Adeno-associated virus (AAV)-delivered anti-HIV-1 broadly neutralizing antibodies (bNAbs) could prevent and treat HIV-1 infection but are limited by host immune responses, specifically anti-drug antibodies (ADA). We tested whether PD-L1-mediated immune shielding could improve the consistency of AAV-delivered bNAb expression from muscle tissue in rhesus macaques. AAV9.PD-L1 co-delivery with AAV9.3BNC117 reduced the occurrence of ADA and T cell responses and improved the durability of 3BNC117 expression for one year post administration. Importantly, 5 of 6 macaques that received co-delivered AAV9.PD-L1 vectors were protected against ten repeated SHIVAD8-EO challenges. Histopathological and spatial transcriptomic profiling showed that AAV9.PD-L1 co-delivery prevented severe local inflammation, muscle injury, and tertiary lymphoid structure formation at the administration site. Thus, immune shielding could serve as a strategy to prolong transgene expression from muscle-directed AAV-delivered biologics.

Individual mammalian mRNAs and proteins are typically believed to originate from single genomic loci, with isoform diversity arising through cis-splicing of pre-mRNA. Whether mRNA from distant genes can undergo trans-splicing to generate functionally relevant chimeric transcripts has remained unclear. Here we develop a pipeline combining long-read direct RNA sequencing with non-targeted and targeted validation to identify chimeric transcripts in macrophages. Chromatin conformation capture studies reveal that inflammation induces interchromosomal DNA interactions, positioning parent genes proximally to facilitate the formation of chimeric mRNA. Notably, we identify a protein-coding chimeric mRNA representing a fusion between the pore-forming protein gasdermin D (GSDMD)1,2 and a C-terminal domain translated out of frame from Tmem106a (Gsdmd-Tmem106a) in mice. We show that inflammasome priming upregulates Gsdmd-Tmem106a, with the protein localizing to the plasma membrane. After activation of the inflammasome, GSDMD-TMEM106A directly interacts with canonical GSDMD N termini to accelerate and enhance pore formation and IL-1β release. Finally, we show that GSDMD-TMEM106A balances host defence and immunopathology in vivo: its loss protects against lethal sepsis but compromises antibacterial defence, whereas overexpression enhances host protection while increasing sepsis lethality. We establish that protein-coding chimeric mRNAs formed by regulated transcript fusion events are operative during inflammation and immunity.

Adeno-associated virus (AAV)-delivered anti-HIV-1 broadly neutralizing antibodies (bNAbs) have demonstrated promise for preventing and treating HIV-1 infection in preclinical models. However, host immune responses, specifically anti-drug antibodies (ADA), limit sustained bNAb expression. We have previously shown that PD-L1-mediated immune shielding improves the consistency of AAV-delivered bNAb 3BNC117 expression from muscle tissue in rhesus macaques. Here, we test the breadth of this approach with another bNAb, 10-1074. We show that AAV9.PD-L1 co-delivery with AAV9.10-1074 reduced the occurrence of ADA responses and improved the durability of bNAb expression for one year post administration. Notably 12 of 12 macaques that received AAV9.10-1074 vectors were protected against ten repeated SHIVAD8-EO challenges. Histopathological profiling showed that AAV9.PD-L1 co-delivery prevented severe local inflammation and tertiary lymphoid structure formation at the administration site. Thus, immune shielding could serve as a broad strategy to prolong transgene expression from muscle-directed AAV-delivered biologics.

Loss of function mutation in the human DPP9 gene causes Hatipoglu syndrome leading to severe inflammasomopathy. A key feature of the disease is pancytopenia and patients require bone marrow transplantation, but the mechanism of cell loss is unclear since Dpp9 mutant mice have normal hematopoiesis, suggesting that a distinct mechanism of disease occurs in humans. Here, we present a model of human DPP9 deficiency leveraging reverse genetics in the MISTRG6 humanized mice. We found that CRISPR editing of human CD34+ hematopoietic stem and progenitor cells (HSPCs) led to very efficient and persistent gene deletion in vivo. Human DPP9 deletion recapitulated cytopenia in peripheral blood and in the bone marrow, and cell loss was cell intrinsic. However, DPP9 deletion led to little transcriptional changes suggesting post-transcriptional regulation in human HSPCs. Mechanistically, DPP9 deficiency led to the activation of the CARD8 inflammasome resulting in HSPC pyroptosis, whereas NLRP1 was dispensable for cell death. Thus, our results reveal a unique human mechanism of disease and offer therapeutic insight for this inflammasomopathy.

