Lymph node migratory dendritic cells modulate HIV-1 transcription through PD-1 engagement.

T-follicular helper (Tfh) cells, co-expressing PD-1 and TIGIT, serve as a major cell reservoir for HIV-1 and are responsible for active and persistent HIV-1 transcription after prolonged antiretroviral therapy (ART). However, the precise mechanisms regulating HIV-1 transcription in lymph nodes (LNs) remain unclear. In the present study, we investigated the potential role of immune checkpoint (IC)/IC-Ligand (IC-L) interactions on HIV-1 transcription in LN-microenvironment. We show that PD-L1 (PD-1-ligand) and CD155 (TIGIT-ligand) are predominantly co-expressed on LN migratory (CD1chighCCR7+CD127+) dendritic cells (DCs), that locate predominantly in extra-follicular areas in ART treated individuals. We demonstrate that TCR-mediated HIV production is suppressed in vitro in the presence of recombinant PD-L1 or CD155 and, more importantly, when LN migratory DCs are co-cultured with PD-1+/Tfh cells. These results indicate that LN migratory DCs expressing IC-Ls may more efficiently restrict HIV-1 transcription in the extra-follicular areas and explain the persistence of HIV transcription in PD-1+/Tfh cells after prolonged ART within germinal centers.

Astroglial-axonal interactions during early stages of myelination in mixed cultures using in vitro and ex vivo imaging techniques.

BACKGROUND: [corrected] Myelination is a very complex process that requires the cross talk between various neural cell types. Previously, using cytosolic or membrane associated GFP tagged neurospheres, we followed the interaction of oligodendrocytes with axons using time-lapse imaging in vitro and ex vivo and demonstrated dynamic changes in cell morphology. In this study we focus on GFP tagged astrocytes differentiated from neurospheres and their interactions with axons. RESULTS: We show the close interaction of astrocyte processes with axons and with oligodendrocytes in mixed mouse spinal cord cultures with formation of membrane blebs as previously seen for oligodendrocytes in the same cultures. When GFP-tagged neurospheres were transplanted into the spinal cord of the dysmyelinated shiverer mouse, confirmation of dynamic changes in cell morphology was provided and a prevalence for astrocyte differentiation compared with oligodendroglial differentiation around the injection site. Furthermore, we were able to image GFP tagged neural cells in vivo after transplantation and the cells exhibited similar membrane changes as cells visualised in vitro and ex vivo. CONCLUSION: These data show that astrocytes exhibit dynamic cell process movement and changes in their membrane topography as they interact with axons and oligodendrocytes during the process of myelination, with the first demonstration of bleb formation in astrocytes.

PD1 inhibits PKCθ-dependent phosphorylation of cytoskeleton-related proteins and immune synapse formation.

ABSTRACT: The inhibitory surface receptor programmed cell death protein 1 (PD1) is a major target for antibody-based cancer immunotherapies. Nevertheless, a substantial number of patients fail to respond to the treatment or experience adverse effects. An improved understanding of intracellular pathways targeted by PD1 is thus needed to develop better predictive and prognostic biomarkers. Here, via unbiased phosphoproteome analysis of primary human T cells, we demonstrate that PD1 triggering inhibited the phosphorylation and physical association with protein kinase Cθ (PKCθ) of a variety of cytoskeleton-related proteins. PD1 blocked activation and recruitment of PKCθ to the forming immune synapse (IS) in a Src homology-2 domain-containing phosphatase-1/2 (SHP1/SHP2)-dependent manner. Consequently, PD1 engagement led to impaired synaptic phosphorylation of cytoskeleton-related proteins and formation of smaller IS. T-cell receptor induced phosphorylation of the PKCθ substrate and binding partner vimentin was long-lasting and it could be durably inhibited by PD1 triggering. Vimentin phosphorylation in intratumoral T cells also inversely correlated with the levels of the PD1 ligand, PDL1, in human lung carcinoma. Thus, PKCθ and its substrate vimentin represent important targets of PD1-mediated T-cell inhibition, and low levels of vimentin phosphorylation may serve as a biomarker for the activation of the PD1 pathway.

Multilevel human secondary lymphoid immune system compartmentalization revealed by complementary imaging approaches.

