CD70 identifies alloreactive T cells and represents a potential target for prevention and treatment of acute GvHD.

Graft-versus-host disease (GvHD) remains a major challenge following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and further understanding of its immunopathology is crucial for developing new treatments. CD70 interacts with CD27 and is upregulated transiently on T cells following recent TCR engagement. Here we investigated the functional and clinical significance of CD70 expression on T cells during the early post-transplantation period. CD70 was expressed on a subset of highly activated memory T cells within the first 2 weeks post-transplant which then gradually declined in most patients. CD70+ T cells exhibited an open chromatin landscape and a transcriptional profile indicative of intense MYC-driven glycolysis and proliferation. CD4+ and CD8+ CD70+ T cell number increased by 9-fold and 4-fold respectively during acute GvHD (aGvHD) and displayed an oligoclonal TCR repertoire. These cells expressed CCR4 and CCR6 chemokine receptors and were markedly increased in aGvHD tissue samples. Furthermore, CD70+ T cells demonstrated alloreactive specificity in vitro and proliferative and inflammatory cytokine responses were markedly attenuated by CD70 blockade. These findings identify CD70 as a marker of highly activated alloreactive T cells and reveal the potential therapeutic importance of inhibiting CD27-CD70 co-stimulation in both the prophylaxis and treatment of aGvHD.

Interplay in galectin expression predicts patient outcomes in a spatially restricted manner in PDAC.

BACKGROUND: Galectins (Gal's) are a family of carbohydrate-binding proteins that are known to support the tumour microenvironment through their immunosuppressive activity and ability to promote metastasis. As such they are attractive therapeutic targets, but little is known about the cellular expression pattern of galectins within the tumour and its neighbouring stromal microenvironment. Here we investigated the cellular expression pattern of Gals within pancreatic ductal adenocarcinoma (PDAC). METHODS: Galectin gene and protein expression were analysed by scRNAseq (n=4) and immunofluorescence imaging (n=19) in fibroblasts and epithelial cells of pancreatic biopsies from PDAC patients. Galectin surface expression was also assessed on tumour adjacent normal fibroblasts and cancer associated primary fibroblasts from PDAC biopsies using flow cytometry. RESULTS: scRNAseq revealed higher Gal-1 expression in fibroblasts and higher Gal-3 and -4 expression in epithelial cells. Both podoplanin (PDPN+, stromal/fibroblast) cells and EpCAM+ epithelial cells expressed Gal-1 protein, with highest expression seen in the stromal compartment. By contrast, significantly more Gal-3 and -4 protein was expressed in ductal cells expressing either EpCAM or PDPN, when compared to the stroma. Ductal Gal-4 cellular expression negatively correlated with ductal Gal-1, but not Gal-3 expression. Higher ductal cellular expression of Gal-1 correlated with smaller tumour size and better patient survival. CONCLUSIONS: In summary, the intricate interplay and cell-specific expression patterns of galectins within the PDAC tissue, particularly the inverse correlation between Gal-1 and Gal-4 in ducts and its significant association with patient survival, highlights the complex molecular landscape underlying PDAC and provides valuable insights for future therapeutic interventions.

Spatial determination and prognostic impact of the fibroblast transcriptome in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma has a poor clinical outcome and responses to immunotherapy are suboptimal. Stromal fibroblasts are a dominant but heterogenous population within the tumor microenvironment and therapeutic targeting of stromal subsets may have therapeutic utility. Here, we combine spatial transcriptomics and scRNA-Seq datasets to define the transcriptome of tumor-proximal and tumor-distal cancer-associated fibroblasts (CAFs) and link this to clinical outcome. Tumor-proximal fibroblasts comprise large populations of myofibroblasts, strongly expressed podoplanin, and were enriched for Wnt ligand signaling. In contrast, inflammatory CAFs were dominant within tumor-distal subsets and expressed complement components and the Wnt-inhibitor SFRP2. Poor clinical outcome was correlated with elevated HIF-1α and podoplanin expression whilst expression of inflammatory and complement genes was predictive of extended survival. These findings demonstrate the extreme transcriptional heterogeneity of CAFs and its determination by apposition to tumor. Selective targeting of tumor-proximal subsets, potentially combined with HIF-1α inhibition and immune stimulation, may offer a multi-modal therapeutic approach for this disease.

