Viral interleukin 10 is critical for the induction of B cell growth transformation by Epstein-Barr virus.

We have used an efficient cDNA subtraction library procedure to identify newly induced genes in human B lymphocytes infected for 6 h with Epstein-Barr virus (EBV). Among the genes identified by automated sequencing of a random subset of clones from this library, one coded the EBV BCRF1 open reading frame, which specifies the viral interleukin 10 gene (vIL-10). This molecule is highly homologous to human (h)IL-10 and was previously thought to represent a "late" viral gene expressed only during the lytic phase of virus replication. Using gene amplification by reverse transcriptase polymerase chain reaction of B cell RNA obtained at varying times after infection, we detected vIL-10 expression within a few hours of EBV infection, followed, 20-30 h later by expression of hIL-10. Expression of both genes continued beyond the initial transformation phase (5-10 d) and was present in all transformed cell lines tested. When added at the time of viral infection, antisense (but not sense) oligonucleotides for vIL-10 mRNA (cytosolic half-life, approximately 6 h) prevented subsequent B cell transformation. The antisense effect was highly specific, leaving the expression levels of other transformation-related genes intact. Addition of exogenous (h)IL-10 rescued the transformation process in antisense-treated cells. Our observations establish vIL-10 as a new latency gene with a directly transformation-prerequisite function.

Independent regulation of Ca2+ entry and release from internal stores in activated B cells.

Addition of crosslinking antibody to B lymphocytes results in a rapid rise in cytoplasmic-free Ca2+ ([Ca2+]i) due to release of Ca2+ from internal stores and uptake of Ca2+ across the plasma membrane. Inositol 1,4,5-trisphosphate is believed to mediate the release of internal Ca2+ stores and has also been proposed to mediate extracellular Ca2+ entry. We have compared the properties of these two pathways for Ca2+ mobilization by dissociating the [Ca2+]i changes in ligand-activated human B cells after loading of the cells with the Ca2+ chelator BAPTA. In the present paper we show that: (a) the sustained increase in [Ca2+]i is due to increased unidirectional influx of external [Ca2+]i; (b) entry of extracellular Ca2+, but not release of internal stores, is sensitive to the transmembrane potential; and (c) entry of extracellular Ca2+, but not release of internal stores, is inhibited by increasing [Ca2+]i. These findings suggest that the permeation pathways mediating the translocation of Ca2+ across the plasma membrane and endoplasmic reticulum membrane are not identical.

Volume regulation by human lymphocytes. Identification of differences between the two major lymphocyte subpopulations.

Following exposure to hypotonic media, human peripheral blood lymphocytes swell initially but restore their isotonic volume within minutes. In contrast, tonsillar lymphocytes demonstrate a similar initial phase of swelling but fail to restore their isotonic volume. We have studied the ionic basis for this second or regulatory volume decrease (RVD) phase using lymphocytes from peripheral blood, tonsil, and thymus. RVD was characterized by 86Rb efflux and a decrease in K+ content. The increase in K+ permeability in response to hypotonic challenge was characteristic for T lymphocytes obtained from peripheral blood, tonsil, or thymus. B lymphocytes showed only a modest increase in K+ permeability and consequently little RVD. The data confirm that the response of peripheral blood and tonsillar lymphocytes to a hypotonic environment can be accounted for by differences in the proportions of T and B cells, and the differential behaviour of B and T lymphocytes is based on differences in membrane permeability to K+ upon swelling.

