Generation of Inducible BCL11B Knockout in TAL1/LMO1 Transgenic Mouse T Cell Leukemia/Lymphoma Model.

The B-cell CLL/lymphoma 11B gene (BCL11B) plays a crucial role in T-cell development, but its role in T-cell malignancies is still unclear. To study its role in the development of T-cell neoplasms, we generated an inducible BCL11B knockout in a murine T cell leukemia/lymphoma model. Mice, bearing human oncogenes TAL BHLH Transcription Factor 1 (TAL1; SCL) or LIM Domain Only 1 (LMO1), responsible for T-cell acute lymphoblastic leukemia (T-ALL) development, were crossed with BCL11B floxed and with CRE-ER/lox mice. The mice with a single oncogene BCL11Bflox/floxCREtg/tgTAL1tg or BCL11Bflox/floxCREtg/tgLMO1tg were healthy, bred normally, and were used to maintain the mice in culture. When crossed with each other, >90% of the double transgenic mice BCL11Bflox/floxCREtg/tgTAL1tgLMO1tg, within 3 to 6 months after birth, spontaneously developed T-cell leukemia/lymphoma. Upon administration of synthetic estrogen (tamoxifen), which binds to the estrogen receptor and activates the Cre recombinase, the BCL11B gene was knocked out by excision of its fourth exon from the genome. The mouse model of inducible BCL11B knockout we generated can be used to study the role of this gene in cancer development and the potential therapeutic effect of BCL11B inhibition in T-cell leukemia and lymphoma.

Allogeneic stem cell transplantation for mantle cell lymphoma-update of the prospective trials of the East German Study Group Hematology/Oncology (OSHO#60 and #74).

Mantle cell lymphoma (MCL) is a non-Hodgkin's lymphoma with an often aggressive course, incurable by chemotherapy. Consolidation with high-dose therapy and autologous stem cell transplantation (autoSCT) has a low transplant-related mortality but does not lead to a survival plateau. Allogeneic stem cell transplantation (alloSCT) is associated with a higher early mortality, but can cure MCL. To investigate alloSCT for therapy of MCL, we conducted two prospective trials for de novo MCL (OSHO#74) and for relapsed or refractory MCL (OSHO#60). Fifteen and 24 patients were recruited, respectively. Induction was mainly R-DHAP alternating with R-CHOP. Conditioning was either Busulfan/Cyclophosphamide or Treosulfan/Fludarabin. Either HLA-identical siblings or matched-unrelated donors with not more than one mismatch were allowed. ATG was mandatory in mismatched or unrelated transplantation. Progression-free survival (PFS) was 62% and overall survival (OS) was 68% after 16.5-year follow-up. Significant differences in PFS and OS between both trials were not observed. Patients below 56 years and patients after myeloablative conditioning had a better outcome compared to patients of the corresponding groups. Nine patients have died between day +8 and 5.9 years after SCT. Data from 7 long-term surviving patients showed an excellent Quality-of-life (QoL) after alloSCT. AlloSCT for MCL delivers excellent long-term survival data. The early mortality is higher than after autoSCT; however, the survival curves after alloSCT indicate the curative potential of this therapy. AlloSCT is a standard of care for all feasible patients with refractory or relapsed MCL and should offer to selected patients with de novo MCL and a poor risk profile. For defining the position of alloSCT in the therapeutic algorithm of MCL therapy, a randomized comparison of autoSCT and alloSCT is mandatory.

Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations.

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B-BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

Easy Expression and Purification of Fluorescent N-Terminal BCL11B CCHC Zinc Finger Domain.

