THE ROLE OF B CELLS IN THE T CELL DEPENDENT RELEASE OF HUMAN B CELL GROWTH FACTOR

Quiescent human B cells are postulated to go through activation and proliferation phases before undergoing differentiative phase for immunoglobulin secretion. The present studies address some of the aspects of activation and proliferation phase of normal human B cells. The definitions of signals responsible for B cell activation and proliferation resulted in the development of a highly specific, reproducible B cell growth factor (BCGF) assay. This BCGF bioassay utilizes activation by rabbit anti-human IgM-antibody. The functional specificity of this assay for measuring BCGF activity was demonstrated by the finding that target B cells proliferated but did not differentiate. The factor specificity was determined by specific absorption of BCGF by anti-IgM activated B cells. This assay was utilized for the studies of T-B cell collaboration and the essential function of monocytes in the production and/or release of B cell growth factor in a syngeneic in vitro system. It is apparent that highly purified T cells are poor producers of BCGF by themselves and require monocytes to secrete significant quantities of BCGF upon PHA stimulation. Macrophage soluble factor, Interleukin 1, is capable of replacing monocyte function for the release of BCGF by activated T cells. In our studies, B cells are incapable to function as accessory cells to replace monocyte function. Normal B cells are also not capable of producing BCGF under our experimental observations. However, the addition of these B cells at an optimum cell density (T:B ratio 1:1) doubles the monocyte dependent release of BCGF by syngeneic T cells. The augmentative role of B cells is expanded to understand the mechanism of BCGF release by T cells. It is observed from our studies that DR antigen of B cell surface is involved in the release of BCGF. The functional difference between DR of B cells and monocytes is observed as IL-1 could replace DR-treated monocytes whereas failed to replace DR-treated B cells for the release of BCGF by T cells. This functional difference may be attributed to the reported microheterogeneity in DR of B cells and monocytes. The addition of irradiated B cells increased the monocyte dependent T cell proliferation, suggesting the increase of T cell pool for BCGF release. In summary, the development of a biological assay specific for B cell growth factor led to the delineation of an interesting role of B cells in the release of its own growth factor by T cells. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Functional and physical associations of beta1 integrins in the activation of human T lymphocytes

Adhesion involves interactions between cells or cells with extracellular matrix components and is a fundamental process for all multicellular organisms as well as many pathogenic microbes. Integrins are heterodimeric transmembrane proteins that function as adhesion molecules and transduce signals between the extracellular environment and the intracellular cytoskeletal machinery. β1 integrin subfamily is highly expressed on T lymphocytes and mediates cell spreading, adhesion and coactivation. T lymphocytes have an important role in the regulation and homeostasis of the immune system therefore, the goals of this study were to first to investigate β1 integrin interaction with fibronectin binding protein A (FnbpA), a surface protein expressed on gram-negative bacteria Staphylococcus aureus. Second, characterize the association and function of a non-integrin surface protein, CD98, with β1 integrins on T lymphocytes. ^ FnbpA binds to fibronectin (FN), also a ligand for α5β1 and α4β1 integrins on T lymphocytes. Since both bacterial proteins FnbpA and T cell integrins utilize FN, it was of interest to determine the effects FnbpA on T cell activation. Results demonstrated that recombinant FnbpA (rFnbpA) coimmobilized with OKT3 mediated T cell coactivation in a soluble FN-dependent manner. Integrin α5β1 was identified as the main integrin utilized by Staphylococcus aureus FnbpA from studies using soluble antibodies to inhibit T cell proliferation and parallel plate flow chamber assays. The mechanism of rFnbpA-mediated coactivation was one that used soluble FN as a bridge between rFnbpA and integrin α5β1 on the T lymphocyte. ^ Since integrins are utilized by T lymphocytes and bacterial proteins, it was of interest to identify proteins involved in integrin regulation. Anti-CD98 mAb 80A10 was identified and characterized from a screen to identify surface proteins involved in integrin signaling and functions. CD98 is a non-integrin protein that was sensitive to integrin inhibition in human T lymphocyte aggregation and activation, thus suggested that CD98 shared a common signaling pathway with integrins. These results led to the question of whether CD98 physically associates with β1 integrins. Fluorescence microscopy and biochemical analysis determined that CD98 is specifically associated with β1 integrin on human T lymphocytes and may be part of a larger multimolecular signaling complex. ^

