Enrique Martín-Gayo

The tyrosine kinase inhibitor dasatinib is prescribed as a first-line treatment for Philadelphia chromosome–positive leukemias and is increasingly used in other clinical settings. While dasatinib is effective and generally well tolerated, patients often experience a transient lymphocytosis attributable to a dasatinib-induced spleen contraction and an acute mobilization of splenocytes, as we have recently demonstrated in murine models and human patients. In contrast, the impact of dasatinib on lymphocyte surveillance of other secondary lymphoid organs, such as peripheral lymph nodes (LNs), has remained unclear. Here, we report that while dasatinib treatment selectively disrupted in vitro chemotaxis of B but not T cells, it caused a substantial, transient reduction of the in vivo interstitial motility in both subsets. The broad in vivo inhibition of T and B cell motility correlated with dasatinib-induced vasoconstriction of blood vessels and increased hypoxia within LNs. Pharmacological inhibition of the contraction-promoting factor Rho-associated coiled-coil kinase (ROCK) reversed dasatinib-induced LN hypoxia and rescued CD8 ⁺ T cell motility. Last, dasatinib treatment delayed but did not inhibit dendritic cell–mediated CD8 ⁺ T cell activation and proliferation within LNs. Overall, our findings uncover a dasatinib-induced transient disruption of the local adaptive immune system, which is distinct from the previously observed spleen contraction. From a clinical point of view, the disruption-recovery cycle of physiological immune surveillance induced by daily dasatinib treatment does not appear to cause a substantial impairment of key pathways underpinning CD8 ⁺ T cell activation.

Despite its effectiveness in suppressing active HIV-1 replication, antiretroviral therapy (ART) does not eliminate the persistent long-lived pool of HIV-1-infected reservoir cells, preventing the eradication of the infection. Lymphoid tissues are key anatomical sites where these reservoirs persist even in the presence of ART, but the mechanisms that are associated with viral persistence in lymphoid tissues and how tissue networks are reshaped in the setting of viral replication remain incompletely understood. Advances in tissue imaging offer a unique opportunity to characterize immune correlates of viral persistence. Here, we used a spatial proteomic method to map immune microenvironments in HIV-1-infected lymph nodes (LNs) at different stages of infection, including with or without ART. LNs from people with HIV-1 (PWH) were characterized by lower CD4+ T cell counts and higher CD8+ T cell counts in both the whole tissue and within follicles compared to people without HIV (PWOH). CD8+ T cells were more abundant in LN samples with active viral replication, defined by detection of the viral protein p24. Further characterization of p24+ LNs showed that CD8+ T cells located inside of B cell follicles exhibited higher levels of markers associated with immune activation and exhaustion, in addition to the inflammasome protein caspase-1. Using a spatial niche detection method, we found that LNs from PWH with varying levels of viral replication were differentially enriched for CD8+ T cells near antigen-presenting cells, myeloid cells, and fibroblasts. Notably, we found that p24+ cells were less enriched near CD8+ T cells but closer to follicular dendritic cells. Finally, comparing LNs from viremic and aviremic donors, where viremia was defined by detectable plasma viral load, we found low levels of activation markers in CD11c+ cells in aviremic donors, including NLRP3 inflammasome activation. Thus, using spatial proteomics to map the immune landscape in LNs, we identified novel markers characterizing immune cell subsets and tissue microenvironments that were differently enriched in PWH with varying levels of viremia, implying that HIV-1 infection confers long-term changes on the immune landscape in LN tissue. Collectively, these data provide new insights into the complex cell networks associated with viral replication at a key tissue reservoir site, which could be relevant for future HIV-1 cure strategies.

Chronic inflammation in people with HIV-1 (PWH) is associated with low CD4/CD8 T-cell ratios and inflammasome activation, which may impair monocyte (Mo) and monocyte-derived dendritic cell (MDDC) function and limit the efficacy of myeloid-based immunotherapies. Here, we investigated the role of inflammasome sensors in regulating immune ligands for TIM3 and TIGIT immune checkpoints in Mo and MDDC from PWH and their capacity to activate HIV-1-specific CD8+ T cells. Following TLR stimulation, galectin-9 (Gal-9), PVR and IL-1β were preferentially upregulated in cells from PWH with low CD4/CD8 ratios. IFI16 and AIM2 expression was increased basally or inducible in Mo and co-localized with caspase-1 in these myeloid cells present in lymphoid tissues of HIV-1-infected humanized mice. Si-RNA or small molecule-mediated inhibition of IFI16/AIM2 reduced Gal-9 and PVR expression in MDDC and enhanced their ability to activate polyfunctional HIV-1-specific CD8+ T-cells displaying improved elimination of infected CD4+ T cells in vitro and in vivo . These findings identify IFI16 and AIM2 as key regulators of myeloid dysfunction and therapeutic targets to enhance HIV-1 immunotherapies.

