Lung influenza virus specific memory CD4 T cell location and optimal cytokine production are dependent on interactions with lung antigen-presenting cells.

Influenza A virus (IAV) infection leads to the formation of mucosal memory CD4 T cells that can protect the host. An in-depth understanding of the signals that shape memory cell development is required for more effective vaccine design. We have examined the formation of memory CD4 T cells in the lung following IAV infection of mice, characterising changes to the lung landscape and immune cell composition. IAV-specific CD4 T cells were found throughout the lung at both primary and memory time points. These cells were found near lung airways and in close contact with a range of immune cells including macrophages, dendritic cells, and B cells. Interactions between lung IAV-specific CD4 T cells and MHCII+ cells during the primary immune response were important in shaping the subsequent memory pool. Treatment with an anti-MHCII blocking antibody increased the proportion of memory CD4 T cells found at lung airways but reduced interferon-γ expression by IAV-specific immunodominant memory CD4 T cells. The immunodominant CD4 T cells expressed higher levels of PD1 than other IAV-specific CD4 T cells and PD1+ memory CD4 T cells were located further away from MHCII+ cells than their PD1-low counterparts. This distinction in location was lost in mice treated with anti-MHCII antibody. These data suggest that sustained antigen presentation in the lung impacts on the formation of memory CD4 T cells by regulating their cytokine production and location.

An easy to use tool for the analysis of subcellular mRNA transcript colocalisation in smFISH data.

Single molecule fluorescence in situ hybridisation (smFISH) has become a valuable tool to investigate the mRNA expression of single cells. However, it requires a considerable amount of programming expertise to use currently available open-source analytical software packages to extract and analyse quantitative data about transcript expression. Here, we present FISHtoFigure, a new software tool developed specifically for the analysis of mRNA abundance and co-expression in QuPath-quantified, multi-labelled smFISH data. FISHtoFigure facilitates the automated spatial analysis of transcripts of interest, allowing users to analyse populations of cells positive for specific combinations of mRNA targets without the need for computational image analysis expertise. As a proof of concept and to demonstrate the capabilities of this new research tool, we have validated FISHtoFigure in multiple biological systems. We used FISHtoFigure to identify an upregulation in the expression of Cd4 by T-cells in the spleens of mice infected with influenza A virus, before analysing more complex data showing crosstalk between microglia and regulatory B-cells in the brains of mice infected with Trypanosoma brucei brucei. These analyses demonstrate the ease of analysing cell expression profiles using FISHtoFigure and the value of this new tool in the field of smFISH data analysis.

Cotransfer of antigen and contextual information harmonizes peripheral and lymph node conventional dendritic cell activation.

T cell responses against infections and cancer are directed by conventional dendritic cells (cDCs) in lymph nodes distant from the site of challenge. Migratory cDCs, which travel from the tissue to the lymph node, not only drive initial T cell activation but also transfer antigen to lymph node-resident cDCs. These resident cells have essential roles defining the character of the resulting T cell response; however, it is unknown how they can appropriately process and present antigens to suitably direct responses given their spatial separation. Here, using a novel strain of influenza A and a modified melanoma model, we show that tissue and lymph node cDC activation is harmonized and that this is driven by cotransfer of contextual cues. In the tumor, incomplete cDC activation in the tumor microenvironment is mirrored by lymph node-resident cDCs, whereas during influenza infection, pathogen-associated molecular patterns cotransferred with antigen drive TLR signaling in resident cDCs and their subsequent robust activation. This cotransfer mechanism explains how individual antigens can be handled distinctly by resident cDCs and how signals driving poor tumoral cDC activation further impact the lymph node. Our findings clarify how tissue context dictates antigenic and, consequently, T cell fate in the lymph node.

Superinfection exclusion creates spatially distinct influenza virus populations.

Interactions between viruses during coinfections can influence viral fitness and population diversity, as seen in the generation of reassortant pandemic influenza A virus (IAV) strains. However, opportunities for interactions between closely related viruses are limited by a process known as superinfection exclusion (SIE), which blocks coinfection shortly after primary infection. Using IAVs, we asked whether SIE, an effect which occurs at the level of individual cells, could limit interactions between populations of viruses as they spread across multiple cells within a host. To address this, we first measured the kinetics of SIE in individual cells by infecting them sequentially with 2 isogenic IAVs, each encoding a different fluorophore. By varying the interval between addition of the 2 IAVs, we showed that early in infection SIE does not prevent coinfection, but that after this initial lag phase the potential for coinfection decreases exponentially. We then asked how the kinetics of SIE onset controlled coinfections as IAVs spread asynchronously across monolayers of cells. We observed that viruses at individual coinfected foci continued to coinfect cells as they spread, because all new infections were of cells that had not yet established SIE. In contrast, viruses spreading towards each other from separately infected foci could only establish minimal regions of coinfection before reaching cells where coinfection was blocked. This created a pattern of separate foci of infection, which was recapitulated in the lungs of infected mice, and which is likely to be applicable to many other viruses that induce SIE. We conclude that the kinetics of SIE onset segregate spreading viral infections into discrete regions, within which interactions between virus populations can occur freely, and between which they are blocked.

