OMIP-089: Cattle T-cell phenotyping by an 8-color panel.

This multiplex staining panel was developed to differentiate cattle T cells into conventional (CD4 and CD8) and unconventional (γδ-TCR) subsets as well as their stage of differentiation and activation. The combination of CD45RO and CD62L allows the identification of naïve (TNaïve ), central memory (TCM ), effector memory (TEM ) and terminal effector (TTE ) T cells. Activated cattle T cells (TAV ) can be identified by the cell surface expression of CD25. This panel was developed using cryopreserved cattle peripheral blood mononuclear cells (PBMCs) and tested on fresh as well as stimulated PBMCs. Therefore, this 8-color, 10-parameter flow cytometry panel simultaneously identifies cattle TNaïve , TAV , TCM , TEM , TTE and γδ-TCR cells. This panel will improve our ability to examine T-cell response to pathogens and vaccines in cattle including the potential to identify previously undescribed subpopulations. Furthermore, this panel can be readily optimized for other bovid species as many of these reagents are likely to cross react.

OMIP-085: Cattle B-cell phenotyping by an 8-color panel.

This 8-color panel has been optimized to distinguish between functionally distinct subsets of cattle B cells in both fresh and cryopreserved peripheral blood mononuclear cells (PBMCs). Existing characterized antibodies against cell surface molecules (immunoglobulin light chain (S-Ig[L]), CD20, CD21, CD40, CD71, and CD138) enabled the discrimination of 24 unique populations within the B-cell population. This allows the identification of five putative functionally distinct B-cell subsets critical to infection and vaccination responses: (1) naïve B cells (BNaïve ), (2) regulatory B cells (BReg ), (3) memory B cells (BMem ), (4) plasmablasts (PB), and (5) plasma cells (PC). Although CD3 and CD8α can be included as an additional dump channel, it does not significantly improve the panel's ability to separate "classical" B cells. This panel will promote better characterization and tracking of B-cell responses in cattle as well as other bovid species as the reagents are likely to cross react.

Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling.

Marek's disease virus (MDV), an alphaherpesvirus of poultry, causes Marek's disease and is characterized by visceral CD4+TCRαß+ T-cell lymphomas in susceptible hosts. Immortal cell lines harbouring the viral genome have been generated from ex vivo cultures of MD tumours. As readily available sources of large numbers of cells, MDV-transformed lymphoblastoid cell lines (LCLs) are extremely valuable for studies of virus-host interaction. While the viral genome in most cells is held in a latent state, minor populations of cells display spontaneous reactivation identifiable by the expression of lytic viral genes. Spontaneous reactivation in these cells presents an opportunity to investigate the biological processes involved in the virus reactivation. For detailed characterization of the molecular events associated with reactivation, we used two lymphoblastoid cell lines derived from lymphomas induced by pRB1B-UL47eGFP, a recombinant MDV engineered to express enhanced green fluorescent protein (EGFP) fused with the UL47. We used fluorescence-activated cell sorting to purify the low-frequency EGFP-positive cells with a spontaneously activating viral genome from the majority EGFP-negative cells and analysed their gene expression profiles by RNA-seq using Illumina HiSeq2500. Ingenuity pathway analysis on more than 2000 differentially expressed genes between the lytically infected (EGFP-positive) and latently infected (EGFP-negative) cell populations identified the biological pathways involved in the reactivation. Virus-reactivating cells exhibited differential expression of a significant number of viral genes, with hierarchical differences in expression levels. Downregulation of a number of host genes including those directly involved in T-cell activation, such as CD3, CD28, ICOS and phospholipase C, was also noticed in the LCL undergoing lytic switch.

Induction of lymphomas by inoculation of Marek's disease virus-derived lymphoblastoid cell lines: prevention by CVI988 vaccination.

Lymphoblastoid cell lines 265(L) and 990(O) are monoclonal lymphomas, derived respectively from liver and ovarian tumours, generated in inbred P-line (MHC B(19)/B(19)) chickens infected with RB-1B strain of Marek's disease virus (MDV) and pRB-1B5 BAC clone respectively. These were inoculated into inbred, MDV-susceptible, P-line chickens by intra-venous or intra-abdominal routes. Additional groups of birds were vaccinated using 1000 plaque-forming units of CVI988 vaccine 8 days prior to inoculation of the cell lines. Non-vaccinated birds developed visceral Marek's disease tumours with an increased rate 30 to 60 days post inoculation. Vaccination prevented tumour and disease development in challenged birds. TCRß repertoire analysis by spectratyping and sequencing of the inoculum was used to track tumour identity in primary tumours and tumour cell lines derived from inoculated birds. These data revealed that the tumours were a consequence of de novo virus infection and not metastasis and expansion of the inoculated tumour cells. Moreover, the data showed that the two MDV-derived cell lines were not transplantable even in syngeneic P-line birds. The data also demonstrated the application of spectratyping as a tool to track tumour identity in lymphoma transplantation studies.

