Pulmonary influenza infection promotes the awakening of dormant metastatic breast cancer cells

Breast cancer is the most common form of cancer and the second cancer-causing death in females. Although remission rates are high if detected early, survival rates drop substantially when breast cancer becomes metastatic. The most common sites of metastatic breast cancer are bone, liver and lung. Respiratory viral infections inflict illnesses on countless people. The latest pandemic caused by the respiratory virus, SARS-CoV-2, has infected more than 600 million worldwide, with documented COVID-related death upward of 1 million in the United States alone. Respiratory viral infections result in increased inflammation with immune cell influx and expansion to facilitate viral clearance. Prior studies have shown that inflammation, including through neutrophils, can contribute to dormant cancer cells reawakening and outgrowth. Moreover, inhibition of IL6 has been shown to decrease breast cancer lung metastasis in mouse models. However, how respiratory viral infections contribute to breast cancer lung metastasis remains to be unraveled. Using MMTV/PyMT and MMTV/NEU mouse models of breast cancer lung metastasis and influenza A virus as a model respiratory virus, we demonstrated that acute influenza infection and the accompanying inflammation and immune cell influx awakens and dramatically increased proliferation and expansion of dormant disseminated cancer cells (DCC) in the lungs. Acute influenza infection leads to immune influx and expansion, including neutrophils and macrophages, with increased proportion of MHCII+ macrophages in early time points, and a sustained decrease in CD206+ macrophages starting 6 days post-infection until 28 days after the initial infection. Additionally, we observed a sustained accumulation of CD4+ T cells around expanding tumor cells for as long as 28 days after the infection. Notably, neutrophil depletion or IL6 knockout reversed the flu-induced dormant cell expansion in the lung. Finally, awakened DCC exhibited downregulation of vimentin immunoreactivity, suggesting a role for phenotypic plasticity in DCC outgrowth following viral infection. In conclusion, we show that respiratory viral infections awaken and increase proliferation of dormant breast cancer cells in the lung, and that depletion of neutrophils or blocking IL6 reverses influenza-induced dormant cell awakening and proliferation.

Expansion of human T cells using xenogeneic free and gamma irradiated human platelet lysate

Commercially available human platelet lysate (hPL) is produced using expired human platelets obtained from accredited blood banks in the United States. These platelets were originally intended for use in patient transfusion. The safety of platelets used in transfusion is managed by the U.S. Food Drug Administration (FDA), as well as the American Association of Blood Banks (AABB). These organizations set standards, including testing for transmissible diseases. The United States record for blood safety is well established, with extremely low rates of disease transmission, making the platelet units used for hPL manufacture low risk. The Covid-19 pandemic has increased awareness of emerging infectious diseases, even though transmission of Covid-19 via blood transfusion has not been documented. For that reason, gamma irradiated hPL offers an additional safety measure in the clinic. Chimeric Antigen Receptor (CAR) expressing T-cells have demonstrated potent clinical efficacy in patients with hematological malignancies. In addition, there are several phase I clinical trials evaluating the use of CAR-T-cells for targeting of solid tumorassociated antigens. Some of the challenging issues found during production of CAR-T cells are the efficiency of T cell transduction to generate CAR-T cells, the expansion of T cells to clinically relevant numbers and the long-term survival in vivo of the therapeutic cells. The use of human platelet lysate has been demonstrated to improve these issues. Our data from experiments performed using human CD3+ from donors demonstrates that human platelet lysates offer an improved performance on T cell expansion versus serum derived products. hPL efficiently promotes T cell expansion, with higher cell yields and lower cell exhaustion rate. Additionally, we efficiently developed a protocol for suspension culture of T cells, which could facilitate the large-scale expansion of allogeneic CAR-T cells.

Development of a Lymphoid Organ-Chip to Evaluate Covid Vaccine Boosting Strategies

