CD66c antigen expression is myeloid restricted in normal bone marrow but is a common feature of CD10+ early-B-cell malignancies

Abstract: CD66c is a surface (and intracellular) molecule bound to the membrane by a glycosyl-phosphatidylinositol anchor. While its expression on peripheral granulocytes is well recognized, less is known about its distribution in early steps of normal and neoplastic hematopoiesis. We analyzed by flow cytometry cell surface expression of CD66c on bone marrow cells from 4 healthy subjects and on bone marrow or peripheral blood cells from 127 patients with newly diagnosed hematologic malignancies: 70 de novo acute myeloid leukemias (AML), 6 refractory anemias with excess of blasts in transformation, 3 myeloid and 3 lymphoid blastic phases of chronic mye-logenous leukemia, 33 B-lineage and 6 T-lineage acute lymphoblastic leukemias (B- and T-ALL), and 3 B-cell and 3 T-cell non-Hodgkin's lymphomas in the leukemic phase. We found that in normal bone marrow CD66c expression was myeloid restricted, reaching its highest level on promyelocytes. As for de novo AML, slight expression of CD66c was found on 6/25 (24%) AML-M4 and only occasionally in other subgroups. In 9 out of 10 cases of acute promyelocytic leukemia, CD66c was totally absent, but antigen expression was easily detectable following in vitro exposure to all- trans retinoic add. Among lymphoid malignancies, CD10⁺ early-B-ALL consistently expressed the molecule (20/23 cases, or 87%) whereas both CD10^- early-B ALL and Smlg⁺ B-ALL completely lacked it. Finally, dual staining with CD66c and CD10 proved to be a suitable tool for distinguishing even low percentages of residual leukemic cells (CD10⁺/CD66c⁺) from normal regenerating early-B cells (CD10⁺/CD66c^-) in CD10⁺ early-B-ALL induced into remission.     

Delineation of the human hematolymphoid system: potential applications of defined cell populations in cellular therapy

Summary: Hematopoietic stem cells (HSC) have the capacity to reconstitute ail the blood cells in the body HSC are rare, representing on average 0.0 5% of the mononuclear cells present in healthy human bone marrow. Due to their capacity for self–renewal and their pluripotent, long–term reconstituting potential. HSC are considered ideal for transplantation to reconstitute the hematopoietic system after treatment for various hematologic disorders or as a target for the delivery of therapeutic genes. Human HSC also have potential applications in restoring the immune system in autoimmune diseases and in the induction of tolerance for allogeneic solid organ transplantation. With the increased interest in human HSC for clinical applications, technology for the isolation of candidate HSC and knowledge of human hematopoiesis have been growing rapidly. In this article, we discuss the functional characterization of a human CD34⁺ Thy-1⁺ HSC population which is essentially free of residual disease, our efforts to generate alternate monoclonal antibodies for the isolation of clinically useful stem or progenitor cell populations, and the identification of a novel lymphoid progenitor as part of an exploration towards defining progenitors with potential application as adjuncts to HSC–based cellular therapy.     

Rapid expression of the CD69 antigen on T cells and natural killer cells upon antigenic stimulation of peripheral blood mononuclear cell suspensions

