Cell surface expression of surrogate light chain (ΨL) in the absence of μ on human pro-B cell lines and normal pro-B cells

Surrogate light chains (ΨL) encoded by λ-like (λ5) and VpreB genes play a critical role in controlling the early steps of B cell differentiation. We prepared new anti-VpreB monoclonal antibodies (mAb) (3C7/6F6) which preferentially recognize the VpreB epitope at the cell surface of human cell lines that do not express the μ chain. These mAb provide the first characterization of human pro-B cell lines expressing surface ΨL. We demonstrate that surface ΨL expression is considerably enhanced upon interleukin-7 stimulation and that the ΨL complex is formed independently of the Igα/Igβ heterodimer. Finally, using these antibodies, we confirm the existence of a normal pro-B cell population in human adult bone marrow. These cells are CD34⁺ CD38⁺ ΨL⁺, do or do not express CD19, CD10, or both epitopes, and may represent the earliest cell population committed to B cell differentiation.     

Characterization of mouse CD6 with novel monoclonal antibodies which enhance the allogeneic mixed leukocyte reaction

Human CD6 is a cell surface protein expressed by thymocytes, mature T cells, a subset of B cells and certain cells of the brain. On human T cells, CD6 has been shown to act as a co-stimulatory molecule which modulates T cell receptor (TCR)-mediated T cell activation. To study further the recently identified mouse CD6 (mCD6), we generated and characterized a set of anti-mCD6 mAb. Anti-mCD6 mAb recognizing the mCD6 scavenger receptor cysteine-rich (SRCR) extracellular domains 1 and 3 were identified. mAb against SRCR domain 3, but not domain 1, inhibited the interaction of CD6 with a recently identified ligand, activated leukocyte cell adhesion molecule (ALCAM). Immunohistochemical analysis indicated that mCD6 expression was largely localized to the T cell areas of lymphoid tissue and, as previously reported in the human, CD6 was also expressed by neurons. CD6 was highly expressed on mouse T cells isolated from the spleen, lymph node and thymus as demonstrated by two-color immunofluorescence analysis. The CD4⁺ and CD8⁺ cells in these lymphoid compartments expressed similar levels of CD6. Immunoprecipitation studies showed that mouse thymocytes predominantly express a CD6 isoform of ～130 kDa, while splenocytes predominantly express a CD6 isoform of ～100 kDa. Anti-mCD6 mAb enhanced allogeneic mixed leukocyte reactions (MLR), indicating that CD6-ALCAM interactions may regulate the proliferative capacity of T cells.     

Flow Cytometric Identification of CD93 Expression on Naive T Lymphocytes (CD4<Superscript>+</Superscript>CD45RA<Superscript>+</Superscript> Cells) in Human Neonatal Umbilical Cord Blood

