Loss of lineage antigens is a common feature of apoptotic lymphocytes.

The analysis of apoptosis in cell populations involves the detection of their specific lineage antigen (LAg) expression. This experimental approach relies on their assumed constant expression, but it is unclear whether such expression is actually maintained during cell death. We examined whether the loss of LAgs is a common feature of apoptotic lymphocytes and whether some might completely lose their LAgs. The changes in the expression of CD3, CD5, CD8, CD4, CD28, CD56, and CD19 were monitored in highly purified lymphocyte populations obtained by negative selection in a fluorescence-activated cell sorter. These were cultured for 24 h with or without phytohemagglutinin or staurosporin. For each LAg-positive subset studied, apoptosis was consistently more common among cells showing partial or total loss of LAg expression compared with cells maintaining their initial LAg levels. The kinetics of expression loss was rapid for CD8, CD56, and CD28, and more than 80% of initial expression was lost in the early stages of apoptosis but was slower for CD3, CD5, and CD4. For CD3 and CD5, expression was dependent on the apoptotic stimulus used. It is interesting that loss of antigen expression was independent of cell size. This phenomenon was also found in nonmanipulated, highly pure CD19 B lymphocytes of peripheral blood mononuclear cells from B chronic lymphocytic leukemia patients. Loss of LAg expression appeared to be a common feature of apoptotic lymphocytes under all the conditions assayed. The different kinetic patterns of LAg loss suggest apoptotic cells might actively regulate this process.

Meics, a novel zinc-finger protein which relocates from nuclei to the central meiotic spindle during Drosophila spermatogenesis.

A Drosophila gene encoding a novel zinc-finger protein, Meics, was cloned using a monoclonal antibody. The predicted amino acid sequence contains 12 zinc-finger motifs of the C2H2-type. During spermatogenesis, Meics distributes intranuclearly at pre- and post-meiotic stages whereas it relocates to central-spindle microtubules at both meiotic divisions.

Cell surface CD28 levels define four CD4+ T cell subsets: abnormal expression in rheumatoid arthritis.

CD28 is a costimulatory receptor expressed in most CD4(+) T cells. Despite the long-standing evidence for up- and downregulation of surface CD28 expression in vitro, and the key regulatory role assigned to the upregulation of CD28 counterreceptor [the CD152 (CTLA-4) molecule], in vivo CD28 induction has attracted little attention. We studied CD28 and CD152 expression and function in 33 rheumatoid arthritis (RA) patients, 20 clinically active and 13 inactive, and in 24 healthy donors. Four subsets of CD28(-), CD28(low), CD28(int), and CD28(high) peripheral blood human CD4(+) T cells were defined using three-color flow cytometry. The three CD28(+) subsets displayed a one-, two-, or threefold quantitative difference in their relative number of CD28 antibody binding sites, respectively (P < 0.01). RA patients, whether active or inactive, showed a distinct phenotype when compared to healthy donors: (i) the percentage of CD4(+)CD28(high) cells was increased twofold and the CD4(+)CD28(low) subset was reduced twofold (P < 0.01) and (ii) the CD4(+)CD28(high) cells from RA patients showed an in vivo activated phenotype, CD45RO(+)CD5(high)IL-2Ralpha(+) (P < 0.01). Active RA patients were different from inactive patients. They showed a twofold increase in mean CD28 expression (P < 0.05), whereas each of the CD28(+) subsets in the inactive RA patients showed reduced expression when compared to healthy donors. Notably, both active and inactive RA patients showed abnormal CD28 upregulation when T cells were activated in vitro with CD3 antibodies, but only inactive RA patients showed a hypoproliferative response to TCR/CD3 triggering when compared to healthy donors (P < 0.01). This defective proliferation was normalized by concurrent crosslinking with CD28 antibody. No differences were noted in the expression of CD152 or CD80, a CD28 and CD152 shared ligand. The disregulated in vivo expression of CD28 was related to the RA patients' disease activity and suggests that modulation of CD28 surface levels may be an additional mechanism to finely tune the delicate responsiveness/tolerance balance.