The CD69 antigen has been identified as the earliest activation marker on the surface of cytokine- or mitogen-activated lymphocytes. The expression of this molecule may be a useful early marker of antigen- or allergen-specific activation of lymphocytes in vitro. We evaluated the expression of the CD69 and CD25 antigens on antigen- or allergen-stimulated lymphocytes and the proliferative responses as detected by thymidine incorporation. Peripheral blood mononuclear cells (PBMC) of allergic patients sensitized to Dermatophagoides pteronyssinus , bovine casein, or nickel sulfate were cultured in the absence or presence of clinically relevant allergens, tetanus toxoid, or recombinant interleukin (IL)-2. The respective binding of CD69 or CD25 antibodies to PBMC and thymidine incorporation were measured. An early expression of CD69, but not of CD25, antigen was detectable after 24-72 h of stimulation on up to 80% of natural killer (NK) cells and up to 10% of CD4⁺ T cells in PBMC cultures. Anti-IL-2 antibodies inhibited these increases of CD69 on NK cells and T cells by up to 60%. After 6 days of antigenic stimulation, the rates of both CD25⁺ and CD69⁺ lymphocytes were higher. Seventy-four percent of the CD25⁺ PBMC but only 55% of the CD69⁺ cells were CD3⁺ T lymphocytes at this time. No qualitative differences were detectable in allergen- or tetanus-toxoid-stimulated PBMC from allergic patients. The high expression of CD69 on NK cells in antigen-stimulated cultures suggests that these cells are easily activated by cytokines from antigen-stimulated T cells. CD69⁺ NK cells may serve as early-indicator cells in cultures with antigen- or allergen-stimulated mononuclear cells.     

Immunophenotypic and Functional Characteristics of Haemopoietic Cells from Human Cord Blood

Cord blood (CB) as a new source for bone marrow transplantation represents advantageous features concerning stem cell and leucocyte compartments and function. We attempted to get more information about the phenotypes and function of CB cells by investigating their cell surface markers and also the production of IL-2, IFN-γ and IL-6 by mitogen and alloantigen stimulation. The CB cells were characterized by a low proportion of CD3⁺ T cells, CD4⁺ T subpopulation, activated T cells and CD3⁺ CD16/CD56⁺ cytotoxic cells, suggesting reduced graft versus host potential. The significant increase of CD19/CD3 double positive cells and decrease of CD19/HLA-DR double positive mature B cells reflect that immature B cells exist in CB. In the functional studies, a 27- and 5-fold reduction was observed in the production of IFN-γ by CB cells stimulated with PHA and allogeneic cells, respectively. The production of IL-2 in PHA-stimulated CB cells also showed a 50% determination. Decrease in the production of these cytokines by CB cells is supported by the decline of the proportion of CD3⁺ T cells. However, an increase was observed in the production of IL-6 by CB cells stimulated with allogeneic cells as compared with the controls. These results suggest a difference in the functional activity of the T helper cell subsets between the CB and peripheral blood and/or differences in the functional maturity of T helper cell subsets and B cells in these compartments.     

NG2-positive cells in the mouse white and grey matter display distinct physiological properties

Cells that express the NG2 proteoglycan are the largest proliferative progenitor population in the postnatal central nervous system (CNS). Although this entire population has long been considered to be oligodendrocyte progenitors, numerous NG2⁺ cells are present in the cerebral cortex, where relatively little myelination occurs, and also persist long after myelination is complete in the CNS. Several studies have alluded to the presence of distinct NG2⁺ cell subtypes based on marker expression, but no experimentally derived hypotheses about the physiological role of these subtypes has been proposed. In the current study, whole-cell patch-clamp data from acutely isolated slices demonstrate that subcortical white matter and cortical NG2⁺ cells display distinct membrane properties in addition to possessing differing K⁺- and Na⁺-channel expression profiles. A striking observation is that a subpopulation of cortical, but not white matter NG2⁺ cells, elicit depolarization-induced spikes that are akin to immature action potentials. Our data demonstrate that a population of cortical NG2⁺ cells display physiological properties that differ from their white matter counterparts.     

