Investigating the role of TCR signalling during T cell development

MD (res)The thymus generates multiple T cell populations from an early common CD4(-) CD8(-) double negative (DN) progenitor. Conventional αβ T cells, such as CD4(+) and CD8(+) T cells undergo thymic positive selection on partial agonist interactions with self-peptide/MHC, whereas unconventional TCRαβ(+)CD8αα(+) intraepithelial lymphocytes (IELs) and regulatory T cells (Tregs), are thought to require full agonist interactions. By contrast, very little is known about how γδ T cells undergo selection. While it has been suggested that IL-17A-secreting γδ T cells rely on strong TCR signals resulting from potential interactions with a ligand, several studies have led to opposite conclusions. This thesis investigates the role of TCR signal strength during the early stages of thymocyte development, and how this dictates subsequent T cell fate. We show that FVB/n mice have relatively few unconventional TCRαβ(+)CD8αα(+) IELs in comparison to C57Bl/6 animals. This depleted IEL compartment correlated with reduced thymic IEL progenitors, an inefficient preTCR-driven DN-to-DP transition, and a relatively increased expression of full length pTαa transcripts in CD44(-)CD25(+) DN3 cells. Transgenic over-expression of pTαa in the absence of pTαb transcripts mimicked the phenotype of FVB/n animals. CD5 levels on DN3 cells from pTαa transgenic mice indicate that preTCR signalling was weaker than in C57Bl/6 mice, suggesting that signal strength at the β-selection checkpoint may influence subsequent selection of mature T cell populations. Consistent with this, we show that OT-II mice, which express a transgenic TCRαβ from an early DN stage of thymocyte 6 development, and which have high levels of CD5 on DN3 cells, have a relatively increased unconventional TCRαβ(+)CD8αα(+)IEL compartment. For γδ T cell development, we also show that weakening signal strength impairs the development of IL-17A-secreting γδ T cells. However, increasing signal strength, through the use of a cross-linking antibody, does not favour their development as predicted. These seemingly paradoxical observations are explored further. Taken together, these data suggest that the quality or quantity of TCR-mediated signals early in T cell development have fundamental consequences for subsequent T cell selection events that differentially generate distinct T cell subsets

Heterogeneous yet stable V delta 2((+)) T-cell profiles define distinct cytotoxic effector potentials in healthy human individuals

The Wellcome Trust [096954/Z/11/Z]; and Bloodwise; grant award 14026 (formally Leukaemia & Lymphoma Research)

Immunomodulation under the lens of real‐time in vivo imaging

International audienceModulation of cells and molecules of the immune system not only represents a major opportunity to treat a variety of diseases including infections, cancer, autoimmune and inflammatory disorders but could also help understand the intricacies of immune responses. A detailed mechanistic understanding of how a specific immune intervention may provide clinical benefit is essential for the rational design of efficient immunomodulators. Visualizing the impact of immunomodulation in real-time and in vivo has emerged as an important approach to achieve this goal. In this review, we aim to illustrate how multiphoton intravital imaging has helped clarify the mode of action of immunomodulatory strategies such as antibodies or cell therapies. We also discuss how optogenetics combined with imaging will further help manipulate and precisely understand immunomodulatory pathways. Combined with other single-cell technologies, in vivo dynamic imaging has therefore a major potential for guiding preclinical development of immunomodulatory drugs

Tumor immunosurveillance and immunotherapies: a fresh look from intravital imaging

International audienceUnderstanding complex interactions between the immune system and the tumor microenvironment is an essential step towards the rational development and optimization of immunotherapies. Several experimental approaches are available to tackle this complexity but most are not designed to address the dynamic features of immune reactions, including cell migration, cellular interactions, and transient signaling events. By providing a unique means to access these precious parameters, intravital imaging offers a fresh look at intratumoral immune responses at the single-cell level. Here, we discuss how in vivo imaging sheds light on fundamental aspects of tumor immunity and helps elucidate modes of action of immunotherapies. We conclude by discussing future developments that may consolidate the unique contribution of intravital imaging for our understanding of tumor immunity

Imaging the mechanisms of anti-CD20 therapy in vivo uncovers spatiotemporal bottlenecks in antibody-dependent phagocytosis

International audienceAnti-CD20 antibody (mAb) represents an effective strategy for the treatment of B cell malignancies, possibly involving complement activity, antibody-dependent cellular cytotoxicity and phagocytosis (ADP). While ADP by Kupffer cells deplete circulating tumors, mechanisms targeting non-circulating tumors remain unclear. Using intravital imaging in a model of B cell lymphoma, we establish here the dominance and limitations of ADP in the bone marrow (BM). We found that tumor cells were stably residing in the BM with little evidence for recirculation. To elucidate the mechanism of depletion, we designed a dual fluorescent reporter to visualize phagocytosis and apoptosis. ADP by BM-associated macrophages was the primary mode of tumor elimination but was no longer active after one hour, resulting in partial depletion. Moreover, macrophages were present at low density in tumor-rich regions, targeting only neighboring tumors. Overcoming spatiotemporal bottlenecks in tumor-targeting Ab therapy thus represents a critical path towards the design of optimized therapies

A crosstalk between CAR T cell subsets and the tumor microenvironment is essential for sustained cytotoxic activity

