Low dose post-transplant cyclophosphamide and sirolimus induce mixed chimerism with CTLA4-Ig or lymphocyte depletion in an MHC-mismatched murine allotransplantation model.

Allogeneic hematopoietic cell transplantation (allo-HCT) offers a curative option for patients with certain non-malignant hematological diseases. High-dose post-transplant cyclophosphamide (PT-Cy) (200 mg/kg) and sirolimus (3 mg/kg), (HiC) synergistically induce stable mixed chimerism. Further, sirolimus and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin (CTLA4-Ig), also known as Abatacept (Aba), promote immune tolerance and allograft survival. Here, in a major histocompatibility complex (MHC)-mismatched allo-HCT murine model, we combined Aba and/or T-cell depleting anti-Thy1.2 (Thy) with a lower dose of PT-Cy (50 mg/kg) and Sirolimus (3 mg/kg), (LoC). While mice in the LoC group showed graft rejection, the addition of Thy to LoC induced similar donor chimerism levels when compared to the HiC group. However, the addition of Aba to LoC led to graft acceptance only in younger mice. When Thy was added to the LoC+Aba setting, graft acceptance was restored in both age groups. Engrafted groups displayed significantly reduced frequencies of recipient-specific interferon-γ-producing T cells as well as an increased frequency in regulatory T cells (Tregs) except in the LoC+Aba group. Splenocytes from engrafted mice showed no proliferation upon restimulation with Balb/c stimulators. Collectively, in combination with Aba or Thy, LoC may be considered to reduce graft rejection in patients who undergo allo-HCT.

Kruppel-like factor 1-GATA1 fusion protein improves the sickle cell disease phenotype in mice both in vitro and in vivo.

Sickle cell disease (SCD) and ß-thalassemia are among the most common genetic disorders worldwide, affecting global health and mortality. Hemoglobin A2 (HbA2, α2δ2) is expressed at a low level in adult blood due to the lack of the Kruppel-like factor 1 (KLF1) binding motif in the δ-globin promoter region. However, HbA2 is fully functional as an oxygen transporter, and could be a valid antisickling agent in SCD, as well as a substitute for hemoglobin A in ß-thalassemia. We have previously demonstrated that KLF1-GATA1 fusion protein could interact with the δ-globin promoter and increase δ-globin expression in human primary CD34+ cells. We report the effects of 2 KLF1-GATA1 fusion proteins on hemoglobin expression, as well as SCD phenotypic correction in vitro and in vivo. Forced expression of KLF1-GATA1 fusion protein enhanced δ-globin gene and HbA2 expression, as well as reduced hypoxia-related sickling, in erythroid cells cultured from both human sickle CD34+ cells and SCD mouse hematopoietic stem cells (HSCs). The fusion proteins had no impact on erythroid cell differentiation, proliferation, and enucleation. Transplantation of highly purified SCD mouse HSCs expressing KLF1-GATA1 fusion protein into SCD mice lessened the severity of the anemia, reduced the sickling of red blood cells, improved SCD-related pathological alterations in spleen, kidney, and liver, and restored urine-concentrating ability in recipient mice. Taken together, these results indicate that the use of KLF1-GATA1 fusion constructs may represent a new gene therapy approach for hemoglobinopathies.

Comparative analysis of the immune response to RFA and cryoablation in a colon cancer mouse model.