Peripheral Immune Response in Pulmonary Tuberculosis

Cellular immune response and delayed-type hypersensitivity reactions are considered to play a major role in the immunopathogenesis of pulmonary tuberculosis (PTB). But the exact mechanism is still to be clarified. Th1 cells are mainly involved in cellular immune responses in PTB and provide a normal healing process with minimal or no sequela whereas Th2 cell and CD8⁺ T lymphocyte responses may lead to more severe type of disease. In this study, we investigated the peripheral blood immune responses in PTB. The study group consisted of acid fast positive young male soldiers with PTB and a negative HIV serology. The control group included healthy young volunteer male soldiers without a history of PTB. Intracytoplasmic cytokine content of CD8⁺ T cells and lymphocytes, including IL-2, IL-4, IL-5, IL-10 and IFN-γ were determined by flow cytometry, and IL-2, IL-4, IL-5, IL-10, IFN-γ and TNF-α serum levels were measured by cytometric bead array (CBA). No difference was observed between the percentages of T, B, NK cells and HLA-DR expression in both groups, however, the number of CD3⁺HLA-DR⁺ activated T cell percentages was higher in PTB group as compared to healthy subjects. IL-2, IL-4, IL-5, IL-10 contents of lymphocytes and IFN-γ⁺CD8⁺ T cells were found to be significantly lower in PTB patients when compared with healthy subjects, and in parallel, serum IL-2, IL-4, IL-5 and TNF-α levels were also significantly lower in PTB patients. In conclusion we suggest that, CD8⁺ T cells producing both Th1 and Th2 type cytokines, may play important role in the peripheral immune response to mycobacteria.     

Lineage commitment and differentiation of T and natural killer lymphocytes in the fetal mouse

Summary: T cells and natural killer (NK) cells are presumed to share a common intrathymic precursor. The development of conventional a|3 T lymphocytes begins within the early fetal thymus, after the colonization of multipotent CDl1 71 precursors. Irrevocable commitment to the T lineage is marked by thymus-induced expression of CD25. However, the contribution of the fetal thymus to NK lineage commitment and differentiation remains largely unappreciated. Recently, we demonstrated that the development of functional mouse NK cells occurs first in the fetal thymus. Moreover, the appearance of mature fetal thymic NK cells (NK1.1⁺/CD 117^-) is preceded by a thymus-induced developmental stage (NKl.1⁺/CD1 17⁺) that marks lineage commitment of multipotent hematopoietic precursors to the T and NK-cell fates. Commitment to the T/NK bipotent stage is induced by fetal thymic stroma, but is not thymus dependent. Recent data indicate that CD90⁺/CD117^lo fetal blood prothymocytes exhibit NK lineage potential and are phenotypically and functionally identical to fetal thymic NK1,1⁺/CD1 17⁺ progenitors. This finding also indicates that full commitment of circulating precursors to the T-cell lineage occurs after thymus colonization. In this review, we discuss recent insights into the cellular and molecular events involved in fetal mouse T and NK lineage commitment and differentiation to unipotent progenitors.     

The CD4+CD8+ and CD4+ Subsets of FOXP3+ Thymocytes Differ in their Response to Growth Factor Deprivation or Stimulation

The timing of thymic regulatory T (Treg) cell commitment remains unclear. Specifically, there is disagreement as to whether the CD4⁺CD8⁺ FOXP3⁺ thymocytes are precursors of mature CD4⁺ FOXP3⁺ Treg cells, or an independent Treg cell lineage. We reasoned that precursors should be more susceptible to apoptosis than mature Treg cells, and tested this by growth factor removal and anti-CD3 stimulation. Both treatments resulted in an increase of CD4⁺ FOXP3⁺ thymocytes, whereas the frequency of CD4⁺CD8⁺ FOXP3⁺ thymocytes decreased significantly. These changes were accompanied by an increase of annexin⁺ apoptotic cells. Both of these FOXP3⁺ subsets expressed higher levels of Bcl-2 and BIM than other thymocytes, and while in our setting expression of BIM seemed to predispose the cells to apoptosis, Bcl-2 had no apparent protective effect. These results indicate that CD4⁺CD8⁺ FOXP3⁺ thymocytes are more susceptible to apoptosis than mature CD4⁺ FOXP3⁺ Treg cells. This is consistent with the view that they are still immature and thus likely to represent a precursor population.     