Secondary human lymphoid tissue immune reactions take place in a highly coordinated environment with compartmentalization representing a fundamental feature of this organization. In situ profiling methodologies are indispensable for the understanding of this compartmentalization. Here, we propose a complementary experimental approach aiming to reveal different aspects of this process. The analysis of human tonsils, using a combination of single cell phenotypic analysis based on flow cytometry and multiplex imaging and mass spectrometry-based methodologies, revealed a compartmentalized organization at the cellular and molecular levels. More specifically, the skewed distribution of highly specialized immune cell subsets and relevant soluble mediators was accompanied by a compartmentalized localization of several lipids across different anatomical areas of the tonsillar tissue. The performance of such combinatorial experimental approaches could lead to the identification of novel in situ interactions and molecular targets for the in vivo manipulation of lymphoid organ, particularly the germinal center, immune reactions.

Astrocyte phenotypes and their relationship to myelination.

Astrocytes are one of the major glial cell types that maintain homeostasis in the undamaged CNS. After injury and disease, astrocytes become reactive and prevent regeneration; however, it has also been suggested that astrocytes can become activated and promote regeneration. Thus, it is hypothesised that astrocytes have an important role in modulating CNS repair. This review will focus on the variable phenotypic state of astrocytes that range from inactive/quiescent to reactive, and relate these to their ability to influence myelination. Using myelinating cultures plated on astrocytes we propose a possible mechanism for oligodendrocyte precursor cell interaction with the axon, leading to myelination. The phenotypic status of astrocytes is an intriguing and widely discussed issue, which is critical for understanding the mechanisms involved in CNS injury and its subsequent repair.

Mutually exclusive lymphangiogenesis or perineural infiltration in human skin squamous-cell carcinoma.

Although tumor-associated lymphangiogenesis correlates with metastasis and poor prognosis in several cancers, it also supports T cell infiltration into the tumor and predicts favorable outcome to immunotherapy. The role of lymphatic vessels in skin squamous-cell carcinoma (sSCC), the second most common form of skin cancer, remains mostly unknown. Although anti-PD-1 therapy is beneficial for some patients with advanced sSCC, a greater understanding of disease mechanisms is still needed to develop better therapies. Using quantitative multiplex immunohistochemistry, we analyzed sSCC sections from 36 patients. CD8+ T cell infiltration showed great differences between patients, whereby these cells were mainly excluded from the tumor mass. Similar to our data in melanoma, sSCC with high density of lymphatic endothelial cells showed increased CD8+ T cell density in tumor areas. An entirely new observation is that sSCC with perineural infiltration but without metastasis was characterized by low lymphatic endothelial cell density. Since both, metastasis and perineural infiltration are known to affect tumor progression and patients' prognosis, it is important to identify the molecular drivers, opening future options for therapeutic targeting. Our data suggest that the mechanisms underlying perineural infiltration may be linked with the biology of lymphatic vessels and thus stroma.

Postmortem Cardiopulmonary Pathology in Patients with COVID-19 Infection: Single-Center Report of 12 Autopsies from Lausanne, Switzerland.

We report postmortem cardio-pulmonary findings including detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in formalin-fixed paraffin embedded tissue in 12 patients with COVID-19. The 5 women and 7 men (median age: 73 years; range 35-96) died 6-38 days after onset of symptoms (median: 14.5 days). Eight patients received mechanical ventilation. Ten patients showed diffuse alveolar damage (DAD), 7 as exudative and 3 as proliferative/organizing DAD. One case presented as acute fibrinous and organizing pneumonia. Seven patients (58%) had acute bronchopneumonia, 1/7 without associated DAD and 1/7 with aspergillosis and necrotic bronchitis. Microthrombi were present in 5 patients, only in exudative DAD. Reverse transcriptase quantitative PCR detected high virus amounts in 6 patients (50%) with exudative DAD and symptom-duration ≤14 days, supported by immunohistochemistry and in-situ RNA hybridization (RNAscope). The 6 patients with low viral copy levels were symptomatic for ≥15 days, comprising all cases with organizing DAD, the patient without DAD and one exudative DAD. We show the high prevalence of DAD as a reaction pattern in COVID-19, the high number of overlying acute bronchopneumonia, and high-level pulmonary virus detection limited to patients who died ≤2 weeks after onset of symptoms, correlating with exudative phase of DAD.

