Discordant expression of human Ia-like antigens on hematopoietic progenitor cells

The expression of HLA-DR, SB, MT2, and DC antigens on human hematopoietic progenitor cells has been determined by using monoclonal antibodies with complement (C)-mediated cell lysis and immune separation techniques. HLA-DR was detected on greater than 85% of CFU-G/M, myeloid clones (MyCl), BFU-E, and CFU-E. CFU-E were less susceptible to C-mediated lysis at suboptimal C concentrations. The polymorphic MT2 and SB antigens were also present on all categories of progenitor cells, although a lesser proportion of cells were positive. Because in most individuals the antigen density of MT2 and SB, as determined by monoclonal antibody staining, was also lower on B cells and monocytes when compared to HLA-DR expression, the lower number of positive progenitor cells probably reflects lower antigen density rather than distinct positive and negative progenitor cell populations. The DC antigen is expressed weakly on monocytes and B cells, although there is considerable individual variation. In some individuals, distinct DC-positive and -negative monocyte populations are detectable. The DC antigen was not detected on myeloid progenitor cells, even in those individuals with moderate DC expression on their monocytes and B cells. This discordant expression of DC and other Ia-like antigens on hematopoietic progenitor cells may be of physiologic significance and may assist in the purification of progenitor cells from blood and marrow.     

The alpha chain,not the beta chain of HLA-DR antigens participates in activation of T cells in autologous mixed lymphocyte reaction

Using antisera against the alpha, the beta or the complex of both chains of HLA-DR antigens, we have studied the role of individual chains of HLA-DR antigens in activation of T cells in autologous mixed lymphocyte reaction (AMLR). Alpha chain-specific antibody, not anti-beta chain serum prevented T cells from acquiring responsiveness to interleukin-2 (IL-2), suppressed the production of 1L-2, and inhibited the T cell proliferative response in both primary and secondary AMLR cultures. However, proliferation of already activated IL-2 reactive T cells supported by IL-2 was not affected by any of the four different types of anti-DR sera used. Fifty to sixty percent of T cells activated by AMLR or by PHA possessed DR antigens and functioned well as stimulator cells in secondary AMLR cultures. Moreover, the stimulatory activity of these DR-positive T cells was suppressed by the anti-alpha chain, not by the beta chain-specific antibody. Since continuous proliferation of T cells requires IL-2 and since nonactivated T cells are not sensitive to IL-2 and are unable to absorb this growth factor, we conclude the following: (1) The alpha, not the beta chain of HLA-DR antigens seems to be the structure responsible for enabling resting T cells to respond to IL-2 and induce production of IL-2 in AMLR. (2) Once T cells have acquired responsiveness to IL-2 and the growth factor has been produced, there is no further requirement for HLA-DR antigens, but the availability of IL-2 determines the level and extent of proliferation of IL-2 sensitive T cells.     

The cell-mediated lympholysis-inducing capacity of highly purified human monocytes and T lymphocytes in primary and secondary mixed leukocyte cultures

In this study the capacity of T cells and monocytes to induce cell-mediated lympholysis (CML) in primary and secondary MLC was investigated. The T lymphocytes were enriched by rosetting with sheep red blood cells (E) and further purified by sedimentation at unit gravity, which completely removed the contaminating monocytes. In addition, a highly purified monocyte population was obtained by 1 X G sedimentation of the non-E rosette-forming cells. These purified T cells have a poor CML-inducing capacity in primary and secondary MLC. In contrast, monocytes were very effective in inducing CML in both primary and secondary MLC. Induction of CML by monocytes in primary MLC was inhibited by heterologous anti-Ia-like antisera, indicating that the induction of CML by monocytes was related to the presence of HLA-DR (Ia-like) antigens on these cells.     

