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

ABSTRACT: Graft-versus-host disease (GVHD) remains a major challenge after 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 after recent T-cell receptor (TCR) engagement. Here, we investigated the functional and clinical significance of CD70 expression on T cells during the early posttransplantation period. CD70 was expressed on a subset of highly activated memory T cells within the first 2 weeks after transplant, which then gradually declined in most patients. CD70+ T cells exhibited an open chromatin landscape and a transcriptional profile indicative of intense Myelocytomatosis oncogene (MYC)-driven glycolysis and proliferation. CD4+ and CD8+CD70+ T-cell numbers increased by ninefold and fourfold, 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 costimulation in both the prophylaxis and treatment of aGVHD.

Age differentially impacts adaptive immune responses induced by adenoviral versus mRNA vaccines against COVID-19.

Adenoviral and mRNA vaccines encoding the viral spike (S) protein have been deployed globally to contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Older individuals are particularly vulnerable to severe infection, probably reflecting age-related changes in the immune system, which can also compromise vaccine efficacy. It is nonetheless unclear to what extent different vaccine platforms are impacted by immunosenescence. Here, we evaluated S protein-specific immune responses elicited by vaccination with two doses of BNT162b2 or ChAdOx1-S and subsequently boosted with a single dose of BNT162b2 or mRNA-1273, comparing age-stratified participants with no evidence of previous infection with SARS-CoV-2. We found that aging profoundly compromised S protein-specific IgG titers and further limited S protein-specific CD4+ and CD8+ T cell immunity as a probable function of progressive erosion of the naive lymphocyte pool in individuals vaccinated initially with BNT162b2. Our results demonstrate that primary vaccination with ChAdOx1-S and subsequent boosting with BNT162b2 or mRNA-1273 promotes sustained immunological memory in older adults and potentially confers optimal protection against coronavirus disease 2019.

Safety of a controlled human infection model of tuberculosis with aerosolised, live-attenuated Mycobacterium bovis BCG versus intradermal BCG in BCG-naive adults in the UK: a dose-escalation, randomised, controlled, phase 1 trial.

BACKGROUND: Mycobacterium tuberculosis is the main causative agent of tuberculosis. BCG, the only licensed vaccine, provides inadequate protection against pulmonary tuberculosis. Controlled human infection models are useful tools for vaccine development. We aimed to determine a safe dose of aerosol-inhaled live-attenuated Mycobacterium bovis BCG as a surrogate for M tuberculosis infection, then compare the safety and tolerability of infection models established using aerosol-inhaled and intradermally administered BCG. METHODS: This phase 1 controlled human infection trial was conducted at two clinical research facilities in the UK. Healthy, immunocompetent adults aged 18-50 years, who were both M tuberculosis-naive and BCG-naive and had no history of asthma or other respiratory diseases, were eligible for the trial. Participants were initially enrolled into group 1 (receiving the BCG Danish strain); the trial was subsequently paused because of a worldwide shortage of BCG Danish and, after protocol amendment, was restarted using the BCG Bulgaria strain (group 2). After a dose-escalation study, during which participants were sequentially allocated to receive either 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 colony-forming units (CFU) of aerosol BCG, the maximum tolerated dose was selected for the randomised controlled trial. Participants in this trial were randomly assigned (9:12), by variable block randomisation and using sequentially numbered sealed envelopes, to receive aerosol BCG (1 × 107 CFU) and intradermal saline or intradermal BCG (1 × 106 CFU) and aerosol saline. Participants were masked to treatment allocation until day 14. The primary outcome was to compare the safety of a controlled human infection model based on aerosol-inhaled BCG versus one based on intradermally administered BCG, and the secondary outcome was to evaluate BCG recovery in the airways of participants who received aerosol BCG or skin biopsies of participants who received intradermal BCG. BCG was detected by culture and by PCR. The trial is registered at ClinicalTrials.gov, NCT02709278, and is complete. FINDINGS: Participants were assessed for eligibility between April 7, 2016, and Sept 29, 2018. For group 1, 15 participants were screened, of whom 13 were enrolled and ten completed the study; for group 2, 60 were screened and 33 enrolled, all of whom completed the study. Doses up to 1 × 107 CFU aerosol-inhaled BCG were sufficiently well tolerated. No significant difference was observed in the frequency of adverse events between aerosol and intradermal groups (median percentage of solicited adverse events per participant, post-aerosol vs post-intradermal BCG: systemic 7% [IQR 2-11] vs 4% [1-13], p=0·62; respiratory 7% [1-19] vs 4% [1-9], p=0·56). More severe systemic adverse events occurred in the 2 weeks after aerosol BCG (15 [12%] of 122 reported systemic adverse events) than after intradermal BCG (one [1%] of 94; difference 11% [95% CI 5-17]; p=0·0013), but no difference was observed in the severity of respiratory adverse events (two [1%] of 144 vs zero [0%] of 97; 1% [-1 to 3]; p=0·52). All adverse events after aerosol BCG resolved spontaneously. One serious adverse event was reported-a participant in group 2 was admitted to hospital to receive analgesia for a pre-existing ovarian cyst, which was deemed unrelated to BCG infection. On day 14, BCG was cultured from bronchoalveolar lavage samples after aerosol infection and from skin biopsy samples after intradermal infection. INTERPRETATION: This first-in-human aerosol BCG controlled human infection model was sufficiently well tolerated. Further work will evaluate the utility of this model in assessing vaccine efficacy and identifying potential correlates of protection. FUNDING: Bill & Melinda Gates Foundation, Wellcome Trust, National Institute for Health Research Oxford Biomedical Research Centre, Thames Valley Clinical Research Network, and TBVAC2020.