Human CD93 has a molecular weight of about 100 kDa and is selectively expressed by myeloid cell lineages in peripheral blood (PB) mononuclear cells. Although CD93 was initially identified as a receptor for complement component 1, subcomponent q phagocytosis (C1qRp) involved in the C1q-mediated enhancement of the phagocytosis of various antigens, several recent studies have reported that CD93 is not a receptor for the C1q-mediated enhancement of phagocytosis. The expression patterns of CD93 have been previously investigated in PB mononuclear cells (lymphocytes, monocytes, and granulocytes) from adult PB and neonatal umbilical cord blood (UCB), and the expression of CD93 was not found on lymphocytes from either normal adult PB or neonatal UCB. However, the detection of CD93 expression in neonatal UCB using CD93 monoclonal antibodies (mAbs) that recognize different antigenic epitopes remains poorly understood. In this study, we examined the expression of CD93 on lymphocytes, monocytes, and granulocytes from neonatal UCB using four different types of CD93 mAb detection probes, mNI-11, R139, R3, and X-2, using flow cytometric and western blot analyses. We found that CD93, as defined using all four mAbs, was expressed on monocytes and granulocytes in PB mononuclear cells from adult PB and neonatal UCB. On the other hand, we observed for the first time that the expression of CD93 on lymphocytes in neonatal UCB can only be detected using the mNI-11 mAb, established in our laboratory, and not with commercially available CD93 mAbs (R139, R3, and X-2). However, CD93 expression on lymphocytes from normal adults was not detected using any of the four CD93 mAbs. Two-color flow cytometric analyses showed that the CD93 recognized by mNI-11 mAb was expressed on CD3⁺ T lymphocytes (mainly CD4⁺ helper T lymphocytes), but not on CD19⁺ B lymphocytes or on CD8⁺ suppressor/cytotoxic T lymphocytes from neonatal UCB. In addition, CD93 was expressed on CD45RA⁺ (naive antigen) lymphocytes from neonatal UCB, but not on CD45RO⁺ (memory antigen) lymphocytes from neonatal UCB or on CD45RA⁺ and CD45RO⁺ lymphocytes from normal adult PB. Three-color flow cytometric analysis showed that CD93 was co-expressed on naive T lymphocytes (CD4⁺CD45RA⁺) from neonatal UCB. In a western blot analysis, the CD93 mAb (mNI-11) immunoprecipitated at a molecular weight of 98 kDa, identified as a CD93 molecule, in the CD4⁺CD45RA⁺ cells from neonatal UCB but not from adult PB, similar to the results in the human monocyte-like cell line U937 (human CD93-positive cells). Taken together, these results provide the first direct evidence of a novel/naive cell population (CD4⁺CD45RA⁺CD93⁺) in neonatal UCB that may have an important role in cell biology, transplantation, and immature/mature immune responses.     

A novel 120-kDa antigen shared by immature human thymocytes and long-term-activated T cells

In this study we report the characterization of monoclonal antibody (mAb) 8B4/20, raised against immature human thymocytes, that identifies a novel leukocyte antigen. The molecular characterization of the antigen by immunoprecipitation and immunoblotting yields, under nonreducing conditions, a specific band of 120 kDa which, under reducing conditions, displays a slightly lower molecular mass (110 kDa). mAb 8B4/20 detects a molecule found on the majority of thymocytes with an inverted gradient of expression when compared to CD3. It appears at high density on the CD3^?/low thymocytes, at reduced density on the CD3^med and double-positive thymocytes, and is absent on CD3^hi and singlepositive thymocytes and on peripheral blood T cells. Immunohistochemistry on frozen sections demonstrates cortical staining of the thymic lobules. Flow cytometric analysis of the different subsets of peripheral blood mononuclear cells shows that mAb 8B4/20 detects an antigen expressed only on CD56⁺/CD16⁺ natural killer cells and on a fraction of CD14⁺ monocytes. T cells, B cells, erythrocytes, granulocytes and platelets are consistently negative. The expression of the molecule on tumor cell lines does not show lineage restriction. Analysis of phytohemagglutinin plus recombinant interleukin-2-activated peripheral blood lymphocytes shows that mAb 8B4/20 identifies an antigen expressed on CD3⁺ cells by week 3 of culture. Thus, it recognizes a very late activation antigen (VLA) on mature T cells. The cell distribution and the electrophoretic pattern of the molecule identified by mAb 8B4/20 is distinct from that of known CD and of integrin/VLA molecules. Its function on thymocytes is so far unknown; however, the binding of mAb 8B4/20 to tumor lines induces changes in the morphology and adhesive properties of the 8B4/20⁺ cells growing in suspension. We suggest that mAb 8B4/20 recognizes a molecule that may be involved in interactions between thymocytes and other thymic structures that may be relevant for the selection process.     

CR3 (CD11b/CD18) expressed by cytotoxic T cells and natural killer cells is upregulated in a manner similar to neutrophil CR3 following stimulation with various activating agents

