Direct measurement of T-cell receptor repertoire diversity with AmpliCot

Many studies require the measurement of nucleic acid sequence diversity. Here we describe a method, called AmpliCot, that measures the sequence diversity of PCR products on the basis of DNA hybridization kinetics, thereby avoiding the time, expense and biases associated with cloning and sequencing. SYBR Green dye is used to measure DNA hybridization kinetics in a homogeneous, automated fashion. PCR products are prepared in wholly double-stranded homoduplex form for a baseline measurement of DNA concentration. The DNA is melted and then reannealed under stringent conditions that allow only homoduplexes to form. The sequence diversity of a sample is proportional to the product of its concentration and the time required for it to anneal. After validating AmpliCot with a library of diverse sequences, we use it to measure the diversity of expressed rearrangements of the gene encoding the T-cell antigen receptor (TCR) beta chain. AmpliCot measurements are in good agreement with previous estimates of murine TCR repertoire diversity that required extensive cloning and sequencing.     

Direct Determination of Serum T-4 by Isotopic Exchange: Comparison with T-3 * and PBI in 1700 Cases

We have studied in detail the procedure for the direct determination of serum thyroxine based on the liberation of ¹²⁵I T-4 from a tagged euthyroid serum reagent, the thyroxine being liberated by alcohol denaturation from 0.5 ml. of patient serum.We have established that there is proceeding simultaneously a second mechanism of T-4 liberation which is in no way associated with patient thyroxine. A simple technique for determining the extent of, and correcting for this second reaction is described.The procedure employs the same equipment used for the ¹²⁵I T-3 and involves only one additional step. No calibration curve is required. Results are not influenced by iodine in any form.The T-4 content is best expressed as a ratio to that of a standard mid-euthyroid serum. The ratio values for the hypothyroid, hyperthyroid and euthyroid states are characteristic and free from overlap. Separation between low normals and hypothyroids is very sharp. The relation between the serum T-4 ratio and the assayed thyroxine content has a correlation coefficient of 0.91. The product of the ¹²⁵I T-3 ratio and the T-4 ratio makes the procedure applicable in pregnancy and during steroid use, and provides a good indication of the free thyroxine present.The values of T-4, the ¹²⁵I T-3 and the PBI have been applied to 1084 patient sera and the T-4 and the ¹²⁵I T-3 to an additional 616. In this series of 1700 patients the serum T-4 was found to indicate the most probable clinical classification with a reliability of at least 95%.     

DNA reassociation kinetics and diversity indices: richness is not rich enough

DNA reassociation kinetics, also known as Cot curves, were recently used by Gans and co-workers to estimate the number of bacterial species present in soil samples. By reanalysing the mathematical model we show that rather than the number of species, Simpson and Shannon diversity indices are encoded in the experimental data. Our main tool to establish this result are the so-called Rényi diversities, closely related to Hill numbers, illustrating the power of these concepts in interpreting ecological data. We argue that the huge diversity encountered in microbial ecology can be quantified more informatively by diversity indices than by number of species.     

Distinct tyrosine phosphorylation sites in ZAP-70 mediate activation and negative regulation of antigen receptor function.

Biochemical and genetic evidence has implicated two families of protein tyrosine kinases (PTKs), the Src- and Syk-PTKs, in T- and B-cell antigen receptor signaling. ZAP-70 is a member of the Syk-PTKs that associates with the T-cell antigen receptor and undergoes tyrosine phosphorylation following receptor activation. Three tyrosine residues, Tyr-292, -492, and -493, have been identified as sites of phosphorylation following T-cell antigen receptor engagement. Utilizing ZAP-70- and Syk-deficient lymphocytes (Syk-DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutations at Tyr-292 or -492 demonstrate hyperactive T- and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both activation and inactivation of antigen receptor signaling.     

Regulation of protein kinase Cnu by the B-cell antigen receptor

Diacylglycerol-dependent signaling plays an important role in signal transduction through T- and B-lymphocyte antigen receptors. Recently, a novel serine-threonine kinase of the protein kinase C (PKC) family has been described and designated as PKCnu. PKCnu has two putative diacylglycerol binding C1 domains, suggesting that it may participate in a novel diacylglycerol-mediated signaling pathway. Here we show that both endogenous and recombinant PKCnu are trans-located to the plasma membrane and activated by the diacylglycerol mimic phorbol 12-myristate 13-acetate. Mutational analysis demonstrates that PKCnu activation is dependent on trans-phosphorylation of two conserved activation loop serine residues. We also find that PKCnu is an important physiologic target of the B-cell receptor (BCR), because PKCnu is found to be abundantly expressed in chicken and human B-cell lines and, in addition, exhibits robust activation after BCR engagement. Genetic and pharmacologic analyses of BCR-mediated PKCnu activation indicate that it requires intact phospholipase Cgamma and PKC signaling pathways. Furthermore, in co-transfection assays, PKCnu can be trans-phosphorylated by the novel PKC isozymes PKCepsilon, PKCeta, or PKCtheta but not the classical PKC enzyme, PKCalpha. Taken together, these results suggest that PKCnu is an important component of signaling pathways downstream from novel PKC enzymes after B-cell receptor engagement.     