Background: Secondary lymphoid organs provide the adequate microenvironment for the development of antigen (Ag)-specific immune responses. The tight collaboration between CD4+ T cells and B cells in germinal centers is crucial to shape B cell fate and optimize antibody maturation. Dissecting these immune interactions remains challenging in humans, and animal models do not always recapitulate human physiology. To address this issue, we developed an in vitro 3D model of a human lymphoid organ. The model relies on a microfluidic device, enabling primary human cells to self-organize in an extracellular matrix (ECM) under continuous fluid perfusion. We applied this Lymphoid Organ-Chip (LO chip) system to the analysis of B cell recall responses to SARS-CoV-2 antigens. Method(s): We used a two-channel microfluidic Chip S1 from Emulate, where the top channel is perfused with antigen (spike protein or SARS-CoV-2 mRNA vaccine), while the bottom channel contains PBMC (n = 14 independent donors) seeded at high-density in a collagen-based ECM. Immune cell division and cluster formation were monitored by confocal imaging, plasmablast differentiation and spike-specific B cell amplification by flow cytometry, antibody secretion by a cell-based binding assay (S-flow). Result(s): Chip perfusion with the SARS-CoV-2 spike protein for 6 days resulted in the induction CD38hiCD27hi plasmablast maturation compared to an irrelevant BSA protein (P< 0.0001). Using fluorescent spike as a probe, we observed a strong amplification of spike-specific B cell (from 3.7 to 140-fold increase). In line with this rapid memory B cell response, spike-specific antibodies production could be detected as early as day 6 of culture. Spike perfusion also induced CD4+ T cell activation (CD38+ ICOS+), which correlated with the level of B cell maturation. The magnitude of specific B cell amplification in the LO chip was higher than in 2D and 3D static cultures at day 6, showing the added value of 3D perfused culture for the induction of recall responses. Interestingly, the perfusion of mRNA-based SARS-CoV-2 vaccines also led to strong B cell maturation and specific B cell amplification, indicating that mRNA-derived spike could be expressed and efficiently presented in the LO chip. Conclusion(s): We developed a versatile Lymphoid Organ-Chip model suitable for the rapid evaluation of B cell recall responses. The model is responsive to protein and mRNA-encoded antigens, highlighting its potential in the evaluation of SARS-CoV-2 vaccine boosting strategies.

HIGH FAT AND SUGAR DIET INDUCES GUT LEUKOCYTE DISRUPTIONS EXACERBATED BY SARS-CoV-2

Background: The disparity in COVID-19 disease burden between European, Asian, and African countries is notable, with considerably higher morbidity and mortality in many European countries as well as the U.S. Dietary differences between regions could play a role in differential COVID-19 pathogenesis, as Western diets high in fat and sugar have been implicated in enhancing gut damage and pathogenesis during viral infections. Here we investigate the effect of diet on gut immunity and SARS-CoV-2 infection. Method(s): Six pigtail macaques were fed a commercial monkey chow diet, then transitioned to a high fat and sugar chow diet (HFD) for approximately two months prior to infection with Delta strain SARS-CoV-2. Animals were sampled prior to HFD initiation, during HFD administration but prior to infection, and for approximately one month post-infection. HFD was maintained following infection and animals were euthanized at the study conclusion. Result(s): Viral RNA was detected for up to 28 days post-infection in nose swabs, with peak viral load at day 2 at a mean of 8.2x109 copies/mL of swab fluid. Subgenomic RNA (sgRNA, indicating viral replication) decayed more rapidly, with all animals having undetectable sgRNA by day 21, and a lower peak of 2.6x109 copies/mL swab fluid on day 2. Viral RNA load was approximately 3.5 logs greater and sgRNA load approximately 3 logs higher at day 2 than in rhesus macaques infected with WA2020 SARS-CoV-2 and fed standard monkey chow. Mucosal rectal biopsies indicated significantly lower B cell frequencies from baseline to approximately two months following HFD administration (p=0.04, Dunn's), and frequencies had not recovered approximately one month postinfection. GI tract-resident IgG+ B cells were nearly absent at necropsy, with mean frequency 0.03% of total B cells. B cell loss was coupled with modest T cell expansion during HFD administration, though frequencies declined following infection. Furthermore, NK cell frequencies tended to decline from baseline throughout HFD administration, and were further depleted at necropsy one month post-infection. Conclusion(s): SARS-CoV-2 infection can induce lymphopenia, and our sampling of gut mucosal tissue indicates B cell depletion and NK cell loss with a HFD that is further exacerbated by SARS-CoV-2 infection. Excess dietary fat and sugar may disrupt gut barrier integrity and immunity, in turn predisposing the tissue to pathology of viral infection.