CR3 (CD11b/CD18) functions both as an iC3b-receptor and as an adhesion molecule for cellular ligands such as ICAM-1. Although CR3 has been well characterized on phagocytic cells, much less is known about CR3 on lymphocytes. In this study, the expression of CR3 was examined on resting and stimulated B, T, and natural killer (NK) cells by three-color flow cytometry. Biotinylated anti-CR3 mAb and streptavidin-FITC were used in combination with anti-CD3 mAb conjugated with peridinin chlorophyll-a protein (PerCP) and phycoerythrinlabeled mAbs to CD4, CD8, CD19, or CD56. Among resting lymphocytes, CR3 was expressed on nearly all NK cells (CD56⁺CD3^–), 1% of CD4⁺CD3⁺ helper T cells, 7% of CD8⁺CD3⁺ cytotoxic T cells, and 20% of B cells (CD19⁺). Among the 5% of T cells (CD3⁺) expressing CR3, the majority was CD56⁺. Incubation of PBMC for 30 min with PMA induced a three- to fivefold increase in CR3 expression on NK cells and a twofold increase on T cells but did not change the expression of CR3 on B cells. This effect of PMA was not blocked by the presence of cycloheximide, suggesting the presence of cytoplasmic (granule) stores of CR3 in these lymphoid cells resembling those previously reported in neutrophils and monocytes. When PBMC were incubated with rIFN-, rIL-2, -glucan, or high concentrations of LPS, expression of CR3 on NK cells increased significantly, but 4 hr of stimulation was required. Other cytokines (rIFN-, rIL-1, rIL-4, rIL-6, TNF-) and rC5a had no significant effect on CR3 expression. Among NK cells, both the CD56^bright and the CD56^dim cells expressed CR3, and the expression of CR3 on both of these NK cell subsets was increased in a similar manner by PMA. However, rIL-2 stimulated a greater increase in CR3 expression on CD56^bright cells than on CD56^dim cells. These studies suggest that CR3 expressed by NK cells or cytotoxic T cells resembles phagocyte CR3 in that cellular activation stimulates increased surface expression of CR3 derived from cytoplasmic reserves of the receptor.     

Properties of mouse CD40: Cellular distribution of CD40 and B cell activation by monoclonal anti-mouse CD40 antibodies

We describe here the derivation of a rat monoclonal antibody (mAb) against mouse CD40 (designated 3/23), which stains 45–50% of spleen cells of adult mice, approximately 90% of which are B cells. Interestingly, some 5–10% of both CD4⁺ and CD8⁺ T cells in the spleens of (some, but not all) adult, unimmunized mice are also CD40⁺, whereas CD40⁺ cells were not detectable in the thymus, even following collagenase digestion. Some 35–40% of lymphoid cells in the bone marrow of adult mice are CD40⁺ and virtually all of these are B220⁺, and hence of the B cell lineage: triple-color flow cytometry showed that CD40 is expressed at low levels on some 30% of pre-B cells, at intermediate levels on 80% of immature B cells and on essentially all mature B cells in the bone marrow. These results, therefore, suggest that in the mouse CD40 is expressed relatively late during the process of B cell differentiation. The mAb induced marked up-regulation of major histocompatibility complex class II molecules, CD23 and B7.2 antigens on mature B cells. It also stimulated modest levels of DNA synthesis in mature B cells by itself: this was markedly enhanced by suboptimal concentrations of mitogenic (but not non-mitogenic) anti-μ and anti-δ mAb, and moderately enhanced by co-stimulation with interleukin-4. Hyper-cross-linking of CD40 (using biotinylated mAb and avidin) also enhanced the proliferative response to anti-CD40.     

CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: Use of multiple peptide sets increases the detectable breadth of the CTL response

Background

The early B lymphopoiesis in mammals is regulated through close interactions with stromal cells and components of the intracellular matrix in the bone marrow (BM) microenvironment. Although B lymphopoiesis has been studied for decades, the factors that are implicated in this process, both autocrine and paracrine, are inadequately explored. Wnt signaling is known to be involved in embryonic development and growth regulation of tissues and cancer. Wnt molecules are produced in the BM, and we here ask whether canonical Wnt signaling has a role in regulating human BM B lymphopoiesis.

