Phenotypic characterisation of bovine alveolar macrophages reveals two major subsets with differential expression of CD163.

Bovine alveolar macrophages (AMs) defend the lungs against pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis. However, little is known about the surface molecules expressed by bovine AMs and whether there is heterogeneity within the population. The purpose of this study was to characterise the bovine AM cell surface phenotype using flow cytometry. Bronchoalveolar lavage samples from four different calves were stained with a combination of antibodies against immune cell molecules prior to flow cytometric analysis. To assess the degree of expression, we considered the distribution and relative intensities of stained and unstained cells. We demonstrated that bovine AMs have high expression of CD172a, ADGRE1, CD206, and CD14, moderate expression of CD80, MHC II, CD1b, and CD40, low expression of CX3CR1 and CD86, and little or no expression of CD16 and CD26. Two distinct subsets of bovine AMs were identified based on CD163 expression. Subsequent analysis showed that the CD163+ subset had greater expression of other typical macrophage molecules compared to the CD163- subset, suggesting that these cells may perform different roles during infection. The characterisation of the uninfected bovine AM phenotype will provide a foundation for the examination of M. bovis-infected AMs.

Unconventional human CD61 pairing with CD103 promotes TCR signaling and antigen-specific T cell cytotoxicity.

Cancer remains one of the leading causes of mortality worldwide, leading to increased interest in utilizing immunotherapy strategies for better cancer treatments. In the past decade, CD103+ T cells have been associated with better clinical prognosis in patients with cancer. However, the specific immune mechanisms contributing toward CD103-mediated protective immunity remain unclear. Here, we show an unexpected and transient CD61 expression, which is paired with CD103 at the synaptic microclusters of T cells. CD61 colocalization with the T cell antigen receptor further modulates downstream T cell antigen receptor signaling, improving antitumor cytotoxicity and promoting physiological control of tumor growth. Clinically, the presence of CD61+ tumor-infiltrating T lymphocytes is associated with improved clinical outcomes, mediated through enhanced effector functions and phenotype with limited evidence of cellular exhaustion. In conclusion, this study identified an unconventional and transient CD61 expression and pairing with CD103 on human immune cells, which potentiates a new target for immune-based cellular therapies.

GTAC enables parallel genotyping of multiple genomic loci with chromatin accessibility profiling in single cells.

Understanding clonal evolution and cancer development requires experimental approaches for characterizing the consequences of somatic mutations on gene regulation. However, no methods currently exist that efficiently link high-content chromatin accessibility with high-confidence genotyping in single cells. To address this, we developed Genotyping with the Assay for Transposase-Accessible Chromatin (GTAC), enabling accurate mutation detection at multiple amplified loci, coupled with robust chromatin accessibility readout. We applied GTAC to primary acute myeloid leukemia, obtaining high-quality chromatin accessibility profiles and clonal identities for multiple mutations in 88% of cells. We traced chromatin variation throughout clonal evolution, showing the restriction of different clones to distinct differentiation stages. Furthermore, we identified switches in transcription factor motif accessibility associated with a specific combination of driver mutations, which biased transformed progenitors toward a leukemia stem cell-like chromatin state. GTAC is a powerful tool to study clonal heterogeneity across a wide spectrum of pre-malignant and neoplastic conditions.

Evaluation of T cell responses to naturally processed variant SARS-CoV-2 spike antigens in individuals following infection or vaccination.

Most existing studies characterizing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell responses are peptide based. This does not allow evaluation of whether tested peptides are processed and presented canonically. In this study, we use recombinant vaccinia virus (rVACV)-mediated expression of SARS-CoV-2 spike protein and SARS-CoV-2 infection of angiotensin-converting enzyme (ACE)-2-transduced B cell lines to evaluate overall T cell responses in a small cohort of recovered COVID-19 patients and uninfected donors vaccinated with ChAdOx1 nCoV-19. We show that rVACV expression of SARS-CoV-2 antigen can be used as an alternative to SARS-CoV-2 infection to evaluate T cell responses to naturally processed spike antigens. In addition, the rVACV system can be used to evaluate the cross-reactivity of memory T cells to variants of concern (VOCs) and to identify epitope escape mutants. Finally, our data show that both natural infection and vaccination could induce multi-functional T cell responses with overall T cell responses remaining despite the identification of escape mutations.