Pancreatic cancer is one of the deadliest and most difficult cancers to treat. It responds poorly to immunotherapy for instance, despite this approach often succeeding in enlisting immune cells to fight tumours in other organs. This may be due, in part, to a type of cell called fibroblasts. Not only do these wrap pancreatic tumours in a dense, protective layer, they also foster complex relationships with the cancerous cells: some fibroblasts may fuel tumour growth, while other may help to contain its spread. These different roles may be linked to spatial location, with fibroblasts adopting different profiles depending on their proximity with cancer calls. For example, certain fibroblasts close to the tumour resemble the myofibroblasts present in healing wounds, while those at the periphery show signs of being involved in inflammation. Being able to specifically eliminate pro-cancer fibroblasts requires a better understanding of the factors that shape the role of these cells, and how to identify them. To examine this problem, Croft et al. relied on tumour samples obtained from pancreatic cancer patients. They mapped out the location of individual fibroblasts in the vicinity of the tumour and analysed their gene activity. These experiments helped to reveal the characteristics of different populations of fibroblasts. For example, they showed that the myofibroblast-like cells closest to the tumour exhibited signs of oxygen deprivation; they also produced podoplanin, a protein known to promote cancer progression. In contrast, cells further from the cancer produced more immune-related proteins. Combining these data with information obtained from patients' clinical records, Croft et al. found that samples from individuals with worse survival outcomes often featured higher levels of podoplanin and hypoxia. Inflammatory markers, however, were more likely to be present in individuals with good outcomes. Overall, these findings could help to develop ways to selectively target fibroblasts that support the growth of pancreatic cancer. Weakening these cells could in turn make the tumour accessible to immune cells, and more vulnerable to immunotherapies.

Tissue-Resident Memory T Cells in Pancreatic Ductal Adenocarcinoma Coexpress PD-1 and TIGIT and Functional Inhibition Is Reversible by Dual Antibody Blockade.

Pancreatic ductal adenocarcinoma (PDAC) has a poor clinical outlook. Responses to immune checkpoint blockade are suboptimal and a much more detailed understanding of the tumor immune microenvironment is needed if this situation is to be improved. Here, we characterized tumor-infiltrating T-cell populations in patients with PDAC using cytometry by time of flight (CyTOF) and single-cell RNA sequencing. T cells were the predominant immune cell subset observed within tumors. Over 30% of CD4+ T cells expressed a CCR6+CD161+ Th17 phenotype and 17% displayed an activated regulatory T-cell profile. Large populations of CD8+ tissue-resident memory (TRM) T cells were also present and expressed high levels of programmed cell death protein 1 (PD-1) and TIGIT. A population of putative tumor-reactive CD103+CD39+ T cells was also observed within the CD8+ tumor-infiltrating lymphocytes population. The expression of PD-1 ligands was limited largely to hemopoietic cells whilst TIGIT ligands were expressed widely within the tumor microenvironment. Programmed death-ligand 1 and CD155 were expressed within the T-cell area of ectopic lymphoid structures and colocalized with PD-1+TIGIT+ CD8+ T cells. Combinatorial anti-PD-1 and TIGIT blockade enhanced IFNγ secretion and proliferation of T cells in the presence of PD-1 and TIGIT ligands. As such, we showed that the PDAC microenvironment is characterized by the presence of substantial populations of TRM cells with an exhausted PD-1+TIGIT+ phenotype where dual checkpoint receptor blockade represents a promising avenue for future immunotherapy.

Early expression of CD94 and loss of CD96 on CD8+ T cells after allogeneic stem cell tranplantation is predictive of subsequent relapse and survival.

Allogeneic stem cell transplantation is used widely in the treatment of hematopoietic malignancy. However, relapse of malignant disease is the primary cause of treatment failure and reflects loss of immunological graft-versus-leukemia effect. We studied the transcriptional and phenotypic profile of CD8+ T cells in the first month following transplantation and related this to risk of subsequent relapse. Single cell transcriptional profiling identified five discrete CD8+ T-cell clusters. High levels of T-cell activation and acquisition of a regulatory transcriptome were apparent in patients who went on to suffer disease relapse. A relapse-associated gene signature of 47 genes was then assessed in a confirmation cohort of 34 patients. High expression of the inhibitory receptor CD94/NKG2A on CD8+ T cells within the first month was associated with 4.8 fold increased risk of relapse and 2.7 fold reduction in survival. Furthermore, reduced expression of the activatory molecule CD96 was associated with 2.2 fold increased risk of relapse and 1.9 fold reduction in survival. This work identifies CD94 and CD96 as potential targets for CD8-directed immunotherapy in the very early phase following allogeneic transplantation with the potential to reduce long term relapse rates and improve patient survival.

