In vivo modulation of CD1 and MHC class II expression by sheep afferent lymph dendritic cells. Comparison of primary and secondary immune responses.

The experiments described in this article characterize the phenotypic and functional changes in afferent lymph cell populations that occur as a result of in vivo immune stimulation. During the primary immune response (in antigen-naive sheep) there are very transient increases in level of CD1 expression by subpopulations of dendritic cells (DC) but no alterations in cell kinetics or MHC class II expression. In contrast, secondary antigenic challenge (in primed sheep) into the drainage area of an afferent lymphatic causes profound changes in the cell output, characterized by a greater than threefold drop in total cell output on days 1-3 followed by an approximate fivefold rise on day 5. There is also a substantial increase in both the proportion of MHC class II-positive T lymphocytes (from 28 to 54%) and in the quantitative expression of class II by both DC and lymphocytes. Class II expression by DC increases five- to sixfold by day 5, while the level of expression of class II on lymphocytes approximately doubles. The increase in CD1 expression during the secondary response is more prolonged than during the primary response, being detectable between days 2 and 6 after challenge. The rise in class II affects the whole DC population, in contrast to CD1 where the increase affects only a subpopulation of cells. In terms of functional properties, afferent lymph DC isolated during a primary response show no alteration of their activity, whereas DC taken 4-5 d after secondary challenge are up to fivefold more active in their ability to present soluble antigen to primed autologous T cells and to antigen-specific cell lines as well as to stimulate in the MLR. The relative expression of class II correlates temporally with an increased capacity of DC to present antigen. Monoclonal anti-class II antibodies totally inhibit the in vitro assays but anti-CD1 antibodies have no effect. The previous paper has demonstrated that afferent DC can associate with antigen in vivo and can present that antigen to antigen-specific T cells. This article extends our knowledge of DC biology and demonstrates that DC, activated during secondary in vivo immune responses, have an enhanced ability to present an antigen, unrelated to that used for challenge, to specific T cell lines. This enhancement correlates directly with quantitative variation of expressed class II and not CD1 and suggests that this variation in class II expression plays a physiological role in in vivo immune regulation.

Characterization of sheep afferent lymph dendritic cells and their role in antigen carriage.

We have ablated peripheral lymph nodes in sheep and subsequently cannulated the pseudo-afferent lymphatic vessel that arises as a consequence of afferent lymphatic vessels reanastomosing with the former efferent duct. This technique allows the collection of lymph with a cellular composition that resembles true afferent fluid, and in particular, containing 1-10% dendritic cells. A 16-h collection of this lymph may contain between 10(6) and 10(7) dendritic cells. This dendritic cell population may be enriched to greater than 75% by a single-density gradient centrifugation step. We have generated a mAb that recognizes sheep CD1. This monoclonal not only reacts with afferent dendritic cells, but with dendritic cells in the skin and paracortical T cell areas of lymph nodes. The expression of CD1 suggests afferent dendritic cells are related to skin Langerhans' cells and other dendritic cells that act as accessory cells for T cell responses. Consistent with this is the high level of expression by dendritic cells of molecules involved in antigen recognition by T cells, including MHC class I and class II. Afferent dendritic cells express high levels of the cellular adhesion molecule LFA-3, and at the same time express a ligand for this molecule, namely CD2. The accessory functions of afferent dendritic cells resemble those displayed by mature Langerhans' cells and by lymph node interdigitating cells. These include clustering with resting T cells and stimulating their proliferation in a primary response to antigen. Afferent dendritic cells are capable of acquiring soluble protein antigen in vivo or in vitro and presenting the material directly to autologous T cells in an antigen-specific manner. We conclude that afferent dendritic cells represent a lymph-borne Langerhans' cell involved in antigen carriage to the lymph node.

Control of gammaherpesvirus latency by latent antigen-specific CD8(+) T cells.

The contribution of the latent antigen-specific CD8(+) T cell response to the control of gammaherpesvirus latency is currently obscure. Some latent antigens induce potent T cell responses, but little is known about their induction or the role they play during the establishment of latency. Here we used the murine gammaherpesvirus system to examine the expression of the latency-associated M2 gene during latency and the induction of the CD8(+) T cell response to this protein. M2, in contrast to the M3 latency-associated antigen, was expressed at day 14 after infection but was undetectable during long-term latency. The induction of the M2(91-99)/K(d) CD8(+) T cell response was B cell dependent, transient, and apparently induced by the rapid increase in latently infected cells around day 14 after intranasal infection. These kinetics were consistent with a role in controlling the initial "burst" of latently infected cells. In support of this hypothesis, adoptive transfer of an M2-specific CD8(+) T cell line reduced the initial load of latently infected cells, although not the long-term load. These data represent the first description of a latent antigen-specific immune response in this model, and suggest that vaccination with latent antigens such as M2 may be capable of modulating latent gammaherpesvirus infection.