An immunodominant NP105-113-B*07:02 cytotoxic T cell response controls viral replication and is associated with less severe COVID-19 disease.

NP105-113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105-113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105-113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105-113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19.

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.

Broad and strong memory CD4 + and CD8 + T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients.

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFNγ based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4 + and/or CD8 + epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8 + T cells than spike-specific CD8 + T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8 + to CD4 + T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.

A Comprehensive Analysis of Key Immune Checkpoint Receptors on Tumor-Infiltrating T Cells From Multiple Types of Cancer.

Background: Cancer patients often display dysfunctional antitumor T-cell responses. Because noteworthy benefits of immune checkpoint pathway blockade, such as programmed cell death protein 1 (PD-1) inhibitors, have been achieved in multiple advanced cancers, the next critical question is which mono-blockade or combinatorial blockade regimens may reinvigorate antitumor T-cell immunity in those cancer patients while limiting immune-related adverse effects. Method: This study recruited, in total, 172 primary cancer patients (131 were blood-tumor-matched patients) who were treatment-naïve prior to the surgeries or biopsies covering the eight most prevalent types of cancer. With access to fresh surgical samples, this study simultaneously investigated the ex vivo expression level of eight known immune checkpoint receptors [PD-1, cytotoxic T-lymphocyte antigen-4 [CTLA-4], T-cell immunoglobulin and mucin-domain containing-3 [Tim-3], 2B4, killer cell lectin like receptor G1 [KLRG-1], TIGIT, B- and T-lymphocyte attenuator [BTLA], and CD160] on tumor-infiltrating T cells (TILs) and paired circulating T cells in blood from a 131-patient cohort. Results: We found increased an expression of PD-1 and Tim-3 but a decreased expression of BTLA on TILs when compared with peripheral blood from multiple types of cancer. Moreover, our co-expression analysis of key immune checkpoint receptors delineates "shared" subsets as PD-1+Tim-3+TIGIT+2B4+KLRG-1-CTLA-4- and PD-1+TIGIT+2B4+Tim-3-KLRG-1-CTLA-4- from bulk CD8 TILs. Furthermore, we found that a higher frequency of advanced differentiation stage T cells (CD27-CCR7-CD45RA-) among the "shared" subset (PD-1+Tim-3+TIGIT+2B4+KLRG-1-CTLA-4-) in bulk CD8 TILs was associated with poorly differentiated cancer type in cervical cancer patients. Conclusions: To our knowledge, our study is the first comprehensive analysis of key immune checkpoint receptors on T cells in treatment-naïve, primary cancer patients from the eight most prevalent types of cancer. These findings might provide useful information for future design of mono-blockade/combinatorial blockades and/or genetically modified T-cell immunotherapy.

SCL/TAL1 cooperates with Polycomb RYBP-PRC1 to suppress alternative lineages in blood-fated cells.

During development, it is unclear if lineage-fated cells derive from multilineage-primed progenitors and whether active mechanisms operate to restrict cell fate. Here we investigate how mesoderm specifies into blood-fated cells. We document temporally restricted co-expression of blood (Scl/Tal1), cardiac (Mesp1) and paraxial (Tbx6) lineage-affiliated transcription factors in single cells, at the onset of blood specification, supporting the existence of common progenitors. At the same time-restricted stage, absence of SCL results in expansion of cardiac/paraxial cell populations and increased cardiac/paraxial gene expression, suggesting active suppression of alternative fates. Indeed, SCL normally activates expression of co-repressor ETO2 and Polycomb-PRC1 subunits (RYBP, PCGF5) and maintains levels of Polycomb-associated histone marks (H2AK119ub/H3K27me3). Genome-wide analyses reveal ETO2 and RYBP co-occupy most SCL target genes, including cardiac/paraxial loci. Reduction of Eto2 or Rybp expression mimics Scl-null cardiac phenotype. Therefore, SCL-mediated transcriptional repression prevents mis-specification of blood-fated cells, establishing active repression as central to fate determination processes.

Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for ß-thalassemia.

ß-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and ß-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with ß-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with ß-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for ß-thalassemia.ß-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.

Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia.

Recent advances in single-cell transcriptomics are ideally placed to unravel intratumoral heterogeneity and selective resistance of cancer stem cell (SC) subpopulations to molecularly targeted cancer therapies. However, current single-cell RNA-sequencing approaches lack the sensitivity required to reliably detect somatic mutations. We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 SCs from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML-SCs, including the identification of a subgroup of CML-SCs with a distinct molecular signature that selectively persisted during prolonged therapy. Analysis of nonleukemic SCs from patients with CML also provided new insights into cell-extrinsic disruption of hematopoiesis in CML associated with clinical outcome. Furthermore, we used this single-cell approach to identify a blast-crisis-specific SC population, which was also present in a subclone of CML-SCs during the chronic phase in a patient who subsequently developed blast crisis. This approach, which might be broadly applied to any malignancy, illustrates how single-cell analysis can identify subpopulations of therapy-resistant SCs that are not apparent through cell-population analysis.

Subset-Specific Expression of Toll-Like Receptors by Bovine Afferent Lymph Dendritic Cells.

Within the ruminant system, several possibilities exist to generate dendritic cells migrating out from the tissue into the regional draining lymph nodes as afferent lymph dendritic cells (ALDCs). Here, we analyzed toll-like receptor (TLR) 1-10 mRNA expression by using quantitative real-time PCR in highly purified subsets of bovine ALDC. As TLR expression may be influenced by pathogens or vaccines and their adjuvant, it is necessary to understand what TLRs are expressed in a steady-state system to elucidate specific differences and to potentially optimize targeted vaccines. In this study, we have assessed the TLR expression profiles of the four main bovine ALDC subsets [cDC1 and cDC2 (subsets 2-4)]. We demonstrate differences in TLR expression between the four subsets that may reflect the ability of these cells to respond to different pathogens or to respond to adjuvants.

Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics.

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.

Natural killer cell number and phenotype in bovine peripheral blood is influenced by age.

Natural killer (NK) cells are critical to the innate defence against intracellular infection. High NK cell frequencies have been detected in human neonates, which may compensate for the relative immaturity of the specific immune response. Additionally, phenotypic subsets of NK cells have been identified in humans with different functional properties. In this study, we examined the age distribution and phenotype of NK populations in bovine peripheral blood, including neonatal animals. We found that the NK cell populations defined by the phenotypes CD3(-)CD2(+) and NKp46(+) largely overlapped, so that the majority of NK cells in bovine peripheral blood were CD3(-)CD2(+)NKp46(+). The remainder of the NK-like cells comprised two minor populations, CD3(-)CD2(+)NKp46(-) and CD3(-)CD2(-)NKp46(+); the relative proportions of these varied with age. The lowest frequency of NK cells was recorded in 1-day-old calves, with the highest frequency in day 0 calves. The phenotypic characteristics of CD3(-)CD2(+) and NKp46(+) NK populations were similar; both populations expressed CD45RO, CD45RB, CD11b, CC84, CD8alphaalpha and CD8alphabeta and did not express CD21, WC1, CD14 or gammadelta TCR. Age-related phenotypic differences were apparent. The phenotypic characteristics of three NK subpopulations were described; a significantly greater proportion of the CD3(-)CD2(-)NKp46(+) population expressed CD8alpha compared to CD3(-)CD2(+)NKp46(+) cells. Furthermore, a significantly greater proportion of the CD3(-)CD2(+)NKp46(-) population expressed CD8 compared to total CD3(-)CD2(+) cells. Adult cattle had a significantly higher proportion of perforin(+) cells compared to calves aged

A toolbox facilitating stable transfection of Eimeria species.