Systems immunology profiling of treatment-naive children with multisystem inflammatory syndrome

Background. Multisystem inflammatory syndrome in children (MIS-C) is a severe post-infectious complication occurring weeks after SARS-CoV-2 infection. The exact mechanisms leading to immune dysregulation and organ damage remain incompletely understood. Progress in understanding the immunopathology underlying MIS-C has been halted by limited availability of pre-treatment patient samples and confounding effects of immunomodulatory treatment on previously studied specimens. Methods. In this study, we have restricted enrollment to treatment-naive patients with MIS-C and used a systems biology approach combining CyTOF, single cell transcriptomics, serum cytokine profiling and T cell receptor sequencing to dissect how immune responses in children with MIS-C differ from children with mild SARS-CoV2 infection, adults with severe COVID-19 and healthy individuals. We also integrated single cell transcriptomics datasets from post-treatment MIS-C samples to study how immune responses change along disease course. Results. We identified increased activation markers and antigen presentation across multiple immune cell types in MIS-C patients from both CyTOF and single cell transcriptomics data. Importantly, in PBMCs of MIS-C patients, we identified a distinct subset of proinflammatory monocytes, with increased expression of interferon gamma response genes combined with a signature of enhanced complement expression, antigen processing and presentation, which was not observed in post-treatment MIS-C samples. Interestingly, this monocyte population bears resemblance to a subset of monocytes that emerges after the BNT162b2 mRNA vaccine booster. In addition, in PBMCs of MIS-C patients, we identify increased proportion of proliferating T/NK cells, suggesting distinct T cell expansions in MIS-C. T and NK cells in MIS-C samples also showed increased cell cytotoxicity markers. Conclusion. Taken together, treatment-naive MIS-C samples display distinct monocyte clusters, activated antigen presentation and complement expression, and increased T and NK cell cytotoxicity, which may account for the clinical presentation of MIS-C.

Abstracts for the 29th Annual Conference of the German Society for Immunogenetics (Deutsche Gesellschaft fur Immungenetik - DGI)

The proceedings contain 15 papers. The topics discussed include: eplet analysis: a tool for risk assessment of de novo donor-specific HLA-DQ-antibodies in patients after lung transplantation;beta-2-microglobulin the neglected edge of HLA class I;temporal loss of HLA heterozygosity in an AML patient prior to stem cell transplantation: does it affect HLA typing strategies?;in-vivo monitoring of CAR-T cell expansion using TaqMan real time PCR;immunogenetic features with SARS-COV-2 breakthrough infections: analyzing 1st/2nd and omicron waves;uterus transplantation from the immunological point of view - experiences from the first center in Germany;follow-up of HLA class I antibodies in neonatal thrombocytopenia;monitoring the in-vivo persistence and expansion of CAR-T cells by TaqMan real-time PCR;and detection of complement-fixing anti-HLA antibodies by a C3D-detecting Luminex technique. is there any additional diagnostic value not available through the use of standard antibody specification at the single antigen level?.

Process Development and Manufacturing: A SUPPLY CHAIN CRISIS STORY: CULTURE BAG SHORTAGE ENFORCED VALIDATION OF AN ALTERNATIVE EXPANSION SYSTEM FOR CAR T CELLS

Background & Aim: The recent supply chain crisis highlights a need to establish alternative manufacturing (MFG) protocols ensuring continuity of existing and new cell therapy (CT) clinical trials. Our academic CT program, and likely others, experienced purchasing delays and restrictions caused by diversion of critical supplies to meet COVID-19- related research demands and/or reduced vendor capacity due to resource constraints, including attrition of skilled workforce. Mitigation strategies aimed at creating process redundancies overcome production challenges resulting from a scarcity of goods. Here, we validated an alternative ex vivo culture system to clinically MFG lentiviral vector (LV) modified CAR T cells due to limited availability of cell expansion culture bags for the Wave bioreactor, a critical unit of operation that we have used to successfully MFG thousands of gene-modified T cell products for 30+ clinical trials. Methods, Results & Conclusion: The disposable G-REX culture vessels were compatible and seamlessly integrated with our closed system platform. Mesothelin CAR T cells were manufactured in parallel via the G-REX or conventional Wave bioreactor using consented patient starting material. Critical quality attributes of the final T cell products, including viability, transduction efficiency, phenotype and function were assessed. Transduction efficiencies assessed by flow cytometry and/or molecular qPCR were lower in products generated in the G-REX compared to the wave using the same multiplicity of infection. However, at least 50-fold expansion was achieved, with cell viabilities greater than 90% and with comparable cellular phenotypes. The Meso CAR T cells generated by either process were capable of eliciting CAR-mediated cytotoxicity and effector cytokine production. Strikingly, 2-4 billion T cells were harvested from a starting seed number of just 50 million T cells in the 1L G-REX, which may be sufficient to meet most protocol- specified cell therapy doses, suggesting that a full apheresis collection may not be needed. Notably, this process required just 1/3 of the starting material, 1/5 of the media and decreased manual effort through culture duration compared to the Wave. Additionally, the reduced reliance on specialized capital equipment combined with a small footprint enables simultaneous MFG of several immunotherapy products. These advantages propose consideration in replacement of current expansion platform as well as validating an alternative process for MFG CAR T cells.