Diversity in T cell costimulation by alpha4beta1 integrin

T cell activation requires antigen-specific T cell receptor signals that spatially and temporally coincide with a second costimulatory signal. CD28 and α4β1 integrin both function as T cell costimulators, but their individual mechanisms remain elusive. By directly comparing CD3-dependent functions and signaling pathways employed by these two costimulatory receptors, aspects of their individual signaling mechanisms are explored. We determined that CD28 and α4β1 integrins both use Src-family kinase Lck and MAPK Erk, but to different extents and functional ends. After identifying functional differences between CD28 and integrin costimulatory pathways, the focus of the study turned to integrin signaling in naïve and memory T cell subsets. CD45RO T cells are fully co-activated by natural β1 integrin ligands fibronectin (FN) and VCAM-1, β1 monoclonal antibody 33B6, as well as α4β1 monoclonal antibody 19H8 which binds a combinatorial epitope of the α4β1 heterodimer. While CD28 fully costimulates CD45RA T cells, the degree of activation from integrin ligands varies. FN costimulates CD3-dependent proliferation, IL-2 secretion, and early activation markers CD25 and CD69. However, β1 antibody 33B6, which binds to the same T cell integrins (α4β1 and α5β1) as natural ligand FN, failed to costimulate proliferation or IL-2 in the CD45RA subset, but retained the ability to regulate CD25 and CD69. Unique aspects of 19H8 signaling involve early Erk activation and IL-2 independent proliferation. Signaling defects through 33B6 ligation correlates with poor adhesion under fluid flow conditions, suggesting a cytoskeletal basis for signaling. All together, these data provide evidence for a mechanism of α4β1 integrin signaling and describe functional differences between naïve and memory T cells. ^

Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^

Activation changes the spectrum but not the diversity of genes expressed by T cells

During activation T cells are thought to change their patterns of gene expression dramatically. To find out whether this is true for T cells activated in animals, the patterns of genes expressed in resting T cells and T cells 8 and 48 hr after activation were examined by using Affymetrix gene arrays. Gene arrays gave accurate comparisons of gene expression in the different cell types because the expression of genes known to vary during activation changed as expected. Of the approximately 6,300 genes assessed by the arrays, about one-third were expressed to appreciable extents in any of the T cells tested. Thus, resting T cells express a surprisingly large diversity of genes. The patterns of gene expression changed considerably within 8 hr of T cell activation but returned to a disposition more like that of resting T cells within 48 hr of exposure to antigen. Not unexpectedly, the activated T cells expressed genes associated with cell division at higher levels than resting T cells. The resting T cells expressed a number of cytokine receptor genes and some genes thought to suppress cell division, suggesting that the state of resting T cells is not a passive failure to respond to extant external stimuli.

Functional domains are specified to single-cell resolution in a Drosophila epithelium

Specification of pattern is fundamental to the development of a multicellular organism. The Malpighian (renal) tubule of Drosophila melanogaster is a simple epithelium that proliferates under the direction of a single tip cell into three morphologically distinct domains. However, systematic analysis of a panel of over 700 P{GAL4} enhancer trap lines reveals unexpected richness for such an apparently simple tissue. Using numerical analysis, it was possible formally to reconcile apparently similar or complementary expression domains and thus to define at least five genetically defined domains and multiple cell types. Remarkably, the positions of domain boundaries and the numbers of both principal and secondary (“stellate”) cell types within each domain are reproducible to near single-cell precision between individual animals. Domains of physiological function were also mapped using transport or expression assays. Invariably, they respect the boundaries defined by enhancer activity. These genetic domains can also be visualized in vivo, both in transgenic and wild-type flies, providing an “identified cell” system for epithelial physiology. Building upon recent advances in Drosophila Malpighian tubule physiology, the present study confirms this tissue as a singular model for integrative physiology.

Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.

Mitochondrial control of calcium-channel gating: A mechanism for sustained signaling and transcriptional activation in T lymphocytes

In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca2+ signals. In many cells, mitochondria act as Ca2+ buffers by taking up and releasing Ca2+, but this simple buffering action by itself often cannot explain the organelle's effects on Ca2+ signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca2+ channels in T lymphocytes as a mechanism of mitochondrial Ca2+ signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca2+ stores causes sustained activation of the store-operated Ca2+ current, ICRAC (CRAC, Ca2+ release-activated Ca2+). Inhibition of mitochondrial Ca2+ uptake by compounds that dissipate the intramitochondrial potential unmasks Ca2+-dependent inactivation of ICRAC. Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca2+ accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca2+ uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca2+]i elevation, indicating a functional link between mitochondrial regulation of ICRAC and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca2+ channel activity and signal transmission from the plasma membrane to the nucleus.

Fully competent dendritic cells as inducers of T cell anergy in autoimmunity

Mature immunologically competent dendritic cells are the most efficient antigen-presenting cells that powerfully activate T cells and initiate and sustain immune responses. Indeed, dendritic cells are able to efficiently capture antigens, express high levels of costimulatory molecules, and produce the combination of cytokines required to create a powerful immune response. They are also considered to be important in initiating autoimmune disease by efficiently presenting autoantigens to self-reactive T cells that, in this case, will mount a pathogenic autoimmune reaction. Triggering T cells is not a simple on–off procedure, as T cell receptor responds to minor changes in ligand with gradations of T cell activation and effector functions. These “misfit” peptides have been called Altered Peptide Ligands, and have been shown to have important biological significance. Here, we show that fully capable dendritic cells may present, upon natural antigen processing, a self-epitope with Altered Peptide Ligands features that can unexpectedly induce anergy in a human autoreactive T cell clone. These results indicate that presentation of a self-epitope by immunologically competent dendritic cells does not always mean “danger” and show a mechanism involved in the fine balance between activation and tolerance induction in humans.

Multiple modes of cellular activation and virus transmission in HIV infection: A role for chronically and latently infected cells in sustaining viral replication

CD4+ T cell activation, required for virus replication in these cells, occurs in local microenvironmental domains in transient bursts. Thus, although most HIV originates from short-lived virus-producing cells, it is unlikely that chronic infection is generally sustained in rapid continuous cycles of productive infection as has been proposed. Such continuity of productive infection cycles would depend on efficient long-range transmission of HIV from one set of domains to another, in turn requiring the maintenance of sufficiently high concentrations of cell-free virus across lymphoid tissues at all times. By contrast, long-lived cellular sources of HIV maintain the capacity to infect newly activated cells at close range despite the temporal and spatial discontinuities of activation events. Such proximal activation and transmission (PAT) involving chronically and latently infected cells may be responsible for sustained infection, particularly when viral loads are low. Once CD4 cells are productively infected through PAT, they can infect other activated cells in their immediate vicinity. Such events propagate locally but generally do not spread systemically, unlike in the acute phase of the infection, because of the early establishment of protective anergy. Importantly, antiretroviral drug treatment is likely to differentially impact long-range transmission and PAT.

The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte–antigen-presenting cell interface

During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav−/− mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav+/− mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav−/− mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.

Decreased ability of HIV-1 Tat protein-treated accessory cells to organize cellular clusters is associated with partial activation of T cells

It has been shown in several animal models that HIV infection of accessory cells (ACs) plays an important role in development of AIDS. Here, we report that ACs treated with HIV-1 Tat protein (Tat-ACs) have a decreased ability to organize cellular aggregates as compared with untreated ACs, resulting in incomplete activation of T cells in responses to anti-CD3 mAb or staphylococcal enterotoxin B stimulation. The T cells failed to up-regulate adhesion molecules CD11a and CD2 on the cell surface and had reduced proliferative responses, as determined by [3H]thymidine incorporation, but they obtained lymphoblast-like morphology and expressed early activation antigens on the cell surface such as Fas and CD69 and interleukin 2 receptor, at comparable levels as those T cells undergoing a maximal proliferation. These results suggest that the Tat-AC-induced defect occurs in the late, but not in the early, phases of T cell activation. Normal expression of cell surface Fas antigen accompanied by defects in late activation thus may result in the susceptibility of these T cells to apoptosis. Our studies suggest that dysfunction, hyperactivation, and susceptibility to apoptosis, as observed with T cells isolated from HIV-infected individuals, may be, at least in part, a consequence of abnormal functions of ACs.