Stable transfection of Eimeria species has been difficult to achieve because of the obligate requirement for in vivo amplification and selection of the parasites. Strategies to generate and stabilise populations of transfected Eimeria tenella are described here, together with the identification of optimal parameters for the transfection process. A series of plasmids expressing selectable markers, including a panel of fluorescent reporter genes and a mutant Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TSm2m3) gene that confers resistance to pyrimethamine, were electroporated into sporozoites of the E. tenella Wisconsin strain and stabilised by selective passage through chickens. Very high transfection efficiencies of up to 25% sporozoites in transient transfection and up to 9% oocysts following a single round of in vivo selection were achieved. Crucial factors include the use of very freshly harvested parasites with the AMAXA nucleofection system (program U33 in a cytomix-buffered reaction) and linearised plasmid DNA. The use of a restriction enzyme mediated integration (REMI) protocol boosted overall efficiency and elevated insertion rate per genome. Successful development of methods to generate and isolate stable populations of transfected Eimeria parasites will now stimulate rapid expansion of reverse genetic studies in this important coccidian.

Cross-reactivity of mAbs to human CD antigens with cells from cattle.

A panel of 377 commercially available mAbs were submitted to the animal homologue section of the 8th International Workshop on Human Leukocyte Differentiation Antigens (HLDA8, Adelaide, Australia) for cross-reactivity studies on different animal species. In this study we describe the results of testing the mAbs on cattle cells by flow cytometry and Western blot. Eight commercial suppliers participated, providing mAbs to a total of 144 CD antigens plus controls. Fifty-two mAbs were identified as potentially staining cattle cells in the first round screen. In the second phase, 38 mAbs were confirmed as staining cattle cells. This included some that may recognise polymorphic determinants and others with atypical distribution patterns compared to humans. mAb to human CD9, CD11a, CD14, CD18, CD21, CD23, CD29, CD44, CD45R, CD47, CD49d and CD172a cross-reacted with bovine cells and mAb to CD22, CD88, CD119 and CD163 stained CD antigens that have not previously been identified in cattle.

Workshop cluster 1+ gammadelta T-cell receptor T cells from calves express high levels of interferon-gamma in response to stimulation with interleukin-12 and -18.

Gammadelta T-cell receptor(+) T lymphocytes are an important element of the innate immune system. Early production of interferon (IFN)-gamma by gammadelta T cells may have a role in linking innate and adaptive immune responses and contribute to T helper-1 bias. We investigated the role of cytokines in the activation and induction of IFN-gamma secretion by bovine workshop cluster 1(+) (WC1(+)) gammadelta T cells. The effects of culture with interleukin (IL)-12, IL-18, IL-15 and IL-2 were investigated; these cytokines are known to influence murine and human gammadelta T cells. We report that bovine WC1(+)gammadelta T cells are synergistically stimulated by IL-12 and IL-18 to secrete large quantities of IFN-gamma. Neonatal calves were shown to have significantly higher numbers of circulating WC1(+)gammadelta T cells than adult animals. In addition, the response of peripheral blood WC1(+)gammadelta T cells was significantly higher in neonatal calves compared with adult animals. However, in adult animals the response of lymph node WC1(+)gammadelta T cells to IL-12/IL-18 was more pronounced than that of peripheral blood WC1(+)gammadelta T cells. We hypothesize that the induction of IFN-gamma secretion from WC1(+)gammadelta T cells by IL-12 and IL-18 is likely to be an important element of the innate response to pathogens such as Mycobacterium bovis. The high numbers of WC1(+)gammadelta T cells in neonatal calves, and their inherent ability to respond to inflammatory cytokines, could be a key factor in the enhanced responses seen in calves to BCG vaccination.

Summary of the animal homologue section of HLDA8.