Nasopharyngeal brush biopsies and detection of nasopharyngeal cancer in a high-risk population.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is an important tumor in many countries. Ethnic and regional factors strongly influence disease risk. NPC is usually diagnosed late in disease development, and 10-year survival rates are as low as 10%. Epstein-Barr virus (EBV), a possibly causative agent, is present in all cells of essentially all undifferentiated NPCs. We wished to determine the following: 1) whether an ambulatory nasopharyngeal brush biopsy could provide sufficient tumor cell DNA for the detection of EBV and 2) whether the detection of EBV in this locale reflects the presence of tumor cells or simply EBV carrier status. METHODS: We collected nasopharyngeal tissue via ambulatory brush biopsies from 21 patients with newly diagnosed NPC and from 157 subjects with other otolaryngologic complaints. The majority of study subjects were from high-risk populations. Sample DNA was analyzed for the presence of EBV genomic sequences by use of the polymerase chain reaction (PCR). RESULTS: Ninety-six percent of samples yielded sufficient DNA for PCR amplification. Nineteen of 21 patients with NPC brushed positive for EBV DNA, while all but two (1.3%) of 149 informative control subjects were negative for EBV (two-sided P<.0001). One of the EBV-positive control subjects had an EBV-positive inverted sinonasal papilloma; the other EBV-positive control subject exhibited no overt clinical disease. CONCLUSION: Demonstration of EBV DNA in nasopharyngeal brush biopsy specimens detects NPC with a sensitivity of at least 90% (95% confidence interval = 89.63%-91.32%) and a specificity of approximately 99% (95% confidence interval = 98.64%-98.68%). This technique merits further testing as a possible ambulatory screening strategy in high-risk populations.

Cloning of human and rat p69 cDNA, a candidate autoimmune target in type 1 diabetes.

Triggering of autoimmunity in insulin-dependent diabetes was linked to dietary bovine serum albumin (BSA). Anti-BSA antibodies from diabetes-prone rats precipitate a protein, p69, from islet cell lysates. We have used these antibodies to identify rat p69 cDNAs. Human p69 cDNA was identified by crosshybridization. The p69 coding regions show 87% nucleotide and 89% amino acid homology. Recombinant p69 is recognized by autoantibody and T cells from diabetic children.

Immunological aspects of nutritional diabetes prevention in NOD mice: a pilot study for the cow's milk-based IDDM prevention trial.

Human epidemiological studies delineated early exposure to intact dietary protein (e.g., most infant formulas) as an environmental risk factor for the development of IDDM. The Trial to Reduce IDDM in the Genetically at Risk (TRIGR), an international IDDM prevention trial, has been designed to determine if avoidance of intact dairy protein in high-risk infants < or =6 months of age can reduce the subsequent diabetes incidence. We here studied the casein hydrolysate-based trial diet (Nutramigen) in NOD mice. When given either continuously or for 10 weeks after weaning, the test diet was highly effective in preventing autoimmune diabetes (32-week incidence: 4.6 vs. 58.8%) and in preserving pancreatic insulin levels, with little effect on islet inflammation. Spleen cells from protected NOD mice failed to adoptively transfer diabetes into irradiated syngeneic recipients. When co-transferred with splenocytes from diabetic donors, cells from diet-protected mice inhibited adoptive diabetes transfer (incidence 50 vs. 94%, P < 0.001). T-cell reactivity to the islet cell autoantigens ICA69 (islet cell antigen 69) and GAD65 developed only in diabetic recipients of spleen cell grafts, indicating that diabetes protection extends to more than one autoantigen. In protected mice, ICA69 T-cell reactivity was not detectable spontaneously nor after priming with this autoantigen; however, priming with the cross-reactive non-self-antigen bovine serum albumin recruited T-cells responsive to ICA69. Thus, diabetes prevention with the clinical trial diet is effective in NOD mice, where it affects some T-cell repertoires and allows development of regulatory cells that interfere with destructive autoimmunity.

Epstein-Barr virus surveillance after renal transplantation.