Tumor-specific cytolytic CD4 T cells mediate immunity against human cancer.

CD4 T cells have been implicated in cancer immunity for their helper functions. Moreover, their direct cytotoxic potential has been shown in some patients with cancer. Here, by mining single-cell RNA-seq datasets, we identified CD4 T cell clusters displaying cytotoxic phenotypes in different human cancers, resembling CD8 T cell profiles. Using the peptide-MHCII-multimer technology, we confirmed ex vivo the presence of cytolytic tumor-specific CD4 T cells. We performed an integrated phenotypic and functional characterization of these cells, down to the single-cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells and machine learning. We demonstrated a direct, contact-, and granzyme-dependent cytotoxic activity against tumors, with delayed kinetics compared to classical cytotoxic lymphocytes. Last, we found that this cytotoxic activity was in part dependent on SLAMF7. Agonistic engagement of SLAMF7 enhanced cytotoxicity of tumor-specific CD4 T cells, suggesting that targeting these cells might prove synergistic with other cancer immunotherapies.

Myeloid antigen-presenting cell niches sustain antitumor T cells and license PD-1 blockade via CD28 costimulation.

The mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer, we show that tumor-specific CD8+ TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1+CD8+ TIL can be, however, polyfunctional. PD-1+ TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8+ TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APC. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.

In Situ Characterization of Follicular Helper CD4 T Cells Using Multiplexed Imaging.

Follicular helper CD4 T (Tfh) cells play an essential role in the formation of germinal centers (GCs), where mature B cells proliferate, differentiate, and provide long-term protective humoral responses. Despite the extensive phenotypic characterization and identification of human Tfh cell subsets, their spatial positioning at tissue level is not well understood. Here, we describe a quantitative multiplexed immunofluorescence approach allowing for the comprehensive in situ characterization of Tfh cells in human tonsils and lymph nodes (LNs) from individuals with angioimmunoblastic T-cell lymphoma (AITL). We have developed eight multiplexed panels comprising a spectrum of Tfh cell markers, like PD-1, CXCR5, and ICOS, along with transcription factors (Bcl6, Tbet, GATA3), to assess their expression, frequencies, spatial distribution and co-localization in a quantitative manner. Combined analysis of relevant markers revealed the presence of several Tfh cell subsets at tissue level based on the differential expression of surface receptors, nuclear factors as well as their distinct localization within the follicular areas. Interestingly, we found a considerable amount of tonsillar Tfh cells expressing high levels of the Th2 regulator GATA3. The co-expression of GATA3, CXCR5, and BCL6, points to an important role of GATA3 for the generation of effector human Tfh cells. Furthermore, our data revealed significantly different Tfh cell profile signatures between health and disease. Therefore, our imaging platform generates meaningful information for the in situ characterization of human Tfh cells and could provide the base for future studies aiming to a comprehensive understanding of Tfh cell tissue heterogeneity.

Low Avidity T Cells Do Not Hinder High Avidity T Cell Responses Against Melanoma.

The efficacy of T cells depends on their functional avidity, i. e., the strength of T cell interaction with cells presenting cognate antigen. The overall T cell response is composed of multiple T cell clonotypes, involving different T cell receptors and variable levels of functional avidity. Recently, it has been proposed that the presence of low avidity tumor antigen-specific CD8 T cells hinder their high avidity counterparts to protect from tumor growth. Here we analyzed human cytotoxic CD8 T cells specific for the melanoma antigen Melan-A/MART-1. We found that the presence of low avidity T cells did not result in reduced cytotoxicity of tumor cells, nor reduced cytokine production, by high avidity T cells. In vivo in NSG-HLA-A2 mice, the anti-tumor effect of high avidity T cells was similar in presence or absence of low avidity T cells. These data indicate that low avidity T cells are not hindering anti-tumor T cell responses, a finding that is reassuring because low avidity T cells are an integrated part of natural T cell responses.

T cell-induced CSF1 promotes melanoma resistance to PD1 blockade.