As the primary receptor for HIV-1 entry, human CD4 is widely recognized as the hallmark of HIV-1 target cells. However, viral negative factor (Nef) and viral protein U (Vpu) down-regulate surface CD4 on infected cells, raising an intriguing question: If an infected CD4 + T cell survives and establishes latency, can CD4 expression recover? By tracking the fate of HIV-1–infected CD4 + T cells, we unexpectedly found that a subset of these cells directly convert to CD8 + T cells. T cell receptor (TCR) sequencing and single-cell RNA sequencing data revealed that these induced CD8 + T cells primarily originated from regulatory CD4 + T cells, which retained a regulatory-like phenotype postconversion. Mechanistically, HIV-1 viral protein R (Vpr) specifically induced this conversion by up-regulating transforming growth factor–β1 (TGF-β1), which, in turn, activates a TGF-β–dependent signaling pathway essential for CD8 lineage reprogramming. Critically, ex vivo analyses confirmed that CD8 + T cells from both untreated and antiretroviral therapy (ART)–suppressed people living with HIV-1 (PLWH) harbor transcriptionally active viral RNA or intact proviral DNA. Furthermore, we identified that human leukocyte antigen (HLA) class II–restricted CD8 + T cells, which were present in PLWH but absent in healthy controls, share TCR clonotypes with their antigen-specific CD4 + T cell counterparts, providing direct evidence for HIV-1–driven CD4-to-CD8 conversion in vivo. Collectively, these findings uncover a viral mechanism of host-cell reprogramming, demonstrate that CD8 + T cells constitute a previously overlooked component of the HIV-1 reservoir, and broaden our understanding of latent reservoir heterogeneity, an essential consideration for future cure strategies.

Reverse genetics approaches in mice are widely used to understand gene functions and their aberrations in diseases. However, limitations exist in translating findings from animal models to human physiology. Humanized mice provide a powerful bridge to understanding human physiology and mechanisms of disease pathogenesis while maintaining the feasibility of working with small animals. Methods for generating humanized mouse models that allow scientists to probe contributions of particular genes have been rudimentary. Here, we established an efficient method for generating genetically modified human cord blood-derived CD34+ cells for transplantation, resulting in humanized mice with near-complete loss of specific gene expression by the human immune system. Mice transplanted with Cas9-edited human CD34+ cells recapitulate functional consequences of specific gene losses in the human immune system. Our approach enables targeted gene knockouts in humanized mice, offering a valuable tool for human gene function studies in vivo.

BACKGROUND:The infant nasal microbiota closely mediates the risks of developing childhood respiratory diseases. However, the primary sources of these early residing bacteria remain largely unknown, preventing the development of microbiome strategies for disease prevention. OBJECTIVE:Our aim was to identify the primary maternal source of bacteria found in the early infant nasal microbiome. METHODS:We conducted a birth cohort study titled the Mother Infant Microbiome International Cohort (MIMIC) study. We recruited 95 mother-newborn dyads from 3 sites (St Louis, Mo; San Juan, Puerto Rico; and Accra, Ghana) and collected samples at 2 time points (at the infants' birth and when they were around 2 months old). We performed analyses of 16S ribosomal RNA gene sequencing data to evaluate the maternal microbiomes (nasal, saliva, breast milk, and areola skin) as sources seeding the infant nasal microbiome. RESULTS:The infant nasal microbiome underwent a major compositional change during the first 2 months of life. The maternal nasal microbiome was identified as the primary source of bacteria in the early nasal microbiome across the 3 study regions. Corynebacterium was predominantly transferred from the maternal nasal microbiome. Infants were more likely to harbor a Corynebacterium-dominant nasal microbiome if the nasal microbiome of their mother was Corynebacterium dominant. CONCLUSIONS:The maternal nasal microbiome is an important source of bacteria in the early nasal microbiome. A large portion of transmitted bacteria from the maternal nasal microbiome belonged to the generally beneficial bacterial genus Corynebacterium. The results from this study will aid in the development of early-life intervention strategies aimed at reducing the incidence of childhood respiratory diseases and asthma.