T-cell receptor and immunoglobulin genes are rearranged together in Abelson virus-transformed pre-B and pre-T cells.

We have assessed the state of rearrangement and expression of B- and T-cell antigen receptor genes in cells of Abelson murine leukemia virus-transformed thymomas and other tumors. We found that unrearranged TcR gamma genes are expressed, as are unrearranged C mu genes, in pre-T, pre-B, and myeloid cells. We also found TcR gamma genes rearranged and expressed in putative pre-T cells and in cells apparently committed to the B-cell lineage. This is in contrast to the data from more mature T- and B-cell tumors. We conclude that in immature lymphoid cells both immunoglobulin and TcR gamma genes are accessible for rearrangement. We discuss the implications of these observations for an understanding of the B-T lymphoid differentiation event.     

The HIV-1 cell entry inhibitor T-20 potently chemoattracts neutrophils by specifically activating the N-formylpeptide receptor

T-20, a synthetic peptide corresponding to the heptad repeat sequence of HIV-1 gp41, blocks HIV-1 entry by targeting gp41, and is currently in clinical trials as an anti-retroviral agent. We recently reported that in vitro T-20 also functions as a phagocyte chemoattractant and a chemotactic agonist at the phagocyte N-formylpeptide receptor (FPR). Here we show that T-20 is also a potent chemotactic agonist in vitro at a related human phagocyte receptor FPRL1R. To test the relative importance of FPR and FPRL1R in primary cells, we identified the corresponding mouse T-20 receptors, mFPR and FPR2, which are both expressed in neutrophils, and compared T-20 action on neutrophils from wild type and mFPR knockout mice. Surprisingly, although T-20 activates mFPR and FPR2 in transfected cells with equal potency and efficacy in both calcium flux and chemotaxis assays, neutrophils from mFPR knockout mice did not respond to T-20. These results provide genetic evidence that FPR is the major phagocyte T-20 receptor in vivo and point to the potential feasibility of studying T-20 effects on immunity in a mouse model. This may help define the cause of local inflammation after T-20 injection that has recently been reported in Phase I clinical trials.     

Redefinition of lymphoid progenitors

Similarities between T and B lymphocytes might have led to the idea that these functionally distinct cells develop from a common lymphoid progenitor. However, investigations with a new clonal assay which allows for T-, B- and myeloid-lineage development indicate that commitment to T-cell and B-cell lineages occurs instead through myeloid/T and myeloid/B bipotential stages, respectively. These findings provide an opportunity to reconsider the ontogeny and phylogeny of T- and B-cell development.     

Deregulated activation of oncoprotein kinase Tpl2/Cot in HTLV-I-transformed T cells

Protein kinase Tpl2/Cot is encoded by a protooncogene that is cis-activated by retroviral insertion in murine T cell lymphomas. It has remained unclear whether this oncoprotein kinase is mutated or post-translationally activated in human cancer cells. We have shown here that Tpl2/Cot is constitutively activated in human leukemia cell lines transformed by the human T cell leukemia virus type I (HTLV-I). The kinase activity of Tpl2/Cot is normally suppressed through its physical interaction with an inhibitor, the NF-kappaB1 precursor protein p105. Interestingly, a large pool of Tpl2/Cot is liberated from p105 and exhibits constitutive kinase activity in HTLV-I-transformed T cells. In contrast to its labile property in normal cells, the pathologically activated Tpl2/Cot is remarkably stable. Further, whereas the physiological activation of Tpl2/Cot involves its long isoform, the HTLV-activated Tpl2/Cot is predominantly the short isoform. We have also shown that the HTLV-I-encoded Tax protein is able to activate Tpl2/Cot in transfected cells. Finally, Tpl2/Cot participates in the activation of NF-kappaB by Tax. These findings indicate that deregulated activation of Tpl2/Cot may occur in human cancer cells.     