Expression of C3d receptors during human B cell differentiation: immunofluorescence analysis with the HB-5 monoclonal antibody

We have examined human B lymphocytes at different stages of differentiation for the expression of surface receptors for the C3d fragment of complement. C3d receptors (C3dR) were identified by indirect immunofluorescence using the HB-5 monoclonal antibody, which recognizes a 145,000 m.w. C3dR molecule on B lymphocytes. Pre-B and immature B cells from fetal bone marrow and liver did not express C3dR, whereas a small subpopulation (25%) of B cells in fetal spleen were C3dR+. Approximately 50% of the B cells in adult bone marrow were C3dR+, whereas the more mature B cells in the blood of newborns and adults and in peripheral lymphoid tissue of adults uniformly expressed the C3dR. Activated B cells responsive to T cell-derived differentiation factors were C3dR+, whereas plasma cells rarely expressed C3dR. T cells, NK cells, erythrocytes, and myelomonocytic cells did not express detectable surface C3dR. These results suggest that in hematopoietic and lymphoid tissues, the expression of C3dR is a specific feature of relatively mature lymphoid cells of B lineage.     

Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.

Human bone marrow cells expressing CD34 but not HLA-DR were isolated by immunofluorescence flow cytometric cell sorting. These cells contained a hematopoietic cell (CFU-B1) capable of producing, in an in vitro semisolid culture system, blast-cell-containing colonies, which possessed the capacity for self-renewal and commitment to multipotential differentiation. In addition, CD34+ HLA-DR- marrow cells contained primitive megakaryocyte progenitor cells, the burst-forming unit-megakaryocyte (BFU-MK). A subset of CD34+ HLA-DR- marrow cells lacking the expression of CD15 and CD71 was obtained by flow cytometric cell sorting and was capable of sustaining in vitro hematopoiesis in suspension culture for up to 8 weeks in the absence of a preestablished adherent marrow cell layer. The combination of IL-3 + IL-1 alpha and IL-3 + IL-6 sustained proliferation of these cells for 8 weeks, induced maximal cellular expansion, and increased the numbers of assayable progenitor cells. These studies demonstrate that human CD34+ HLA-DR- marrow cells and their subsets contain primitive multipotential hematopoietic cells capable of self-renewal and of differentiation into multiple hematopoietic lineages.     

Subpopulations of mouse Lyt-2+ T cells defined by the expression of an Ly-6-linked antigen, B4B2

The production and characterization of a rat mu,kappa monoclonal anti-mouse T cell subset antibody, B4B2, is reported in this paper. B4B2 typing of lymphoid tissues of commonly used inbred mouse strains revealed two types of reactivity patterns. They can be characterized as C57BL/6-like (B6-like) or C3H/He-like (C3H-like). Among B6-like strains, B4B2 recognizes 5 to 10% of spleen cells, 30 to 50% of bone marrow cells, and less than 2 to 3% of thymocytes. In C3H-like strains, B4B2 reacts with less than 1% of spleen cells, 2 to 8% of bone marrow cells, and less than 1% of thymocytes. B4B2 recognizes a T cell subset differentiation antigen expressed by B6-like strains but not by C3H-like strains. Typing of BXH recombinant inbred strains showed linked expression of B4B2 and the Ly-6 antigen. The expression of B4B2 antigen appears to be under codominant control as the median fluorescence distribution of B4B2+ cells in C57BL/6 was approximately twice that of (C57BL/6xC3H)F1. B4B2 was shown to react with approximately 40 to 50% of Lyt-2+ T cells and less than 1% of L3T4+ T cells. No staining of resting or activated B cells by B4B2 was detected. The ratio of B4B2+:Lyt-2+ cells was similar for resting T cells and activated T cells obtained from mitogen-stimulated cultures or mixed lymphocyte cultures. In neonatal spleen, substantially more B4B2+ than Lyt-2+ cells were found. With increasing age, however, a rapid decline in B4B2+ cells and a corresponding increase of Lyt-2+ cells was observed. By approximately 1 mo of age, the relative proportion of these subsets had reversed so that Lyt-2+ cells became more numerous than B4B2+ cells.     