Results

Examination of the mRNA expression pattern of Wnt ligands, Fzd receptors and Wnt antagonists revealed that BM B progenitor cells and stromal cells express a set of ligands and receptors available for induction of Wnt signaling as well as antagonists for fine tuning of this signaling. Furthermore, different B progenitor maturation stages showed differential expression of Wnt receptors and co-receptors, β-catenin, plakoglobin, LEF-1 and TCF-4 mRNAs, suggesting canonical Wnt signaling as a regulator of early B lymphopoiesis. Exogenous Wnt3A induced stabilization and nuclear accumulation of β-catenin in primary lineage restricted B progenitor cells. Also, Wnt3A inhibited B lymphopoiesis of CD133⁺CD10^- hematopoietic progenitor cells and CD10⁺ B progenitor cells in coculture assays using a supportive layer of stromal cells. This effect was blocked by the Wnt antagonists sFRP1 or Dkk1. Examination of early events in the coculture showed that Wnt3A inhibits cell division of B progenitor cells.

Conclusion

These results indicate that canonical Wnt signaling is involved in human BM B lymphopoiesis where it acts as a negative regulator of cell proliferation in a direct or stroma dependent manner.     

A Novel Molecular Complex Expressed on Immature B Cells: A Possible Role in T Cell-Independent B Cell Development

To identify surface molecules that may play a role in regulating ileal Peyer''s patch (PP) B cell growth, we generated monoclonal antibodies (mAbs) and then selected them for a unique reactivity with ileal PP B cells. Flow cytometric analysis identified a mAb (SIC4.8R) that labeled 97% of ileal and 50–60% of jejunal PP sIgM⁺B cells. SIC4.8R also labeled a subpopulation of cortical thymocytes but few B or T cells in other lymphoid tissues, including bone marrow. Immunohistochemistry revealed intense SIC4.8R staining of B cells in the cortex of ileal PP follicles. SIC4.8R also labeled bovine PP B cells, a murine pro-B cell line, and pre-B cells in human bone marrow. Protein chemistry revealed that a structurally similar molecular complex was expressed on sheep ileal PP B cells and thymocytes and murine pro-B cells. Addition of soluble SIC4.8R to cultured ileal PP B cells reduced apoptotic cell death, elevated proliferative responses, partially inhibited anti-Ig-induced cell death, and induced IL-4 responsiveness. In contrast, soluble SIC4.8R had an antiproliferative effect on a mouse pro-B cell line. Finally, SIC4.8R labeling declined following the stimulation of ileal PP B cells with CD40 ligand. In conclusion, the present investigation determined that SIC4.8R identified a novel molecular complex that is expressed at several stages of T cell-independent B cell development in a variety of mammalian species. This observation confirmed that PP B cells are developmentally distinct from other B cell populations in sheep and suggested that the bone marrow may not be a site of B lymphopoiesis in young lambs.     

Regulatory role of CD95 ligation on human B cells induced in vivo capable of spontaneous and highrate Ig secretion

CD95 ligation elicits apoptotic signals in many cell systems. This study analyzes the effect of anti-CD95 mAb on human cells capable of spontaneous and highrate Ig secretion. Such cells have been induced in vivo and represent a highly mature B cell stage. Addition of the anti-CD95 monoclonal antibody (mAb) CH11 to tonsil B cells inhibited 50–60% of their spontaneous Ig secretion. The effect was exerted early in the culture and could be reversed by a pre-treatment with a neutralizing mAb. N-acetyl-D-sphingosine (C2-ceramide), although not a close analog, also reduced Ig secretion to a similar extent. The inclusion of a tetrapeptide inhibitor for certain interleukin-1β-converting enzyme proteases prevented the inhibitory effect of CH11 mAb on tonsil B cells. B cells capable of spontaneous Ab secretion obtained from blood of recently-immunized volunteers were also inhibited by CH11 mAb and C2-ceramide. In contrast, bone marrow (BM) B cells capable of spontaneous Ig secretion were unaffected by these agents. This CD95 ligation-mediated inhibition of tonsil and blood Ig-secreting B cells could not be reversed by cytokines with demonstrated activity on these B cells. Human mature B cells induced in vivo are identifiable as CD38^hi cells. Flow cytometric analysis revealed that a fraction of tonsil CD38^hi cells expressed low levels of CD95. Moreover, about 20% of these cells exhibited basal apoptosis, as defined by annexin V binding. This phenomenon was markedly increased by CD95 ligation. On the other hand, BM CD38^hi cells showed neither CD95 expression nor CD95-induced annexin V binding. These data suggest that CD95 ligation might play a role in the control of human humoral responses by inducing apoptosis in susceptible mature B cells.     