Highly Expandable Human Alveolar Organoids to Study Respiratory Diseases

Organoids are emerging to be an excellent tool for studying human development and disease. The COVID-19 pandemic has highlighted the importance of physiologically relevant alveolar infection models that include both alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. To address the need for an alveolar organoid culture system for respiratory research, we developed the PneumaCult™ Alveolar Organoid Expansion and Differentiation Media for the highly efficient expansion of isolated primary human AT2 cells and subsequent differentiation into AT1 cells. Alveolar organoids were established from a panel of various donors (n=5) by culturing purified human AT2 cells in Corning® Matrigel® domes with serum-free PneumaCult™ Alveolar Organoid Expansion Medium. Typically by day 10-14 the organoids are fully established and display a spherical morphology. Alveolar organoids can then be either expanded long-term by passaging cultures as single cells in Expansion Medium or differentiated into AT1 cells using the PneumaCult™ Alveolar Organoid Differentiation Medium. Organoids in PneumaCult™ Alveolar Organoid Expansion Medium contain self-renewing AT2 cells marked by the expression of HT2-280 in 89.9 +/- 14.5 (mean +/- SD;n=5 donors) of cells and the presence of Pro-SPC, demonstrate a great expansion potential of > 10,000-fold with more than 13 population doublings within 10 passages (n=5 donors). Alveolar organoids differentiated for 10 days in the PneumaCult™ Alveolar Organoid Differentiation Medium downregulate AT2 markers HT2-280 and Pro-SPC and start expressing AT1 markers HT1-56 in 93.8 +/- 7.2 (mean +/- SD;n=5 donors) of cells and are positive for RAGE and GPRC5a. Furthermore, we assessed the expression of SARS-CoV-2 entry receptor ACE2, which is present in both undifferentiated and differentiated alveolar organoids.To investigate the use of these alveolar organoids for infectious disease modeling, AT2-containing alveolar organoids were transduced with a GFP-labelled Respiratory Syncytial Virus (RSV). Alveolar organoids were susceptible to viral infection and replication was confirmed by fluorescence microscopy and quantitative PCR. In summary, the PneumaCult™ Alveolar Organoid Expansion and Differentiation Media are highly efficient and reproducible tools for the feeder-free expansion of AT2 cells and robust differentiation into AT1 cells, which can be used for infectious disease modeling.

Single cell profiling in COVID-19 associated acute kidney injury reveals patterns of tubule injury and repair in human

Background: Acute Kidney Injury (AKI) affects up to one in two critically ill patients. The cellular mechanisms of kidney tubule repair after acute kidney injury are poorly characterized in humans. Methods: We recruited 5 patients admitted to the Geneva University Hospital's Intensive Care Unit for severe COVID19 and experiencing AKI. For each of them, a kidney biopsy was performed before the planned withdrawal of resuscitation measures. We further applied single-cell RNA sequencing to analyze the kidney in the first days after acute injury. Results: After data processing and quality control, we obtained 20,165 single-cell transcriptomes. The most prominent finding in the snRNAseq analyses was in the proximal tubule (PT) compartment. We defined two cell populations corresponding to mature and undifferentiated PT cells, connected by two cell state transitions (Figure 1). Undifferentiated PT cells display an injured pattern characterized by metabolic impairment, reduction of the tubule transport function, and expression of injury markers confirmed in immunochemistry. We found that tubule repair follows two converging patterns involving the plasticity of mature tubule cells and the expansion and differentiation of progenitor-like cells. Tubule repair by cell plasticity displayed substantial similarities among mice and men and determined the transient expansion of undifferentiated tubule cells with altered functional and metabolic properties. Progenitorlike cells marked by PROM1 proliferated in response to injury and followed a differentiation process characterized by the sequential activation of the WNT, NOTCH, and HIPPO signaling pathways. Conclusions: Here we generated the first map of PT injury and repair in humans. Taken together, our analyses reveal cell states transitions and fundamental cellular hierarchies underlying kidney injury and repair in patients.