HIV persists in diverse tissues, with distinct cellular reservoirs presenting a major barrier to a cure and requiring targeted therapeutic strategies to address this heterogeneity. Here, we develop tissue models of HIV latency using human tonsillar, intestinal and cervicovaginal tissues. These models reveal differential HIV infection across CD4⁺ T-cell subpopulations, with ART partially restoring CD4⁺ T cells and reducing intact HIV DNA. T follicular helper cells (T_{FH CD69+ CCR7-}) are the primary inducible reservoir in tonsils, while tissue-resident memory cells (T_{RM CD69+ CD49a+}) dominate in the intestine. Identification of markers for inducible reservoirs shows that CD69, CD45RO, and PD-1 are shared across tissues, while CXCR5 in tonsils and CD49a in the intestine act as tissue-specific markers. Furthermore, using different latency reversal agents (LRAs) demonstrates that Histone Deacetylase Inhibitors (HDACis) fail to induce HIV in any tissue, the SMAC mimetic AZD5582 is effective only in a resident-memory CD4⁺ T-cell subpopulation in the intestine, and IL-15 exhibits the broadest reactivation potential across tissues and CD4⁺ T-cell subsets. These models provide insights into the inducible reservoir’s composition in different tissues and inform strategies for its elimination.

The Interferon-Stimulated Gene 15 (ISG15) has a key role during viral infections, since it can modify and regulate the expression of proteins from the host and from viruses. Isg15 ^−/− mice are more susceptible to infectious diseases. ISG15 is induced by type I Interferons (IFN-I) and apart from defense against pathogens, it can also play an important function in cancer or immune-mediated inflammatory diseases. Previous studies reported that ISG15 expression is increased in post-synaptic dendritic cells (DCs) upon interaction with CD4 + T cells. However, data regarding the role of ISG15 in DCs are scarce. The present study assesses the function of ISG15 in DCs activation, migration and ability to mediate T cell activation using bone marrow-derived DCs (BMDCs) and stimulation with lipopolysaccharide (LPS). Activation of DCs was not impaired but tended to be lower in Isg15 -deficient mice, as observed by reduced CD40 induction in Isg15 ^−/− BMDCs. Isg15 ^−/− BMDCs induced less proliferation and Granzyme B expression in co-cultured CD8 + T cells. Interestingly, Isg15 ^−/− BMDCs secreted reduced levels of the pro-inflammatory cytokines IL-1β and IL-12 upon LPS stimulation. Mechanistically, our data suggest that ISG15 regulates Caspase-1 activity in DCs leading to lower IL-1 β secretion. In conclusion, this study reveals an essential role for ISG15 modulating DCs inflammatory activity and raises new questions regarding the specific mechanisms of how this protein regulates innate immune responses.

Expression of TIGIT and KLRG1 has been associated to an exhausted, dysfunctional state in natural killer (NK) and CD8+ T cells from people with HIV-1 (PWH), limiting the efficacy of immunotherapies aiming at achieving a functional cure of the infection. Antiretroviral therapy (ART) does not completely reverse this immune exhaustion, and its combination with blockade of immune checkpoint receptors such as TIGIT and KLRG1 could be a promising strategy to promote control of viral replication in PWH. However, the impact of targeting these two immune checkpoint receptors has not been evaluated in vivo . In this study, we used a humanized Bone Marrow, Liver and Thymus (hBLT) mouse model of HIV-1 infection to study the impact of ART in combination with aTIGIT or aKLRG1 or a bispecific aTIGIT/aKLRG1 mAbs. Our results indicated that combination of ART with either aTIGIT or aKLRG1 mAbs led to faster reduction of HIV-1 pVL. Furthermore, viral rebound after ART interruption (ATI) was delayed in mice treated with aTIGIT and aKLRG1 mAbs. Histological detection of HIV-1 p24 in the spleen was restricted to the white pulp in the aKLRG1 mAb-treated group, which correlated with higher infiltration of IFNγ+ CD8+ T cells in these histological regions and with increased cytotoxic CD107a+ Granzyme B+ CD8+ T cells in the spleen. In contrast, control of HIV-1 replication induced by the aTIGIT mAb was associated with an increased splenic CD107a+ IFNγ+ NKG2C+ CD57-adaptive NK cells. In contrast, combination of ART with a bispecific aTIGIT/aKLRG1 mAb was unable to efficiently suppress viral replication or delay viral rebound after ATI, potentially by inducing apoptosis of adaptive NKG2C+ NK. Together, these results suggest that combination of ART with individual TIGIT or KLRG1 blockade may be a promising immunotherapy strategy against HIV-1 by eliciting differential immune control mechanisms.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy arising from the neoplastic transformation of immature T cells during their development in the thymus. Deciphering the developmental programs whose dysregulation leads to T-ALL pathogenesis is critical for the development of novel targeted therapies, which remain an urgent unmet need for the treatment of this disease. MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of numerous physiological processes and cancer. However, the specific role of miRNAs in human T-cell development and T-ALL pathogenesis remains largely unexplored. In this study, we comprehensively evaluated miRNA expression profiles across human T-cell development by microarray analysis and identified a dynamic expression pattern of miR-16-2, which is upregulated across early pre-T cell proliferative stages up to the resting stage of immature thymocytes immediately preceding TCRαβ expression, and decreased thereafter. We confirmed the coordinated regulation of miR-15b expression, consistent with the reported clustered genomic location of both miRNAs. Notably, functional studies identified the miR-15b/16-2 cluster as a negative regulator of early thymocyte proliferation, and showed that overexpression of miR-15b/16-2 in T-ALL cells impaired leukemic growth in vitro and tumor progression in patient-derived xenotransplantation assays. Mechanistically, miR-15b/16-2 expression represses the genes encoding BCL-2 and CYCLIN D3, leading to T-ALL apoptosis and cell cycle dysregulation, with an accumulation of G0-phase cells and a defective transition to the G2/M phase. Overall, these findings support a novel function for miR-15b/16-2 as tumor suppressors in T-ALL, highlighting their role as promising targets for T-ALL therapy. KEY POINTS: miR-15b/16-2 overexpression impairs the proliferation of human thymic progenitors leading to defective T-cell production. miR-15b/16-2 represses BCL-2 and CYCLIN D3 expression and impairs tumor progression in human T-ALL, revealing its role as tumor suppressor.