A new method for the simultaneous analysis of growth and death of immunophenotypically defined cells in culture.

BACKGROUND: Internal standards have been used in flow cytometry methods to enumerate lymphoid subsets and hemopoietic progenitor cells ex vivo. However, the currently available methods cannot be readily applied to the analysis of cultured cells because of the frequent occurrence of cell death during in vitro assays. METHODS: This paper reports a new method for the enumeration of both viable and nonviable cells in culture. Cells were counted with the aid of an internal reference standard of microbeads, and live versus dead cell discrimination was performed using 7-amino-actinomycin D which allows the double staining of surface antigens. RESULTS: The method is more precise, accurate and sensitive than either conventional light microscopy-based or automated cell counting. Additionally, it may be used to accurately measure the number of apoptotic cells in a culture. RESULTS: Through the enumeration of surviving cells it is demonstrated that, when applied to the study of mitogen-activated T lymphocytes, current flow cytometry techniques (which do not use internal standards) for the study of the viability and apoptosis overestimate the fraction of viable cells and underestimate both the fraction of dead and apoptotic cells. CONCLUSIONS: The new method overcomes these limitations and is of use in the in vitro study of cell growth and apoptosis.

Abnormal functional behavior of peripheral blood mononuclear cells from Hashimoto's disease patients. Immunomodulatory effects of cyclosporin A.

In vitro studies of activation and proliferation induced by mitogens in the presence of Cyclosporin A (CsA) and or cytokines were carried out to determine the effects of CsA and cytokines on mitogen activated peripheral blood mononuclear cells (PBMC) from thirteen Hashimoto's disease patients (HP) and ten healthy controls. The proliferative response (PR) of PBMC from HP to mitogens at 7 days of culture was higher than in controls. Interleukin 2 (IL-2) addition significantly increased the PR in phytohemagglutinin-stimulated PBMC from HP, but not in controls. CsA inhibited in a dose dependent manner the PR, as well as the expression of activation antigens induced by mitogens in both groups of subjects, but PBMC from HP were sensitive to CsA at lower doses than those that were effective on PBMC from controls. Both IL-2 or IL-4 overcame the inhibitory effect of CsA on PBMC from HP and controls. Conversely, IL-10 or IFN-alpha addition increases the inhibitory effect of CsA on the PR of PBMC from both HP and controls. We conclude that PBMC from Hashimoto's disease patients shown an abnormal pattern of PR that is associated to increased PR to mitogens and higher sensitivity to immunomodulatory effects of IL-2 and CsA.

Multivalent structure of an alphabetaT cell receptor.

Whether there is one or multiple alphabetaT cell antigen receptor (TCR) recognition modules in a given TCR/CD3 complex is a long-standing controversy in immunology. We show that T cells from transgenic mice that coexpress comparable amounts of two distinct TCRbeta chains incorporate at least two alphabetaTCRs in a single TCR/CD3 complex. Evidence for bispecific alphabetaTCRs was obtained by immunoprecipitation and immunoblotting and confirmed on the surface of living cells both by fluorescence resonance energy transfer and comodulation assays by using antibodies specific for TCRbeta-variable regions. Such (alphabeta)2TCR/CD3 or higher-order complexes were evident in T cells studied either ex vivo or after expansion in vitro. T cell activation is thought by many, but not all, to require TCR cross-linking by its antigen/major histocompatibility complex ligand. The implications of a multivalent (alphabeta)2TCR/CD3 complex stoichiometry for the ordered docking of specific antigen/major histocompatibility complex, CD4, or CD8 coreceptors and additional TCRs are discussed.

In vitro reconstitution of human B-cell ontogeny: from CD34(+) multipotent progenitors to Ig-secreting cells.