Human tonsil,blood and bone marrow in vivo-induced B cells capable of spontaneous and high-rate immunoglobulin secretion in vitro: Differences in the requirements for factors and for adherent and bone marrow stromal cells,as well as distinctive adhesion molecule expression

Human B cells capable of spontaneous IgG secretion are commonly found in circulation and in lymphoid tissues such as tonsil and bone marrow (BM). The present study compares the mechanisms that regulate tonsil, blood and BM B cells capable of spontaneous IgG secretion. The BM cell subset produced IgG during a markedly longer period of time (14 days) than did tonsil and blood cell subsets (2–3 days). Blood and BM, but not tonsil, B cell IgG secretion depended on the presence of adherent cells, as demonstrated by adherent cell depletion and re-addition experiments. Stromal BM cells supported linear IgG secretion by non-adherent BM cells for 2 weeks, but were unable to prolong the short-term IgG secretion by tonsil and blood cells. Different factors induced IgG secretion in each of the three B cell populations as optimal IgG secretion by tonsil, blood or BM cell subsets required either tumor necrosis factor-α, interleukin-6 or fibronectin + interleukin-6, respectively. Finally, these populations also showed differences in the expression of adhesion molecules; the tonsilar cell subset was PNA^+/? CD44⁺ CD49d⁺ CD49e^? Leu-8^+/?, the blood cell subset was PNA^? CD44^+/? CD49d⁺ CD49e^? Leu-8⁺ and the BM cell subset was PNA^? CD44^+/? CD49d⁺ CD49e^? Leu-8^?. These results suggest that the mechanisms controlling the final differentiation and the expression of adhesion molecules in these B lymphocytes exhibit territorial specificity.     

CD73 expression and fyn-dependent signaling on murine lymphocytes

CD73 is a glycosyl phosphatidylinositol-anchored protein with both ecto-enzyme activity (ecto-5′-nucleotidase) and signal transducing capabilities for human T lymphocytes. We now report an analysis of the distribution and function of CD73 in murine lymphoid tissues made possible by the development of the first monoclonal antibodies (mAb) specific for murine CD73. Subsets of T and B lymphocytes are CD73⁺ and the level of expression increases with lymphocyte maturation in both species. Among B cells, CD73 is largely restricted to cells which have undergone isotype switching. The signal transmitting function of CD73 is also conserved, as splenic T cells treated with anti-CD73 mAb plus phorbol 12-myristate 13-acetate proliferate and secrete IL-2. Fyn^−/– mice are unresponsive to CD73 ligation, however, demonstrating the requirement for this tyrosine kinase in CD73-mediated signal transduction. CD73 is down-regulated after mAb plus cross-linking, suggesting that expression may be controlled by interaction with a ligand. Only small numbers of thymocytes are CD73⁺, so CD73 receptor functions are unlikely to be important for developing T cells. However, immunohistochemical analysis reveals that reticular and vascular cells throughout the thymus and other lymphoid tissues are markedly CD73⁺. Therefore, CD73 might mediate lymphocyte-stromal cell interactions or condition the local microenvironment to facilitate lymphocyte development and / or function.     

Pre-pro-B cell growth-stimulating factor (PPBSF) upregulates IL-7Ralpha chain expression and enables pro-B cells to respond to monomeric IL-7.