Natural Killer (NK) cells are promising candidates for targeting persistently infected CD4 + T cells in people with HIV-1 (PWH). However, chronicity of HIV-1 infection impairs NK cell functionality, requiring additional strategies to potentiate their cytotoxic activity. This study demonstrates that dendritic cells primed with nanoparticles containing Poly I:C (Nano-PIC-MDDC) enhance the natural cytotoxic function of NK cells from effective responder PWH. These NK cells exhibit increased proportions of NKG2C+ cell subsets capable of eliminating HIV-1 infected CD4 + T cells through the TRAIL receptor. In contrast, in non-responder PWH, elevated expression of the inhibitory receptor TIGIT is associated with reduced frequencies of NKG2C + NK cells and diminished TRAIL expression. TIGIT blockade restores cytotoxicity of NK cells from non-responder PWH against HIV-1-infected cells by upregulating TRAIL. Furthermore, combining Nano-PIC-MDDC-primed NK cells with anti-TIGIT immunotherapy in humanized NSG mice reduces the expansion of HIV-1 infected cells, preserves NKG2C + NK cell precursors and increases TRAIL expression in tissue. Collectively, these findings support the combined use of Nano-PIC-MDDC and TIGIT blockade as a promising immunotherapeutic strategy toward an HIV-1 cure.

Enhancing effective antiviral responses within tissue compartments supporting HIV persistence at the time of antiretroviral therapy interruption will be necessary to limit viral rebound. With the hypothesis that CD8 ⁺ tissue resident memory T cell (T _RM ) phenotypes might be more competent at controlling tissue viral recrudescence, we examined their capacity to control HIV after reactivation and the benefit of inducing T _RM -like phenotypes through cytokine stimulation from blood. CD8 ⁺ T _RM derived from cervical tissue were more efficient at eliminating reactivated HIV-infected CD4 ⁺ T cells compared to circulating effector CD8 ⁺ T cells. Expansion of CD8 ⁺ T _RM -like phenotypes from blood through IL-15/TGF-β1 stimulation recovered functional HIV-specific CD8 ⁺ T cells displaying residency features, increased clonotypic diversity and mitochondrial function, and were the most efficient phenotypes at eliminating intact viruses after reactivation. Altogether, we provide a relevant therapeutic strategy to enhance elimination of persistent antigens like HIV by generating functional CD8 ⁺ T _RM -like phenotypes.

HIV infection remains incurable as the virus persists within a latent reservoir of CD4⁺T cells. Novel approaches to enhance immune responses against HIV are essential for effective control and potential cure of the infection. In this study, we designed a novel tetravalent bispecific antibody (Bi-Ab32/16) to simultaneously target the gp120 viral protein on infected cells, and the CD16a receptor on NK cells. In vitro, Bi-Ab32/16 triggered a potent, specific, and polyfunctional NK-dependent response against HIV-infected cells. Moreover, addition of the Bi-Ab32/16 significantly reduced the latent HIV reservoir after viral reactivation and mediated the clearance of cells harboring intact proviruses in samples from people with HIV (PWH). However, the in vivo preclinical evaluation of Bi-Ab32/16 in humanized mice expressing IL-15 (NSG-Hu-IL-15) revealed a significant decline of NK cells associated with poor virological control after ART interruption. Our study underscores the need to carefully evaluating strategies for sustained NK cell stimulation during ART withdrawal.