Characterization of T-Cell Receptor Vβ Repertoire in Ovarian Tumour-Reacting CD3+ CD8+ CD4− CTL Lines

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumour responses. To characterize the T-cell antigen receptor (TCR) Vβ expression in autologous cytotoxic effectors we isolated CD3⁺ CD8⁺ CD4⁻ cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR Vβ repertoire of CD3⁺ CD8⁺ CD4⁻ lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cellsensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8⁺ CTL lines expressed a broad distribution of TCR Vβ repertoire which was dominated by particular groups of Vβ families in each CTL line. However, no predominant expression of one or the same Vβ segment in all CTL lines was observed although statistical correlations between Vβ family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR Vβ families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR Vβ repertoire of human ovarian tumour-reactive CD3⁺ CD8⁺ CD4⁻ CTL from different individuals of known HLA types.     

Requirement of CD4+ Lymphocytes in IL-2-Stimulated NK Cell Proliferation

Growth requirements of human natural killer cells in IL-2-supplemented cultures were studied, NK cell proliferation was monitored by the MAC (morphology antibody chromosomes) technique and subset specific cell cycle analysis, which both enable direct determination of cell growth in specific lymphocyte subsets among heterogeneous lymphocyte populations. Our results show that even in the presence of saturating concentrations of IL-2, the proliferative capacity of purified CD16⁺ cells is quite low, but can be stimulated in a dose dependent manner by CD4⁺ cells. CD4⁺ cells could partially be replaced by IL-4 but not by various other commercially available cytokines. These results provide further evidence of the requirement of accessory stimuli in NK cell proliferation, and support the interpretation that NK cells have a direct regulatory role in specific T cell responses.     

Intrathymic maturation of CD4+ T-lymphocytes in an MHC class II deficient transplant model

Major histocompatibility complex (MHC) class II knockout (class II^-) mice fail to generate CD4⁺ CD8^- T-lymphocytes. We were interested in determining whether these class II^- mice could be reconstituted with CD4⁺ CD8^- T-lymphocytes following marrow transplantation from normal (class II⁺) donors. Transplantation of class II⁺ marrow into lethally irradiated class II^- recipients failed to generate peripheral CD4⁺ CD8^- T-lymphocytes. Unexpectedly, however, transplantation of class II^- marrow into class II⁺ recipients also resulted in a deficiency of CD4⁺ CD8^- cells. Analysis of intrathymic T cells showed normal distribution of CD4 and CD8 single and double positive or negative thymocytes in normal recipients, while class II^- recipients always lacked CD4⁺ CD8^- T cells intrathymically. These results suggest, therefore, that T-cell maturation in mice requires the presence of MHC class II antigens not only in the thymus but also on immature, marrow-derived pre-thymocytes.     

A lymphocyte differentiation and activation antigen, CZ-1, that distinguishes between CD8+ and unstimulated CD4+ T lymphocytes.

We report the generation and cellular reactivity of a novel rat IgM monoclonal antibody (mAb), CZ-1, made against mouse natural killer (NK) cells activated in vivo. mAb CZ-1 recognizes a molecule whose properties are consistent with that of a trypsin-sensitive, non-phosphatidyl inositol-linked sialoglycoprotein. The expression of the antigen recognized by mAb CZ-1 is restricted mostly to cells of the lymphoid lineage. The antigen is expressed on 10%-25% of bone marrow cells and 3%-5% of thymocytes. Analysis of thymocyte subpopulations indicates expression of the CZ-1 antigen on 100% of the NK1.1+, 27% of the CD4-CD8-, 1.1% of the CD4+CD8+, 1.1% of the CD4+CD8-, and 33% of the CD4-CD8+ cells. In the spleen, the CZ-1 antigen is expressed on B lymphocytes, NK cells, and virtually all CD8+ T lymphocytes. Most unstimulated CD4+ splenic T lymphocytes, monocytes and polymorphonuclear cells, with the notable exception of basophils, do not react with mAb CZ-1. CD4+ T cells activated in vivo by virus infection or in vitro by anti-CD3 and interleukin-2 express the CZ-1 antigen. These results indicate that mAb CZ-1 identifies a novel inducible lymphocyte activation/differentiation antigen that distinguishes between thymic and unstimulated splenic CD4+ and CD8+ T lymphocytes. This mAb will be a useful tool in the identification of lymphocyte subpopulations and in the study of the ontogeny and activation of these cells.     