Early inflammatory markers as prognostic indicators following allogeneic stem cell transplantation.

Allogeneic stem cell transplantation is used widely in the treatment of hematopoietic malignancy although graft versus host disease and relapse remain major complications. We measured the serum protein expression of 92 inflammation-related markers from 49 patients at Day 0 (D0) and 154 patients at Day 14 (D14) following transplantation and related values to subsequent clinical outcomes. Low levels of 7 proteins at D0 were linked to GvHD whilst high levels of 7 proteins were associated with relapse. The concentration of 38 proteins increased over 14 days and higher inflammatory response at D14 was strongly correlated with patient age. A marked increment in protein concentration during this period associated with GvHD but reduced risk of disease relapse, indicating a link with alloreactive immunity. In contrast, patients who demonstrated low dynamic elevation of inflammatory markers during the first 14 days were at increased risk of subsequent disease relapse. Multivariate time-to-event analysis revealed that high CCL23 at D14 was associative of AGvHD, CXCL10 with reduced rate of relapse, and high PD-L1 with reduced overall survival. This work identifies a dynamic pattern of inflammatory biomarkers in the very early post-transplantation period and reveals early protein markers that may help to guide patient management.

The single cell transcriptional landscape of esophageal adenocarcinoma and its modulation by neoadjuvant chemotherapy.

Immune checkpoint blockade has recently proven effective in subsets of patients with esophageal adenocarcinoma (EAC) but little is known regarding the EAC immune microenvironment. We determined the single cell transcriptional profile of EAC in 8 patients who were treatment-naive (n = 4) or had received neoadjuvant chemotherapy (n = 4). Analysis of 52,387 cells revealed 10 major cell subsets of tumor, immune and stromal cells. Prior to chemotherapy tumors were heavy infiltrated by T regulatory cells and exhausted effector T cells whilst plasmacytoid dendritic cells were markedly expanded. Two dominant cancer-associated fibroblast populations were also observed whilst endothelial populations were suppressed. Pathological remission following chemotherapy associated with broad reversal of immune abnormalities together with fibroblast transition and an increase in endothelial cells whilst a chemoresistant epithelial stem cell population correlated with poor response. These findings reveal features that underlie and limit the response to current immunotherapy and identify a range of novel opportunities for targeted therapy.

Machine learning of COVID-19 clinical data identifies population structures with therapeutic potential.

Clinical outcomes for patients with COVID-19 are heterogeneous and there is interest in defining subgroups for prognostic modeling and development of treatment algorithms. We obtained 28 demographic and laboratory variables in patients admitted to hospital with COVID-19. These comprised a training cohort (n = 6099) and two validation cohorts during the first and second waves of the pandemic (n = 996; n = 1011). Uniform manifold approximation and projection (UMAP) dimension reduction and Gaussian mixture model (GMM) analysis was used to define patient clusters. 29 clusters were defined in the training cohort and associated with markedly different mortality rates, which were predictive within confirmation datasets. Deconvolution of clinical features within clusters identified unexpected relationships between variables. Integration of large datasets using UMAP-assisted clustering can therefore identify patient subgroups with prognostic information and uncovers unexpected interactions between clinical variables. This application of machine learning represents a powerful approach for delineating disease pathogenesis and potential therapeutic interventions.

DNA and modified vaccinia Ankara prime-boost vaccination generates strong CD8+ T cell responses against minor histocompatibility antigen HA-1.

Allogeneic immune responses underlie the graft-versus-leukaemia effect of stem cell transplantation, but disease relapse occurs in many patients. Minor histocompatibility antigen (mHAg) peptides mediate alloreactive T cell responses and induce graft-versus-leukaemia responses when expressed on patient haematopoietic tissue. We vaccinated nine HA-1-negative donors against HA-1 with a 'prime-boost' protocol of either two or three DNA 'priming' vaccinations prior to 'boost' with modified vaccinia Ankara (MVA). HA-1-specific CD8+ T cell responses were observed in seven donors with magnitude up to 1·5% of total CD8+ T cell repertoire. HA-1-specific responses peaked two weeks post-MVA challenge and were measurable in most donors after 12 months. HA-1-specific T cells demonstrated strong cytotoxic activity and lysed target cells with endogenous HA-1 protein expression. The pattern of T cell receptor (TCR) usage by HA-1-specific T cells revealed strong conservation of T cell receptor beta variable 7-9 (TRBV7-9) usage between donors. These findings describe one of the strongest primary peptide-specific CD8+ T cell responses yet recorded to a DNA-MVA prime-boost regimen and this may reflect the strong immunogenicity of mHAg peptides. Prime-boost vaccination in donors or patients may prove of substantial benefit in boosting graft-versus-leukaemia responses.