The latent viral reservoir remains the major barrier to HIV cure, placing the burden of strict adherence to antiretroviral therapy (ART) on people living with HIV to prevent recrudescence of viremia. For infants with perinatally acquired HIV, adherence is anticipated to be a lifelong need. In this study, we tested the hypothesis that administration of ART and viral Envelope-specific rhesus-derived IgG1 monoclonal antibodies (RhmAbs) with or without the IL-15 superagonist N-803 early in infection would limit viral reservoir establishment in SIV-infected infant rhesus macaques. Following initiation of ART at 1-2 weeks after oral SIVmac251 infection, we observed biphasic decay of viremia, with first phase decay significantly faster in the ART + SIV RhmAbs-treated group compared to controls that received only ART. In contrast, the addition of N-803 to ART + SIV RhmAbs significantly slowed both the first and second phase viral decay compared to the ART only group. Treatment with a single dose of N-803 resulted in increased frequency of Ki67 expressing NK, CD8+, and CD4+ T cells. Levels of intact SIV proviruses in CD4+ T cells from blood, lymph nodes, and rectum at week 48 of ART did not differ across groups. Similarly, the time to viral rebound following ART interruption was not impacted by the experimental treatments. These results support the concept that the rebound-competent viral reservoir is formed within days after infection and that targeting only productively infected cells for clearance near the time of ART initiation, even during acute infection, may be insufficient to limit reservoir establishment.

Despite antiretroviral therapy, HIV-1 mainly persists in memory CD4+ T cells in people living with HIV-1. Most long-lived viral reservoir cells are infected by the virus near the time of therapy initiation. A better understanding of the early events in viral reservoir seeding presents opportunities for preventing latent reservoir formation. Here, we demonstrate that CD4+ T cells expressing CCR5, permissive to HIV-1 infection, are effector or terminally differentiated cells. BTB domain and CNC homolog 2 (BACH2) is expressed by a small subset of CCR5+ cells and reverses their terminal differentiation. BACH2-mediated memory differentiation is impeded due to heightened inflammation before treatment initiation. Mice with a BACH2-knockout human immune system have a reduced frequency of HIV-1 reservoir cells and do not experience virus rebound after treatment discontinuation. Our study reveals that BACH2 is essential to the seeding and establishment of long-lived HIV-1 reservoir in memory CD4+ T cells.