Expansion and activation of NK cell populations in a gammaherpesvirus infection.

NK cells are an important component of the innate immune response to many virus infections. In particular, they play a major role in control of alpha and beta herpesvirus infections in humans and mice and there is evidence for a protective role in Epstein-Barr virus infection. MHV-68 has been widely used to study gammaherpesvirus pathogenesis and provides a tractable means of investigating the role of NK cells in gammaherpesvirus infections. We have shown that, following MHV-68 infection of mice, the NK cell population is expanded and activated and capable of cytotoxic killing in vitro. However, depletion of NK cells prior to MHV-68 infection did not affect viral loads in vivo. To investigate the possibility that MHV-68 was downregulating NK cell activity in vivo and evading the NK cell response, we infected NK cell-depleted mice with the related virus, MHV-76, which lacks a 9.5 kb region of the genome known to be involved in modulating the host immune response. Infection of NK cell-depleted mice with MHV-76 did not result in increased viral loads indicating that genes within this region do not encode products which modulate NK cell activity.

Development of an enzyme-linked immunosorbent assay for the detection of antibodies against malignant catarrhal fever viruses in cattle serum.

Malignant catarrhal fever (MCF) is a sporadic but fatal lymphoproliferative viral disease of cattle, deer and other ruminants. The causative agents are highly-cell-associated herpesviruses of the subfamily gammaherpesvirinae. In this study, an ELISA (WC11-ELISA) was developed to detect antibody to malignant catarrhal fever virus (MCFV) in cattle serum and compared to the commercially produced competitive-inhibition ELISA (CI-ELISA). Crude lysate antigen from alcelaphine herpesvirus-1 strain WC11 was bound to 96-well microplates and used to capture antibodies to MCFV. Dilutions of test sera were added to wells containing bound MCF antigen and control wells containing uninfected cell lysates. A horseradish peroxidase-labelled rabbit-anti-bovine IgG conjugate detected antibodies to MCF, and the results were expressed as absorbance readings at 450 nm. Samples were selected blind from cattle sera which had been sent to the laboratory for diagnostic testing for MCFV antibodies and were tested in both the WC11-ELISA and the CI-ELISA. Good agreement between the WC11-ELISA and CI-ELISA test (k=0.86, n=95) results was found.

Sequence of the sheep interleukin-10-encoding cDNA.

The ovine interleukin-10 (oIL-10)-encoding cDNA has been cloned and sequenced using gene amplification by the polymerase chain reaction (PCR). We present the complete coding sequence of the ovine IL-10 gene, as well as the predicted amino acid (aa) sequence. The oIL10 DNA coding sequence is 531 nucleotides long and the mature protein product is predicted to be 18,367 Da, consisting of 158 aa, excluding a 19-aa N-terminal hydrophobic signal peptide. The oIL-10 protein is > 77% identical to pig and human IL-10, > 71% identical to rodent IL-10 and > 68% identical to viral IL-10.

Improved method for the measurement of ribonucleotide reductase activity.

An improved method for the measurement of herpes simplex virus type 1 encoded ribonucleotide reductase has been developed. The enzyme which catalyses the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates was determined by first converting the ribonucleotide substrate and deoxyribonucleotide product to the corresponding nucleosides by treatment with snake venom phosphodiesterase. Then nucleosides were separated by HPLC and measured by flow through scintillation counting and by monitoring their absorbance at 254 nm. Under the conditions used in the experiment cytidine and deoxcytidine, the derivitised substrate and product respectively, eluted from the column at approximately 4 min 33 s and 6 min 24 s. Peak heights and areas were automatically calculated by computer to ascertain the amount of product formed and thus quantitate the assay. Automation of the assay from sample injection to analysis provides a significant saving in time and an improvement in the efficiency of measurement of ribonucleotide reductase activity over other published methods.

Analysis of the CD1 cluster in sheep.

The anti-CD1 monoclonal antibodies submitted to the 1st International Workshop on Leucocyte Differentiation Antigens of Cattle, Sheep and Goats were tested for their reactivity on sheep skin, thymus and lymph node and for their reactivity with sheep efferent and afferent lymph and peripheral blood. With the exception of 20-27 they all stained that same cell populations. The antibodies precipitated molecules with a heavy chain of 46,000 apparent molecular weight and a light chain of 14,000 apparent molecular weight. VPM5 and CC14 antigens were purified by affinity chromatography. All the antibodies cross-reacted with these molecules. The results show that 20-27 recognises the same molecules as the other antibodies and suggest that 20-27 is a pan CD1 monoclonal antibody and the other monoclonal antibodies are homologues of the human CD1b molecules.