At least 1% of organ transplant recipients develop Epstein-Barr virus-positive, often fatal lymphomas. EBV-positive cells accumulating in some organ transplant recipients were suggested to predict EBV+ lymphoma risk but no prospective study has been reported. We used the polymerase chain reaction (PCR) to detect EBV genomic sequences in successive blood samples of 60 kidney recipients before and up to 11 years after renal transplantation. Xenotransplantation of EBV-positive patient and -negative control samples into mice with severe combined immunodeficiency (SCID) was used to assess the tumor risk inherent in these samples. Despite single EBV+ cell detection sensitivity, none of the control samples was positive for EBV genomic sequences. In nearly 2/3 of patients EBV genomic DNA was detectable 3-6 months after transplantation for about 3 months. No patient developed lymphoma. Lymphocytes from 8 EBV-genome positive patients and 10 healthy donors were engrafted into 38 SCID mice. Human B cell lymphoma developed in 75% of the control grafts within about 3 months. In striking contrast, none of the patient grafts developed lymphoma despite the large numbers of EBV+ cells initially transplanted. Patient lymphocyte grafts were resistant to injection of live EBV, while in control lymphocyte grafts this caused lymphoma development within 3 weeks. We conclude that a 100-1000-fold expansion of circulating EBV+ B cell pools occurs frequently after organ transplantation and that it is balanced by effective EBV immunosurveillant functions resistant to immunosuppression. The mere detection of EBV genomic material was not predictive of lymphoma development.

Detection of viral sequences by internally calibrated gene amplification.

Inherent pitfalls of the polymerase chain reaction (PCR) can become serious difficulties when transferring research applications to high-volume routine procedures such as biofermentation process control and clinical diagnostics. Difficulties include 1) the danger of accidental sample contamination with positive control templates; 2) variable amplification due to positional effects in thermocycler blocks and unequal primer efficiency for sense/anti-sense strands; and 3) the need for reliable controls, which provide confidence for reporting negative reactions. Using the PCR detection system for Epstein-Barr virus as a model, we have developed a quick process to generate mutant internal co-amplification templates. These can be used for titration of amplification sensitivity. More importantly, single tube co-amplification without titrations allows determination of the minimum sensitivity achieved in each individual reaction; critical information when reporting negative diagnostic results. Mutant and native fragments are easy to distinguish by size, and sample cross contamination can be readily identified. The system should be easily adaptable to gene amplification procedures, which aim to routinely detect the presence of a given gene fragment in a controlled fashion.

Volume regulation of natural killer cells under hypotonic stress: comparison with T and B cell subpopulations.

In response to hypotonic stress, human T and B cells vary in the ability to adjust their volume. Whereas T cells exhibit a regulatory volume decrease (RVD) under these conditions, B cells do not. We studied the ability of peripheral blood-derived natural killer (NK) cells to regulate their volume in this way. Percoll density gradient purified NK subpopulation showed variable RVD which suggested the presence of mixtures of regulatory and non-regulatory cells within these subgroups. Further purification by flow cytometry into Leu 7+ and Leu 11+ cells showed that these NK cells displayed RVD similar to thymocytes but distinct from B cells and more mature T cells. These data support the hypothesis that NK cells may be derived from T cell precursors which, upon differentiation to NK cells, retain the RVD characteristic of pre-T cells. This finding may also be useful in further characterizing neoplastic clones which display NK-like activity but phenotypic heterogeneity.

The physiologic reservoir of Epstein-Barr virus does not map to upper aerodigestive tissues.

The human Epstein-Barr herpesvirus (EBV) has distinct oncogenic potential, but with over 90% of the adult world population infected, malignancy is a rare outcome of carrier status. However, EBV's association with over half of Hodgkin's and non-Hodgkin's lymphomas as well as several solid tumors, notably nasopharyngeal carcinoma, makes EBV-linked malignancies one of the largest single cancer entities. EBV is a B-lymphotropic virus, well controlled by surveillant T cells in immunocompetent hosts. To determine the presence and site of principal virus reservoirs is a likely prerequisite for understanding the etiology of EBV-associated tumors. Its near 100% association with nasopharyngeal carcinoma led to postulates that the upper aerodigestive tract tissue may be common sites of persistent latent or low-grade replicating infection. Using a protocol designed to avoid viral crosscontamination, the authors employed polymerase chain reaction to detect genomic EBV DNA sequences in 231 biopsies from different mucosal sites in the upper aerodigestive tract, as well as from salivary gland tissue and neck nodes in individuals not suspected to have EBV-related malignancy. Only two samples, one from oral cavity mucosa and one from parotid gland tissue, were positive for EBV. The observation that oropharyngeal tissue is not the principal EBV reservoir has mechanistic implications for the development of EBV-positive tumors in that locale.