We describe a long-term, in vitro culture system initiated with CD34(+) or CD34(+)CD38(-) umbilical cord blood hematopoietic progenitors that supports normal human B-lineage development, including the production of mature Ig-secreting B cells. In the first stage (human B-progenitor long-term culture [HB-LTC]), CD34(+) hematopoietic progenitors are cultured on the murine stromal cell line, S17, leading to the sustained production of large numbers of CD10(+), CD19(+) early B progenitors. Reverse transcriptase-polymerase chain reaction (RT-PCR) and three-parameter flow cytometry for VpreB (surrogate light chain), cytoplasmic mu chain, and surface IgM expression were used to characterize the CD19(+) B progenitors present within these cultures. This analysis showed distinct B-lineage subpopulations, including pro-B cells, cycling pre-B cells, and IgM+, IgD-/+ immature B cells. The limited expansion of IgM+ B cells and the immature surface phenotype of this population (IgM+, IgD+, CD10(+), CD38(+)) suggested that HB-LTC conditions were unable to provide appropriate signals for further differentiation. A second culture stage was used to determine if these immature B cells were functionally competent. Purified CD19(+) cells were transferred onto fibroblasts expressing human CD40-ligand in the presence of IL-10 and IL-4. This lead to cell proliferation, modulation of the IgM+ cell surface phenotype to one consistent with an activated mature B cell, secretion of Ig, and isotype switching. Notably, IgM and IgG producing B cells were also generated using two-stage cultures established with highly purified multipotent CD34(+)CD38(-) hematopoietic stem cell progenitors. This culture model should permit detailed in vitro analysis and genetic manipulation of the major transition points in human B ontogeny, beginning with commitment to the B lineage and leading to development and activation of mature B cells.

PUMA1: a novel protein that associates with the centrosomes, spindle and centromeres in the nematode Parascaris.

We have identified a 227 kDa spindle- and centromere-associated protein in Parascaris, designated PUMA1 (Parascaris univalens mitotic apparatus), using a monoclonal antibody (mAb403) generated against Parascaris embryonic extracts. PUMA1 distribution was studied by immunofluorescence microscopy in mitotic and meiotic Parascaris cells, where centromere organization differs greatly. In mitosis, PUMA1 associates throughout cell division with the centrosomes and kinetochore-microtubules, and it concentrates at the continuous centromere region of the holocentric chromosomes. PUMA1 also localizes to the spindle mid-zone region during anaphase and at the midbody during telophase. In meiosis, PUMA1 associates with the centrosomes and with the discrete centromeric regions lacking kinetochore structures. The analysis of colchicine-treated embryos indicated that the association of PUMA1 with the centromeric region depends on microtubule integrity. mAb403 also recognizes spindle components in Drosophila. A series of overlapping cDNAs encoding the gene were isolated from a Parascaris embryonic expression library. Analysis of the nucleotide sequence identified an open reading frame capable of encoding a protein of 227 kDa. Analysis of the protein sequence indicated that PUMA1 is predicted to be a coiled-coil protein containing a large central alpha-helical domain flanked by nonhelical terminal domains. The structural features and cellular distribution of PUMA1 suggest that it may play a role in the organization of the spindle apparatus and in its interaction with the centromere in Parascaris.

CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage.

The CD3 complex found associated with the T cell receptor (TCR) is essential for signal transduction following TCR engagement. During T cell development, TCR-mediated signalling promotes the transition from one developmental stage to the next and controls whether a thymocyte undergoes positive or negative selection. The roles of particular CD3 components in these events remain unclear. Indeed, it is unknown whether they have specialized or overlapping roles. However, the multiplicity of CD3 components and their evolutionary conservation suggest that they serve distinct functions. Here the developmental requirement for the CD3 delta chain is analyzed by generating a mouse line specifically lacking this component (delta-/- mice). Strikingly, CD3 delta is shown to be differentially required during development. In particular, CD3 delta is not needed for steps in development mediated by pre-TCR or gamma delta TCR, but is required for further development of thymocytes expressing alpha beta TCR. Absence of CD3 delta specifically blocks the thymic selection processes that mediate the transition from the double-positive to single-positive stages of development.

Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci.

CD19+CD10+ human B lineage bone marrow cells were separated into cycling or resting cells, which differ in their expression of CD34, VpreB, recombination activating gene (RAG-1), and terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for DHJH and VkJk, VkJkK(de) and VkK(de) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B lymphocyte pathway of development as follows: CD34+VpreB+RAG-1+TdT+, DHJH-rearranged, kL germline cycling pre-B I cells-->CD34-VpreB+microH chain+ (pre-B receptor+) RAG-1-TdT-, VHDHJH-rearranged, kL germline, cycling pre-B II cells-->CD34-VpreB-, intracytoplasmic microH chain+ (pre-B receptor-) RAG-1+/-TdT-, VHDHJH-rearranged, mainly kL germline cycling pre-B II cells-->CD34-VpreB-intracytoplasmic microH chain+, RAG-1+TdT-, VHDHJH-rearranged, VkJk-rearranged, IgM-, resting pre-B II cells CD34+VpreB-, sIgM+, RAG-1+TdT-, VHDHJH- and VkJk-rearranged IgM+ immature B cells-->CD34-, CD10-, sIgM+/sIgD+ mature B cells. This order, for the first time established for human B lineage cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.

A novel anti-Vpre-B antibody identifies immunoglobulin-surrogate receptors on the surface of human pro-B cells.

Vpre-B and lambda 5 genes, respectively, encode V-like and C-like domains of a surrogate immunoglobulin light chain (psi L). Such psi L complex is expressed in early progenitor B (pro-B) cells, before conventional immunoglobulin heavy (microH) and light (L) chains are produced. We raised a wide panel of monoclonal antibodies (mAbs) against soluble recombinant Vpre-B proteins to study early events in human B cell development. One of these antibodies, B-MAD688, labeled surrogate Ig-complexes on the surface of microH- pro-B cell lines and normal bone marrow cells in immunofluorescence assays. Immunoprecipitations using surface-labeled pro-B cells and B-MAD688 mAb indicated that human psi L is associated with high molecular weight components homologous to the surrogate heavy (psi H) chains described in mouse. Using B-MAD688 and SLC2 mAbs, we were able to distinguish between psi H psi L and microH psi L complexes on the surface of human pro-B and later precursor, pre-B, cells. The finding of psi H psi L complexes in mouse and man lead us to hypothesize a role for psi H-containing receptors in B cell development.

Expanded CD4+CD45RO+ phenotype and defective proliferative response in T lymphocytes from patients with Crohn's disease.

BACKGROUND & AIMS: An abnormal immune response may play a pathogenic role in Crohn's disease. The aim of this study was to determine the role of regulatory T cells in Crohn's disease. METHODS: T-cell phenotype and function were studied in blood lymphocytes from patients with Crohn's disease and a control group consisting of healthy donors and patients with ulcerative colitis. RESULTS: Flow cytometric studies showed a significant increase in the percentage of CD3+DR+ and CD4+CD45RO+ T cells in patients with Crohn's disease. T cells from patients with Crohn's disease and ulcerative colitis showed a defective proliferative response after stimulation with surface mitogenic ligands (phytohemagglutinin and anti-CD28 or anti-CD3 antibodies). Soluble interleukin-2 receptor alpha was augmented in the Crohn's disease and ulcerative colitis groups. In the Crohn's disease group, impairment of T-lymphocyte proliferation was normalized by exogenous interleukin 2, although endogenous interleukin-2 production and interleukin-2 receptor alpha expression were normal. CONCLUSIONS: An in vivo expansion of CD4+ T lymphocytes with memory phenotype and impaired T-cell proliferation that can be restored by pharmacological amounts of interleukin 2 was found in patients with Crohn's disease. There is a severe immunodisturbance in the T-cell compartment of patients with either clinically active or inactive Crohn's disease.