Background: Human immunodeficiency virus (HIV) remains a significant life-threatening agent and burden on public health. Lesser studied and understood aspects of HIV include HIV-associated inflammatory arthritis and the role NK cells and cytokines like GM-CSF have on its development. By utilizing humanized mice with human cytokine knockins that better support a human immune system than other models, we studied HIV-associated arthritis to better understand its development and progression. Objectives: Determine the etiology of HIV-associated inflammatory arthritis including cellular involvement and cytokines which can be used as therapeutic targets. Methods: MISTRG-IL15 mice[1] (human M-CSF/GM-CSF, IL-3, SIRPα, TPO, RAG2 -/-, IL2rg-/-, and IL-15 knocked into the respective endogenous mouse loci) were reconstituted with CD34+ hematopoietic stem cells obtained from human fetal cord blood. After checking for engraftment after 6-8 weeks, we infected reconstituted mice with HIV. Synovial tissues were obtained from the knee joints of the hind limbs at different time points and processed into single cell suspensions. Intercellular staining was performed on cells incubated with PMA/Ionomycin for 4 hours and then permeabilized and fixed for flow cytometry. NK cells were defined as CD3-CD56+. Histochemistry was also performed on hind paws that were fixed in 4% paraformaldehyde. Samples were then sectioned and stained with H&E for histologic analysis. Studies also involved depletion of NK cells and macrophages, Antiretroviral therapy (ART), and viral strains with unique tropisms. Results: The majority of humanized MISTRG-IL15 mice developed inflammatory arthritis after HIV infection. Significant increases in total inflammatory cells and NK cells were seen in the synovial tissue throughout the course of infection. GM-CSF producing NK cells also increased throughout the course of HIV infection. Late in the course of infection, IFNγ- and granzyme B-producing NK cells increased, along with a pro-inflammatory environment in the synovial tissue. Histologic analysis showed increased cellular infiltrate in the synovium by at 8 weeks of infection with erosive changes. ART eliminated cellular infiltration while cessation of therapy resulted in return of cellular infiltration in synovial tissue. Use of a chemokine receptor-tropic HIV virus that does not infect macrophages (X4-tropic) or depletion of macrophages showed reduced pro-inflammatory cytokine production by NK cells, strongly suggesting HIV-infected macrophages induced the NK cell responses. Depletion of NK cells resulted in blockade of development of inflammatory arthritis. Conclusion: We have found a unique role of GM-CSF produced by NK cells contributing to the development of HIV-associated inflammatory arthritis in our unique mouse model. In addition to providing a model to study arthritis induced by other human pathogens, findings from such studies will provide mechanistic understanding of inflammatory arthritis. REFERENCES: [1] Sungur CM, Wang Q, Ozantürk AN, Gao H, Schmitz AJ, Cella M, Yokoyama WM, Shan L. Human NK cells confer protection against HIV-1 infection in humanized mice. J Clin Invest. 2022 Dec 15;132(24):e162694. doi: 10.1172/JCI162694. PMID: 36282589; PMCID: PMC9753998. Acknowledgements: Dr. Wayne Yokoyama and Dr. Liang Shan from Washington University. Disclosure of Interests: None declared.

Murine and human immune systems differ significantly, particularly within the myeloid lineage. Humanized mice, generated by transplanting human hematopoietic stem, progenitor cells into genetically modified mice, are invaluable to study human immune development and function in vivo. However, a major limitation of current models is suboptimal myelopoiesis, particularly lack of functional human neutrophils, hampering the modeling of human immune responses and chronic diseases. Here, we describe a humanized mouse model, named MaGIC for genes replaced, in the C57Bl/6 N strain, which improves human myelopoiesis and enables development of functional human neutrophils. In MaGIC mice, human cytokines M-CSF/CSF1(M), GM-CSF/CSF2(G) and IL-6(I) are knocked-in replacing mouse genes and murine IL2rg and Rag1(a) are deleted. Human THPO in these mice supports human hematopoiesis. More importantly, insertion of human CD47 (C) under the control of endogenous mouse CD47 promoter enables xenotransplantation and human neutrophil development. MaGIC mice support all human neutrophil subsets found in human bone marrow and blood, a major improvement. This is achieved by creating a niche postirradiation for human granulocyte-macrophage progenitors via reduced murine CD47 and physiological levels of human CD47. These mice also have mature human monocytes, tissue macrophages, alveolar macrophages, dendritic cells, and NK cells, enabled by humanized M-CSF and GM-CSF. Human neutrophils in MaGIC mice are fully functional in chemotaxis, phagocytosis, reactive oxygen species production, and neutrophil extracellular trap formation in response to inflammation. MaGIC mice address critical gaps in current models and enable incisive translational research on human neutrophils, advancing studies in infectious, autoimmune, and inflammatory diseases.