Clonal structure of rapid-onset MDV-driven CD4+ lymphomas and responding CD8+ T cells.

Lymphoid oncogenesis is a life threatening complication associated with a number of persistent viral infections (e.g. EBV and HTLV-1 in humans). With many of these infections it is difficult to study their natural history and the dynamics of tumor formation. Marek's Disease Virus (MDV) is a prevalent α-herpesvirus of poultry, inducing CD4+ TCRαß+ T cell tumors in susceptible hosts. The high penetrance and temporal predictability of tumor induction raises issues related to the clonal structure of these lymphomas. Similarly, the clonality of responding CD8 T cells that infiltrate the tumor sites is unknown. Using TCRß repertoire analysis tools, we demonstrated that MDV driven CD4+ T cell tumors were dominated by one to three large clones within an oligoclonal framework of smaller clones of CD4+ T cells. Individual birds had multiple tumor sites, some the result of metastasis (i.e. shared dominant clones) and others derived from distinct clones of transformed cells. The smaller oligoclonal CD4+ cells may represent an anti-tumor response, although on one occasion a low frequency clone was transformed and expanded after culture. Metastatic tumor clones were detected in the blood early during infection and dominated the circulating T cell repertoire, leading to MDV associated immune suppression. We also demonstrated that the tumor-infiltrating CD8+ T cell response was dominated by large oligoclonal expansions containing both "public" and "private" CDR3 sequences. The frequency of CD8+ T cell CDR3 sequences suggests initial stimulation during the early phases of infection. Collectively, our results indicate that MDV driven tumors are dominated by a highly restricted number of CD4+ clones. Moreover, the responding CD8+ T cell infiltrate is oligoclonal indicating recognition of a limited number of MDV antigens. These studies improve our understanding of the biology of MDV, an important poultry pathogen and a natural infection model of virus-induced tumor formation.

Regional and global changes in TCRalphabeta T cell repertoires in the gut are dependent upon the complexity of the enteric microflora.

The repertoire of gut associated T cells is shaped by exposure to microbes, including the natural enteric microflora. Previous studies compared the repertoire of gut associated T cell populations in germ free (GF) and conventional mammals often focussing on intra-epithelial lymphocyte compartments. Using GF, conventional and monocolonised (gnotobiotic) chickens and chicken TCRbeta-repertoire analysis techniques, we determined the influence of microbial status on global and regional enteric TCRbeta repertoires. The gut of conventionally reared chickens exhibited non-Gaussian distributions of CDR3-lengths with some shared over-represented peaks in neighbouring gut segments. Sequence analysis revealed local clonal over-representation. Germ-free chickens exhibited a polyclonal, non-selected population of T cells in the spleen and in the gut. In contrast, gnotobiotic chickens exhibited a biased repertoire with shared clones evident throughout the gut. These data indicate the dramatic influence of enteric microflora complexity on the profile of TCRbeta repertoire in the gut at local and global levels.

The blood contains multiple distinct progenitor populations with clonogenic B and T lineage potential.

The thymus is seeded by bone marrow-derived progenitors that circulate in the blood. Multiple cell types can be found in the thymus early after i.v. administration or in steady state, but most fail to satisfy the known characteristics of true T progenitors. Cells that do conform to classical definitions retain multilineage potential, but surprisingly, cannot make B cells. Because acquisition of the T lineage fate among noncommitted progenitors is a lengthy process, the absence of B cell potential in early thymocytes suggests that B and T lineages diverge prethymically. To test this suggestion, we screened numerous presumptive progenitor populations for T cell growth and differentiation potential, as well as for clonogenic T or B cell development. We find that blood and marrow each contain multiple distinct subsets that display growth and differentiation potential consistent with being canonical T progenitors. Assessment of clonogenic potential further shows that although all blood and marrow populations have high T cell cloning potential, no T/non-B cells are apparent. These data suggest that either true thymic reconstitution potential derives from a small T/non-B cell subset of one of these populations, or that most of the cells defined as canonical progenitors within the thymus do not, in fact, reside in the mainstream of T progenitor differentiation.