Zinc finger proteins play pivotal roles in health and disease and exert critical functions in various cellular processes. A majority of zinc finger proteins bind DNA and act as transcription factors. B-cell lymphoma/leukemia 11B (BCL11B) represents one member of the large family of zinc finger proteins. The N-terminal domain of BCL11B was shown to be crucial for BCL11B to exert its proper function by homodimerization. Here, we describe an easy and fast preparation protocol to yield the fluorescently tagged protein of the recombinant N-terminal BCL11B zinc finger domain (BCL11B42-94) for in vitro studies. First, we expressed fluorescently tagged BCL11B42-94 in E. coli and described the subsequent purification utilizing immobilized metal ion affinity chromatography to achieve very high yields of a purified fusion protein of 200 mg/L culture. We proceeded with characterizing the atypical zinc finger domain using circular dichroism and size exclusion chromatography. Validation of the functional fluorescent pair CyPet-/EYFP-BCL11B42-94 was achieved with Förster resonance energy transfer. Our protocol can be utilized to study other zinc finger domains to expand the knowledge in this field.

Targeting IL-11 in the treatment of BK virus-associated haemorrhagic cystitis-A promising new approach.

The BK polyomavirus (BKPyV) has pathogenic relevance especially in immunocompromised patients. No causal therapy has been established yet. Therefore, new therapeutic targets need to be identified in experimental studies. A 3D organotypic cell culture model with primary urothelial cells and fibroblasts was used as infection model. The detection of virus replication was performed with quantitative polymerase chain reaction (qPCR), and immunohistochemistry (IHC) was also used for analysis. Interleukin levels were measured by enzyme-linked immunosorbent assay (ELISA). Interestingly, the signal transducer and activator of transcription 3 (STAT3) pathway seems to be activated during infection with BKPyV, for example phosphorylated STAT3 is significantly (P < 0.0001) elevated on day 6 following infection. Therefore, we performed ELISAs for involved interleukins in STAT3 pathway. Interleukin 11 (IL-11) was significantly (P = 0.026) elevated at day 9. Subsequently, 3D cultures were treated with IL-11 neutralizing antibody. At day 9 following infection, the median virus replication rate is 4.4 × 106 copies/ml. The difference to replication rate without treatment was significantly lower at day 6 (P < 0.0001) and at day 9 (P < 0.0001), respectively. STAT3 pathways seem to be involved during BKPyV infection and need further investigation in experimental studies. A very promising target for treatment might be IL-11.

Recovery from hypogonadism and male health in adult allogeneic stem cell transplantation.

OBJECTIVE: There is a substantial lack of data about men`s health in adult allogeneic stem cell transplantation. METHODS: We conducted prospective unicentric non-interventional clinical study on men's health with a follow-up time of 1 year. RESULTS: Between 11/2013 and 12/2015, we were able to include 27 patients. AML was the most frequent underlying disease (25.9%), and we mainly used intermediate intense conditioning protocols (77.8%). Erectile dysfunction, loss of libido, and loss of efficiency were the most frequent symptoms of hypogonadism. At inclusion of the study, hypogonadism was already frequent. Primary hypogonadism was found in eight cases (29.6%) and secondary hypogonadism in one case (3.7%). We did not observe hypogonadism 6 months after inpatient treatment anymore, but there might still be the impairment of fertility because of still rising FSH levels at the end of the observation period. There were no significant associations of hypogonadism with myeloablative conditioning or kind of donor. Interestingly, there is a significant association with nicotine abuse (P = .049). CONCLUSIONS: On the whole, male hypogonadism was found in one-third of the patients who underwent allogeneic stem cell transplantation.

Hypothermic, initially oxygen-free, controlled limb reperfusion for acute limb ischemia.

