD8+ cells from 8 AA patients with CD8+CD57+ expansion in the ILLumina Hiseq 2000 sequencer. For the CD4+ compartment, patients were similar to healthy control CD4+ cells: frequencies of the most abundant clones less than 6%, CDR3 size profiles with Gaussian distribution and low degree of diversity (Simpson's indexes range 0-1: 0 means infinite diversity, and 1 means no diversity). For CD8+ cells, 6 of 8 patients displayed a "skyscraper" Vβ/Jβ plot due to the presence of 1 to 3 dominant clones, with frequency greater than 10%, predominant classes in CDR3 size profile, and Simpson's index similar to the CD8+CD57+ subset (p=0.1914). Analyzing CDR3 amino acid sequences for homology, we found that all the dominant sequences from patients were present at very low frequency in a healthy donor CD8+ cell pool (range, 0.12% - 53.6% vs 0.01% - 1.64%, p=0.0037), but no matches were described for the healthy CD8+CD57+ CDR3 repertoire. Only three CDR3 sequences were shared between patients. One of these CDR3 sequences (CSARDPPVSGTRGTDTQYF) was present in all patients and controls at very low frequency (mean expression, 5.01%), and it was the dominant clone in one patient, carried by TRBV20-1/TRBJ2-3 rearrangement at a frequency of 53.6%. Shared sequences were not found in reported viral TCR repertoires, nor in T cells recognizing peptide of myelin basic protein in multiple sclerosis, or synovial T cells from rheumatoid arthritis patients, nor matches were identified in T-large granular lymphocyte leukemia, AA, cancer or melanoma-derived TCR repertoires. Our data support the hypothesis of autologous immune-mediated attack leading to bone marrow destruction, likely triggered by autoantigens. Our results highlight the role of effector memory CD8+CD57+ T cells in sustaining an aberrant immune response during active disease. CD8+CD57+ cell expansion mirrors Vβ oligoclonal expansion, and Vβ flow cytometry should be useful in diagnosis and periodic evaluation of AA patients. Disclosures Fernandez Ibanez: GSK/Novartis: Research Funding. Young:Novartis: Research Funding.

Oligoclonal Expansion of TCR Vδ T Cells May be a Potential Immune Biomarker for AML Outcome

Background: Recent data showed that gamma delta T cells can act as a vast functional setting of immune defense against tumors. Our previous study found that persist clonally expanded TRDV4 T cells might be relatively to better outcome for the patient with T-cell acute lymphoblastic leukemia (T-ALL) after hematopoietic stem cell transplantation (HSCT). However, little is known, the distribution and clonality of TRDV repertoire of gamma delta T cells in peripheral blood (PB) and its potential effect for clinical outcome in acute myeloid leukemia (AML) patients. Methods: Gamma delta T cells were sorted from peripheral blood of 30 patients with untreated de novo AML (AML-M0 6 cases, M1 1 case, M2 5 cases, M3 10 cases, M4 3 cases and M5 5 cases) and were observed the distribution of TRDV repertoire, while 12 healthy individuals served as normal controls. Then the TRDV repertoire were further analyzed by Genescan technique to determine the size of complementarity determining region 3 (CDR3) of TRDV (1-8) subfamilies genes and to evaluate the clonality of the detectable subfamilies of gamma delta cells. The binary Logistic regression analysis was used to explore the associations between clonal expansion of gamma delta T cells and outcome of AML patients. Results: The frequencies of TRDV1, TRDV3, TRDV4 and TRDV6 in gamma delta T cells from AML patients were significantly lower than that from healthy individuals (HI) (Figure A). The most frequently expanded TRDV subfamilies in AML patients were TRDV8 (56.67%) and TRDV4 (40%). Furthermore, the most frequently oligoclonally expanded TRDV subfamilies from AML patients were TRDV2 and TRDV4 subfamilies, and the clonal expansion frequencies were significant higher than that from healthy individuals, however, a significant lower clonal expansion frequency of TRDV1 was observed in AML patients (Figure B). Importantly, oligoclonalities of TRDV4 and TRDV8subfamilies were the independent protective factors for complete remission. In addition, the oligoclonal expansion frequencies of TRDV5 and TRDV6 subfamilies from the recurrence cases were significantly higher than that from non-recurrence cases. Significantly, oligoclonal TRDV5 and TRDV6subfamilies were found in the patients who developed AML relapse in different time point after first complete remission. Conclusions: The distribution and clonality of TRDV subfamilies significantly changed in gamma delta T cells from AML patients. Clonally expanded TRDV4 and TRDV8 T cells might contribute to immune response against AML, while oligoclonal TRDV5 and TRDV6 may be accompanied to patients who underwent AML relapse, the function of such oligoclones of gamma delta T cells is needed further investigation. Overall, these TRDV gamma delta T cell clones might be a potential immune biomarker for AML outcome. Foundation: This study was supported by grants from Natural Science Foundation of China (No.81200388), Natural Science Foundation of Guangdong Province (NO.2014A030313380) and the Project of the Zhujiang Science & Technology Star of Guangzhou City (No. 2013J2200046). Figure Figure. Disclosures No relevant conflicts of interest to declare.