A strategy to functionally cure AIDS by eliminating latent HIV-1 reservoirs involves non-nucleoside reverse transcriptase inhibitors (NNRTIs) that promote pyroptosis of HIV-1 infected cells. These NNRTIs stimulate dimerization of the Gag-Pol polyprotein, resulting in premature HIV-1 protease (PR) dimerization and cleavage of intracellular CARD8. A unique cell-based high-throughput screen was developed to identify potent compounds activating the CARD8 inflammasome through Gag-Pol dimerization. Our in-house library of NNRTIs was evaluated, including a series of catechol diethers, which are potent, nontoxic antivirals. JLJ648 was identified as a promising dual-function antiviral and Gag-Pol dimerizer. Cryo-EM studies of HIV reverse transcriptase p66 bound to JLJ648 revealed populations of homodimers and, surprisingly, a homotetramer. This novel homotetramer structure resembling an ‘infinity knot’ revealed two JLJ648-bound homodimers forming an extensive interface and nucleated around a dimer of JLJ648 molecules. Structure-guided mutagenesis studies indicate that Gag-Pol homotetramerization may play a critical role in facilitating PR self-cleavage and triggering pyroptosis.

A critical determinant for early post-entry events, the HIV-1 capsid (CA) protein forms the conical core when it rearranges around the dimeric RNA genome and associated viral proteins. Although mutations in CA have been reported to alter innate immune sensing of HIV-1, a direct link between core stability and sensing of HIV-1 nucleic acids has not been established. Herein, we assessed how manipulating the stability of the CA lattice through chemical and genetic approaches affects innate immune recognition of HIV-1. We found that destabilization of the CA lattice resulted in potent sensing of reverse transcription products when destabilization per se does not completely block reverse transcription. Surprisingly, due to the combined effects of enhanced reverse transcription and defects in nuclear entry, two separate CA mutants that form hyperstable cores induced innate immune sensing more potently than destabilizing CA mutations. At low concentrations that allowed the accumulation of reverse transcription products, CA-targeting compounds GS-CA1 and lenacapavir measurably impacted CA lattice stability in cells and modestly enhanced innate immune sensing of HIV. Interestingly, innate immune activation observed with viruses containing unstable cores was abolished by low doses of lenacapavir. Innate immune activation observed with both hyperstable and unstable CA mutants was dependent on the cGAS-STING DNA-sensing pathway and reverse transcription. Overall, our findings demonstrate that CA lattice stability and reverse transcription are finely balanced to support reverse transcription and minimize cGAS-STING-mediated sensing of the resulting viral DNA. IMPORTANCE:In HIV-1 particles, the dimeric RNA genome and associated viral proteins and enzymes are encased in a proteinaceous lattice composed of the viral capsid protein. Herein, we assessed how altering the stability of this capsid lattice through orthogonal genetic and chemical approaches impacts the induction of innate immune responses. Specifically, we found that decreasing capsid lattice stability results in more potent sensing of viral reverse transcription products, but not the genomic RNA, in a cGAS-STING-dependent manner. The recently developed capsid inhibitors lenacapavir and GS-CA1 enhanced the innate immune sensing of HIV-1. Unexpectedly, due to increased levels of reverse transcription and cytosolic accumulation of the resulting viral cDNA, capsid mutants with hyperstable cores also resulted in the potent induction of type I interferon-mediated innate immunity. Our findings suggest that HIV-1 capsid lattice stability and reverse transcription are finely balanced to minimize exposure of reverse transcription products in the cytosol of host cells.

Human immunodeficiency virus (HIV) continues to be a significant global health challenge despite decades of research and advances in treatment. Substantial gaps in our understanding of the mechanisms of HIV pathogenesis and the host immune responses still exist. The interaction between HIV and these immune responses is pivotal in the disease progression to acquired immunodeficiency syndrome (AIDS). Recently, the caspase recruitment domain-containing protein 8 (CARD8) inflammasome has emerged as a crucial factor in orchestrating innate immune responses to HIV infection and exerting a substantial impact on viral pathogenesis. CARD8 restricts viral replication by detecting the activity of HIV protease. Conversely, it also contributes to the depletion of CD4+ T cells, a key feature of disease progression towards AIDS. The purpose of this review is to summarize the role of the CARD8 inflammasome in HIV pathogenesis, delving into its mechanisms of action and potential implications for the development of therapeutic strategies.