Although pro-B cells are well represented in IL-7 knockout (KO) mice, they express abnormally low concentrations of the interleukin-7 receptor alpha-chain (IL-7Ralpha) and do not generate pre-B cells. Here, we demonstrate that pro-B cells from IL-7 KO mice can be induced to generate pre-B cells and immature B cells by exposure to recombinant IL-7 (rIL-7) in vivo but not in vitro. Experiments in recombinant activation gene-1 (RAG-1) KO mice indicate that the in vitro unresponsiveness of IL-7(-/-) pro-B cells to rIL-7 is unrelated to the absence of a functional pre-B cell receptor (pre-BCR). Rather, it appears to be due to the suboptimal expression of the IL-7Ralpha chain. Thus, IL-7(-/-) pro-B cells readily respond to rIL-7 in vitro if IL-7Ralpha chain expression is first upregulated by exposure to IL-7 in vivo or to IL-7(+/+) bone marrow (BM) stromal cells or conditioned medium (CM) therefrom in vitro. Similar results were obtained when pro-B cells from IL-7 KO mice were cultured on IL-7(-/-) BM stromal cells in the presence of rIL-7. This suggested that the recently described pre-pro-B cell growth-stimulating factor (PPBSF), a self-assembling hybrid cytokine comprising IL-7 and the stromal cell-derived hepatocyte growth factor beta-chain (HGFbeta), is required to stimulate pro-B cells from IL-7 KO mice. This inference was verified by demonstrating that purified PPBSF upregulates IL-7Ralpha chain expression on IL-7(-/-) pro-B cells in vitro and enables them to respond to rIL-7 in a stepwise manner. We, therefore, postulate that PPBSF is the operative form of IL-7 that normally induces IL-7Ralpha(lo) pre-pro-B cells to proliferate and differentiate into IL-7Ralpha(hi) pro-B cells, which then proliferate and differentiate into pre-B cells on stimulation with monomeric IL-7.     

Localization in situ of the co-stimulatory molecules B7.1, B7.2, CD40 and their ligands in normal human lymphoid tissue

Functional interactions between B and T lymphocytes are known to depend on the expression of co-stimulatory molecules B7.1/CD80, B7.2/CD86 and their counter-receptors CD28 and CTLA4, as well as CD40 and its ligand CD40L. To study the role of these molecules in situ, an immunohistochemical analysis was carried out on normal human lymphoid tissue. In the germinal centers (GC), B7.1 and B7.2 were differentially expressed. In the dark zone, centroblasts were predominantly B7.1⁺, while centrocytes in the light zone were B7-2⁺, resulting in reversed gradients of both markers in GC. Follicle mantle cells were negative for B7.1 and B7.2. Macrophages and interdigitating dendritic cells (IDC) in T cell zones both expressed B7.1 and B7.2. Moreover, clusters of B7.2⁺ T cells were demonstrated in interfollicular areas. Intrafollicular CD4⁺ T cells in GC, predominantly in the apical light zone, expressed CD28 and CTLA4, as did the majority of interfollicular T cells. CTLA4 showed a striking excentric cytoplasmic staining, which was also seen on T cells activated in vitro. CD40 was expressed on all B cells and more strongly on macrophages and IDC. Moreover, small clusters of T cells in a rim outside the GC showed CD40 expression. CD40L was expressed both on intrafollicular CD4⁺ T cells as well as on T cells in T cell zones. The differential distribution of co-stimulatory molecules in different compartments of normal human lymphoid tissue in situ indicates that these interactions play a distinctive role in different stages of B cell differentiation and in the immune response.     

Mechanisms involved in the differential bone marrow homing of CD45 subsets in 5T murine models of myeloma

Multiple myeloma (MM) is an incurable plasma cell cancer, localized in the bone marrow (BM). The mechanisms used by these cells to (re-)enter this organ remain largely unknown. Recently, we reported that both CD45⁺ and CD45⁻ myeloma cells home to the BM and induce myeloma disease. In this work, we investigated the underlying mechanisms involved in the homing of CD45⁺ and CD45⁻ myeloma cells in the experimental 5T2MM and 5T33MM murine models. In vivo tracing of flow cytometric sorted and radioactively labeled CD45 subsets revealed a reduced homing of the CD45⁻ 5TMM cells to the BM as compared to the CD45⁺ 5TMM cells. Migration assays demonstrated an impaired chemotaxis towards BM endothelial cell conditioned medium, BM stromal cell conditioned medium and towards the basement membrane component laminin-1 of the CD45⁻ 5TMM cells compared to the CD45⁺ subset. Matrix metalloproteinase-9 (MMP-9) and urokinase type plasminogen activator (uPA) are key extracellular matrix proteases involved in the invasion of cancer cells. Inhibitor and antibody blocking experiments demonstrated the involvement of both in the invasion of the 5TMM cells. CD45⁻ 5TMM cells had a low secretion of MMP-9 and (for the non-aggressive line 5T2MM only) a low cell surface expression of uPA receptor, as revealed by gelatin zymography and flow cytometric analysis, respectively. Accordingly, the synthetic basement membrane invasive capacity of the CD45⁻ 5TMM subpopulations was also impaired. Our results indicate that CD45⁺ and CD45⁻ 5T myeloma cells have a differential BM homing attributable to differential migratory and invasive capacities. This revised version was published online in July 2006 with corrections to the Cover Date.     