BACKGROUND: Controlled limb reperfusion has been shown to prevent the deleterious effects of ischemia-reperfusion (IR) syndrome following revascularization of acute limb ischemia (ALI). To reduce the production of cell-toxic oxygen-free radicals, we have established a new initially oxygen-free, hypothermic, heparin-coated perfusion and hemofiltration system and report on our first results. METHODS: In a retrospective single-center study, controlled limb reperfusion was applied in 36 patients (64.7 ± 15 years) with ALI of category IIA to III (33.7 ± 20.7 hr ischemic time). 52.8% had central (aortic and bifurcation) and 47.2% had peripheral (common iliac artery and distal) vascular occlusions. The common femoral artery and vein were cannulated, and a hypothermic (22°C), initially oxygen-free, potassium-free ringer's solution was perfused using a heparin-coated extracorporeal membrane oxygenation (ECMO) and hemofiltration system with low-dose heparinization. Thirty-day mortality, clinical recovery of neurological dysfunction, limb amputation, and fasciotomy rate were analyzed. Laboratory parameters associated with ischemia and IR injury were determined. RESULTS: Average perfusion time was 94 ± 35 min. Thirty-day mortality was 27.8%. 55.5% of patients showed complete recovery of motor and sensory dysfunction. A total of 27.8% of patients developed a compartment syndrome and required fasciotomy. Lower leg amputation was necessary in 11.1% of patients. Lactate levels were reduced in ischemic limbs by 25.3% within 60 min (P < 0.05). Preoperative negative base excess of -1.96 ± 0.96 mmol/L was equalized after 12 hr (P < 0.05), while pH stayed balanced at 7.4. Serum potassium stayed within normal limits throughout 24 hr, and therefore systemic hyperkalemia was prevented and imminent metabolic acidosis was corrected. CONCLUSIONS: An initially oxygen-free, hypothermic, heparin-coated ECMO counteracts local and systemic effects of IR injury. Reduced mortality and morbidity might result from this new treatment, although this could not be conclusively proven in our study. A prospective, randomized controlled trial is needed to prove superiority of this new concept.

Submicroscopic genomic rearrangements change gene expression in T-cell large granular lymphocyte leukemia.

OBJECTIVES: To better understand the molecular pathogenesis of T-cell large granular lymphocyte leukemia (T-LGL), we decided to search for those genetic alterations in T-LGL patients and MOTN-1 cell line (established from T-LGL patient) that have an impact on gene expression and as a result can influence cell biology. METHODS: Multicolor fluorescence in situ hybridization (mFISH) analysis of the MOTN-1 cell line was performed as well as paired-end next-generation sequencing (NGS; Illumina HiSeq2000) of this cell line and one T-LGL patient. In addition, chosen 6q region was characterized in three T-LGL patients using high-resolution comparative genomic hybridization (FT-CGH) and LM-PCR. Gene expression was studied by RNA sequencing (RNAseq; SOLID5500). RESULTS: Rearrangements were detected within 1p and 2q in MOTN-1 affecting expression of FGR, ZEB2, and CASP8, and within 6q in MOTN-1 and one T-LGL patient affecting MAP3K5 and IFNGR1. Nineteen genes, among them FOXN3, RIN3, AKT1, PPP2R5C, were overexpressed as a result of an amplification in 14q in one T-LGL patient. Two novel fusion transcripts were identified: CASP8-ERBB4 in MOTN-1 and SBF1-PKHD1L1 in T-LGL patient. CONCLUSIONS: This study showed that submicroscopic genomic rearrangements change gene expression in T-LGL. Several genes involved in rearrangements were previously linked to cancer and survival pattern that characterizes T-LGL cells.

Paraincisional subcutaneous infusion of ropivacaine after open abdominal vascular surgery shows significant advantages.

BACKGROUND: Opiates are widely used for postoperative pain relief. Unfortunately, their side effects such as inhibited gastrointestinal motility and respiratory depression may compromise or delay postoperative recovery after laparotomy. We used paraincisional subcutaneous catheters (PSCs) and applied 0.25% ropivacaine infusion to improve pain relief and decrease postoperative morphine consumption in patients after open surgery for aortic aneurysm. METHODS: A retrospective single-center study including 58 patients treated by open surgery for aortic aneurysm between October 2006 and June 2012. Overall, 28 patients (control group) received standard postoperative pain management including opiates, and 30 patients (PSC group) were treated with paraincisional continuous local analgesia with 0.25% ropivacaine administrated via bilateral subcutaneous catheters along with additional ad libitum opiates administration, at first intravenously and then orally. RESULTS: Patients characteristics as well as perioperative and postoperative outcomes were comparable between the groups during the first 5 days after surgery. Patients of the PSC group received significantly less morphine, although the patients in both groups reported a similar pain intensity. Neither wound-healing disorder nor catheter-associated subcutaneous infection was reported. High serum concentration of ropivacaine was detected in 2 patients (6%) with end-stage renal disease, who developed temporary neurologic symptoms. Length of intensive care unit (ICU) stay was significantly shorter in the PSC group (2 [0-23] vs. 4.5 [0-32] ICU days). CONCLUSIONS: This is the first report about PSCs for analgesia after laparotomy. This case/control study shows that continuous paraincisional subcutaneous infusion of 0.25% ropivacaine after open surgery for aortic aneurysm repair is a feasible method of postoperative analgesia. This technique allows sustained pain relief with significant reduction of opiate requirement and faster recovery after surgery. Prospective randomized controlled trial is necessary for the assessment of safety and efficacy of this method.