The growth transformation of human B cells involves superinduction of hsp70 and hsp90.

Epstein-Barr virus (EBV) is a latent human herpes virus associated with a range of malignant and non-malignant disorders. EBV binds to CD21 virus receptors on B lymphocytes and growth transforms these cells; in susceptible (e.g., immunodeficient) hosts such cells rapidly expand into fatal lymphomas. Virus binding and infection trigger a cascade of cellular events which are transformation prerequisite and analogous to non-oncogenic cell activation events but which differ in several quantitative or qualitative respects. Unique trans-membrane Ca2+ currents, Na+/H+ exchange, as well as tyrosine phosphorylation and p56lck-gene induction suggest that even early on the transformation process has oncogenic specificity. In this report we describe that two additional cellular gene families, the stress proteins hsp70 and hsp90, are coordinately induced at mRNA and protein levels and, quite different from hsp induction by thermal stress, this induction is dependent on EBV-induced trans-membrane Ca2+ currents. Blockade of hsp induction prevents transformation. The kinetics and induction prerequisites set this response well apart from reported responses to thermal or viral stress protein induction. Like p56lck-, hsp induction is purely a post-receptor binding event and not dependent on expression of any viral gene. The induction kinetics, with a peak at approximately 12-16 hr and subsequent decline to control levels, considerably extend the chronological map of elements in the CD21-dependent branch of the transformation pathway and suggest a specific role of induced hsp different from the cell cycle-related functions observed in other cell systems.

Dissociation of unidirectional influx of external Ca2+ and release from internal stores in activated human T lymphocytes.

Addition of lectin or antibody to the T cell receptor complex of human T cells results in a rapid increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This response is biphasic and results from contributions of Ca2+ from internal stores, uptake of Ca2+ across the plasma membrane and possibly a decrease in Ca2+ efflux. These responses have been linked through the activity of inositol 1,4,5-trisphosphate in releasing Ca2+ from internal stores and potentially mediating Ca2+ uptake across the plasma membrane. Following addition of phytohemagglutinin or anti-CD3 antibody to resting T cells or Jurkat cells, we have been able to dissociate the [Ca2+]i responses by loading cells with the Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA). In BAPTA-loaded T cells, we have shown that Ca2+ mobilized from intracellular stores following activation is effectively buffered, while stimulated Ca2+ uptake and associated changes in [Ca2+]i were relatively unaffected. In this report, we show that the sustained increase in [Ca2+]i is due to increased unidirectional influx of external Ca2+ without changes in efflux and that it is the entry of extracellular Ca2+ which is sensitive to the transmembrane potential.

The tyrosine kinase lck is critically involved in the growth transformation of human B lymphocytes.

Epstein-Barr virus (EBV) exposure of human B lymphocytes induces rapid, Ca(2+)-dependent tyrosine phosphorylation of two cytosolic proteins, one likely the CD21 EBV receptor and another unknown species of 55-60 kDa. We now identify the latter protein as the tyrosine kinase lck (p56lck). In T cells many activation events reduce the high constitutive p56lck expression levels typical for that lineage, and they induce the appearance of a 60-kDa lck species. We now demonstrate that in B cells exposed to EBV the at best low constitutive p56lck expression levels are rapidly and transiently up-regulated without generation of 60-kDa lck. lck-specific antisense oligonucleotides block p56lck induction and prevent subsequent B cell activation and immortalization whereas B cell activation by nononcogenic agents was unaffected. We propose that p56lck superinduction is a transformation prerequisite which signals entry into the oncogenic growth transformation process.