The immune response to radiofrequency ablation (RFA) and cryoablation (CRA) was characterized and compared in a colon cancer mouse model. All studies were conducted under a research protocol approved by the National Institutes of Health, Clinical Center, Animal Care and Use Committee. BALB/cJ mice were inoculated with CT26 cells, and randomized to RFA, CRA, or sham treatment. Mice were sacrificed 3 days post-treatment, and tumor, spleen, and serum were harvested. Cell death was determined by Caspase-3 immunohistochemical and TUNEL stains. Immune response was analyzed using flow cytometry, serum cytokine assay and immunohistochemistry. Cell death, necrosis, and apoptosis induced by ablation were comparable in RFA and CRA. Decreased frequency of systemic T-regulatory cells was found in the CRA group. Both RFA and CRA reduced frequencies of several myeloid-derived suppressor cell (MDSC) subpopulations. RFA induced pro-inflammatory cytokine secretion including TNF-α and IL-12 as well as anti-inflammatory cytokines IL-5, and IL-10. CRA augmented secretion of a wider array of cytokines compared to RFA with both pro- and anti-inflammatory properties including IL-1ß, IL-5, IL-6, IL-10, and KC GRO. In the tumor microenvironment, RFA reduced the number of T-regulatory cells, a finding not observed with CRA. Reduction of immune suppression via decreases in T-regulatory cells and MDSC was found to be induced by RFA or CRA. CRA augmented a wider range of cytokines than RFA, which were mainly pro-inflammatory, but also anti-inflammatory. In the tumor microenvironment, RFA demonstrated more pronounced anti-tumoral immunity. Further delineation of specific immunomodulation induced by ablation could inform drug-device development and may play a role in future hypothesis-driven immunomodulatory paradigms that combine immunotherapy drugs with tumor destruction for the treatment of metastatic colon cancer.

Early Myeloid Derived Suppressor Cells (eMDSCs) Are Associated With High Donor Myeloid Chimerism Following Haploidentical HSCT for Sickle Cell Disease.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC<20%) groups along with engrafted and rejected groups. We found that early myeloid-derived suppressor cell (eMDSC) frequencies were elevated in engrafted patients and patients with HDMC at PT-Day 30 (P< 0.04 & P< 0.003, respectively). 9 of 20 patients were evaluated for the source of eMDSCs. The HDMC group had high mixed chimeric eMDSCs as compared to the LDMC group (P< 0.00001). We found a positive correlation between the frequencies of eMDSCs and Tregs at PT-Day 100 (r=0.72, P <0.0007); eMDSCs at BSL and Tregs at PT-Day 100 (r=0.63, P <0.004). Of 10 immune regulatory cells and 50 cytokines, we observed mixed chimeric eMDSCs and IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, TGFb1 as potential hits which could serve as prognostic markers in predicting allograft outcome towards engraftment following haploidentical HSCT employing post-transplant cyclophosphamide. The current findings need to be replicated and further explored in a larger cohort.

Identification of candida albicans and nonalbicans candida resistant species in tobacco users and oral squamous cell carcinoma patients: Comparison of HiCrome agar and automated VITEK 2 system.

Introduction: Candida is most common fungal pathogen in the immunocompromised and medically ill patients. Higher prevalence of Candida albicans has been reported in tobacco users and oral squamous cell carcinoma (OSCC) patients which may be due to immunosuppression. Recently, emergence of nonalbicans candida (NAC) species resistant to conventional antifungal treatment has been observed that requires accurate identification of organisms at species level for reduction of progression of suspicious oral lesions toward malignancy. Aims and Objectives: To detect and compare the prevalence of C. albicans and NAC species smokeless tobacco chewers, histopathologically confirmed oral squamous cell carcinoma patients and the normal individuals. Effectiveness of automated Vitek 2 system in comparison to HiCrome agar color media in the identification of the candida species was also evaluated. Methodology: One hundred and fifty patients (90 males, 60 females) aged between 20 and 76 years were divided into three groups: Group I individuals with habit of chewing Gutka, and betel quid/pan masala with or without tobacco, Group II individuals with clinically and histopathologically confirmed oral squamous cell carcinoma and Group III comprised of controls. Salivary samples were cultured on HiCrome agar color media and results were compared with those of Vitek 2 system in the accurate identification of candida species. Data were statistically analyzed and Chi-square test was used to estimate the effectiveness of color and Vitek method in the identification of candida species in all the three groups. P < 0.05 was considered to be statistically significant. RESULTS: HiCrome agar color method identified six candida isolates C. albicans, Candida tropicalis, Candida krusei and Candida glabrata isolates in all the three groups, with 0.00 unidentified organisms (P = 0.00001) whereas VITEK 2 system identified five isolates of candida; C. albicans, Candida famat, Candida ciferri, Candida gulleri, C. tropicalis, unidentified organisms were observed in 26% of subjects. Further confirmation by supplemental tests indicated the presence of two or three organisms of different species/or subspecies with low reactivity biopattern. Higher incidence of opportunistic infections was seen in Group II OSCC patients (P = 0.00001). Conclusion: The results suggested that there is shift toward NAC species, with higher species diversity in OSCC patients followed by gutka, betel quid/pan masala with or without tobacco users. Conventional agar media culture methods of species identification should be used in conjunction with automated Vitek 2 method for better control of candida-associated oral cancer.