Implication of protein kinase C in IL-2-mediated proliferation and apoptosis in a murine T cell clone.

Several reports have suggested that protein kinase C plays an essential role in T cell activation and apoptosis. The recent synthesis of the selective protein kinase C inhibitor, GF109203X, has made it possible to test the relevance of protein kinase C in T cell proliferation and apoptosis. We report that the use of GF109203X, in concentrations that are below its toxicity limits, inhibits IL-2-dependent proliferation in murine T cells expressing intermediate or high-affinity IL-2R (TS1 beta and TS1 alpha beta). In addition, GF109203X reverts the suppression of apoptosis mediated by IL-2 or IL-2+ dexamethasone. The use of the phorbol ester PMA, a protein kinase C activator, allows a bypass of the IL-2/IL-2R interaction in the suppression of apoptosis mediated by dexamethasone or IL-2 withdrawal in TS1 beta cells but not in TS1 alpha beta cells. Taken together, our data indicate that activation of protein kinase C is an important step in IL-2-mediated proliferation and in suppression of apoptosis.

Self-limited autoimmune disease related to transient donor B cell activation in mice neonatally injected with semi-allogeneic F1 cells.

BALB/c mice injected at birth with 10(8) semi-allogeneic (C57BL/6 x BALB.IgHb)F1 spleen cells develop a lupus-like syndrome in which autoantibodies bear exclusively the donor allotype. We have analyzed the evolution of donor B cell chimerism and the autoimmune manifestations during the first year of life in these mice. Anti-DNA, -histone, and -cardiolipin IgG antibodies as well as circulating immune complexes appeared in the second week of life, reached the highest values around the sixth week, and then progressively dropped to normal values after the sixth month in most mice. The kinetics of the evolution of the autoimmune manifestations, as well as the kinetics of serum donor Ig allotype, were parallel to the kinetics of donor B cell chimerism, which was particularly prominent in the spleens in early weeks of life, and progressively decreased after remission of the autoimmune syndrome. Membrane-proliferative glomerulonephritis, which was followed as the more representative histological abnormality in this model, was particularly evident after 10 weeks of life, but disappeared by the end of the follow-up. Interestingly, when mice with a self-limited disease were re-injected with 10(8) F1 spleen cells i.v., a flare in the serological manifestations was observed. In these re-injected mice a predominance of anti-DNA, IgG1 antibodies bearing exclusively the donor allotype was also observed, as in the early weeks of life.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21).

Multivalent but not monovalent CR2 ligands are required to elicit Raji cell proliferation as well as other B cell responses. It has been reported (C. Servis and J. D. Lambris, J. Immunol. 1989. 142: 2207) that the tetrameric peptide T-(C31202-1214)4, which represents the CR2-binding site in C3d, was able to support Raji cell growth. We show here that the tetrameric peptide T-(gp350(19-30)4, which contains the CR2-binding site in gp350 protein of EBV also induces Raji cell growth and this effect is inhibited by the monomeric peptides gp350(19-30) and C3(1201-1214). We also investigated the nature of the interaction between C3 fragment and CR2 in order to explain the Raji cell growth-supporting effect exerted by C3. The following findings suggest that there are multiple sites in the C3 molecule able to interact with CR2: (1) both C3c and C3d immobilized on microspheres are able to bind to Raji cells through CR2. (2) soluble C3d inhibits to a greater extent the binding of CR2 to fixed C3d than to fixed C3b, which suggests the existence of additional CR2-binding sites within C3b not present in the C3d portion of the molecule; (3) synthetic peptides C3(1187-1214), C3(741-757) and C3(295-307) which represents regions of similarity in the C3 molecule bind specifically to CR2 on Raji cells and compete with each other for binding to the receptor and (4) preincubation of microtiter plate-fixed C3b with monoclonal or polyclonal anti-peptide antibodies (C3-9, anti-C3(727-768) recognize the N terminus of the alpha chain of C3 (including residues 741-757) inhibited CR2 binding. Therefore, these data suggest that the N terminus of the alpha chain of C3 is involved in binding to CR2.