Asialogangliosides as differentiation markers of rat erythropoietic and granulopoietic cells in rat bone marrow. Flow cytometric analysis of rat bone marrow cells using anti-asialoganglioside antibodies

Flow cytometric analysis using anti-glycolipid antiserum was used on rat bone marrow cells to determine the relation between the glycolipid species expressed on cell surfaces and cell differentiation. Four kinds of antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (IV2 alpha Fuc-Gg4Cer) and IV3 alpha Gal-fucogangliotetraosylceramide (IV3 alpha GalIV2 alpha Fuc-Gg4Cer, blood group B lipid) were used. The cells sorted out by each anti-glycolipid antiserum were stained with May-Grünwald-Giemsa reagent and identified by microscopy. In the erythropoietic group, only polychromatic erythroblasts had these four glycolipids on their cell surfaces; none appeared on differentiated erythrocytes. These glycolipids were expressed during the early stages of immature granulocytes, especially in the promyelocyte and myelocyte stages of eosinophilic and neutrophilic granulocytes. Very limited populations of lymphocytes were sorted out as asialoganglioside-expressing cells. We concluded that asialogangliosides are useful differentiation markers for the erythropoietic and granulopoietic cells of rat bone marrow, and that anti-asialoganglioside antibody-flow cytometry is a very useful technique with which to isolate immature granulocytes and erythropoietic cells from rat bone marrow cells.     

Purification of immunologically functional subsets of human Ia-like antigens on a monoclonal antibody (Q5/13) immunoadsorbent

Serologic and immunochemical asays have shown that the monoclonal antibody Q5/13 recognizes an antigenic determinant expressed on a subset of human Ia-like antigens. Testing with a panel of HLA typed B lymphoid cells has shown that this determinant is different from those defining the serologic polymorphism of HLA-DR antigens. The monoclonal antibody Q5/13 has been used to purify subsets of human Ia-like antigens, which are immunologically functional. These reagents should facilitate the characterization of structural and functional properties of human Ia-like antigens.     

The Chinese hamster gene map. Assignment of four genes (DTS, PGM2, 6PGD, EnoI) to chromosome 2

Mouse morulae and blastocysts express cell surface antigens that fortuitously cross-react with antisera to human chorionic gonadotropin (hCG). In the present study, the cell surface and cytoplasmic expression of these antigens was followed in mouse unfertilized oocytes, different stages of preimplantation embryos and in early post-implantation embryos cultured from blastocysts. In addition to their known stage-dependent cell surface expression on morulae and blastocysts, these antigens (1) were already present in the cytoplasm of mature unfertilized oocytes and pre-morula stages of embryos; (2) remained expressed as cell surface antigens on cells of the inner cell mass (ICM), but not on the surface of trophectodermal cells with further blastocyst development although (3) they persisted as cytoplasmic antigens in trophectodermal cells. In addition, these antigens were also detectable by antiserum to the alpha subunit of hCG.     

Fibronectin increases the migration induced by stromal cell-derived factor-1 alpha (SDF-1 alpha) in pre-B acute lymphoblastic leukemia cells