Hyperlipidaemia and IFNgamma/TNFalpha Synergism are associated with cholesterol crystal formation in Endothelial cells partly through modulation of Lysosomal pH and Cholesterol homeostasis.

BACKGROUND: Inflammation plays an important role in the development of cardiovascular disease (CVD). Patients with chronic inflammation diseases have high levels of inflammation and early fatal myocardial infarction due to early, unstable coronary plaques. Cholesterol crystals (CC) play a key role in atherogenesis. However, the underlying mechanisms of endothelial cell (EC)-derived CC formation are not well understood in chronic inflammation. METHODS: We utilized a combination of a mouse psoriasis model (K14-Rac1V12 mouse model) and human psoriasis patients to study the effect of inflammatory cytokines on CC formation in ECs. Lysosomal pH, alterations in lipid load and inflammatory proteins were evaluated as potential mechanisms linking inflammatory cytokines to CC formation. Coronary CT angiography was performed (n = 224) to characterize potential IFNγ and TNFα synergism on vascular diseases in vivo. FINDINGS: We detected CC presence in the aorta of K14-Rac1V12 mice on chow diet. IFNγ and TNFα were found to synergistically increase LDL-induced CC formation by almost 2-fold. There was an increase in lysosomal pH accompanied by a 28% loss in pH-dependent lysosomal signal and altered vATPaseV1E1 expression patterns. In parallel, we found that LDL+IFNγ/TNFα treatments increased free cholesterol content within EC and led to a decrease in SOAT-1 expression, an enzyme critically involved cholesterol homeostasis. Finally, the product of IFNγ and TNFα positively associated with early non-calcified coronary burden in patients with psoriasis (n = 224; ß = 0.28, p < 0.001). INTERPRETATION: Our results provide evidence that IFNγ and TNFα accelerate CC formation in endothelial cells in part by altering lysosomal pH and free cholesterol load. These changes promote early atherogenesis and contribute to understanding the burden of CVD in psoriasis. FUNDING: Funding was provided by the Intramural Research Program at NIH (NNM) and the National Psoriasis Foundation (NNM and YB).

Dendritic Cell-Restricted Progenitors Contribute to Obesity-Associated Airway Inflammation via Adam17-p38 MAPK-Dependent Pathway.

Proliferation of dendritic cell (DC)-restricted progenitor cells in bone marrow compartment is tightly regulated at steady state and responds to multiple tissue-specific triggers during disturbed homeostasis such as obesity. DCs in the lung stem from a rapidly dividing DC-restricted progenitor cells and are effective at generating adaptive immune responses in allergic airway inflammation. Precisely, how DC-restricted progenitor expansion and differentiation are influenced by airway inflammation to maintain constant supply of myeloid DCs is poorly understood. Here we show that a high fat diet (HFD) induces oxidative stress and accelerates the expansion of DC- restricted progenitor cells in bone marrow and correlates with persistent induction of p38 mitogen activated protein kinase (MAPK), which is blocked with a selective p38α/ß MAPK inhibitor. Mice fed a HFD and sensitized to inhaled allergen house dust mite (HDM) led to alterations of DC- restricted progenitor cells that were characterized by increased expansion and seeding of lung DCs in airway inflammation. Mechanistically, we establish that the expansion induced by HFD dysregulates the expression of a disintegrin and metallopeptidase domain 17 (Adam17) and is required for p38 MAPK activation in DC-restricted progenitors. These results demonstrates that obesity produces persistent changes in DC precursors and that elevation of Adam17 expression is tightly coupled to p38 MAPK and is a key driver of proliferation. Altogether, these data provide phenotypic and mechanistic insight into dendritic cell supply chain in obesity-associated airway inflammation.