Regulation of the natural killer activity of lymphocytes from healthy donors and patients with chronic lympholeukemia through the interaction of T- and non-T cells

The role of T- and B-cell cooperative interaction in the regulation of natural killer (NK) activity in human peripheral blood lymphocytes was studied. It has been shown that preincubation of normal donor mononuclear cells (MNC) for 48 h is followed by the loss of NK activity, while the incubation of the isolated T- and non-T-cell subsets does not lead to an analogous fall in the killer lymphocyte function. NK activity of MNC and isolated lymphocyte subsets in normal donors is shown to exceed that of CLL patients. The absence of preincubation effect on NK activity level of MNC, T- and non-T-cells in CLL patients has been also found. The findings obtained suggest that as a result of T- and B-cell interaction during preincubation differentiation of young T lymphocytes with NK cellular properties takes place. It is followed by the loss in NK activity. B-cell defect in CLL patients might cause the absence of preincubation effect on NK activity of T cells.     

General analysis of receptor-mediated viral attachment to cell surfaces.

Viruses are multivalent particles that attach to cells through one or more bonds between viral attachment proteins (VAP) and specific cellular receptors. Three modes of virus binding are presented that can explain the diversity in binding data observed among viruses. They are based on multivalency of attachment and spatial versus receptor saturation effects which are easily distinguished based upon simple criteria. Mode 1 involves only monovalent virus/receptor binding. Modes 2 and 3 involve multivalent bonds between the virus and cell; however, in mode 3 space on the cell surface becomes saturated before receptors. A model is developed for viral attachment that accounts for nonspecific binding, receptor/virus interactions, and spatial saturation effects. The model can describe each mode in different limits and can be applied to virus binding data to extract key physical information such as receptor number and affinity. These values are used to postulate the type of VAP/receptor interaction involved and to predict binding at different parameter values. For the mode 2 binding of Adenovirus 2, the model predicts a receptor number of 4-15 x 10(3) on HeLa cells and an affinity of 2-6 x 10(7) M-1 which closely approximate experimental estimates. For the binding of three, broad-host-range, enveloped viruses, Semliki Forest virus, Vesicular Stomatitis virus, and the baculovirus, Autographa californica nuclear polyhedrosis virus, the model predicts receptor numbers of 10(5) or greater and affinities in the range of 10(4) to 10(5) M-1. These values are indicative of a VAP/oligosaccharide interaction which has been documented for a number of other viruses. Experimental evidence is presented that is the first to demonstrate that baculovirus binding is mediated by a cell surface receptor.     

Comparative migration of guinea pig T and B lymphocytes from capillary tubes

Using a capillary tube migration technique, a comparison was made between the random mobility of separated guinea pig T- and B-lymph node lymphocyte subpopulations, and macrophage-rich peritoneal exudate cells. A decrease in random movement was found in that order, which would fit with the hypothesis that migration on a substrate is an adherence-dependent phenomenon. In order to characterize a possible dichotomy between T-and B-cell locomotion, the effects of several factors which might interfere with their migration were studied. These factors included drugs which affect cell metabolism, cell surface ligands, and some factors which may play a role in inflammatory foci (acidity, immune complexes, and lymphokines). The results emphasize the similarity in the mode of locomotion of T and B lymphocytes. However, a remarkable difference was found in the stronger inhibition of B-cell migration by pH values below pH 7.0. The relevance of these findings to the migration of T and B lymphocytes into inflammatory foci is discussed.     

ALVAC-mediated gene transfer is efficient in lymphoid malignancies of T-and early B-cell origin, but not in tumors arising from mature B-cells

Natural attenuation of ALVAC virus in mammals makes it an attractive vector for cancer vaccine therapy of immunocompromised hosts, such as patients with lymphoid malignancies. However, the transduction efficiency of ALVAC constructs in lymphoid tumors has not yet been characterized. We studied a wide spectrum of human T- and B-cell leukemia and lymphomas and found significant heterogeneity of the ALVAC-mediated gene product expression in these tumors. While ALVAC-B7.1, ALVAC-B7.2, or ALVAC-luciferase vectors effectively expressed recombinant genes in malignancies arising from T- or early B-cell precursors, negative or low expression of ALVAC recombinant genes occurred in tumors arising from mature B-cells. We showed that ALVAC-encoded B7.1 or B7.2 was continuously expressed on the infected, and subsequently irradiated, leukemia cells, and only cells with ALVAC-mediated expression of costimulatory molecules (but not unmodified leukemia cells or those infected with the ALVAC-parental vector) induced significant proliferation and IFN-gamma production by alloreactive T-cells. These data provide the rationale for clinical studies using the ALVAC vector system for gene transfer into lymphoid tumors of T- and early B-cell origin to render them more immunogenic, while alternative strategies should be considered for immunotherapy of mature B-cell malignancies.     

