Follicular helper CD4^hi T cells (T_FH) are a major cellular pool for the maintenance of the HIV reservoir. Therefore, the delineation of the follicular (F)/germinal center (GC) immune landscape will significantly advance our understanding of HIV pathogenesis. We have applied multiplex confocal imaging, in combination with the relevant computational tools, to investigate F/GC in situ immune dynamics in viremic (vir-HIV), antiretroviral-treated (cART HIV) People Living With HIV (PLWH) and compare them to reactive, non-infected controls. Lymph nodes (LNs) from viremic and cART PLWH could be further grouped based on their T_FH cell densities in high-T_FH and low-T_FH subgroups. These subgroups were also characterized by different in situ distributions of PD1^hi T_FH cells. Furthermore, a significant accumulation of follicular FOXP3^hiCD4^hi T cells, which were characterized by a low scattering in situ distribution profile and strongly correlated with the cell density of CD8^hi T cells, was found in the cART-HIV low-TFH group. An inverse correlation between plasma viral load and LN GrzB^hiCD8^hi T and CD16^hiCD15^lo cells was found. Our data reveal the complex GC immune landscaping in HIV infection and suggest that follicular FOXP3^hiCD4^hi T cells could be negative regulators of T_FH cell prevalence in cART-HIV.

Follicular (F) and germinal center (GC) immune dynamics are crucial in generating pathogen-specific antibodies with high affinity and neutralizing activity capable of combating infections. GCs are populated by highly differentiated CD4 T-cell and B-cell populations with unique phenotypes, functions, and molecular signatures. Aging, which is associated with a state of "immunosenescence," is characterized by compromised B-cell responses to infections and reduced vaccine efficacy, pointing to altered GC immunoreactivity and function. Therefore, there is a need for novel, improved approaches to strengthen antibody responses in these individuals and mitigate morbidity and mortality. Despite the importance of mouse models, studies focusing on human F/GC immune dynamics are of great interest. Furthermore, studying these dynamics in various human diseases could provide important insights into the cellular and molecular mechanisms that regulate GC development under different local microenvironmental conditions. The development and application of cutting-edge methodologies allowing for the comprehensive analysis of relevant cell types and a better understanding of the spatial organization of the immune system in these anatomical sites are essential to delineate the impact of molecular targets and pathways that could be used in designing in vivo immune interventions aiming to strengthen B-cell responses, especially in aging.

Follicular helper CD4+ T cells (TFH) are a major cellular pool for the maintenance of HIV reservoir. Therefore, the delineation of the follicular (F)/germinal center (GC) immune landscape will significantly advance our understanding of HIV pathogenesis. We have applied multiplex confocal imaging, in combination with relevant computational tools, to investigate, F/GC in situ immune dynamics in viremic (vir-HIV), antiretroviral treated (cART HIV) People Living With HIV (PLWH) and compare them to reactive, non-infected controls. Lymph nodes (LNs) from viremic and cART PLWH could be further grouped based on their TFH cell densities in high-TFH and low-TFH subgroups. These subgroups were also characterized by different in situ distribution of PD1hi TFH cells. The significantly accumulated follicular, compared to extrafollicular, FOXP3hi CD4+ T cells found in the low-TFH cART-HIV group were characterized by a less scattered in situ distribution and strongly correlated with the cell density of CD8+ T cells in this group. An inverse correlation between plasma viral load and LN GrzBhiCD8+ T and CD16hiCD15lo cells was found. Our data reveal the complex GC immune landscaping in HIV infection and suggest that follicular FOXP3hi CD4+ T cells could be negative regulators of TFH cell prevalence in cART-HIV.

Follicles (Fs)/Germinal Centers (GCs) in tonsils and lymph nodes are dynamic microenvironments where diverse immune cell populations interact for the development of antibody responses against pathogens. The accurate in situ phenotypic analysis of these immune cells is a prerequisite for the comphehensive understanding of GC development. In this study, we explore unsupervised clustering approaches for distinguishing cell populations within F/GCs using marker expression data. We evaluate multiple clustering algorithms and find that k-means clustering provides the most effective separation of distinct cell subsets. Additionally, we investigate the predictive potential of common GC markers (CD3, CD4, CD20 and BCL6) for PD-1 expression, an important immune checkpoint regulator. Our analysis demonstrates that PD-1 expression can be reliably inferred using these markers, suggesting potential applications for automated cell classification in immunological studies. This approach enhances our ability to analyze immune cell heterogeneity and may contribute to improved understanding of GC dynamics in health and disease. Our findings support the use of computational clustering for high-dimensional immune profiling.

The regulation of follicular (F) and germinal center (GC) immune reactivity in human lymph nodes (LNs), particularly during the acute stages of viral infection, remains poorly understood. We have analyzed lung-draining lymph nodes (LD-LNs) from COVID-19 autopsies using multiplex imaging and spatial transcriptomics to examine the immune landscape with respect to follicular immune reactivity. We identified 3 groups of donors based on the Bcl6 prevalence of their reactive follicles (RFs): RF-Bcl6 ^no/lo , RF-Bcl6 ^int , and RF-Bcl6 ^hi . A distinct B/Tfh immune landscape, associated with increased prevalence of proliferating B cell and Tfh cell subsets, was found in RF-Bcl6 ^hi LD-LNs. The comparison between LD-LNs and subdiaphragmatic (SD) LNs from the same donor revealed a divergent Bcl6 expression between the 2 anatomical sites. LD-LN Bcl6 expression was also associated with a distinct spatial transcriptomic profile. TH1-associated genes/pathways (e.g., CXCR3 , STAT5 , TNF signaling) were significantly upregulated in RF-Bcl6 ^no/lo tissues, while the RF-Bcl6 ^hi tissues exhibited significant upregulation of GC-promoting genes/pathways (e.g., CXCL13 , B-cell receptor signaling). Our findings reveal a heterogeneous F/GC landscape in COVID-19 LD-LNs, highlighting specific molecular targets and pathways that could regulate human F/GC immune dynamics during acute viral infections.

The unique ability of chimeric antigen receptor (CAR) T cells to infiltrate tissues is revolutionizing our perspectives for tackling severe, refractory and otherwise untreatable diseases. In HIV, CAR-T cells have been designed to target viral biomarkers, with limited success so far. Here, we investigated the possibility of redirecting CAR-T cells against a cellular biomarker of the HIV reservoir, the programmed cell death protein 1 (PD-1). We designed two second-generation 4-1BB-CARs using the scFv of either a blocking (bPD1-CAR) or a nonblocking (nbPD1-CAR) anti–PD-1 monoclonal antibody. The CAR avidity modulated T cell sensitivity, trogocytosis, and effector functions, independently of the PD-1 signaling domain. Both anti–PD-1 CAR-T cells could persist for 70 days in HIV-infected humanized mice, correlating with viral protection and a disruption of the lymphoid architecture in the white pulp of the spleen. Together, our results open strategic avenues for reducing the HIV reservoir by demonstrating the feasibility of depleting specific T cell subpopulations.