Histone Deacetylase Inhibitor Modulates NKG2D Receptor Expression and Memory Phenotype of Human Gamma/Delta T Cells Upon Interaction With Tumor Cells.

The functional plasticity and anti-tumor potential of human γδ T cells have been widely studied. However, the epigenetic regulation of γδ T-cell/tumor cell interactions has been poorly investigated. In the present study, we show that treatment with the histone deacetylase inhibitor Valproic acid (VPA) significantly enhanced the expression and/or release of the NKG2D ligands MICA, MICB and ULBP-2, but not ULBP-1 in the pancreatic carcinoma cell line Panc89 and the prostate carcinoma cell line PC-3. Under in vitro tumor co-culture conditions, the expression of full length and the truncated form of the NKG2D receptor in γδ T cells was significantly downregulated. Furthermore, using a newly established flow cytometry-based method to analyze histone acetylation (H3K9ac) in γδ T cells, we showed constitutive H3K9aclow and inducible H3K9achigh expression in Vδ2 T cells. The detailed analysis of H3K9aclow Vδ2 T cells revealed a significant reversion of TEMRA to TEM phenotype during in vitro co-culture with pancreatic ductal adenocarcinoma cells. Our study uncovers novel mechanisms of how epigenetic modifiers modulate γδ T-cell differentiation during interaction with tumor cells. This information is important when considering combination therapy of VPA with the γδ T-cell-based immunotherapy for the treatment of certain types of cancer.

Ultrasensitive Quantification of Cytokine Proteins in Single Lymphocytes From Human Blood Following ex-vivo Stimulation.

In this study we demonstrate the feasibility of direct, quantitative measurement of cytokine proteins in single human CD8 lymphocytes from fresh peripheral blood of healthy donors following a brief ex vivo stimulation. Cytokine-secreting cells were identified using cell surface "catch" reagents and single cell data were obtained by sorting of individual cytokine-secreting cells into 96 well plates containing lysis buffer followed by analysis using ultrasensitive immunoassays for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). CD8 cells negative for cytokine production, as determined by the cell surface catch reagents were used as negative controls. Furthermore, studies were undertaken to compare the mean fluorescence intensity (MFI) values of cytokine staining by flow cytometry with the quantification of cytokines using the current method. This study demonstrates that it is feasible to quantify cytokines from individual primary cells. A shift from qualitative to quantitative determinations of cytokine protein levels in single cells will permit more precise and reproducible studies of heterogeneity in the immune system and can be accomplished with readily available instrumentation.

Low-density lipoprotein receptor-related protein 1 attenuates house dust mite-induced eosinophilic airway inflammation by suppressing dendritic cell-mediated adaptive immune responses.

BACKGROUND: Low-density lipoprotein receptor-related protein 1 (LRP-1) is a scavenger receptor that regulates adaptive immunity and inflammation. LRP-1 is not known to modulate the pathogenesis of allergic asthma. OBJECTIVE: We sought to assess whether LRP-1 expression by dendritic cells (DCs) modulates adaptive immune responses in patients with house dust mite (HDM)-induced airways disease. METHODS: LRP-1 expression on peripheral blood DCs was quantified by using flow cytometry. The role of LRP-1 in modulating HDM-induced airways disease was assessed in mice with deletion of LRP-1 in CD11c+ cells (Lrp1fl/fl; CD11c-Cre) and by adoptive transfer of HDM-pulsed CD11b+ DCs from Lrp1fl/fl; CD11c-Cre mice to wild-type (WT) mice. RESULTS: Human peripheral blood myeloid DC subsets from patients with eosinophilic asthma have lower LRP-1 expression than cells from healthy nonasthmatic subjects. Similarly, LRP-1 expression by CD11b+ lung DCs was significantly reduced in HDM-challenged WT mice. HDM-challenged Lrp1fl/fl; CD11c-Cre mice have a phenotype of increased eosinophilic airway inflammation, allergic sensitization, TH2 cytokine production, and mucous cell metaplasia. The adoptive transfer of HDM-pulsed LRP-1-deficient CD11b+ DCs into WT mice generated a similar phenotype of enhanced eosinophilic inflammation and allergic sensitization. Furthermore, CD11b+ DCs in the lungs of Lrp1fl/fl; CD11c-Cre mice have an increased ability to take up HDM antigen, whereas bone marrow-derived DCs display enhanced antigen presentation capabilities. CONCLUSION: This identifies a novel role for LRP-1 as a negative regulator of DC-mediated adaptive immune responses in the setting of HDM-induced eosinophilic airway inflammation. Furthermore, the reduced LRP-1 expression by circulating myeloid DCs in patients with eosinophilic asthma suggests a possible role for LRP-1 in modulating type 2-high asthma.