The chemokine, stromal cell-derived factor-1 alpha (SDF-1 alpha) and its receptor CXCR-4 (fusin, LESTR) are thought to be involved in the trafficking of hematopoietic progenitors and stem cells, as suggested by the chemotactic effect of SDF-1 alpha on these cells. Gene inactivation studies have shown that both SDF-1 alpha and CXCR-4 are essential for B lymphopoiesis. Migration of leukemic cells may also be dependent on SDF-1 alpha and CXCR-4. Fibronectin (FN) is a component of the extracellular matrix (ECM), and one of the natural supports for cell movement in their bone hematopoietic environment. In the present study, we examined the influence of FN on the chemotactic effect of SDF-1 alpha and on the CXCR-4 expression and function on human precursor-B acute lymphoblastic leukemia (pre-B ALL) cells at sequential stages of development. Fourteen children with pre-B ALL were studied. Their immunophenotypes belonged to the first three stages of B cell differentiation. Despite relatively high levels of CXCR-4 expression at all stages, the responsiveness to SDF-1 alpha, measured as the percentage of migrating cells in the transwell culture system, varied with patients and seems to be less significant for pre-B3 (and pre-B1) than for pre-B2. There was no correlation (r = 0.2) between the SDF-1 alpha induced migration (range: 2.5-39%) and the cell surface density of CXCR-4 (range: 46.5-97.5%). The extracellular matrix protein FN, either coated on the filter (for more than 18 hours) or in soluble form, enhanced the SDF-1 alpha induced migration of pre-B ALL respectively (2 fold and 1.6 fold) without influencing CXCR-4 expression in short term cultures. Therefore, we analyzed the expression of the FN receptors, VLA-4 (CD49d) and VLA-5 (CD49e), by direct immunofluorescence, on these leukemic cells. VLA-4 was strongly expressed in all stages of pre-B ALL (range: 77-97%) while VLA-5 expression was more variable (range: 14-94%), but no correlation with the FN-dependent increased SDF-1 alpha chemotactic effect was noted. In conclusion, the migratory behavior of pre-B leukemic cells in response to SDF-1 alpha partly depends upon the stage of differentiation, and partly upon unexplained patient variability. Our results suggest that several molecules from the extracellular matrix, such as FN, may be implicated in this phenomenon.     

Cytofluorometric isolation of I937, an Ia antigen-bearing variant of the Ia-negative human monocytic cell line U937

Cells of the human monocyte cell line U937 are generally considered devoid of any Ia antigens on their surface. In analyzing U937 cells with a large panel of monoclonal anti-human Ia antibodies by flow cytometry, we detected a small number of cells that appeared to react with antibodies to HLA-DR and HLA-DS/DC molecules. These Ia-positive cells were isolated and were cloned, resulting in a human monocyte cell line that expresses high levels of Ia antigens. We analyzed these antigens by one- and two-dimensional polyacrylamide gel electrophoresis, after radiolabeling and immunoprecipitation. Three distinct Ia molecules, alpha 1 beta 1, alpha 1 beta 3 (HLA-DR-like), and alpha 2 beta 2 (HLA-DC/DS-like) are synthesized by I937 cells, alpha 1 beta 3 molecule being the predominant species. The Ia antigen-bearing human monocyte cell line is expected to be useful for studying events involved in antigen presentation.     

Expression of HLA-DR, MB, MT and SB antigens on human mononuclear cells: identification of two phenotypically distinct monocyte populations

The expression of HLA-DR, SB, MB, and MT antigens in different populations of human mononuclear cells was investigated with the use of monoclonal antibodies that recognize distinct human Ia-like antigens. Our results indicate that in man, as previously reported in other species, two phenotypically distinct populations of monocytes or macrophages can be identified on the basis of expression of Class II MHC antigens. Virtually all circulating monocytes displayed determinants associated with HLA-DR, SB, and MT. In addition, a subpopulation of human monocytes expressed MB/DS-associated antigens, as detected with monoclonal antibodies specific for MB1, MB3, and DS-framework determinants. Most B lymphocytes expressed antigens associated with HLA-DR, and the specificities SB2, SB3, MB1, MB3, MT2, and MT3 were also present. Resting T lymphocytes were unreactive with antibodies that recognize all of the Class II MHC antigens tested. T lymphocytes activated by soluble antigen or alloantigens, and expanded in culture, expressed DR, SB, MB, and MT. The majority of the MB/DS+ cells present in the adherent population were monocytes, because they were phagocytic and had the monocyte-specific marker 63D3. The rest of the cells were not identified. They are likely to include mostly B lymphocytes. The presence of other cells, such as dendritic cells, in this subset needs to be determined.     