Polyclonal expansion of TCR VBETA 21.3+ CD4+ and CD8+ T CELLS is a hallmark of multisystem inflammatory syndrome in children

Introduction: At the end of April 2020, European clinicians warned the Public Health Agencies about an abnormal increase of Kawasakilike diseases and myocarditis requiring critical care support in the context of the ongoing COVID-19 epidemic in children. American clinicians also reported a large outbreak of severe inflammation in children following COVID-19 infection, a condition that is now named Pediatric Inflammatory Multisystemic Syndrome (PIMS) or Multisystem Inflammatory Syndrome in children (MIS-C). Objectives: As MIS-C combines clinical features of Kawasaki disease (KD) and Toxic Shock Syndrome (TSS), we aimed to compare the immunological profile of pediatric patients with these different conditions. Methods: We analysed blood cytokine expression, and the T cell repertoire and phenotype in 36 MIS-C cases, which were compared to 16 KD, 58 TSS, and 42 COVID-19 cases. Results: We observed an increase of serum inflammatory cytokines (IL-6, IL-10, IL-18, TNF-a, IFNg, CD25s, MCP1, IL-1RA) in MIS-C, TSS and KD, contrasting with low expression of HLA-DR in monocytes. We detected a specific expansion of activated T cells expressing the Vβ21.3 T cell receptor β chain variable region in both CD4 and CD8 subsets in 75% of MIS-C patients and not in any patient with TSS, KD, or acute COVID-19;this correlated with the cytokine storm detected. The T cell repertoire returned to baseline within weeks after MIS-C resolution. Vβ21.3+ T cells from MIS-C patients expressed high levels of HLA-DR, CD38 and CX3CR1 but had weak responses to SARS-CoV- 2 peptides in vitro. Consistently, the T cell expansion was not associated with specific classical HLA alleles. Conclusion: Thus, our data suggested that MIS-C is characterized by a polyclonal Vβ21.3 T cell expansion not directed against SARS-CoV-2 antigenic peptides, which is not seen in KD, TSS and acute COVID-19.

Human Hematopoietic Stem, Progenitor, and Immune Cells Respond Ex Vivo to SARS-CoV-2 Spike Protein.

Despite evidence that SARS-CoV-2 infection is systemic in nature, there is little known about the effects that SARS-CoV-2 infection or exposure has on many host cell types, including primitive and mature hematopoietic cells. The hematopoietic system is responsible for giving rise to the very immune cells that defend against viral infection and is a source of hematopoietic stem cells (HSCs) and progenitor cells (HPCs) which are used for hematopoietic cell transplantation (HCT) to treat hematologic disorders, thus there is a strong need to understand how exposure to the virus may affect hematopoietic cell functions. We examined the expression of ACE2, to which SARS-CoV-2 Spike (S) protein binds to facilitate viral entry, in cord blood derived HSCs/HPCs and in peripheral blood derived immune cell subtypes. ACE2 is expressed in low numbers of immune cells, higher numbers of HPCs, and up to 65% of rigorously defined HSCs. We also examined effects of exposing HSCs/HPCs and immune cells to SARS-CoV-2 S protein ex vivo. HSCs and HPCs expand less effectively and have less functional colony forming capacity when grown with S protein, while peripheral blood monocytes upregulate CD14 expression and show distinct changes in size and granularity. That these effects are induced by recombinant S protein alone and not the infectious viral particle suggests that simple exposure to SARS-CoV-2 may impact HSCs/HPCs and immune cells via S protein interactions with the cells, regardless of whether they can be infected. These data have implications for immune response to SARS-CoV-2 and for HCT. Graphical Abstract • Human HSCs, HPCs, and immune cells express ACE2 on the cell surface, making them potentially susceptible to SARS-CoV-2 infection. • SARS-CoV-2 S protein, which binds to ACE2, induces defects in the colony forming capacity of human HPC and inhibits the expansion of HSC/HPC subpopulations ex vivo. These effects can be at least partially neutralized by treatment with SARS-CoV-2 targeting antibody, recombinant human ACE2, or Angiotensin1-7. • S protein also induces aberrant morphological changes in peripheral blood derived monocytes ex vivo. • Thus, there are many different manners in which SARS-CoV-2 virus may impact the functional hematopoietic system, which has important implications for hematological manifestations of COVID-19 (i.e. thrombocytopenia and lymphopenia), immune response, and hematopoietic stem cell transplant in the era of COVID-19.