Eva María García-Cuesta

HIV-1 entry into susceptible cells requires the dynamic interaction of its envelope (Env) glycoprotein with the host cell receptor CD4 and a co-receptor, either CCR5 or CXCR4. While the core molecular mechanisms driving Env-receptor interactions and subsequent membrane fusion are well characterized, the precise nanoscale spatial reorganization of these co-receptors at the viral binding site remains poorly defined. In this study, we employed single-particle tracking total internal reflection fluorescence (SPT-TIRF) microscopy to quantitatively analyze nanoscale organizational changes of CXCR4 on the surface of human CD4 ⁺ T cells following binding by X4-tropic HIV-1. Our data reveal that both recombinant X4-gp120 and virus-like particles expressing physiological levels of X4 Env proteins (gp120 and gp41) promote CXCR4 clustering, a phenomenon linked to cell infection. Furthermore, these ligands induced oligomerization of CXCR4 ^R334X , a naturally occurring mutant associated with WHIM syndrome that supports HIV-1 infection, but fails to oligomerize in response to CXCL12. Our findings establish a link between CXCR4 clustering and HIV-1 infection, enhancing our understanding of the initial events in viral attachment and entry. These results further suggest that HIV-1 depends on a specific spatial arrangement of co-receptors, distinct from that induced by their natural chemokine ligands, highlighting the critical role of cell-surface receptor spatial organization in dictating cellular function.

We previously described GMC, a graphene-based nanomaterial obtained from carbon nanofibers (CNFs), to be biologically compatible and functional for therapeutic purposes. GMC can reduce triglycerides’ content in vitro and in vivo and has other potential bio-functional effects on systemic cells and the potential utility to be used in living systems. Here, immunoreactivity was evaluated by adding GMC in suspension at the biologically functional concentrations, ranging from 10 to 60 µg/mL, for one or several days, to cultured lymphocytes (T, B, NK), either in basal or under stimulating conditions, and monocytes that were derived under culture conditions to pro-inflammatory (GM-MØ) or anti-inflammatory (M-MØ) macrophages. All stirpes were obtained from human peripheral mononuclear cells (PBMCs) from anonymized healthy donors. The viability (necrosis, apoptosis) and immunological activity of each progeny was analyzed using either flow cytometry and/or other analytical determinations. A concentration of 10 to 60 µg/mL GMC did not affect lymphocytes’ viability, either in basal or active conditions, during one or more days of treatment. The viability and expression of the inflammatory interleukin IL-1β in the monocyte cell line THP-1 were not affected. Treatments with 10 or 20 µg/mL GMC on GM-MØ or M-MØ during or after their differentiation process promoted phagocytosis, but their viability and the release of the inflammatory marker activin A by GM-MØ were not affected. A concentration of 60 µg/mL GMC slightly increased macrophages’ death and activity in some culture conditions. The present work demonstrates that GMC is safe or has minimal immunological activity when used in suspension at low concentrations for pre-clinical or clinical settings. Its biocompatibility will depend on the dose, formulation or way of administration and opens up the possibility to consider GMC or other CNF-based biomaterials for innovative therapeutic strategies.

Significance: The ability of the T cell receptor (TCR) to convey signals of different intensity is essential for the generation of a diverse, protecting, and self-tolerant T cell repertoire. We provide evidence that pre-TCR signaling during the first stage of T cell differentiation, thought to only check for in-frame rearrangement of TCRβ gene segments, determines the degree of diversity in a signaling intensity–dependent manner and controls the diversity of the TCR repertoire available for subsequent thymic positive and negative selection. Pre-TCR signaling intensity is regulated by the transmembrane region of its associated CD3ζ chains, possibly by organizing pre-TCRs into nanoclusters. Our data provide insights into immune receptor signaling mechanisms and reveal an additional checkpoint of T cell repertoire diversity.