Veno-venous perfusion to cool and rewarm in thoracic and thoracoabdominal aortic aneurysm repair.

BACKGROUND: Femoro-femoral veno-arterial perfusion is an established circulatory support and cooling method for thoracic- and/or thoracoabdominal aortic aneurysm repair. However, retrograde perfusion through femoral arteries can lead to retrograde cerebral embolization and neurologic dysfunction after surgery. To avoid these complications, we have established a femoro-femoral veno-venous perfusion technique and evaluated its safety and effectiveness in elective and nonelective patients. METHODS: Common femoral veins were cannulated bilaterally percutaneously following systemic low-dose heparinization (100 IU/kg body weight). Venous blood was drained from drainage of the inferior vena cava, and venous return followed through the superior vena cava. After proximal aortic cross-clamping, veno-venous perfusion was switched to veno-arterial antegrade perfusion through the distal descending thoracic aorta to achieve spinal and visceral perfusion or through iliac arteries for distal perfusion combined with selective renovisceral blood perfusion. After completion of aortic repair, the arterial cannula was removed and the patient rewarmed just by switching back to veno-venous perfusion. Gas and temperature exchange as well as relevant hemodynamic parameters were recorded prospectively and analyzed retrospectively in 25 consecutive patients including 15 nonelective cases. RESULTS: Percutaneous insertion of outflow (28F cannula) and inflow (18F cannula) venous cannulae was complication-free and allowed unrestricted perfusion in all 25 patients. Veno-venous perfusion allowed effective cooling (mean body temperature 36.6 ± 0.6°C to 31.6 ± 2.1°C, P = .001 compared with start of cooling) and re-warming (mean body temperature 30.5 ± 3°C to 36.3 ± 0.8°C, P = .03 compared with start of re-warming). Hemodynamic as well as pulmonary parameters remained remarkably stable during surgical dissection and single lung ventilation even in nonelective cases. There was no complication associated with the perfusion technique during surgery. CONCLUSIONS: Transfemoral veno-venous cooling and re-warming results in remarkable hemodynamic stability during open repair of thoracic- and/or thoracoabdominal aortic aneurysms and eliminates the need for retrograde arterial perfusion and its inherent risks.

Endoluminal stent-graft relining of visceral artery bypass grafts to treat perigraft seroma.

PURPOSE: To describe the endovascular treatment of intra-abdominal perigraft seromas associated with small-caliber expanded polytetrafluoroethylene (ePTFE) grafts. CASE REPORTS: Two patients who underwent hybrid repair of thoracoabdominal aortic aneurysms in which renovisceral bypass grafts were implanted presented with large, symptomatic perigraft seromas. The 5- to 8-mm-diameter ePTFE bypass grafts believed to be involved in the seromas were successfully relined with self-expanding Viabahn stent-grafts in percutaneous procedures. The patients' symptoms were relieved, and imaging follow-up (18 and 10 months, respectively) has shown near complete resorption of the seromas. CONCLUSION: It is expected that this minimally invasive technique could be very valuable in treating aortic, renovisceral, and peripheral perigraft seroma.