CD52/GPI- T-Cells Are Enriched for Alloreactive Specificity and Predict Acute Graft-Versus-Host-Disease After Stem Cell Transplantation.

Alemtuzumab is a CD52-specific lympho-depleting antibody. CD52- T cells emerge under alemtuzumab selection pressure. We sought to investigate the phenotype and function of the CD52- T cell fraction and related their presence to clinical outcome. We obtained longitudinal peripheral blood samples from 67 consecutive patients undergoing allo-HSCT between 2013-2016. Forty-seven patients (70%) had a myeloid disease (acute myelogenous leukemia or myelodysplastic syndrome) whereas 20 patients had lymphoid disease. All patients received in vivo alemtuzumab (10 mg/d from day -5 for 5 days) as part of their conditioning protocol. Sixty-three (94%) received reduced-intensity conditioning chemotherapy, whereas 4 (6%) received a myeloablative regimen. All patients received post-transplantation cyclosporine A for graft-versus-host disease (GVHD) prophylaxis. Six (9%) also received methotrexate, whereas 2 (3%) patients also received mycophenolate mofetil. Overall survival at 2 years was 68%, and relapse-free survival was 48%. Twenty-none percent of patients experienced acute GVHD (grade 2 or above), and 15% developed chronic GVHD. CD52- T cells were detectable in 66 of 67 consecutive patients. CD52- T cells demonstrated low binding of fluorescent aerolysin, indicating downregulation of the glycophosphatidylinositol anchor, although we did not detect any mutations in the PIG-A gene as is typically seen in patients with paroxysmal nocturnal hemoglobinuria. CD52- T cells were almost exclusively CD4+ and exhibited a dominant memory phenotype with only small numbers of CD25+ CD127low Foxp3+ regulatory T cells. CD52- T cells exhibited alloreactive specificity in vitro and have a distinct TCR repertoire to CD52+ T cells. Early after allo-hematopoietic stem cell transplantation, the presence of a significant population of CD52- T cells (comprising >51% of the T cell fraction) was found to be an independent risk factor for acute GvHD. This was confirmed in a validation cohort of 28 patients obtained between 2017-2018. These data suggest that the CD52- T cell fraction may represent a residual "footprint" of an early CD4+ T cell alloreactive response and may have been rescued from alemtuzumab-mediated lysis by antigen engagement in vivo. These data help to delineate the nature of T cell escape from alemtuzumab surveillance and contribute to increasing interest in the importance of CD4+ T cells in alloreactive immune responses, which could help inform immunotherapy protocols.

PD-1 is imprinted on cytomegalovirus-specific CD4+ T cells and attenuates Th1 cytokine production whilst maintaining cytotoxicity.

PD-1 is expressed on exhausted T cells in cancer patients but its physiological role remains uncertain. We determined the phenotype, function and transcriptional correlates of PD-1 expression on cytomegalovirus-specific CD4+ T cells during latent infection. PD-1 expression ranged from 10-85% and remained stable over time within individual donors. This 'setpoint' was correlated with viral load at primary infection. PD-1+ CD4+ T cells display strong cytotoxic function but generate low levels of Th1 cytokines which is only partially reversed by PD-1 blockade. TCR clonotypes showed variable sharing between PD-1+ and PD-1- CMV-specific cells indicating that PD-1 status is defined either during T cell priming or subsequent clonal expansion. Physiological PD-1+ CD4+ T cells therefore display a unique 'high cytotoxicity-low cytokine' phenotype and may act to suppress viral reactivation whilst minimizing tissue inflammation. Improved understanding of the physiological role of PD-1 will help to delineate the mechanisms, and potential reversal, of PD-1+ CD4+ T cell exhaustion in patients with malignant disease.

Homeostatic Cytokines Drive Epigenetic Reprogramming of Activated T Cells into a "Naive-Memory" Phenotype.