Expression of markers shared between human natural killer cells and neuroblastoma lines

Summary Neuroblastoma is a tumor of neuroectodermal origin arising most commonly from the adrenal medulla. We have examined the ability of several monoclonal antibodies which recognize markers predominantly expressed on human natural killer (NK) cells to react with neuroblastoma cell lines in vivo derived sections of tumor. HNK-1 (Leu 7) is a monoclonal IgM antibody which recognizes a carbohydrate epitope on NK cells and a wide range of tumor cell types. We have shown that HNK-1 recognizes the human neuroblastoma lines SMS-KCNR, SMS-KAN, NMB/N7, and IMR/5. Expression of this antigen on cell lines can be slightly increased by retinoic acid-induced differentiation of the cells. N901 (NKH1), a monoclonal antibody raised against interleukin 2-dependent human NK cell lines also recognizes all human neuroblastoma cell lines examined. This expression is independent of differentiation induction and levels remain unaltered following retinoic acid treatment of the cell lines. Lastly, with monoclonal antibody 49H.8, it has been found that reactivity of the lines is weak until induction of differentiation, after which highly significant increases of reactivity are seen. 49H.8 recognizes several cryptic carbohydrate antigens with varying affinities, shown to identify mouse and rat NK cells. In contrast to other NK markers, human neuroblastoma cell lines did not express significant reactivity with B73.1, Leu 11b, or Leu 18. Immunohistochemical staining of sections of human neuroblastoma tumors correlated with the in vitro findings; however, staining with N901 and 49H.8 was only seen on frozen sections, not paraffin-embedded. The significance of shared NK cell-neuroblastoma/neuron antigens is currently under investigation.     

Ontogeny of human hematopoietic cells: analysis utilizing monoclonal antibodies

Lymphoid and myeloid cells isolated from second trimester fetal lymphoid organs were characterized by utilizing a panel of monoclonal antibodies that define human lineage-restricted, differentiation, histocompatibility, and activation antigens. At distinct gestational stages, the appearance of morphologically identifiable lymphoid and myeloid cells paralleled the appearance of cells expressing definable lymphoid and myeloid antigens. The proportion of cells in fetal liver, bone marrow, and spleen that expressed histocompatibility, myeloid, and B cell antigens increased with fetal maturation. In contrast, even the earliest fetal thymuses studied were of a phenotype no different than that seen during later stages of ontogeny. Although the cellular lineage of most fetal hematopoietic cells could be identified by this panel of reagents, a considerable number of fetal liver and bone marrow cells did not express any of these antigens, suggesting the possibility that they might represent early hematopoietic progenitor cells. These studies support the notion that the adult cellular phenotype is the result of both an orderly acquisition of differentiation antigens and the migration of these primitive cellular populations to specific fetal organs. Identification of hematopoietic progenitors in fetal tissues may facilitate the identification and isolation of early lymphoid and myeloid progenitor cells in adults.     

Antigens on human monocytes identified by monoclonal antibodies

Two antigens (Mo1 and Mo2) present on human peripheral blood monocytes have been defined by lytic IgM monoclonal antibodies. Both antigens are present on greater than 70% of adherent mononuclear cells (predominantly monocytes). Mo1 is expressed by monocytes, granulocytes, and Null cells, but is absent from T and B lymphocytes. Mo2, on the other hand, appears specific for peripheral blood monocytes. Neither antigen is present on Ia-positive B cell lines or on tumor cells from patients with B cell lymphoproliferative malignancies, further excluding the possibility that Mo1 and Mo2 are Ia antigens. Mo1 and Mo2 are, however, present on a significant number of blast cells from patients with monocytic leukemia (both myelomonocytic and pure monocytic variants), but relatively infrequently expressed by cells from patients with acute granulocytic leukemia. These results indicate that Mo1 and Mo2 are unique antigens that may represent distinct stages of late monocyte-granulocyte differentiation.     