New insights into antigen specific immunotherapy for chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a stem cell disease in which BCR/ABL plays an important role as an oncoprotein and a molecular and immunogenic target. Despite the success of targeted therapy using tyrosine kinase inhibitors (TKIs), CML remains largely incurable, most likely due to the treatment resistance of leukemic stem cells. Several immunotherapies have been developed for CML in different stages and relapse after allogeneic stem cell transplantation. In the this review, several specific immunotherapeutic approaches for CML, including vaccination and adoptive cellular immunotherapy, are discussed along with results from clinical trials, and the value of such immunotherapies in the era of imatinib and leukemia-associated antigens (LAAs), which are capable of inducing specific T cell responses and are appropriate target structures for the immunological targeting of CML cells, are also summarized.

Toxoplasmosis after allogeneic stem cell transplantation--a single centre experience.

Toxoplasmosis is a rare but possibly underestimated complication following allogeneic stem cell transplantation with a high mortality rate. One reason might be the limitation of the diagnostic instruments relying mainly on imaging and molecular-based techniques. In this report, we present three cases of toxoplasmosis identified among 155 allograft recipients treated at Greifswald University Hospital. Widely disseminated toxoplasmosis was detected post-mortem in two patients allografted for high-risk multiple myeloma. Clinical signs suspicious for toxoplasmosis occurred after days +32 and +75, respectively. In one case, serology and conventional Toxoplasma gondii PCR, targeting the B1 gene, revealed negative results, while in the other patient, toxoplasmosis was not investigated. Both patients received pentamidine for Pneumocystis jirovecii pneumonia (PcP) prophylaxis. The third patient, a 68-year-old woman allografted for AML, developed cerebral toxoplasmosis from day +395 after allogeneic SCT with typical signs in magnetic resonance tomography. Toxoplasma DNA was amplified from one of two samples of cerebrospinal fluid. The patient died of disseminated toxoplasmosis despite immediate initiation of therapy. Retrospective comparative testing of clinical specimens by the conventional T. gondii PCR and by a real-time PCR targeting a 529-bp genomic fragment suggests a higher sensitivity of the latter method in our patients. In conclusion, we suggest a rigorous real-time PCR monitoring for high-risk patients or patients with signs of infections suspicious for toxoplasmosis, even though low-copy results are presently difficult to interpret. Our reported cases might also encourage the use of trimethoprim-sufmethoxazole instead of pentamidine for PcP prophylaxis in those patients.

BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood αß CD8+ T cells.

BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

Implementation of Palliative Care in Clinical Practice in German Units for Allogeneic Stem Cell Transplantation: A Nationwide Survey.

Allogeneic stem cell transplantation (alloSCT) is a curative therapy for otherwise fatal diseases, however it is associated with a considerable morbidity and mortality. In consequence, it can be assumed that a considerable percentage of patients would benefit from high-quality palliative care (PC) during their course of disease. To assess the standard of PC in German transplant centers, a questionnaire was sent out to all German centers recognized from the EBMT membership list and the annually ZKRD report (n = 52). The response rate was not as high as expected with n = 27 (51,9%), even after reminding by phone calls or by e-mails. In brief, palliative care after allogeneic stem cell transplantation shows a wide variation in Germany. This is true for structures, processes and measures. A national standard for SCT-patients has not been established so far and there are no pre-conditions concerning palliative care after alloSCT for a certification by the EBMT according the JACIE standards. There is a considerable need for a crosslinking of alloSCT with PC. Clear standards should be established by the scientific societies concerning personnel, structure and processes.

Proteome analysis reveals new mechanisms of Bcl11b-loss driven apoptosis.

The Bcl11b protein was shown to be important for a variety of functions such as T cell differentiation, normal development of central nervous system, and DNA damage response. Malignant T cells undergo apoptotic cell death upon BCL11B down-regulation, however, the detailed mechanism of cell death is not fully understood yet. Here we employed two-dimensional difference in-gel electrophoresis (2D-DIGE), mass spectrometry and cell biological experiments to investigate the role of Bcl11b in malignant T cell lines such as Jurkat and huT78. We provide evidence for the involvement of the mitochondrial apoptotic pathway and observed cleavage and fragments of known caspase targets such as myosin, spectrin, and vimentin. Our findings suggest an involvement of ERM proteins, which were up-regulated and phosphorylated upon Bcl11b down-regulation. Moreover, the levels of several proteins implicated in cell cycle entry, including DUT-N, CDK6, MCM4, MCM6, and MAT1 were elevated. Thus, the proteome data presented here confirm previous findings concerning the consequences of BCL11B knock-down and provide new insight into the mechanisms of cell death and cell cycle disturbances induced by Bcl11b depletion.