COX2 expression and Erk1/Erk2 activity mediate Cot-induced cell migration

The MAPKKK8 Cot/tpl-2, identified as an oncogene (Cot-T), participates in the intracellular signaling activated by members of the TLR and TNFalpha receptor superfamilies. Here we demonstrate that Cot promotes cell migration by regulating different steps involved in this process, such as cell adhesion and metalloproteinase activity. Indeed, Cot also regulates the cytoskeleton and Cot-T overexpression provokes the polarization of microtubules and the loss of stress fibers. Moreover, and in accordance with the increased Rac-GTP levels observed, Cot-T overexpressing cells develop more lamellipodia than control cells. Conversely, depletion of endogenous Cot increases the formation of stress fibers which is correlated with the high levels of Rho-GTP observed in these cells. In addition, the increase in COX2 expression and the activation of Erk1/2 regulated by Cot are essential for the induction of cell migration. Together, these data provide evidence of a new role for both proto-oncogenic and oncogenic Cot.     

Normal development but differentially altered proliferative responses of lymphocytes in mice lacking CD81.

CD81 (TAPA-1) is a member of the transmembrane 4 superfamily (TM4SF) which is expressed on the cell surface of most cells of the body throughout their cellular differentiation. It has been recognized in several cell surface complexes of lymphocytes, suggesting that it may have diverse roles in lymphocyte development and activation regulation. Mice with a CD81 null mutation revealed normal T- and conventional B-cell development, although CD19 expression on B cells was dull and B-1 cells were reduced in number. However, both T and B cells of mutant mice exhibited strikingly enhanced proliferation in response to various types of stimuli. Interestingly, while proliferative responses of T cells following T-cell antigen receptor (TCR) engagement was enhanced in the absence of CD81, B-cell proliferation in response to B-cell antigen-receptor (BCR) cross-linking was severely impaired. Despite these altered proliferative responses, both tyrosine phosphorylation and intracellular calcium flux in response to cross-linking of cell surface antigen receptors were normal in mutant mice, reflecting apparently normal initial signaling of antigen receptors. In conclusion, though CD81 is not essential for normal T- and conventional B-cell development, it plays key roles in controlling lymphocyte homeostasis by regulating lymphocyte proliferation in distinct manners, dependent on the context of stimulation.     

Trade-offs in antibody repertoires to complex antigens

Pathogens vary in their antigenic complexity. While some pathogens such as measles present a few relatively invariant targets to the immune system, others such as malaria display considerable antigenic diversity. How the immune response copes in the presence of multiple antigens, and whether a trade-off exists between the breadth and efficacy of antibody (Ab)-mediated immune responses, are unsolved problems. We present a theoretical model of affinity maturation of B-cell receptors (BCRs) during a primary infection and examine how variation in the number of accessible antigenic sites alters the Ab repertoire. Naive B cells with randomly generated receptor sequences initiate the germinal centre (GC) reaction. The binding affinity of a BCR to an antigen is quantified via a genotype–phenotype map, based on a random energy landscape, that combines local and distant interactions between residues. In the presence of numerous antigens or epitopes, B-cell clones with different specificities compete for stimulation during rounds of mutation within GCs. We find that the availability of many epitopes reduces the affinity and relative breadth of the Ab repertoire. Despite the stochasticity of somatic hypermutation, patterns of immunodominance are strongly shaped by chance selection of naive B cells with specificities for particular epitopes. Our model provides a mechanistic basis for the diversity of Ab repertoires and the evolutionary advantage of antigenically complex pathogens.     

15-Deoxy-Delta12,14-prostaglandin J2 regulates endogenous Cot MAPK kinase kinase 1 activity induced by lipopolysaccharide

Cot is a MAPK kinase kinase that has been implicated in cellular activation and proliferation. Here, we show that the addition of lipopolysaccharide (LPS) to RAW264 macrophages induces a 10-fold increase of endogenous Cot activity, measured as MAPK kinase kinase 1 activity. Taxol, but not phorbol 12-myristate 13-acetate (PMA), induces a similar activation of Cot. A tyrosine kinase activity is involved in Cot activation by LPS. 15-Deoxy-Delta12,14-prostaglandin J2, but not rosiglitazone, blocks Cot activation by LPS. Furthermore, 15-deoxy-Delta12,14-prostaglandin J2 also inhibited the LPS-induced Cot in vitro. However, 15-deoxy-Delta12,14-prostaglandin J2 does not inhibit MAPK kinase 1 or ERK1/ERK2 activation/phosphorylation induced by PMA and mediated by c-Raf. Considering these data, we propose that the inhibition of LPS-induced Cot activation is one mechanism by which 15-deoxy-Delta12,14-prostaglandin J2 acts as an anti-inflammatory.