Primary stimulation of T cells is believed to trigger unidirectional differentiation from naive to effector and memory subsets. Here we demonstrate that IL-7 can drive the phenotypic reversion of recently differentiated human central and effector memory CD8+ T cells into a naive-like phenotype. These "naive-revertant" cells display a phenotype similar to that of previously reported stem cell memory populations and undergo rapid differentiation and functional response following secondary challenge. The chromatin landscape of reverted cells undergoes substantial epigenetic reorganization with increased accessibility for cytokine-induced mediators such as STAT and closure of BATF-dependent sites that drive terminal differentiation. Phenotypic reversion may at least partly explain the generation of "stem cell memory" CD8+ T cells and reveals cells within the phenotypically naive CD8+ T cell pool that are epigenetically primed for secondary stimulation. This information provides insight into mechanisms that support maintenance of T cell memory and may guide therapeutic manipulation of T cell differentiation.

CD117 (c-Kit) Is Expressed During CD8+ T Cell Priming and Stratifies Sensitivity to Apoptosis According to Strength of TCR Engagement.

CD117 (cKit) is the receptor for stem cell factor (SCF) and plays an important role in early haemopoiesis. We show that CD117 is also expressed following priming of mature human CD8+ T cells in vitro and is detectable following primary infection in vivo. CD117 expression is mediated through an intrinsic pathway and is suppressed by IL-12. Importantly, the extent of CD117 expression is inversely related to the strength of the activating stimulus and subsequent engagement with cell-bound SCF markedly increases susceptibility to apoptosis. CD117 is therefore likely to shape the pattern of CD8+ T cell immunodominance during a primary immune response by rendering cells with low avidity for antigen more prone to apoptosis. Furthermore, CD117+ T cells are highly sensitive to apoptosis mediated by galectin-1, a molecule commonly expressed within the tumor microenvironment, and CD117 expression may therefore represent a novel and potentially targetable mechanism of tumor immune evasion.

NK cells produce high levels of IL-10 early after allogeneic stem cell transplantation and suppress development of acute GVHD.

Natural killer (NK) cells rapidly reconstitute following allogeneic stem cell transplantation (allo-SCT), at the time when alloreactive T cell immunity is being established. We investigated very early NK cell reconstitution in 82 patients following T cell-depleted allo-SCT. NK cell number rapidly increased, exceeding T cell reconstitution such that the NK:T cell ratio was over 40 by day 14. NK cells at day 14 (NK-14) were donor-derived, intensely proliferating and expressed chemokine receptors targeted to lymphoid and peripheral tissue. Spontaneous production of the immunoregulatory cytokine IL-10 was observed in over 70% of cells and transcription of cytokines and growth factors was augmented. NK-14 cell number was inversely correlated with the incidence of grade II-IV acute graft versus host disease (GVHD). These findings reveal that robust reconstitution of immunoregulatory NK cells by day 14 after allo-SCT is an important determinant of the clinical outcome, suggesting that NK cells may suppress the development of the T cell-mediated alloreactive immune response through production of IL-10.

A Bayesian approach to modelling heterogeneous calcium responses in cell populations.

Calcium responses have been observed as spikes of the whole-cell calcium concentration in numerous cell types and are essential for translating extracellular stimuli into cellular responses. While there are several suggestions for how this encoding is achieved, we still lack a comprehensive theory. To achieve this goal it is necessary to reliably predict the temporal evolution of calcium spike sequences for a given stimulus. Here, we propose a modelling framework that allows us to quantitatively describe the timing of calcium spikes. Using a Bayesian approach, we show that Gaussian processes model calcium spike rates with high fidelity and perform better than standard tools such as peri-stimulus time histograms and kernel smoothing. We employ our modelling concept to analyse calcium spike sequences from dynamically-stimulated HEK293T cells. Under these conditions, different cells often experience diverse stimulus time courses, which is a situation likely to occur in vivo. This single cell variability and the concomitant small number of calcium spikes per cell pose a significant modelling challenge, but we demonstrate that Gaussian processes can successfully describe calcium spike rates in these circumstances. Our results therefore pave the way towards a statistical description of heterogeneous calcium oscillations in a dynamic environment.

Probabilistic encoding of stimulus strength in astrocyte global calcium signals.