Involvement of the Gut Microbiome in the Local and Systemic Immune Response to Pancreatic Ductal Adenocarcinoma.

The systemic and local immunosuppression exhibited by pancreatic ductal adenocarcinoma (PDAC) contributes significantly to its aggressive nature. There is a need for a greater understanding of the mechanisms behind this profound immune evasion, which makes it one of the most challenging malignancies to treat and thus one of the leading causes of cancer death worldwide. The gut microbiome is now thought to be the largest immune organ in the body and has been shown to play an important role in multiple immune-mediated diseases. By summarizing the current literature, this review examines the mechanisms by which the gut microbiome may modulate the immune response to PDAC. Evidence suggests that the gut microbiome can alter immune cell populations both in the peripheral blood and within the tumour itself in PDAC patients. In addition, evidence suggests that the gut microbiome influences the composition of the PDAC tumour microbiome, which exerts a local effect on PDAC tumour immune infiltration. Put together, this promotes the gut microbiome as a promising route for future therapies to improve immune responses in PDAC patients.

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.

CD36 regulates macrophage and endothelial cell activation and multinucleate giant cell formation in anti neutrophil cytoplasm antibody vasculitis.

OBJECTIVE: To investigate CD36 in ANCA-associated vasculitis (AAV), a condition characterized by monocyte/macrophage activation and vascular damage. METHODS: CD36 expression was assessed in AAV patients and healthy controls (HC). The impact of palmitic acid (PA) stimulation on multinucleate giant cell (MNGC) formation, macrophage, and endothelial cell activation, with or without CD36 knockdown, was examined. RESULTS: CD36 was overexpressed on AAV patients' monocytes compared to HC, regardless of disease activity. AAV patients exhibited elevated soluble CD36 levels in serum and plasma and PR3-ANCA patients' monocytes demonstrated increased MNGC formation following PA stimulation compared to HC. PA stimulation of macrophages or endothelial cells resulted in heightened CD36 expression, cell activation, increased macrophage migration inhibitory factor (MIF) production, and c-Myc expression, with attenuation upon CD36 knockdown. CONCLUSION: CD36 participates in macrophage and endothelial cell activation and MNGC formation, features of AAV pathogenesis. AAV treatment may involve targeting CD36 or MIF.

