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Journal: Cell reports

Article Title: Anatomical, subset, and HIV-dependent expression of viral sensors and restriction factors

doi: 10.1016/j.celrep.2024.115202

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD4 , Standard BioTools , Cat3174004B; RRID:AB_3661864.

Techniques: Virus, Recombinant, Electron Microscopy, Clinical Proteomics, Modification, Antibody Labeling, Staining, Software

Journal: Cell reports

Article Title: Anatomical, subset, and HIV-dependent expression of viral sensors and restriction factors

doi: 10.1016/j.celrep.2024.115202

Figure Lengend Snippet: (A) Spleen, lymph node (LN), gut, male reproductive tract, kidney, liver, bone marrow (BM), heart, and lung were harvested postmortem from antiretroviral therapy (ART)-treated people with HIV (PWH; N = 7) and analyzed by VISOR-CyTOF. Sample sizes for individual tissues are indicated. Total numbers of immune, myeloid, B cells, CD4 + T, and CD8 + T cells analyzed are shown. Created with BioRender.com . (B and C) VISOR-CyTOF distinguishes myeloid, B, and naive and memory CD4 + and CD8 + T cells from tissues and blood. Immune cells from the tissues of PWH as described in panel (A) (B) and PBMCs from people without HIV (PWOH; N = 4 participants) (C) were phenotyped by VISOR-CyTOF and visualized by tSNE using all markers in the panel. (D and E) tSNE analysis using only VISOR markers of VISOR-CyTOF separates myeloid, B, and T cells from one another. Data from the tissues from PWH (D) and PBMCs from PWOH (E) are shown. In (B)–(E), overlaid tSNEs are shown on the left, while tSNEs separated out by each immune subset are shown on the right. Abbreviations: Tm, memory T cells; Tn, naive T cells.

Article Snippet: CD4 , Standard BioTools , Cat#3174004B; RRID:AB_3661864.

Techniques:

Journal: Cell reports

Article Title: Anatomical, subset, and HIV-dependent expression of viral sensors and restriction factors

doi: 10.1016/j.celrep.2024.115202

Figure Lengend Snippet: (A and B) VISORs are expressed at the highest and lowest levels in myeloid and B cells, respectively, with T cells exhibiting intermediate expression. Results from tissues of PWH (A) and PBMCs of PWOH (B) are shown. (C–E) VISOR expression patterns differ in CD4 + T cell subsets. VISORs are more weakly expressed in CD4 + Tem relative to Tcm across tissues of PWH (C). In LN of PWH, VISORs are highly expressed in CD4 + Tfh, Treg, and Tcm and lowly expressed in Tem, Ttm, and CD69 + Tm (D). In PBMCs of PWOH, VISORs are highly expressed in CD4 + Treg, Tfh, and Tcm and lowly expressed in Ttm and Tem; low VISOR expression among CD69 + Tm likely reflects their being activated cell instead of true Trm (E). For all heatmaps, color intensity (blue to red) denotes the column-normalized mean-scaled expression of each indicated VISOR within the indicated cell subset. Data were generated by unsupervised clustering. Abbreviations: CD4 + Tcm, CD4 + T central memory; CD4 + Tem, CD4 + T effector memory; CD4 + Ttm, CD4 + T transitional memory; CD4 + Tfh, CD4 + T follicular helper; CD4 + Treg, CD4 + T regulatory; CD4 + CD69 + Tm, CD69 + T memory as a proxy for T resident memory (Trm) cells.

Article Snippet: CD4 , Standard BioTools , Cat#3174004B; RRID:AB_3661864.

Techniques: Expressing, Generated

Journal: Cell reports

Article Title: Anatomical, subset, and HIV-dependent expression of viral sensors and restriction factors

doi: 10.1016/j.celrep.2024.115202

Figure Lengend Snippet: (A) In LN, restriction factor TRIM28, which blocks HIV integration, is lowly expressed in HIV-permissive CD4 + Tfh as compared to non-Tfh, while HIV transcription inhibitors (BRD4 and IFI16) and HIV translation inhibitors (IFITM1 and IFIT1) exhibit the opposite expression pattern. (B) In PBMCs, TRIM28, BRD4, IFI16, IFITM1, and IFIT1 are all more highly expressed in CD4 + Tfh as compared to non-Tfh. * p < 0.05, ** p < 0.01, as assessed using Student’s two-sided paired t test. Error bars correspond to SD. (C) Proposed model of restriction factor-mediated HIV latency in LN CD4 + Tfh. Low expression of TRIM28 in LN CD4 + Tfh facilitates HIV integration, while high levels of BRD4, IFI16, IFITM1, and IFIT1 limit HIV gene expression thereby promoting latency. Created with BioRender.com .