PURPOSE OF REVIEW:HIV requires lifelong antiviral treatment due to the persistence of a reservoir of latently infected cells. Multiple strategies have been pursued to promote the death of infected cells. RECENT FINDINGS:Several groups have focused on multipronged approaches to induce apoptosis of infected cells. One approach is to combine latency reversal agents with proapoptotic compounds and cytotoxic T cells to first reactivate and then clear infected cells. Other strategies include using natural killer cells or chimeric antigen receptor cells to decrease the size of the reservoir.A novel strategy is to promote cell death by pyroptosis. This mechanism relies on the activation of the caspase recruitment domain-containing protein 8 (CARD8) inflammasome by the HIV protease and can be potentiated by nonnucleoside reverse transcriptase inhibitors. SUMMARY:The achievement of a clinically significant reduction in the size of the reservoir will likely require a combination strategy since none of the approaches pursued so far has been successful on its own in clinical trials. This discrepancy between promising in vitro findings and modest in vivo results highlights the hurdles of identifying a universally effective strategy given the wide heterogeneity of the HIV reservoirs in terms of tissue location, capability to undergo latency reversal and susceptibility to cell death.

The pattern recognition receptor CARD8 is an inflammasome sensor for intracellular HIV-1 protease activity. Previously, the only method for studying the CARD8 inflammasome has been through utilizing DPP8/DPP9 inhibitors including Val-boroPro (VbP) to modestly and nonspecifically activate the CARD8 inflammasome. The identification of HIV-1 protease as a target for sensing by CARD8 has opened the door for a new method of studying the underlying mechanism of CARD8 inflammasome activation. Additionally, triggering the CARD8 inflammasome offers a promising strategy for reducing HIV-1 latent reservoirs. Here we describe the methods to study CARD8 sensing of HIV-1 protease activity through non-nucleoside reverse transcriptase inhibitor (NNRTI)-mediated pyroptosis of HIV-1-infected immune cells and through an HIV and CARD8 co-transfection model.

The biggest challenge to immune control of HIV infection is the rapid within-host viral evolution, which allows selection of viral variants that escape from T cell and antibody recognition. Thus, it is impossible to clear HIV infection without targeting "immutable" components of the virus. Unlike the adaptive immune system that recognizes cognate epitopes, the CARD8 inflammasome senses the essential enzymatic activity of the HIV-1 protease, which is immutable for the virus. Hence, all subtypes of HIV clinical isolates can be recognized by CARD8. In HIV-infected cells, the viral protease is expressed as a subunit of the viral Gag-Pol polyprotein and remains functionally inactive prior to viral budding. A class of anti-HIV drugs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs), can promote Gag-pol dimerization and subsequent premature intracellular activation of the viral protease. NNRTI treatment triggers CARD8 inflammasome activation, which leads to pyroptosis of HIV-infected CD4+ T cells and macrophages. Targeting the CARD8 inflammasome can be a potent and broadly effective strategy for HIV eradication.