Significance: New imaging-based approaches are incorporating new concepts to our knowledge of biological processes. The analysis of receptor dynamics involved in cell movement using single-particle tracking demonstrates that cells require chemokine-mediated receptor clustering to sense appropriately chemoattractant gradients. Here, we report that this process does not occur in T cells expressing CXCR4 ^R334X , a mutant form of CXCR4 linked to WHIM syndrome (warts, hypogammaglobulinemia, infections, myelokathexis). The underlaying molecular mechanism involves inappropriate actin cytoskeleton remodeling due to the inadequate β-arrestin1 activation by CXCR4 ^R334X , which alters its lateral mobility and spatial organization. These defects, associated to CXCR4 ^R334X expression, contribute to the retention of hematopoietic precursors in bone marrow niches and explain the severe immunological symptoms associated with WHIM syndrome.

High grade non-muscle-invasive bladder tumours are treated with transurethral resection followed by recurrent intravesical instillations of Bacillus Calmette Guérin (BCG). Although bladder cancer patients respond well to BCG, important questions remain unanswered, including how to identify at early stages non-responder patients and patients at risk to abandon the treatment. Here, we analysed the cells released into the urine of bladder cancer patients longitudinally 3-7 days after BCG instillations. Mass cytometry (CyTOF) analyses revealed that most cells were granulocytes and monocytes rather than effector lymphocytes, and most expressed activation markers. A novel population of CD15 ⁺ CD66b ⁺ CD14 ⁺ CD16 ⁺ cells was very abundant in several samples and expression of these markers was confirmed using flow cytometry and qPCR. Samples of patients with a stronger inflammatory response contained more cells in urine; however, this was not due to haematuria, as the proportions of the cell populations observed were different from blood. We provide the proof-of-concept for a new approach to analyse samples that may help classify patients and identify those at risk of BCG infection and other unwanted BCG-related events.

Signaling via pre-TCR is essential for initial T cell differentiation. How the signal is triggered and whether its alteration impacts on lymphocyte repertoires is nevertheless debated. We show that a mutation in the transmembrane domain of the CD3ζ chain impairs murine thymocyte development at the double negative (DN) stage coinciding with reduced levels of steady-state pre-TCR signaling. Single particle tracking and TIRF microscopy on mouse primary DN3/DN4 thymocytes shows that wild-type pre-TCR form particles with a broad range of fluorescent intensities whereas mutant complexes are less intensely labeled and move faster, suggesting reduced pre-TCR nanoclustering. Notably, the mutant early-pre-TCR-selected-DP population shows less TCRβ repertoire diversity. We thus propose that the pre-TCR forms stable nanoclusters, the size of which determines signaling efficacy. This signaling does not merely promote maturation of any thymocyte expressing a rearranged TCRβ chain, but shapes the repertoire of DP thymocytes that can audition for positive and negative selection.

Background: Intra-vesical instillation of Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is an effective therapy for high-grade non-muscle invasive bladder cancer (NMIBC), which provokes a local immune response resulting in 70% of patients free of relapse after three years. Because non-responder patients usually have a bad prognosis, the early identification of treatment failure is crucial. We hypothesized that, if an effective immune response was taking place in the bladder, soluble factors would be released to the urine many days after BCG instillations. Methods: An extensive panel of cytokines and chemokines released into the urine seven days after every BCG instillation was screened in a cohort of NMIBC patients over three years. Results: The determinations of the urinary concentrations of cytokines, chemokines, and creatinine showed that increasing concentrations of C-X-C motif chemokine 10 (CXCL10) also known as interferon-inducible protein 10 (IP10) could be detected during the six-week induction cycle of BCG-treated patients released into the urine by CD14⁺ cells. In vitro, CXCL10 facilitated the recruitment of effector immune cells after the BCG-mediated upregulation of CXCR3 in both T- and natural killer (NK)-cells. Conclusions: The high concentrations of chemokine detected one week after the encounter with mycobacteria suggest that the CXCL10 axis might be related to the intensity of the immune anti-tumor response.