Role of IL-17 in the pathogenesis of psoriatic arthritis and axial spondyloarthritis.

Th17 cells are a discrete subset of T cell subpopulation, which produce IL-17 and certain other pro-inflammatory cytokines. A regulatory role of Th17 cells have been proposed in several autoimmune diseases including psoriasis, psoriatic arthritis (PsA), ankylosing spondylitis (AS), rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, and multiple sclerosis. Psoriatic disease is an autoimmune disease which mainly involves skin and joints. Until recently, psoriasis and PsA were thought to be Th1 mediated disease, but after the discovery of IL-17 and IL-17 knockout animal studies as well as human experimental data indicate a crucial role of the Th17 cells in the pathogenesis of psoriasis and PsA. Our research group have not only found abundance of CD4(+)IL-17(+) T cells, mainly the memory phenotype (CD4RO(+)CD45RA(-)CD11a(+)) in the synovial fluid, but also have shown the existence of a functional IL-17 receptor in synovial fibroblast of psoriatic arthritis patients. Similarly, both animal and human studies indicate a regulatory role of the Th17 cells in AS; most critical observations are that Th17 cytokines (IL-17 and IL-22) can contribute to bone erosion, osteitis and new bone formation the hall mark skeletal features associated with the pathophysiology of AS. In this review article, we have discussed the contributing role of the IL-23/IL-17 axis in the pathogenesis of PsA and AS.

Double negative (DN) αß T cells: misperception and overdue recognition.

CD4(-)CD8(-)double negative (DN) αß T cells are legitimate components of the normal immune system. However, they are poorly understood and largely ignored by immunologists because of their historical association with the lymphoproliferation that occurs in mice (lpr and gld) and humans (autoimmune lymphoproliferative syndromes patients) with impaired Fas-mediated apoptosis where they are considered abnormal T cells. We believe that the traditional view that DN T cells that cause lymphoproliferation (hereafter referred to as lpr DN T cells) are CD4 and CD8 T cells that lost their coreceptor, conceived more than two decades ago, is flawed and that conflating lpr DN T cells with DN T cells found in normal immune system (hereafter referred to as nDN T cells) is unnecessarily dampening interest of this potentially important cell type. To begin rectifying these misperceptions, we will revisit the traditional view of lpr DN T cells and show that it does not hold true in light of recent immunological advances. In lieu of it, we offer a new model proposing that Fas-mediated apoptosis actively removes normally existing DN T cells from the periphery and that impaired Fas-mediated apoptosis leads to accumulation of these cells rather than de novo generation of DN T cells from activated CD4 or CD8 T cells. By doing so, we hope to provoke a new discussion that may lead to a consensus about the origin of lpr DN T cells and regulation of their homeostasis by the Fas pathway and reignite wider interest in nDN T cells.

Fabrication of nanoadjuvant with poly-ε-caprolactone (PCL) for developing a single-shot vaccine providing prolonged immunity.