Colony-stimulating factor 1 (CSF1) is a key regulator of monocyte/macrophage differentiation that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). We show that CSF1 is expressed in human melanoma, and patients with metastatic melanoma have increased CSF1 in blood compared to healthy subjects. In tumors, CSF1 expression correlated with the abundance of CD8+ T cells and CD163+ TAMs. Human melanoma cell lines consistently produced CSF1 after exposure to melanoma-specific CD8+ T cells or T cell-derived cytokines in vitro, reflecting a broadly conserved mechanism of CSF1 induction by activated CD8+ T cells. Mining of publicly available transcriptomic data sets suggested co-enrichment of CD8+ T cells with CSF1 or various TAM-specific markers in human melanoma, which was associated with nonresponsiveness to programmed cell death protein 1 (PD1) checkpoint blockade in a smaller patient cohort. Combination of anti-PD1 and anti-CSF1 receptor (CSF1R) antibodies induced the regression of BRAFV600E -driven, transplant mouse melanomas, a result that was dependent on the effective elimination of TAMs. Collectively, these data implicate CSF1 induction as a CD8+ T cell-dependent adaptive resistance mechanism and show that simultaneous CSF1R targeting may be beneficial in melanomas refractory to immune checkpoint blockade and, possibly, other T cell-based therapies.

Lymphatic vessel density is associated with CD8+ T cell infiltration and immunosuppressive factors in human melanoma.

Increased density of tumor-associated lymphatic vessels correlates with poor patient survival in melanoma and other cancers, yet lymphatic drainage is essential for initiating an immune response. Here we asked whether and how lymphatic vessel density (LVD) correlates with immune cell infiltration in primary tumors and lymph nodes (LNs) from patients with cutaneous melanoma. Using immunohistochemistry and quantitative image analysis, we found significant positive correlations between LVD and CD8+ T cell infiltration as well as expression of the immunosuppressive molecules inducible nitric oxide synthase (iNOS) and 2,3-dioxygénase (IDO). Interestingly, similar associations were seen in tumor-free LNs adjacent to metastatic ones, indicating loco-regional effects of tumors. Our data suggest that lymphatic vessels play multiple roles at tumor sites and LNs, promoting both T cell infiltration and adaptive immunosuppressive mechanisms. Lymph vessel associated T cell infiltration may increase immunotherapy success rates provided that the treatment overcomes adaptive immune resistance.

Heterogeneity assessment of functional T cell avidity.

The potency of cellular immune responses strongly depends on T cell avidity to antigen. Yet, functional avidity measurements are rarely performed in patients, mainly due to the technical challenges of characterizing heterogeneous T cells. The mean functional T cell avidity can be determined by the IFN-γ Elispot assay, with titrated amounts of peptide. Using this assay, we developed a method revealing the heterogeneity of functional avidity, represented by the steepness/hillslope of the peptide titration curve, documented by proof of principle experiments and mathematical modeling. Our data show that not only natural polyclonal CD8 T cell populations from cancer patients, but also monoclonal T cells differ strongly in their heterogeneity of functional avidity. Interestingly, clones and polyclonal cells displayed comparable ranges of heterogeneity. We conclude that besides the mean functional avidity, it is feasible and useful to determine its heterogeneity (hillslope) for characterizing T cell responses in basic research and patient investigation.

Time-Lapse Imaging of Glial-Axonal Interactions.

In the central nervous system (CNS), myelin is formed by oligodendrocytes that are derived from precursor cells, known as oligodendrocyte precursor cells (OPCs). Successive stages of OPC interactions with the axons can be visualized in vitro and ex vivo using mixed neural cell cultures and pieces of intact spinal cord, respectively. OPCs and their differentiation can be imaged using cell-type-specific markers or green fluorescent protein (GFP) tags. This protocol describes methodology for generating these two systems for time-lapse imaging of dynamic cell interactions using fluorescent and 2-photon microscopy.

Time-lapse imaging of the dynamics of CNS glial-axonal interactions in vitro and ex vivo.

BACKGROUND: Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination. METHODOLOGY/PRINCIPAL FINDINGS: We took two approaches to follow myelination over time: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin. CONCLUSION: These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data.