Monoclonal antibodies to the sheep analogues of human CD45 (leucocyte common antigen), MHC class I and CD5. Differential expression after lymphocyte activation in vivo.

This paper describes three anti-sheep monoclonal antibodies. The tissue distribution and apparent molecular weight of the antigens detected by these antibodies is consistent with them reacting with sheep leucocyte common antigen (CD45 (VPM18], MHC class I (VPM19) and CD5 (VPM29). An ELISA method is described that permits the cross-reactivity of different antibodies to be assessed, this confirms the identity of the antigens detected by VPM18, VPM19 and VPM29. This method is also of value as either a positive or a negative screen in the construction of further monoclonals. A study of the expression of these three antigens on efferent lymph small lymphocytes and antigen-activated lymphoblasts shows that the density of CD45 on lymphoblasts (activated either in vivo or in vitro) is approximately half that of small lymphocytes whereas the density of MHC class I is the same in both populations. Furthermore, about 75% of small lymphocytes express CD5 but less than 10% of lymphoblasts are positive. Cell membrane CD5 expression is lost on lymphocyte activation. It does not seem to be linked to cell membranes via phosphatidylinositol and the loss is not due to the breaking of that link.

Comparison of workshop CD45R monoclonal antibodies with OvCD45R monoclonal antibodies in sheep.

The reactivity of the antibovine monoclonal antibodies (mAbs) comprising temporary cluster TC1 was compared with that of two OvCD45R mAbs on sheep cells. Three of the mAbs--CC31, CC99 and CC103--did not cross-react with sheep cells. All the workshop mAbs precipitated two molecules of apparent molecular weight (MW) 200 kDa and 220 kDa while the antisheep CD45R mAb 20-96 precipitated a single band of 220 kDa. Cell surface expression was examined by single colour FACS (fluorescence activated cell sorting) analysis of efferent and afferent lymph cells and peripheral blood lymphocytes and the distribution of the antigens on CD4+, CD8+ and T19+ (WC1) and B cells was determined by two colour fluorescence staining. By cellular distribution and immunohistology the TC1 mAbs could be divided into four distinct groups which differed from a fifth group comprising the two OvCD45R antibodies.

Analysis of the monoclonal antibodies comprising WC6.

The WC6 antibovine monoclonal antibodies (mAbs) CC98, IL-A114 and IL-A53 were investigated for reactivity in sheep by (fluorescence activated cell sorting) FACS analysis, immunoprecipitation and immunohistology. The mAbs behave identically by all criteria although IL-A114 reacts more weakly than the other mAbs. This probably reflects limited cross-species reactivity. The mAbs stain < 30% of lymphocytes from blood, efferent and afferent lymph and the majority of afferent lymph dendritic cells. They also weakly stain granulocytes. They precipitate molecules of apparent molecular weight 220 kD and 180-190 kD. Sequential immunoprecipitation shows that CC98 antigen is not related to CD45. Immunohistology indicates staining of B cell areas and macrophages in Peyer's patch and lamina propria. The data show that these monoclonal antibodies react with a molecule distinct from OvCD45R.

Sheep CD1 genes and proteins.

Interest in CD1 genes and proteins was initially stimulated by their close evolutionary and structural relationship to MHC class I molecules. The demonstration that CD1b and c molecules present novel non-peptide antigens to T-cells and play a role in protection against mycobacterial infection then focused attention on the functional role of CD1 proteins. Sheep possess at least seven CD1 genes, including CD1B, D and E, which is the most complex genetic arrangement identified so far in any animal. OvCD1B consists of at least three distinct genes, with the probability of limited polymorphism and the existence of splice variants. Most anti-sheep CD1-specific monoclonal antibodies react with OvCDlb and phenotypic and immunochemical data suggests the existence of two variants. CD1D genes have been identified in all species studied, suggesting a conserved role for CDld proteins across mammalian species. Presumptive evidence for the existence of OvCDIE has been obtained by NH2-terminal sequencing of protein precipitated by the mAb 20.27 (SBU-T6). Confirmatory evidence from gene cloning experiments is currently being sought. Collectively, these factors make the sheep CD1 family a highly relevant model for investigating the in vivo role of CD1 molecules. In this survey, the properties of monoclonal antibodies specific for sheep CD1, the cellular distribution and physicochemical characteristics of sheep CD1 molecules and the current state of knowledge on sheep CD1 genetics are reviewed.

Discrimination of two subsets of CD1 molecules in the sheep.