Abstract The immune milieu within tumors, consisting of diverse cell types including adaptive immune cells as well as macrophages, dendritic cells, natural killer and other innate immune cells, is critical to determining cancer outcome. However, the immune tumor microenvironment (TME) has been challenging to model, owing to inherent inter-species differences. While humanized mice can support human immune cells, the hematopoietic stem and progenitor cells (HSPCs) used for transplantation have been largely limited to fetal or neonatal stem cell sources, necessitating allogeneic experiments with limited applicability. We sought to develop a method to pre-clinically model an individual adult cancer patient, capturing the unique features of an individual such as germline genetic determinants of immune function and somatic tumor heterogeneity, and creating an autologous system. MISTRG6 may be engrafted with low numbers of HSPCs. When engrafted with equivalent numbers of CD34+ cells from human fetal liver (FL), neonatal cord blood (CB), adult mobilized peripheral blood (MPB), or adult bone marrow (BM), MISTRG6 mice harbored greatly increased human hematopoietic cells as a proportion of total hematopoietic cells in peripheral blood compared with NOD-scid-gamma (NSG) and MISTRG mice (p<0.0001). We found that MISTRG6 mice could be engrafted with as few as 1,000 human HSPCs, arguably 100x more efficient than other models, and achieve robust hematopoietic transplantation after 10-12 weeks, indicating the efficiency of this strain in supporting the growth of hematopoietic cells. To better elucidate the mechanism responsible for this enhanced human engraftment, we enumerated human and mouse hematopoietic progenitors in BM of NSG, MISTRG, and MISTRG6 mice. Human progenitors, including CD34+ and CD34+CD38+ cells, were significantly increased in both frequency and absolute numbers in MISTRG and MISTRG6 mice compared with NSG mice (p<0.001), and mouse hematopoietic lin(-)cKit+ (LK) and lin(-)Sca1+cKit+ (LSK) progenitor populations were significantly diminished (p<0.0001), suggesting that the enhanced hematopoietic engraftment observed in MISTRG6 is, in part, a consequence of increased human progenitor frequency and reduced mouse competition. MISTRG6 allows efficient engraftment of patient derived HSPCs. We sought to apply this improved engraftment prospectively to model individual patients’ TME through collection of BM-derived CD34+ cells from patients under active treatment along with tumor tissue from the same patient. At two cancer centers, we enrolled patients with melanoma, NSCLC, PDAC, and HNSCC to provide BM aspirate, peripheral blood, and tumor tissue. CD34+ cells were isolated from BM aspirates and tumor tissue was utilized to generate PDXs. Overall, 71 patients were enrolled, 46 melanoma, 19 NSCLC, 4 PDAC, 2 HNSCC, ages 22-85, 39% females. These yielded autologous, immune-reconstituted MISTRG6 hosts from 14 melanoma, 5 NSCLC, 2 PDAC, and 1 HNSCC patients. Autologously engrafted MISTRG6 mice displayed the gamut of human immune cells of adaptive and innate types in PB at 7 weeks of age. Notably, this included CD33+ myeloid cells such as CD14+CD16− classical, CD14+CD16+ intermediate, and CD14−CD16+ non-classical monocytes. Moreover, human dendritic cells (DCs), key innate immune cells for initiation of anti-tumor responses were readily detected by flow cytometry in spleens of autologously-engrafted mice, including cDC1, cDC2, and pDC cells. MISTRG6 mice bearing a patient’s hematopoietic cells support autologous PDX growth. Having achieved successful engraftment of patient hematopoietic systems in MISTRG6 hosts, we next subcutaneously introduced the patient’s matched PDX tumor tissue to generate autologously engrafted PDX mice. For most patients, tumors grown in autologous HSPC-engrafted hosts were significantly larger than in non-engrafted hosts. Multicolor immunofluorescence staining of PDX tumors demonstrated that human immune cells, including CD3+ T cells, CD14+ and HLA-DR+ myeloid cells, penetrated deeply into the tumor and co-localized with tumor cells as well as with other engrafted immune cells. Indeed, HLA-DR+CD14+macrophages and HLA-DR+CD14(-) dendritic cells were present, and direct physical interaction between T cells and macrophages was evident. Using whole-exome sequencing, we found that 225 somatic changes were shared between patient Mel738’s surgical resection sample, two PDX tumors from non-engrafted mice lacking human immune cells, and two PDX tumors from mice with autologous engraftment. 