Suppression of Experimental Autoimmune Myasthenia Gravis After CD8 Depletion is Associated with Decreased IFN-γ and IL-4

CDS⁺ T cells can perform both Th1 - and Th2-like functions by producing cytokines such as interferonγ (IFN-γ) and interleukin-4 (IL-4), as well as the immune response down-regulating transforming growth factor-β (TGF-β), which are all involved in the development of experimental autoimmune myasthenia gravis (EAMG), a model for human MG. We have reported that depletion of CD8⁺ T cells results in the suppression of EAMG accompanied by the down-regulation of AChR-specific B cell responses and AChR-reactive IFN-γ secreting Th1-like cells. To identify the involvement of IFN-γ, IL-4 and TGF-β in the development of EAMG after CD8⁺ T cell depletion, the expression of mRNA for these cytokines was studied in mononuclear cells from popliteal, inguinal and mesenteric lymph nodes, spleen and thymus by adopting in situ hybridization with complementary DNA oligonucleotide probes. Depletion of CD8⁺ T cells resulted in decreased levels of IFN-7 and IL-4 mRNA expressing cells in different lymphoid organs except thymus, but no change in the numbers of TGF-β mRNA expressing cells. The results imply that the suppression of EAMG after depletion of CD8⁺ T cells is caused by decreasing the effector factors but not by increasing the suppressor factor(s).     

Activation Signal Induces the Expression of B Cell-Specific CD45R Epitope (6B2) on Murine T Cells

Tlymphocytes express multiple forms of the leukocyte-common antigen CD45, transcribed by alternative usage of leukocyte-common antigen exon 4 6. The various isoforms of CD45R expressed differentially on T cells are involved in different stages of development and activation. The monoclonal antibody (MoAb) RA3-6B2 is established as a B cell-type isoform (B220)-specific marker. However, it reacts with certain activated T cells although the relationship between 6B2 expression and T-cell activation is unclear. We have examined the 6B2 expression on activated T cells and found that concanavalin A, anti-CD3 antibody and staphylococcal enterotoxin B (SEB) induced 6B2 expression on T cells. The expression was found on both CD4⁺ and CD8⁺ T cells and also was induced by SEB in vivo predominantly on CD8⁺ T cells. The 6B2⁺ T cells are IL-2R⁺ and blasted cells according to flow cytometry analysis. Therefore, the 6B2⁺ T cells are supposed to be in an activated stage. Enzymatic analysis demonstrated that trypsin treatment decreased the 6B2 expression, whereas neuraminidase increased the intensity on activated T cells. Neither endo-D or endo-H have any effect on the expression and there are no differences, in the results of immunoprecipitation and RT-PCR analysis, between control T cells and activated T celts. Taken together, the 6B2 epitope is presumed to be the product of CD45R modification and is expressed on activated T cells. These results illustrate a novel classification of a T-cell subpopuiation bearing a 6B2 epitope.     