PURPOSE: The aim of the study was to load a model antigen, tetanus toxoid (TT), in poly-ε-caprolactone nanoparticles (PCL NPs) of two size ranges, ie, mean 61.2 nm (small) and 467.6 nm (large), and study its effect on macrophage polarization as well as antigen presentation in human monocyte-derived macrophages in vitro, along with humoral and cell-mediated immune (CMI) response generated in Swiss albino mice following immunization with the TT-loaded NPs. MATERIALS AND METHODS: PCL NPs were synthesized by solvent evaporation. The antigen-loaded PCL NPs were characterized for size, zeta potential, and protein-release kinetics. Swiss albino mice were immunized with the antigen-loaded PCL NPs. Flow cytometry was used to quantify interferon-γ- and interleukin-4-secreting cluster of differentiation (CD)4(+) and CD8(+) T cells in the spleen, and enzyme-linked immunosorbent assay was used to quantify anti-TT antibody levels in the serum of immunized mice. RESULTS: Small PCL NPs generated an M1/M2 type polarization of human blood monocyte-derived macrophages and T helper (Th)1/Th2 polarization of autologous CD4(+) T cells. Efficient CD8(+) T-cell responses were also elicited. Large PCL NPs failed to cause any type of macrophage polarization. They did not elicit efficient CD8(+) T-cell responses. CONCLUSION: TT-loaded small PCL NPs were able to generate persistent and strong CMI and humoral responses against TT 2 months after single injection in mice without booster dose. This biodegradable nanoadjuvant system may help to develop single-shot immunization for prolonged immunity without booster doses. The capability of enhanced CMI response may have high translational potential for immunization against intracellular infection.

Association of Intracellular T(H)1-T(H)2 Balance in CD4+ T-cells and MIP-1α in CD8+ T-cells with Disease Severity in Adults with Dengue.

BACKGROUND: We tested the hypothesis that dengue haemorrhagic fever (DHF) is associated with a T(H)1-skewed immune response as opposed to dengue fever (DF). METHODS: We estimated intracellular (in T-cells) and serum levels of designate T(H)1/T(H)2 cytokines [interferon-γ (IFN-γ), interleukin-4 (IL-4), and tumor necrosis factor-α] and macrophage inflammatory protein-1α (MIP-1α) at admission, 48 h, and day 5 in 20 adults with dengue (DF=10, DHF=10) and 10 dengue-naïve healthy controls. RESULTS: At admission, intracellular IFN-γ/IL-4 ratio in CD4+ T-cells and proportion of MIP-1α-positive CD8+ T-cells were significantly higher in patients with DHF [7.21 (5.36~10.81) vs. 3.04 (1.75~4.02); p=0.011 and 6.2% (3.2~8.2%) vs. 2.4% (2.0~3.6%); p=0.023]. The latter showed a significant positive correlation with IFN-γ/IL-4 ratio in CD4+ T-cells (Spearman's rho=0.64; p=0.003), percentage-change in haematocrit (rho=0.47; p=0.048), and serum alanine aminotransferase level (rho=0.61; p=0.009). CONCLUSION: We conclude that DHF is associated with a T(H)1-skewed immune response. Further, MIP-1α in CD8+ T-cells is an important immunologic correlate of disease severity in dengue.

Expression of CD13/aminopeptidase N in precursor B-cell leukemia: role in growth regulation of B cells.

Expression of cell surface CD13 in acute B-cell leukemia (ALL-B) is often viewed, as an aberrant expression of a myeloid lineage marker. Here, we attempted to study the stage specific expression of CD13 on ALL-B blasts and understand its role in leukemogenesis as pertaining to stage of B-cell ontogeny. A total of 355 cases of different hematological malignancies were diagnosed by immunophenotyping. Among 68 cases of early B-cell ALL, 22 cases with distinct immunophenotype was identified as immature B-cell ALL. Blasts from these ALL-B patients demonstrated prominent expression of CD10, CD19, CD22, but neither cytoplasmic nor surface IgM receptors. This strongly indicates leukemogenesis at an early stage of B-cell development. We also identified, the existence of a subpopulation of cells with remarkably similar phenotype in non-leukemic marrow from healthy subjects (expressing CD10, CD19, CD22, CD24, Tdt together with the co-expression of CD13). This sub-population of B cells concomitantly expressing CD13 appeared to be a highly proliferating group. By blocking their cell surface CD13 in leukemic blasts with monoclonal antibody we were able to inhibit their proliferation. We hypothesized that neoplastic transformation at this stage may be facilitated by CD13. CD13 may thus be an important target for novel molecular therapy of early stage acute B-cell leukemia.