Think global but act local: Tuning the dendritic cell response in cancer.

Despite their low abundance in tumours conventional dendritic cells play an outsized role in initiating and perpetuating anti-tumour immunity; however progressively growing tumours suppress dendritic cell function in a range of ways preventing effective anti-tumour T cell responses. While the success of immune checkpoint blockade has focused attention on T-cell directed therapies, activating tumour dendritic cells has been shown to be critical for the efficacy of several immunotherapies and other conventional therapies owing to their ability to activate and restimulate anti-tumour T-cells. As such, the importance of understanding the mechanisms by which dendritic cell function is impaired are being investigated further. Yet, while much attention has been paid to the tumour microenvironment less has been given to the macroenvironment including effects in the bone marrow and the lymph node. It is now clear that dendritic cell function can be impaired in a variety of ways at different anatomical sites and understanding these mechanisms will be critical for developing effective strategies to tune the dendritic cell response in cancer.

Metalloproteinase inhibition reduces AML growth, prevents stem cell loss, and improves chemotherapy effectiveness.

Acute myeloid leukemia (AML) is a blood cancer of the myeloid lineage. Its prognosis remains poor, highlighting the need for new therapeutic and precision medicine approaches. AML symptoms often include cytopenias linked to loss of healthy hematopoietic stem and progenitor cells (HSPCs). The mechanisms behind HSPC decline are complex and still poorly understood. Here, intravital microscopy (IVM) of a well-established experimental model of AML allows direct observation of the interactions between healthy and malignant cells in the bone marrow (BM), suggesting that physical dislodgment of healthy cells by AML through damaged vasculature may play an important role. Multiple matrix metalloproteinases (MMPs), known to remodel extracellular matrix, are expressed by AML cells and the BM microenvironment. We reason MMPs could be involved in cell displacement and vascular leakiness; therefore, we evaluate the therapeutic potential of MMP pharmacological inhibition using the broad-spectrum inhibitor prinomastat. IVM analyses of prinomastat-treated mice reveal reduced vascular permeability and healthy cell clusters in circulation and lower AML infiltration, proliferation, and cell migration. Furthermore, treated mice have increased retention of healthy HSPCs in the BM and increased survival following chemotherapy. Analysis of a human AML transcriptomic database reveals widespread MMP deregulation, and human AML cells show susceptibility to MMP inhibition. Overall, our results suggest that MMP inhibition could be a promising complementary therapy to reduce AML growth and limit HSPC loss and BM vascular damage caused by MLL-AF9 and possibly other AML subtypes.

Manipulating niche composition limits damage to haematopoietic stem cells during Plasmodium infection.

Severe infections are a major stress on haematopoiesis, where the consequences for haematopoietic stem cells (HSCs) have only recently started to emerge. HSC function critically depends on the integrity of complex bone marrow (BM) niches; however, what role the BM microenvironment plays in mediating the effects of infection on HSCs remains an open question. Here, using a murine model of malaria and combining single-cell RNA sequencing, mathematical modelling, transplantation assays and intravital microscopy, we show that haematopoiesis is reprogrammed upon infection, whereby the HSC compartment turns over substantially faster than at steady-state and HSC function is drastically affected. Interferon is found to affect both haematopoietic and mesenchymal BM cells and we specifically identify a dramatic loss of osteoblasts and alterations in endothelial cell function. Osteo-active parathyroid hormone treatment abolishes infection-triggered HSC proliferation and-coupled with reactive oxygen species quenching-enables partial rescuing of HSC function.

Effect of the CXCR4 antagonist plerixafor on endogenous neutrophil dynamics in the bone marrow, lung and spleen.

Treatment with the CXCR4 antagonist, plerixafor (AMD3100), has been proposed for clinical use in patients with WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome and in pulmonary fibrosis. However, there is controversy with respect to the impact of plerixafor on neutrophil dynamics in the lung, which may affect its safety profile. In this study, we investigated the kinetics of endogenous neutrophils by direct imaging, using confocal intravital microscopy in mouse bone marrow, spleen, and lungs. Neutrophils are observed increasing their velocity and exiting the bone marrow following plerixafor administration, with a concomitant increase in neutrophil numbers in the blood and spleen, while the marginated pool of neutrophils in the lung microvasculature remained unchanged in terms of numbers and cell velocity. Use of autologous radiolabeled neutrophils and SPECT/CT imaging in healthy volunteers showed that plerixafor did not affect GM-CSF-primed neutrophil entrapment or release in the lungs. Taken together, these data suggest that plerixafor causes neutrophil mobilization from the bone marrow but does not impact on lung marginated neutrophil dynamics and thus is unlikely to compromise respiratory host defense both in humans and mice.