The cyclosporins inhibit lymphocyte activation at more than one site.

Cyclosporin A (CsA), a potent immunosuppressive agent, acts primarily by inhibiting T cell function. Although several potential sites of action have been identified, the mechanisms whereby CsA mediates its immunosuppressive properties have not been fully delineated. We have examined the effects of the immunosuppressive cyclosporins, CsA, dihydrocyclosporin D, and cyclosporin G, and a nonimmunosuppressive analog, cyclosporin H, on early events associated with activation of human T cells. Interleukin 2 (IL 2) receptor expression, as measured by immunofluorescence, was unaffected by CsA. Despite this, in the continuous presence of CsA, exogenous IL 2 did not bypass CsA inhibition of phytohemagglutinin (PHA)-induced proliferation. Thus, one site of activity of CsA is on IL 2-induced proliferation of IL 2 receptor-expressing cells. In addition, several potential mechanisms for inhibiting IL 2 secretion were identified. Changes in cytosolic free Ca2+ ([Ca2+]i), an obligatory event for PHA-induced IL 2 secretion, were inhibited by a 30-min preincubation with the immunosuppressive cyclosporins but not the inactive analog. In this action, the drug effects cannot be distinguished from that of Ca2+ channel blockers. The active compounds also resulted in membrane depolarization, an effect which may, in part, explain the reduction in PHA-induced changes in [Ca2+]i. These results identify multiple sites of action of the immunosuppressive cyclosporins, the combination of which likely accounts for their selective inhibition of T cell function in vitro and in vivo.

Calcium-dependent intracellular acidification dominates the pH response to mitogen in human T cells.

The effects of a phorol ester and a mitogenic lectin on the intracellular pH (pHi) of human T lymphocytes was investigated. In contrast to the cytoplasmic alkalinization induced by 12-0-tetradecanoylphorbol-13-acetate, an acidification was recorded in cells treated with phytohemagglutinin. This decrease in pHi was magnified in Na+-free medium or in the presence of amiloride analogues, suggesting that activation of Na+/H+ exchange partially counteracts the phytohemagglutinin-induced acidification. The decrease in pHi was dependent on a sustained increase in cytosolic free Ca2+ and could be mimicked by addition of the divalent cation ionophore, ionomycin. The elevation of cytosolic free Ca2+ leads to metabolic H+ (equivalent) generation with consequent cytoplasmic acidification, which in human T cells predominates over the concurrent activation of the Na+/H+ antiport. These findings argue against the notion that activation of Na+/H+ exchange is a signal for the initiation of proliferation.

Mitogenic and non-mitogenic ligands trigger a calcium-dependent cytosolic acidification in human T lymphocytes.

We have investigated the patterns of cytosolic pH and Ca2+ ([Ca2+]i) changes after exposure of human peripheral blood T cells to different mitogenic and non-mitogenic ligands. Using ligands that have different accessory cell requirements and varying effect on [Ca2+]i or cell proliferation, we observed that intracellular acidification occurred only with agents that increased [Ca2+]i. However, treatment of the cells with the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, results in significant cytosolic alkalinization without detectable acidification, but did not affect the proliferative responses to mitogenic ligands and was a potent co-mitogen with non-mitogenic ligands. These data indicate that initial acidification or alkalinization responses are not essential for early activation or triggering of DNA synthesis.

Role of membrane potential in the regulation of lectin-induced calcium uptake.

Incubation of lymphocytes with mitogenic lectins triggers Ca2+ uptake. This increase in free cytoplasmic Ca2+ is postulated to be an important signal in the initiation of DNA synthesis. Transmembrane fluxes of monovalent ions and changes in membrane potential are also associated with lectin-induced activation of lymphocytes. We have examined the relationship between extra-cellular monovalent ion substitution, the associated electrical potential changes (measured with cyanine dyes), phytohemagglutinin-induced Ca2+ uptake (measured with Quin-2) and proliferation in human T cells. The results show that (1) the magnitude of the increase in free cytoplasmic Ca2+ concentration is correlated with the extent of the lymphoproliferative response, (2) lectin-induced Ca2+ fluxes are sensitive to membrane potential, decreasing with depolarization, and are likely conductive, and (3) the presence of extra-cellular Na+ during incubation with phytohemagglutinin is not essential to mitogenic triggering.