Expression of human NKRP1A by CD34+ immature thymocytes: NKRP1A-mediated regulation of proliferation and cytolytic activity

In this study, we show that NKRP1A is expressed and functions on a subset of immature human thymocytes. We took advantage of the monoclonal antibody (mAb) 191B8 that was obtained by immunizing mice with cultured human thymocytes characterized by an immature surface phenotype [CD2^? CD3^? CD4^? CD8^? stem cell factor receptor (SCFR)⁺] and expressing cytoplasmic CD3? chain. The 191B8 antibody homogeneously reacted with the immunizing population but not with most unfractionated thymocytes. It stained a minor population of resting immature thymocytes co-expressing CD34, SCFR, or both. Following culture of the CD34⁺ or CD34^? fractions of CD2^? CD3^? CD4^? CD8^? purified immature thymocytes with recombinant interleukin-2 (rIL-2), the 191B8-defined antigen was expressed on virtually all cells even when 191B8⁺ cells were removed from the starting population. On the other hand, no 191B8⁺ cells were detected in fresh or cultured thymocytes expressing a more mature phenotype. Biochemical analysis of 191B8 mAb-reactive molecules revealed, under non-reducing conditions, two bands displaying apparent molecular masses of 80 and 44 kDa and a single band of 44 kDa under reducing conditions. Digestion with proteases indicated that the 80-kDa form represented a homodimeric form of two 44-kDa molecules, while deglycosylation with N-glycanase suggested the existence of four N-glycosylation sites. Transfection of COS7 or NIH3T3 cells with hNKRP1A cDNA showed that the 191B8 mAb recognized NKRP1A as shown by both immunofluorescence analysis and immu-noprecipitation experiments. Functional studies showed that the 191B8/NKRP1A molecule mediated strong inhibition of the cytolytic activity of culturd CD2^? CD3^? immature thymocytes against a panel of tumor target cells. More importantly, 191B8 mAb induced proliferation of CD2^? CD3^? fresh thymocytes which was not increased by rIL-2. Thus, we propose that NKRP1A molecules, which are expressed in highly immature thymocytes, may play a regulatory role in their growth and function.     

Accelerated thymopoiesis and improved T‐cell responses in HLA‐A2/‐DR2 transgenic BRGS‐based human immune system mice

Human immune system (HIS) mouse models provide a robust in vivo platform to study human immunity. Nevertheless, the signals that guide human lymphocyte differentiation in HIS mice remain poorly understood. Here, we have developed a novel Balb/c Rag2^?/? Il2rg^?/? Sirpa^NOD (BRGS) HIS mouse model expressing human HLA‐A2 and ‐DR2 transgenes (BRGSA2DR2). When comparing BRGS and BRGSA2DR2 HIS mice engrafted with human CD34⁺ stem cells, a more rapid emergence of T cells in the circulation of hosts bearing human HLA was shown, which may reflect a more efficient human T‐cell development in the mouse thymus. Development of CD4⁺ and CD8⁺ T cells was accelerated in BRGSA2DR2 HIS mice and generated more balanced B and T‐cell compartments in peripheral lymphoid organs. Both B‐ and T‐cell function appeared enhanced in the presence of human HLA transgenes with higher levels of class switched Ig, increased percentages of polyfunctional T cells and clear evidence for antigen‐specific T‐cell responses following immunization. Taken together, the presence of human HLA class I and II molecules can improve multiple aspects of human B‐ and T‐cell homeostasis and function in the BRGS‐based HIS mouse model.