Influence of Burkitt's lymphoma and primary B cells on latent gene expression by the nonimmortalizing P3J-HR-1 strain of Epstein-Barr virus.

The Epstein-Barr virus (EBV) genes expressed in B lymphocytes immortalized in vitro or in Burkitt's lymphoma (BL) cells infected in vivo have been characterized previously; however, the viral products which are essential for immortalization or for establishment of EBV latency are still not known. To approach this question, we compared the kinetics of expression of EBV nuclear antigens and the two EBV-encoded small RNAs, EBER1 and EBER2, after infection of primary B cells or EBV genome-negative BL cells with either an immortalizing EBV strain (B95-8) or the nonimmortalizing deletion mutant (HR-1). Following infection of primary cells with B95-8 virus, EBV nuclear antigen (EBNA)-2 was expressed first, followed by EBNA-1, -3, and -4 (also called leader protein [LP]) and the two small RNAs. Infection of EBV genome-negative BL cells with the same strain of virus resulted in a similar pattern of gene expression, except that the EBNAs appeared together and more rapidly. EBERs were not apparent in one BL cell line converted by B95-8. The only products detected after infection of primary B lymphocytes with the HR-1 deletion mutant were the EBNA-4 (LP) family and trace amounts of EBER1. Although HR-1 could express neither EBNA-1, EBNA-3, nor EBER2 in primary cells, all these products were expressed rapidly after HR-1 infection of EBV genome-negative BL cell lines. The results indicate that the mutation in HR-1 virus affects immortalization not only through failure to express EBNA-2, a gene which is deleted, but also indirectly by curtailing expression of several other EBV genes whose coding regions are intact in the HR-1 virus and normally expressed during latency. The pattern of latent EBV gene expression after HR-1 infection is dependent on the host cell, perhaps through products specific for the cell cycle or the state of B-cell differentiation.     

Relationship between cytokine-dependent cell cycle progression and MHC class II antigen expression by human CD34+ HLA-DR- bone marrow cells.

Human CD34+ HLA-DR- bone marrow cells constitute a phenotypically homogeneous population of quiescent cells. More than 97% of CD34+ HLA-DR- cells reside in the G0/G1 phase of the cell cycle. The in vitro effects of two cytokines, IL-1 alpha and IL-3, alone or in combination, on the viability, cell cycle status and acquisition of HLA-DR by this cell population were examined. Cell viability was preserved in cultures receiving cytokines, but declined steadily in cultures deprived of exogenous IL. Over a period of 4 days, IL-3 progressively induced the expression of HLA-DR although driving corresponding numbers of cells into S and G2 + M. Although IL-1 alpha induced the expression of HLA-DR, it was not as effective as IL-3 in promoting the exit of these cells from G0/G1. Combinations of IL-1 alpha and IL-3, however, exerted an even greater effect on promoting both HLA-DR expression and entry of cells into active phases of the cell cycle. Simultaneous measurement of HLA-DR expression and cell cycle status in response to IL-1 alpha and IL-3 indicated that the majority of de novo expression of HLA-DR occurred in cells that remained in G0/G1. CD34+ HLA-DR- cells cultured with IL-1 alpha and IL-3 but arrested in G0/G1 by hydroxyurea were still capable of expressing HLA-DR, demonstrating that the acquisition of HLA-DR was independent of the entry of these cells into active phases of the cell cycle. These data indicate that the survival, HLA-DR expression, and cell cycle status of human CD34+ HLA-DR- bone marrow cells are governed by regulatory cytokines such as IL-1 alpha and IL-3. In addition, the entry of these cells into active phases of the cell cycle does not seem to be a prerequisite for the expression of HLA-DR, nor does it seem that the acquisition of HLA-DR by hematopoietic progenitor cells is a marker of cells entering the S phase of the cell cycle.