αβ T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization

Cellular immunity is mediated by the interaction of an αβ T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have αβ TCRs that can recognize both self- and foreign peptide–MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive αβ T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired αβ heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10−4 to 10−6M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the α and β chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.

Mechanism of activation for Zap-70 catalytic activity.

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a zeta-associated protein of M(r) 70,000 (Zap-70) as a critical protein tyrosine kinase in T-cell activation and development. During T-cell activation, Zap-70 becomes associated via its src homology type 2 (SH2) domains with tyrosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) sequences in the cytoplasmic zeta chain of the T-cell receptor. An intriguing conundrum is how Zap-70 is catalytically activated for downstream phosphorylation events. To address this question, we have used purified Zap-70, tyrosine phosphorylated glutathione S-transferase (GST)-Zeta, and GST-Zeta-1 cytoplasmic domains, and various forms of ITAM-containing peptides to see what effect binding of zeta had upon Zap-70 tyrosine kinase activity. The catalytic activity of Zap-70 with respect to autophosphorylation increased approximately 5-fold in the presence of 125 nM phosphorylated GST-Zeta or GST-Zeta-1 cytoplasmic domain. A 20-fold activity increase was observed for phosphorylation of an exogenous substrate. Both activity increases showed a GST-Zeta concentration dependence. The increase in activity was not produced with nonphosphorylated GST-Zeta, phosphorylated zeta, or phosphorylated ITAM-containing peptides. The increase in Zap-70 activity was SH2 mediated and was inhibited by phenylphosphate, Zap-70 SH2, and an antibody specific for Zap-70 SH2 domains. Since GST-Zeta and GST-Zeta-1 exist as dimers, the data suggest Zap-70 is activated upon binding a dimeric form of phosphorylated zeta and not by peptide fragments containing a single phosphorylated ITAM. Taken together, these data indicate that the catalytic activity of Zap-70 is most likely activated by a trans-phosphorylation mechanism.

An essential role for Bruton's [corrected] tyrosine kinase in the regulation of B-cell apoptosis.

Mutations of the Bruton's tyrosine kinase (btk) gene cause X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (Xid) in mice. To establish the BTK role in B-cell activation we examined the responses of wild-type and Xid B cells to stimulation through surface IgM and CD40, the transducers of thymus independent-type 2 and thymus-dependent activation, respectively. Wild-type BTK was necessary for proliferation induced by soluble anti-IgM (a prototype for thymus independent-type 2 antigen), but not for responses to soluble CD40 ligand (CD40L, the B-cell activating ligand expressed on T-helper cells). In the absence of wild-type BTK, B cells underwent apoptotic death after stimulation with anti-IgM. In the presence of wild-type but not mutated BTK, anti-IgM stimulation reduced apoptotic cell death. In contrast, CD40L increased viability of both wild-type and Xid B cells. Importantly, viability after stimulation correlated with the induced expression of bcl-XL. In fresh ex vivo small resting B cells from wild-type mice there was only barely detectable bcl-XL protein, but there was more in the larger, low-density ("activated") splenic B cells and peritoneal B cells. In vitro Bcl-XL induction following ligation of sIgM-required BTK, was cyclosporin A (CsA)-sensitive and dependent on extracellular Ca2+. CD40-mediated induction of bcl-x required neither wild-type BTK nor extracellular Ca2+ and was insensitive to CsA. These results indicate that BTK lies upstream of bcl-XL in the sIgM but not the CD40 activation pathway. bcl-XL is the first induced protein to be placed downstream of BTK.