This paper examines the expression of CD1 in the sheep utilising the monoclonal antibodies (mAbs) which were assigned to OvCD1 in the First and Second Workshops on Ruminant Leukocyte Differentiation Antigens along with those primarily clustered as Bov/OvCD1 in the Third Workshop. Detailed immunohistological studies of both lymphoid and non-lymphoid tissues and flow cytometry of isolated cell populations revealed two distinct patterns of CD1 expression in the sheep. The mAbs assigned to the sub-cluster BovCD1w1 (SBU-T6) and BovCD1w3 (IAH-CC43 and IAH-CC118) were much more widely distributed than those of the sub-cluster BovCD1w2. In addition to cortical thymocytes and dendritic cells (DC) the CD1w1 and w3 molecules are expressed by peripheral blood B lymphocytes, monocytes and many tissue macrophages.

Ribonucleotide reductase induced by herpes simplex virus has a virus-specified constituent.

Ribonucleotide reductase, an enzyme found in all prokaryotic and eukaryotic cells that synthesize DNA, is induced by herpes simplex virus (HSV). In this study the effect of anti-HSV antiserum on the induced ribonucleotide reductase has been examined and the ability of different temperature-sensitive (ts) mutants of HSV-1 to induce the enzyme has been investigated. The HSV-1-induced ribonucleotide reductase was inhibited by antiserum raised against infected cell lysates but not by preimmune serum. The wild-type (ts+) virus induced similar levels of ribonucleotide reductase at 31 degrees C and 38.5 degrees C (the permissive and non-permissive temperatures respectively for the ts mutants). All ts mutants induced approximately wild-type levels of the enzyme at 31 degrees C. At 38.5 degrees C, two of the four ts mutants studied also induced wild-type levels of enzyme but ts G failed to induce any activity while ts K induced variable but low levels. The enzyme activity induced by ts G at 31 degrees C was thermolabile both in vivo and in vitro. These results provide the first strong evidence that the induced ribonucleotide reductase activity is at least partially virus-coded.

The ribonucleotide reductase induced by herpes simplex virus type 1 involves minimally a complex of two polypeptides (136K and 38K).

Herpes simplex virus type 1 (HSV-1) encodes a polypeptide of apparent mol. wt. 136 000 (Vmw136) known to be a component of the virus-specified ribonucleotide reductase. Monoclonal antibodies that precipitate this polypeptide also precipitate a polypeptide of mol. wt. 38 000 (Vmw38) from extracts of HSV-1-infected cells. The basis for this co-precipitation has been investigated using a monoclonal antibody directed against Vmw136 and an oligopeptide-induced antiserum directed against the carboxy terminus of Vmw38. We have also made use of a temperature-sensitive (ts) mutant of HSV-1 which maps within the sequences encoding Vmw136 and which induces a thermolabile ribonucleotide reductase. Our experiments show (i) Vmw136 and Vmw38 form a complex in infected cells and (ii) the mutation in the ts mutant results in the two polypeptides being unable to form the complex at the non-permissive temperature. We speculate that association of the two polypeptides is necessary for ribonucleotide reductase activity. No evidence was found for involvement of host proteins in the proposed virus-induced ribonucleotide reductase complex. The terms RR1 and RR2 are suggested for the large and small subunits of the HSV-induced enzyme.

The m4 gene of murine gammaherpesvirus modulates productive and latent infection in vivo.

Murine gammaherpesvirus 68 (MHV-68) infection of mice represents a viable small-animal model for the study of gammaherpesvirus pathogenesis. MHV-76 is a deletion mutant of MHV-68, which lacks four MHV-68-specific genes (M1 to M4) and eight viral tRNA-like sequences at the 5' end of the genome. These genes are implicated in latency and/or immune evasion. Consequently, MHV-76 is attenuated in the acute phase of in vivo infection with respect to MHV-68. Little is known about the role of M4 in viral infection, except that it is expressed as an immediate-early/early transcript during lytic replication of MHV-68 in vitro. To elucidate the contribution M4 makes to in vivo pathogenesis, we created a novel MHV-76 mutant (MHV-76inM4), in which the region of MHV-68 coding for M4 and accompanying putative promoter elements were inserted into the 5' region of the MHV-76 genome. The growth of MHV-76inM4 in vitro was indistinguishable from that of MHV-76 and MHV-68. However, virus titers from MHV-76inM4-infected BALB/c mice were significantly increased with respect to MHV-76 at early times in the lung. In addition, at days 17 and 21 postinfection, there was a significant elevation in latent viral load in splenocytes of MHV-76inM4-infected mice compared to MHV-76. Like MHV-76-infected mice, MHV-76inM4-infected mice display no evidence of overt splenomegaly, a finding characteristic of MHV-68 infection. M4 expression in vivo was detectable during productive infection in the lung and during the establishment of latency in the spleen, but in general M4 was not detectable during long-term latency (day 100 postinfection).