Impact of COVID-19 on immunocompromised populations during the Omicron era: insights from the observational population-based INFORM study.

Background: Immunocompromised individuals are not optimally protected by COVID-19 vaccines and potentially require additional preventive interventions to mitigate the risk of severe COVID-19. We aimed to characterise and describe the risk of severe COVID-19 across immunocompromised groups as the pandemic began to transition to an endemic phase. Methods: COVID-19-related hospitalisations, intensive care unit (ICU) admissions, and deaths (01/01/2022-31/12/2022) were compared among different groups of immunocompromised individuals vs the general population, using a retrospective cohort design and electronic health data from a random 25% sample of the English population aged ≥12 years (Registration number: ISRCTN53375662). Findings: Overall, immunocompromised individuals accounted for 3.9% of the study population, but 22% (4585/20,910) of COVID-19 hospitalisations, 28% (125/440) of COVID-19 ICU admissions, and 24% (1145/4810) of COVID-19 deaths in 2022. Restricting to those vaccinated with ≥3 doses of COVID-19 vaccine (∼84% of immunocompromised and 51% of the general population), all immunocompromised groups remained at increased risk of severe COVID-19 outcomes, with adjusted incidence rate ratios (aIRR) for hospitalisation ranging from 1.3 to 13.1. At highest risk for COVID-19 hospitalisation were individuals with: solid organ transplant (aIRR 13.1, 95% confidence interval [95% CI] 11.2-15.3), moderate to severe primary immunodeficiency (aIRR 9.7, 95% CI 6.3-14.9), stem cell transplant (aIRR 11.0, 95% CI 6.8-17.6), and recent treatment for haematological malignancy (aIRR 10.6, 95% CI 9.5-11.9). Results were similar for COVID-19 ICU admissions and deaths. Interpretation: Immunocompromised individuals continue to be impacted disproportionately by COVID-19 and have an urgent need for additional preventive measures beyond current vaccination programmes. These data can help determine the immunocompromised groups for which targeted prevention strategies may have the highest impact. Funding: This study was funded by AstraZeneca UK.

Pancreatic Exocrine Insufficiency and the Gut Microbiome in Pancreatic Cancer: A Target for Future Diagnostic Tests and Therapies?

Pancreatic exocrine insufficiency (PEI) is common amongst pancreatic cancer patients and is associated with poorer treatment outcomes. Pancreatic enzyme replacement therapy (PERT) is known to improve outcomes in pancreatic cancer, but the mechanisms are not fully understood. The aim of this narrative literature review is to summarise the current evidence linking PEI with microbiome dysbiosis, assess how microbiome composition may be impacted by PERT treatment, and look towards possible future diagnostic and therapeutic targets in this area. Early evidence in the literature reveals that there are complex mechanisms by which pancreatic secretions modulate the gut microbiome, so when these are disturbed, as in PEI, gut microbiome dysbiosis occurs. PERT has been shown to return the gut microbiome towards normal, so called rebiosis, in animal studies. Gut microbiome dysbiosis has multiple downstream effects in pancreatic cancer such as modulation of the immune response and the response to chemotherapeutic agents. It therefore represents a possible future target for future therapies. In conclusion, it is likely that the gut microbiome of pancreatic cancer patients with PEI exhibits dysbiosis and that this may potentially be reversible with PERT. However, further human studies are required to determine if this is indeed the case.

Protocol for a multicentre randomised controlled trial examining the effects of temporarily pausing Bruton tyrosine kinase inhibitor therapy to coincide with SARS-CoV-2 vaccination and its impact on immune responses in patients with chronic lymphocytic leukaemia.