Development of reagents against leukocyte differentiation antigens in veterinary immunology is slower compared to humans and mice. Cross-reactivity studies with monoclonal antibodies (mAb) generated against human molecules represent an excellent approach for the detection of new reagents for the minor characterised species. Three hundred seventy-seven commercially available mAb from different companies were tested for their reactivity with cells from 17 species--including non-human primates, ruminants, swine, horse, carnivores, rabbit, guinea pig, chicken and fish. In a first round of testing by flow cytometry (FCM) 182 mAb showed reactivity with atleast one of the species described above. Most of the cross-reactivity was found against non-human primate leukocytes, but also species in evolutionarily more distant from humans showed in some cases a clear staining pattern in flow cytometry (FCM). In a second round these FCM-results were confirmed by molecular analyses, by immunoprecipitation studies and analyses on transfectants. Interesting was the broad species-overlapping reactivity of mAb directed against CD9 (11 out of 17 species), CD11a (11/17), CD14 (11/17), CD18 (13/17), CD21 (7/17), CD29 (10/17), CD44 (13/17), CD45 (9/17), CD47 (10/17), and CD49d (13/17), CD61 (6/17), CD86 (7/17), CD91 (5/17), and CD172a (10/17), indicating evolutionary highly conserved epitopes on these surface molecules. Our results suggest the suitability of crossreactive mAb for the animal model studies. Moreover, these findings contribute to our understanding of the evolution of the immune system.

Phenotypic and functional characterization of intraepithelial lymphocytes in a bovine ligated intestinal loop model of enterohaemorrhagic Escherichia coli infection.

Ruminants are a major reservoir of enterohaemorrhagic Escherichia coli (EHEC), which cause acute gastroenteritis in humans with potentially life-threatening sequelae. The mechanisms underlying EHEC persistence in ruminant hosts are poorly understood. EHEC produce several cytotoxins that inhibit the proliferation of bovine lymphocytes in vitro and influence EHEC persistence in calves, suggesting that bacterial suppression of mucosal inflammation may be important in vivo. In order to address this hypothesis, intraepithelial lymphocytes (IEL) obtained from ligated intestinal loops of five 9-14 day old calves were characterized 12 h after inoculation with E. coli strains. Loops were inoculated with an EHEC O103 : H2 strain, an isogenic Deltastx1 mutant incapable of producing Shiga toxin 1 (Stx1) and a porcine non-pathogenic E. coli strain. The IEL mainly comprised activated CD2(+) CD3(+) CD6(+) CD8alpha(+) T cells and resembled IEL obtained from the intestinal mucosa of orally challenged calves. Forty per cent of all IEL were potentially sensitive to Stx1 in that they expressed the receptor for Stx1. Nevertheless, analysis of IEL from inoculated loops failed to detect a significant effect of the different E. coli strains on proliferative capacity, natural killer cell activity or the cytokine mRNA profile. However, the EHEC wild-type strain reduced the percentage of CD8alpha(+) T cells in the ileal mucosa compared with loops inoculated with the Deltastx1 mutant. This shift in IEL composition was not associated with inhibition of IEL proliferation in situ, since the majority of the IEL from all loops were in the G(0)/G(1) phase of the cell cycle. These studies indicate that the ligated ileal loop model will be a useful tool to dissect the mechanisms underlying suppression of mucosal inflammation by EHEC in the reservoir host.

A tumour necrosis factor alpha autocrine loop contributes to proliferation and nuclear factor-kappaB activation of Theileria parva-transformed B cells.

Theileria infection of bovine leucocytes induces uncontrolled proliferation and a transformed phenotype comparable to tumour cells. Infected cells have many characteristics of activated leucocytes and use autocrine loops to augment proliferation. We have shown previously that, in infected B cells, PI3-K controls a granulocyte-macrophage colony-stimulating factor (GM-CSF) autocrine loop to increase both proliferation and activation of the activator protein 1 (AP-1) transcription factor. We show here that the same infected B cells also use a tumour necrosis factor (TNF) alpha autocrine loop that again contributes to proliferation and augments nuclear factor (NF)-kappaB activation. Interestingly, both pharmacological inhibition of TNF synthesis and neutralizing anti-TNF antibodies lead to a reduction in proliferation and a 50% drop in NF-kappaB activation, without inducing apoptosis.