Inhibition of Endosteal Vascular Niche Remodeling Rescues Hematopoietic Stem Cell Loss in AML.

Bone marrow vascular niches sustain hematopoietic stem cells (HSCs) and are drastically remodeled in leukemia to support pathological functions. Acute myeloid leukemia (AML) cells produce angiogenic factors, which likely contribute to this remodeling, but anti-angiogenic therapies do not improve AML patient outcomes. Using intravital microscopy, we found that AML progression leads to differential remodeling of vasculature in central and endosteal bone marrow regions. Endosteal AML cells produce pro-inflammatory and anti-angiogenic cytokines and gradually degrade endosteal endothelium, stromal cells, and osteoblastic cells, whereas central marrow remains vascularized and splenic vascular niches expand. Remodeled endosteal regions have reduced capacity to support non-leukemic HSCs, correlating with loss of normal hematopoiesis. Preserving endosteal endothelium with the small molecule deferoxamine or a genetic approach rescues HSCs loss, promotes chemotherapeutic efficacy, and enhances survival. These findings suggest that preventing degradation of the endosteal vasculature may improve current paradigms for treating AML.

Different repair kinetic of DSBs induced by mitomycin C in peripheral lymphocytes of obese and normal weight adolescents.

In 2013, 42 million children under the age of 5 years were overweight or obese. In the context of obesity, we recently showed that (1) peripheral lymphocytes of obese children/adolescents had an 8-fold increase in double strand breaks (DSBs), expressed as g-H2AX foci, than normal weight adolescents, and (2) 30% of the damage was retained into chromosome mutations. Thus, we investigated DSBs repair efficiency in a group of obese adolescents assessing the kinetic of H2AX phosphorylation in mitomycin C (MMC)-treated lymphocytes harvested 2 h- or 4 h-post mutagen treatment. According to our previous studies, these harvesting times represent the peak of DSBs induction and the time in which an appreciable DSBs reduction was observed. In addition, we evaluated the expression of the high mobility group box-1 protein (HMGB1), a chromatin remodelling protein involved in DSBs repair and obesity. Compared to normal weight adolescents, obese subjects 1) showed higher levels of g-H2AX foci at either 2 h- (0.239±0.041 vs. 0.473±0.048, P=0.0016) or 4 h- (0.150±0.026 vs. 0.255±0.030, P=0.0198) post mutagen treatment, and 2) have repaired a greater amount of the initial lesions (0.088±0.033 vs. 0.218±0.045, P=0.0408). Concordantly, 1) HMGB1 levels of obese individuals increased and decreased at 2h- or 4 h-post mutagen treatment, respectively, and 2) the opposite occurred for the normal weight adolescents where the protein was down-expressed at 2h and over-expressed at 4h. In conclusion, lymphocytes of obese and normal weight adolescents showed a distinct temporal kinetic of repairing MMC-induced DSBs, together with a different expression of HMGB1. The finding that obesity may modulate the repair of DNA damage induced in lymphocytes by genotoxic agents should be confirmed by further experiments.

Kinetics of nuclear phosphorylation (γ-H2AX) in human lymphocytes treated in vitro with UVB, bleomycin and mitomycin C.

After double-strand break induction, formation of γ-H2AX foci due to phosphorylation at Ser-139 of histone H2AX represents an early event of the DNA damage response (DDR). γ-H2AX foci are then rapidly dephosphorylated as signal for the subsequent recruitment of effector proteins. The induction and disappearance of the foci can be, therefore, used to monitor the functioning of the DDR machinery in a cell population exposed to genotoxic stress. Here, we investigated the time-course of γ-H2AX in unstimulated or cultured peripheral lymphocytes in vitro treated with UVB, bleomycin and mitomycin C (MMC). Once the mutagen exposure was performed, cells were harvested at different interval times from 0.5 to 5h. The results show that (i) in 20-h stimulated peripheral lymphocytes, UVB irradiation caused extensive and dose-dependent increases in nuclear phosphorylation, and disappearance of γ-H2AX foci progressed, proportionally to the UV fluence, with increasing the harvesting time; (ii) UVB-exposed G0 cells cultured for 20-h post-irradiation displayed low amounts of DNA phosphorylation, depicting a time-course in which the maximum effect was reached at 0.5h and dephosphorylation started after 1h; (iii) treatment of unstimulated lymphocytes with bleomycin sulphate induced an increase in nuclear phosphorylation of several folds higher than that of untreated cells, depicting kinetics comparable to those observed for UVB-exposed G1 cells; (iv) in stimulated cells, MMC caused a severe and dose-dependent high degree of H2AX phosphorylation together with a very slower kinetic of dephosphorylation with respect to the other experimental treatments. This study confirms the feasibility of the γ-H2AX focus assay as a genotoxic end-point and supports the view that the proposed type of analysis should be introduced in biomonitoring studies of human populations. This could also represent a feasible and useful tool in the screening and diagnosis of precancerous states or very early stages of other diseases.