INTRODUCTION: People who are immunocompromised have a poor biological response to vaccinations. This study aims to determine in patients with chronic lymphocytic leukaemia (CLL) if a 3-week pause in Bruton tyrosine kinase inhibitor therapy (BTKi) starting 1 week before delivery of SARS-CoV-2 vaccine booster, improves vaccine immune response when compared with continuation of BTKi. METHODS AND ANALYSIS: An open-label, randomised controlled superiority trial will be conducted in haematology clinics in approximately 10 UK National Health Service (NHS) hospitals. The sample size is 120, randomised 1:1 to intervention and usual care arms. The primary outcome is anti-spike-receptor binding domain (RBD) antibody level at 3 weeks post-SARS-CoV-2 booster vaccination. Secondary outcomes are RBD antibody levels at 12 weeks postbooster vaccination, participant global assessments of disease activity, blood films, full blood count and lactate dehydrogenase levels, impact on quality of life, self-reported adherence with request to temporarily pause or continue BTKi, T cell response against spike protein and relative neutralising antibody titre against SARS-CoV-2 viral variants. Additionally, there will be an investigation of any effects in those given influenza vaccination contemporaneously versus COVID-19 alone.The primary analysis will be performed on the as randomised groups ('intention to treat'). The difference between the study arms in anti-spike-RBD antibody level will be estimated using a mixed effects regression model, allowing for repeated measures clustered within participants. The model will be adjusted for randomisation factor (first line or subsequent line of therapy), and prior infection status obtained from prerandomisation antinucleocapsid antibodies as fixed effects. ETHICS AND DISSEMINATION: This study has been approved by Leeds East Research Ethics Committee and Health Research Authority (REC Reference:22/YH/0226, IRAS ID: 319057). Dissemination will be via peer-review publications, newsletters and conferences. Results will be communicated to participants, the CLL patient and clinical communities and health policy-makers. TRIAL REGISTRATION NUMBER: ISRCTN14197181.

Defective T-cell response to COVID-19 vaccination in acute myeloid leukaemia and myelodysplastic syndromes.

Limited data exist on COVID-19 vaccination efficacy in patients with acute myeloid leukemia and myelodysplasia with excess blasts (AML/MDS-EB2). We report results from a prospective study, PACE (Patients with AML and COVID-19 Epidemiology). 93 patients provided samples post-vaccine 2 or 3 (PV2, PV3). Antibodies against SARS-COV-2 spike antigen were detectable in all samples. Neutralization of the omicron variant was poorer than ancestral variants but improved PV3. In contrast, adequate T-cell reactivity to SARS-COV-2 spike protein was seen in only 16/47 (34%) patients PV2 and 23/52 (44%) PV3. Using regression models, disease response (not in CR/Cri), and increasing age predicted poor T cell response.

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.

CMV reactivation initiates long-term expansion and differentiation of the NK cell repertoire.

Introduction: NK cells play an important role in suppression of viral replication and are critical for effective control of persistent infections such as herpesviruses. Cytomegalovirus infection is associated with expansion of 'adaptive-memory' NK cells with a characteristic CD56dimCD16bright NKG2C+ phenotype but the mechanisms by which this population is maintained remain uncertain. Methods: We studied NK cell reconstitution in patients undergoing haemopoietic stem cell transplantation and related this to CMV reactivation. Results: NK cells expanded in the early post-transplant period but then remained stable in the absence of viral reactivation. However, CMV reactivation led to a rapid and sustained 10-fold increase in NK cell number. The proportion of NKG2C-expressing cells increases on all NK subsets although the kinetics of expansion peaked at 6 months on immature CD56bright cells whilst continuing to rise on the mature CD56dim pool. Phenotypic maturation was observed by acquisition of CD57 expression. Effective control of viral reactivation was seen when the peripheral NK cell count reached 20,000/ml. Discussion: These data show that short term CMV reactivation acts to reprogramme hemopoiesis to drive a sustained modulation and expansion of the NK cell pool and reveal further insight into long term regulation of the innate immune repertoire by infectious challenge.

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.