5 additional changes were shared among the tumor samples and absent from the cell line, with 36 additional mutations being specific to the cell line. These data underscore the capacity of the autologous PDX method to recapitulate the somatic heterogeneity that the patient tumor possesses. Autologous MISTRG6 mice display diverse human immune cell populations and recapitulate an immunosuppressive TME. To fully characterize the autologous MISTRG6 model and investigate mechanisms by which autologous human immune cells enhance tumor growth, we performed single cell transcriptomics on hCD45+-enriched cells from blood and tumor isolated from autologous mice. This revealed 16 distinct cell subtypes, including 3 myeloid, 2 NK cell, 2 CD8 T cell, 3 CD4 T cell, 2 cycling lymphocyte, 1 B cell, and 3 melanoma cell clusters. Subclustering of myeloid cells revealed 9 distinct clusters including 4 monocyte, 4 macrophage, and 1 DC cluster. Comparing CD8 T cells present in blood versus tumor revealed that the most differentially expressed genes (DEGs) found in blood were characteristic of naïve T cells, while genes present in the TME were consistent with activated T cell phenotypes. In addition, sub-clustering revealed 3 distinct CD8 T cell types that included two activated-like populations, with one of these populations also expressing an activated/exhausted program typified by expression of PDCD1, LAG3, and GZMA. Naïve-like T cells were most highly represented in the blood, while activated and activated/exhausted-like genes were more present in the TME. Inhibiting the actions of human VEGF-A blocks the enhanced tumor growth in autologously engrafted mice. Notably, IPA Upstream Regulator Analysis identified VEGFA, a central player in tumor growth and vascularization, as a key upstream inducer of genes in the TME (FDR p= 5.65 × 10−13). Indeed, expression VEGFA itself was nearly absent in blood but induced in the TME, especially in macrophages and VEGFA targets were highly represented among the DEGs between tumor and blood.To test the relevance of VEGF-A in the TME, we selectively blocked human VEGF-A by treating autologous mice humanized from Mel2 with the anti-hVEGF-A antibody bevacizumab that has high affinity for human VEGF-A yet low affinity for mouse VEGF-A. PDXs grown in untreated autologously engrafted MISTRG6 mice grew significantly larger than those in non-engrafted littermate control hosts (p<0.05). When treated with bevacizumab, the enhanced tumor growth was significantly abrogated, with bevacizumab-treated mice bearing significantly smaller tumors compared with controls (p<0.001). Future Directions: Thus, these in silico and in vivo results suggest that human VEGF-A production in the autologous TME enhances tumor growth in MISTRG6 PDX models and underscores the utility of the MISTRG6 system for pre-clinical testing of drugs that act on human immune components of the TME. By engrafting mice with bone marrow derived stem cells followed by implantation of tumor derived from the same donor, we have demonstrated that autologous MISTRG6 models recapitulate important features of the human TME, including sufficient immunosuppression to prevent tumor clearance, presence of activated/exhausted T cells, and innate immune cells including DCs, monocytes, NK cells, and macrophages, the latter especially relevant to the production of VEGF-A. Citation Format: Michael Chiorazzi, Jan Martinek, Bradley Krasnick, Yunjiang Zheng, Keenan Robbins, Rihao Qu, Gabriel Kaufmann, Zachary Skidmore, Laura Henze, Frederic Brösecke, Adam Adonyi, Jun Zhao, Liang Shan, Esen Sefik, Jacqueline Mudd, Ye Bi, S Peter Goedegebuure, Malachi Griffith, Obi Griffith, Abimbola Oyedeji, Sofia Fertuzinhos, Roland Garcia-Milian, Daniel Boffa, Frank Detterbeck, Andrew Dhanasopon, Justin Blasberg, Benjamin Judson, Scott Gettinger, Katerina Politi, Yuval Kluger, A Karolina Palucka, Ryan Fields, Richard A. Flavell. Autologous humanized PDX modeling for immuno-oncology recapitulates the human tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr NG11.

CD4 + T-cell depletion is the root cause of acquired immunodeficiency syndrome (AIDS). How HIV depletes CD4 + T cells in humans remains unknown because vast majority of the dying CD4 + T cells in patients are uninfected. Burgeoning evidence supports the hypothesis that non-productive HIV-1 infection triggers CD4 + T-cell depletion in the course of pathogenic HIV and SIV infections. Here, we report that the CARD8 inflammasome is activated immediately after HIV-1 entry by the viral protease encapsulated in the incoming HIV-1 particles. Sensing of HIV-1 protease activity by the CARD8 inflammasome leads to rapid pyroptosis of quiescent CD4 + T cells without productive viral infection. In humanized mice reconstituted with a CARD8-deficient immune system, CD4 + T-cell loss is delayed despite increased levels of HIV-1 replication. Our study suggests that the CARD8 inflammasome drives CD4 + T-cell depletion and disease progression through rapid sensing of HIV-1 particles.