Increased Expression of Activation-Induced Cytidine Deaminase is Associated with Anti-CCP and Rheumatoid Factor in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is associated with higher levels of autoantibodies and IL-17. Here, we investigated if ectopic lymphoid follicles and peripheral blood mononuclear cells (PBMCs) from RA patients exhibit increased activation-induced cytidine deaminase (AID), and if increased AID is correlated with serum levels of autoantibodies and IL-17. The results of immunohistochemical staining showed that organized AID⁺ germinal centres were observed in six of the 12 RA synovial samples, and AID⁺ cells were found almost exclusively in the B-cell areas of these follicles. Aggregated but not organized lymphoid follicles were found in only one OA synovial sample without AID⁺ cells. Significantly higher levels of AID mRNA ( Aicda ) detected by RT-PCR were found in the PBMCs from RA patients than PBMCs from normal controls ( P  < 0.01). In the PBMCs from RA patients, AID was expressed predominately by the CD10⁺IgM⁺CD20⁺ B-cell population and the percentage of these cells that expressed AID was significantly higher than in normal controls ( P  < 0.01). AID expression in the PBMCs correlated significantly and positively with the serum levels of rheumatoid factor (RF) ( P  ≤ 0.0001) and anti-cyclic citrullinated peptide (CCP) ( P  = 0.0005). Serum levels of IFN-γ ( P  = 0.0005) and IL-17 ( P  = 0.007), but not IL-4, also exhibited positive correlation with the expression of AID. These results suggest that the higher levels of AID expression in B cells of RA patients correlate with, and may be associated with the higher levels of T helper cell cytokines IFN-γ and IL-17, leading to the development of anti-CCP and RF.     

Peripheral Lymphoid Hyperplasia and Central Lymphoid Depletion in Mice Treated with a Bacterial B-Cell Mitogen (F3'EP-Si/p90)

In order to further understand the mechanism mediating the mitogenic and immunosuppressor effects of p90, a protein produced by Streptococcus intermedius , flow cytometric studies were performed on peripheral and central lymphoid organs of mice treated with this protein. p90 induced a strong blastogenic B-cell response in the spleen and lymph nodes, followed by a slight but significant polyclonal T-cell activation. B-cell repertoire analysis indicated that polyclonal B-cell responses affected similarly both CD5⁺ and conventional (CD5⁻) B cells in the spleen. Repertoire analysis of T cells failed to reveal any preferential stimulation of the Vβ T-cell receptor (Vβ-TcR) families studied. Peripheral lymphoid hyperplasia was observed concomitantly with central lymphoid depiction. In the bone marrow, pre-B and B cells were profoundly depleted, with a more pronounced effect on small pre-B cells. In the thymus, double-positive (CD4⁺ CD8⁺) thymocytes were preferentially eliminated, with a relative enrichment of single positive (either CD4⁺ or CD8⁺) and double-negative (CD4⁻CD8⁻) thymocytes.     

Lymphocytic Choriomeningitis Virus Infection is Associated with Long-Standing Perturbation of LFA-1 Expression on CD8+ T Cells

Flow cytometric analysis of splenocytes from mice infected with lymphocytic Choriomeningitis virus revealed marked and long-standing up-regulation of LFA-1 expression on CD8⁺, but not on CD4⁺ T cells. Appearance of CD8⁺ T cells with a changed expression of adhesion molecules reflected polyclonal activation and expansion which was demonstrated not to depend on CD4⁺ T cells or their products. Cell sorting experiments defined virus-specific CTL to be included in this population (LFA-1^hiMEL-14^lo), but since about 80% of splenic CD8⁺ T cells have a changed phenotype, extensive bystander activation must take place; this is indicated also by the finding that CD8⁺LFA-l^hi cells transiently express several markers of cellular activation, e. g. transferrin receptor, IL-2Rα and β. Analysis of cells from the cerebrospinal fluid of mice infected intracerebrally showed that virtually all T cells present belonged to the CD8LFA-lhi subset and, correspondingly, the ligand ICAM-1 was found to be up-regulated on endothelial cells in the inflamed meninges. Preincubation of LCMV-primed donor splenocytes with anti-LFA-1 markedly inhibited the transfer of virus-specific delayed-type hypersensitivity to naive recipients. Together, these findings indicate that up-regulation of LFA-1 expression is a critical factor involved in directing activated CD8⁺ T cells to sites of viral infection.