International audienceChimeric antigen receptor (CAR) T cell therapy relies on the activity of a large pool of tumor-targeting cytotoxic effectors. Whether CAR T cells act autonomously or require interactions with the tumor microenvironment (TME) remains incompletely understood. Here, we report an essential cross-talk between CAR T cell subsets and the TME for tumor control in an immunocompetent mouse B cell lymphoma model of anti-CD19 CAR T cell therapy. Using single-cell RNA sequencing, we revealed substantial modification of the TME during CAR T cell therapy. Interferon-γ (IFN-γ) produced by CAR T cells not only enhanced endogenous T and natural killer cell activity but was also essential for sustaining CAR T cell cytotoxicity, as revealed by intravital imaging. CAR T cell-derived IFN-γ facilitated host interleukin-12 production that supported host immune and CAR T cell responses. Compared with CD8+ CAR T cells, CD4+ CAR T cells were more efficient at host immune activation but less capable of direct tumor killing. In summary, CAR T cells do not act independently in vivo but rely instead on cytokine-mediated cross-talk with the TME for optimal activity. Invigorating CAR T cell interplay with the host represents an attractive strategy to prevent relapses after therapy

Intravital imaging reveals improved Kupffer cell-mediated phagocytosis as a mode of action of glycoengineered anti-CD20 antibodies

International audienceAnti-CD20 monoclonal antibodies (mAbs) represent an effective treatment for a number of B cell malignancies and autoimmune disorders. Glycoengineering of anti-CD20mAb may contribute to increased anti-tumor efficacy through enhanced antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADP) as reported by in vitro studies. However, where and how glycoengineered Ab may potentiate therapeutic responses in vivo is yet to be elucidated. Here, we have performed mouse liver transplants to demonstrate that the liver is sufficient to mediate systemic B cells depletion after anti-CD20 treatment. Relying on intravital two-photon imaging of human CD20-expressing mice, we provide evidence that ADP by Kupffer cells (KC) is a major mechanism for rituximab-mediated B cell depletion. Notably, a glycoengineered anti-mouse CD20 Ab but not its wild-type counterpart triggered potent KC-mediated B cell depletion at low doses. Finally, distinct thresholds for KC phagocytosis were also observed for GA101 (obinutuzumab), a humanized glycoengineered type II anti-CD20 Ab and rituximab. Thus, we propose that enhanced phagocytosis of circulating B cells by KC represents an important in vivo mechanism underlying the improved activity of glycoengineered anti-CD20 mAbs

Biomedical detection dogs for the identification of SARS-CoV-2 infections from axillary sweat and breath samples **

International audienceAbstract A Polymerase Chain Reaction (PCR) test of a nasal swab is still the ‘gold standard’ for detecting a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, PCR testing could be usefully complemented by non-invasive, fast, reliable, cheap methods for detecting infected individuals in busy areas (e.g. airports and railway stations) or remote areas. Detection of the volatile, semivolatile and non-volatile compound signature of SARS-CoV-2 infection by trained sniffer dogs might meet these requirements. Previous studies have shown that well-trained dogs can detect SARS-CoV-2 in sweat, saliva and urine samples. The objective of the present study was to assess the performance of dogs trained to detect the presence of SARS-CoV-2 in axillary-sweat-stained gauzes and on expired breath trapped in surgical masks. The samples were provided by individuals suffering from mild-to-severe coronavirus disease 2019 (COVID-19), asymptomatic individuals, and individuals vaccinated against COVID-19. Results: Seven trained dogs tested on 886 presentations of sweat samples from 241 subjects and detected SARS-CoV-2 with a diagnostic sensitivity (relative to the PCR test result) of 89.6% (95% confidence interval (CI): 86.4%–92.2%) and a specificity of 83.9% (95% CI: 80.3%–87.0%)—even when people with a low viral load were included in the analysis. When considering the 207 presentations of sweat samples from vaccinated individuals, the sensitivity and specificity were respectively 85.7% (95% CI: 68.5%–94.3%) and 86.0% (95% CI: 80.2%–90.3%). The likelihood of a false-positive result was greater in the two weeks immediately after COVID-19 vaccination. Four of the seven dogs also tested on 262 presentations of mask samples from 98 subjects; the diagnostic sensitivity was 83.1% (95% CI: 73.2%–89.9%) and the specificity was 88.6% (95% CI: 83.3%–92.4%). There was no difference (McNemar’s test P = 0.999) in the dogs’ abilities to detect the presence of SARS-CoV-2 in paired samples of sweat-stained gauzes vs surgical masks worn for only 10 min. Conclusion: Our findings confirm the promise of SARS-CoV-2 screening by detection dogs and broaden the method’s scope to vaccinated individuals and easy-to-obtain face masks, and suggest that a ‘dogs + confirmatory rapid antigen detection tests’ screening strategy might be worth investigating

Tumor-intrinsic sensitivity to the pro-apoptotic effects of IFN-γ is a major determinant of CD4+ CAR T-cell antitumor activity

International audienceAbstract CD4 + T cells and CD4 + chimeric antigen receptor (CAR) T cells display highly variable antitumor activity in preclinical models and in patients; however, the mechanisms dictating how and when CD4 + T cells promote tumor regression are incompletely understood. With the help of functional intravital imaging, we report that interferon (IFN)-γ production but not perforin-mediated cytotoxicity was the dominant mechanism for tumor elimination by anti-CD19 CD4 + CAR T cells. Mechanistically, mouse or human CD4 + CAR T-cell-derived IFN-γ diffused extensively to act on tumor cells at distance selectively killing tumors sensitive to cytokine-induced apoptosis, including antigen-negative variants. In anti-CD19 CAR T-cell-treated patients exhibiting elevated CAR CD4:CD8 ratios, strong induction of serum IFN-γ was associated with increased survival. We propose that the sensitivity of tumor cells to the pro-apoptotic activity of IFN-γ is a major determinant of CD4 + CAR T-cell efficacy and may be considered to guide the use of CD4 + T cells during immunotherapy