Two subsets of naive T helper cells with distinct T cell receptor excision circle content in human adult peripheral blood.

During ageing thymic function declines and is unable to meet the demand for peripheral T helper (Th) cell replenishment. Therefore, population maintenance of naive Th cells must be at least partly peripherally based. Such peripheral postthymic expansion of recent thymic emigrants (RTEs) during ageing consequently should lead to loss or dilution of T cell receptor excision circles (TRECs) from a subset of naive T cells. We have identified two subsets of naive Th cells in human adult peripheral blood characterized by a striking unequal content of TRECs, indicating different peripheral proliferative histories. TRECs are highly enriched in peripheral naive CD45RA(+) Th cells coexpressing CD31 compared with peripheral naive CD45RA(+) Th cells lacking CD31 expression, in which TRECs can hardly be detected. Furthermore we show that CD31(-)CD45RA(+) Th cells account for increasing percentages of the naive peripheral Th cell pool during ageing but retain phenotypic and functional features of naive Th cells. As CD31 is lost upon T cell receptor (TCR) engagement in vitro, we hypothesize that TCR triggering is a prerequisite for homeostatically driven peripheral postthymic expansion of human naive RTEs. We describe here the identification of peripherally expanded naive Th cells in human adult blood characterized by the loss of CD31 expression and a highly reduced TREC content.

Developmental stage, phenotype, and migration distinguish naive- and effector/memory-like CD4+ regulatory T cells.

Regulatory T cells (Tregs) fulfill a central role in immune regulation. We reported previously that the integrin alphaEbeta7 discriminates distinct subsets of murine CD4+ regulatory T cells. Use of this marker has now helped to unravel a fundamental dichotomy among regulatory T cells. alphaE-CD25+ cells expressed L-selectin and CCR7, enabling recirculation through lymphoid tissues. In contrast, alphaE -positive subsets (CD25+ and CD25-) displayed an effector/memory phenotype expressing high levels of E/P-selectin-binding ligands, multiple adhesion molecules as well as receptors for inflammatory chemokines, allowing efficient migration into inflamed sites. Accordingly, alphaE -expressing cells were found to be the most potent suppressors of inflammatory processes in disease models such as antigen-induced arthritis.

Decreased level of recent thymic emigrants in CD4+ and CD8+T cells from CML patients.

BACKGROUND: T-cell immunodeficiency is a common feature in cancer patients, which may relate to initiation and development of tumor. Based on our previous finding, to further characterize the immune status, T cell proliferative history was analyzed in CD4+ and CD8+ T cells from chronic myeloid leukemia (CML) patients. METHODS: Quantitative analysis of deltaRec-psiJalpha signal joint T cell receptor excision circles (sjTRECs) was performed in PBMCs, CD3+, CD4+ and CD8+T cells by real-time PCR, and the analysis of 23 TRBV-D1 sjTRECs was performed by semi-nested PCR. Forty eight CML cases in chronic phase (CML-CP) were selected for this study and 17 healthy individuals served as controls. RESULTS: The levels of deltaRec-psiJalpha sjTRECs in PBMCs, CD3+, CD4+, and CD8+ T cells were significantly decreased in CML patients, compared with control groups. Moreover, the numbers of detectable TRBV subfamily sjTRECs, as well as the frequency of particular TRBV-BD1 sjTRECs in patients with CML were significantly lower than those from healthy individuals. CONCLUSIONS: We observed decreased levels of recent thymic emigrants in CD4+ and CD8+ T cells that may underlay the persistent immunodeficiency in CML patients.