Permissive role of calcium in the inhibition of T cell mitogenesis by calmodulin antagonists.

The importance of Ca++ in the initiation of lymphocyte activation and mitogenesis has been supported by several studies. Because calmodulin functions as the intracellular mediator of the effects of Ca++, it likely plays a major role in the regulation of lymphocyte function. We have examined the effects of known calmodulin antagonists, the phenothiazines, on lectin-induced T cell mitogenesis and have shown a central role for Ca++ uptake in the expression of a phenothiazine-sensitive stage after lectin activation. The drug effects were observed only if the cells were previously activated by PHA or the ionophore A23187, and only in the presence of Ca++. These effects were restricted to a defined time period (5 hr) after lectin activation. The data support the concept that calmodulin is the target for the phenothiazine effects and demonstrate the permissive role of Ca++ in the mediation of these events.

Mitogen-induced changes in Ca2+ permeability are not mediated by voltage-gated K+ channels.

Binding of mitogenic lectins to T lymphocytes results in elevated cytoplasmic Ca2+ concentrations ([Ca2+]i). This change in [Ca2+]i is thought to be essential for cellular proliferation. In addition, the lectins increase the conductance to K+ through voltage-sensitive channels. Based on the inhibitory effect of K+ channel blockers on lectin-induced mitogenesis, it has been suggested that Ca2+ could enter the cells through these activated K+ channels (Chandy, K. G., De Coursey, T. E., Cahalan, M. D., McLaughlin, C., and Gupta, S. (1984) J. Exp. Med. 160, 369-385; Chandy, K. G., De Coursey, T. E., Cahalan, M. D., and Gupta, S. (1985) J. Clin. Immunol. 5, 1-5). This hypothesis was tested experimentally by measuring the effect of activation or blockade of K+ channels on [Ca2+]i using quin-2 and indo-1 and by determining the effect of K+ channel blockers on lectin-induced proliferation. We found that: depolarization of the membrane, which is expected to open the K+ channels, failed to increase [Ca2+]i, K+ channel blockers such as tetraethylammonium and 4-aminopyridine had only a marginal effect on the lectin-induced increase in [Ca2+]i, and the inhibitory effect of K+ channel blockers on proliferation was found to be nonspecific, occurring also when proliferation was triggered by phorbol esters under conditions where [Ca2+]i is not elevated. It is concluded that the lectin-induced changes in [Ca2+]i are not mediated by the opening of voltage-gated K+ channels.

Unexpected patterns of Epstein-Barr virus gene expression during early stages of B cell transformation.

Following Epstein-Barr virus (EBV) binding to its CD21 cell surface receptor, virus internalization, nuclear translocation, and circularization of the viral episome were found to occur within 30 min, immediately preceding the expression of EB nuclear antigen (EBNA)-1 and -2 and latent membrane protein (LMP)-1 and -2 genes. Early viral gene expression was unaffected by blockade of the virus induced, transformation-prerequisite cellular activation pathway (Ca2+ currents, tyrosine phosphorylation, induction of p56lck, hsp70, and hsp90). Despite life times of only 3 h, antisense (but not sense) oligonucleotides for the above latency genes prevented subsequent transformation. Any one antisense oligonucleotide dramatically depleted transcripts not only of the target gene, but of all other latency genes. The blocking effect of antisense oligonucleotides allowed us to identify a new transformation-prerequisite latency gene near the fused termini. The concerted regulation of EBV gene expression is highly unusual and unexplained but our results imply critical, perhaps regulatory roles for initial latency gene transcripts themselves.