Astrocyte calcium signals can range in size from subcellular microdomains to waves that spread through the whole cell (and into connected cells). The differential roles of such local or global calcium signaling are under intense investigation, but the mechanisms by which local signals evolve into global signals in astrocytes are not well understood, nor are the computational rules by which physiological stimuli are transduced into a global signal. To investigate these questions, we transiently applied receptor agonists linked to calcium signaling to primary cultures of cerebellar astrocytes. Astrocytes repetitively tested with the same stimulus responded with global signals intermittently, indicating that each stimulus had a defined probability for triggering a response. The response probability varied between agonists, increased with agonist concentration, and could be positively and negatively modulated by crosstalk with other signaling pathways. To better understand the processes determining the evolution of a global signal, we recorded subcellular calcium "puffs" throughout the whole cell during stimulation. The key requirement for puffs to trigger a global calcium wave following receptor activation appeared to be the synchronous release of calcium from three or more sites, rather than an increasing calcium load accumulating in the cytosol due to increased puff size, amplitude, or frequency. These results suggest that the concentration of transient stimuli will be encoded into a probability of generating a global calcium response, determined by the likelihood of synchronous release from multiple subcellular sites.

Plasticity of Neuron-Glial Transmission: Equipping Glia for Long-Term Integration of Network Activity.

The capacity of synaptic networks to express activity-dependent changes in strength and connectivity is essential for learning and memory processes. In recent years, glial cells (most notably astrocytes) have been recognized as active participants in the modulation of synaptic transmission and synaptic plasticity, implicating these electrically nonexcitable cells in information processing in the brain. While the concept of bidirectional communication between neurons and glia and the mechanisms by which gliotransmission can modulate neuronal function are well established, less attention has been focussed on the computational potential of neuron-glial transmission itself. In particular, whether neuron-glial transmission is itself subject to activity-dependent plasticity and what the computational properties of such plasticity might be has not been explored in detail. In this review, we summarize current examples of plasticity in neuron-glial transmission, in many brain regions and neurotransmitter pathways. We argue that induction of glial plasticity typically requires repetitive neuronal firing over long time periods (minutes-hours) rather than the short-lived, stereotyped trigger typical of canonical long-term potentiation. We speculate that this equips glia with a mechanism for monitoring average firing rates in the synaptic network, which is suited to the longer term roles proposed for astrocytes in neurophysiology.

Parameter identification problems in the modelling of cell motility.

We present a novel parameter identification algorithm for the estimation of parameters in models of cell motility using imaging data of migrating cells. Two alternative formulations of the objective functional that measures the difference between the computed and observed data are proposed and the parameter identification problem is formulated as a minimisation problem of nonlinear least squares type. A Levenberg-Marquardt based optimisation method is applied to the solution of the minimisation problem and the details of the implementation are discussed. A number of numerical experiments are presented which illustrate the robustness of the algorithm to parameter identification in the presence of large deformations and noisy data and parameter identification in three dimensional models of cell motility. An application to experimental data is also presented in which we seek to identify parameters in a model for the monopolar growth of fission yeast cells using experimental imaging data. Our numerical tests allow us to compare the method with the two different formulations of the objective functional and we conclude that the results with both objective functionals seem to agree.

Cellular mRNAs access second ORFs using a novel amino acid sequence-dependent coupled translation termination-reinitiation mechanism.

Polycistronic transcripts are considered rare in the human genome. Initiation of translation of internal ORFs of eukaryotic genes has been shown to use either leaky scanning or highly structured IRES regions to access initiation codons. Studies on mammalian viruses identified a mechanism of coupled translation termination-reinitiation that allows translation of an additional ORF. Here, the ribosome terminating translation of ORF-1 translocates upstream to reinitiate translation of ORF-2. We have devised an algorithm to identify mRNAs in the human transcriptome in which the major ORF-1 overlaps a second ORF capable of encoding a product of at least 50 aa in length. This identified 4368 transcripts representing 2214 genes. We investigated 24 transcripts, 22 of which were shown to express a protein from ORF-2 highlighting that 3' UTRs contain protein-coding potential more frequently than previously suspected. Five transcripts accessed ORF-2 using a process of coupled translation termination-reinitiation. Analysis of one transcript, encoding the CASQ2 protein, showed that the mechanism by which the coupling process of the cellular mRNAs was achieved was novel. This process was not directed by the mRNA sequence but required an aspartate-rich repeat region at the carboxyl terminus of the terminating ORF-1 protein. Introduction of wobble mutations for the aspartate codon had no effect, whereas replacing aspartate for glutamate repeats eliminated translational coupling. This is the first description of a coordinated expression of two proteins from cellular mRNAs using a coupled translation termination-reinitiation process and is the first example of such a process being determined at the amino acid level.