Two different maturational stages of natural killer lymphocytes in human newborn infants.

The objective of this study was to assess the basis for the diminished natural killer (NK) lymphocyte activity of neonates. We found either severely reduced (63% of 68 neonates) or normal (similar to healthy adult) levels of NK activity. The percentages of cord blood mononuclear cells from the two groups of infants that expressed CD16, a differentiation antigen found in NK cells, were similar and within the range found in peripheral blood mononuclear cells of adults. However, infants with low NK activity had reduced numbers of cells in the CD16+56+ subpopulation, whereas the number of these effector cells present in cord blood mononuclear cells from infants with normal NK activity was within the range found in adults. Recombinant interleukin-2, but not recombinant interferon-gamma, normalized the low NK activity of infants in a dose- and time-dependent manner. Analysis of the pattern of target cell susceptibility to lysis, together with the CD16+CD3- phenotype of the precursor and effector lymphocytes, demonstrated that the induced cytotoxicity was mediated by NK cells. In contrast, NK cells from infants with normal cytotoxic levels exhibited a functional response to interleukin-2 and interferon-gamma similar to that of adults. Our results indicate that NK cells in human neonates go through two different maturational stages.

Structure of the T cell antigen receptor (TCR): two CD3 epsilon subunits in a functional TCR/CD3 complex.

Transgenic mice carrying and expressing the human CD3 epsilon gene incorporate the corresponding protein product into T cell receptor (TCR)/CD3 complexes on thymocyte and T cell surfaces. The chimeric antigen receptors allow normal T cell development and selection of repertoires in vivo and are able to transduce activation signals in vitro. We have exploited the ability to distinguish mouse (m) and human (h)CD3 epsilon chains to analyze the stoichiometry of CD3 epsilon in transgenic mouse TCRs. Immunoprecipitation and fluorescence resonance energy transfer experiments demonstrate that such TCRs can contain both h- and mCD3 epsilon chains, implying that more than one CD3 epsilon subunit occurs per TCR. Antigen comodulation studies are consistent with a stochastic use of h- or mCD3 epsilon during receptor assembly, and further suggest a structure for the TCR/CD3 complex with two CD3 epsilon chains. The determination of CD3 epsilon subunit stoichiometry, together with existing biochemical data, allows the generation of a minimal model for the structure of the TCR and illustrates the potential value of the transgenic approach to the analysis of complex receptors.

Identity of PB76 differentiation antigen and lymphocyte alkaline phosphatase.

Alkaline phosphatases (APases, EC 3.1.3.1) are ecto-enzymes bound to cell membranes by a phosphatidyl-inositol anchor. We have previously shown that APase is present on activated murine B cells and its expression correlates with the process of B cell differentiation into immunoglobulin secretion. Recently, a monoclonal antibody (mAb), G-5-2, that recognizes a 76-kDa molecule preferentially expressed on the surface of pre-B and plasma cells (PB76) was described. Some features shared by APase and PB76 differentiation antigen suggest that the G-5-2 mAb might be specific for lymphocyte APase. Here, we have analyzed this possibility and found an absolute correlation between PB76 expression in cells and their APase activity. Although PB76 has been described as a B cell-restricted marker, PB76 is also expressed on some T cells, such as the YAC-1 T cell lymphoma, that are known to bear APase. Treatment of YAC-1 cells with phosphatidylinositol-specific phospholipase C resulted in a quantitatively correlated removal of both APase and PB76 antigens. Moreover, we demonstrate that PB76 antigen has APase activity using an enzyme-antigen immunoassay with the G-5-2 mAb. We conclude that PB76 and lymphocyte APase are one and the same antigen.