Article Snippet: CD4 , Standard BioTools , Cat#3174004B; RRID:AB_3661864.

Techniques: Expressing, Gene Expression

Journal: Cell reports

Article Title: Anatomical, subset, and HIV-dependent expression of viral sensors and restriction factors

doi: 10.1016/j.celrep.2024.115202

Figure Lengend Snippet: (A) Fusion assay experimental design. PHA-stimulated, CCF2-loaded PBMCs ( N = 4) were mock-treated or incubated for 2 h with HIV-F4.HSA virions packaged with Blam-Vpr. Cleavage of CCF2 in HIV-fused cells by Blam-Vpr results in shift from green to blue fluorescence, detectable by FACS. Created with BioRender.com . (B) CCF2 profiles of uninfected and HIV-exposed cultures, which were sorted for unfused and HIV-fused cells and then analyzed by CyTOF-VISOR. Events were pre-gated on live, singlet cells. Gating strategy in . (C and D) tSNE plots of unfused and HIV-fused CD4 + Tm (C) or myeloid (D) cells analyzed by VISOR-CyTOF, highlighting dissimilarity of HIV-fused cells to their unfused counterparts. tSNE plots were generated using all markers in the CyTOF panel. (E and F) HIV-fused CD4 + Tm and myeloid cells express low levels of multiple VISORs, relative to their unfused counterparts. HIV-fused CD4 + Tm (E) and myeloid (F) cells lowly express sensors of HIV capsid (PQBP1 and MX2), RNA (RIGI), and DNA (AIM2, cGAS, and pIRF3). Additionally, HIV DNA sensor IFI16 is lowly expressed in HIV-fused CD4 + Tm (E), while HIV DNA sensors TLR9 and pSTING are lowly expressed in HIV-fused myeloid cells (F). With regards to restriction factors, HIV-fused CD4 + Tm express low levels of factors targeting HIV reverse transcription (SAMHD1 and PAF1), nuclear import (MX2), integration (TRIM28), transcription (TRIM28, IFI16, and BRD4), and translation (IFITM1) (E). HIV-fused myeloid cells express low levels of restriction factors targeting HIV nuclear import (MX2), transcription (BRD4), and translation (IFITM1) (F). (G) HIV-fused myeloid cells express high levels of SAMHD1 and TRIM28. The phosphorylated and inactive form of SAMHD1 (pSAMHD1) exhibits the opposite expression pattern. (H and I) HIV-fused and unfused cells express equivalent levels of HIV receptor and co-receptor. Mean expression levels of CD4 and CCR5 in unfused and HIV-fused CD4 + Tm (H) and myeloid (I) cells. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., non-significant, as assessed using Student’s two-sided paired t tests. Error bars correspond to SD.

Article Snippet: CD4 , Standard BioTools , Cat#3174004B; RRID:AB_3661864.

Techniques: Single Vesicle Fusion Assay, Incubation, Fluorescence, Generated, Reverse Transcription, Expressing

Journal: Cell reports

Article Title: Anatomical, subset, and HIV-dependent expression of viral sensors and restriction factors

doi: 10.1016/j.celrep.2024.115202

Figure Lengend Snippet: (A) Productive infection assays experimental design. PHA-stimulated PBMCs ( N = 4) were mock-treated or exposed to HIV-F4.HSA for 3 days and then analyzed by VISOR-CyTOF. Analyses were performed on events corresponding to bystander or productively infected cells, which were defined based on the expression of long terminal repeat-driven reporter gene HSA. Gating strategies are shown in and . Created with BioRender.com . (B) Identification of productively infected cells by VISOR-CyTOF. Events were pre-gated on live, singlet, CD19 – CD8 – cells. (C and D) tSNE plots of bystander and productively infected CD4 + T (C) or myeloid (D) cells analyzed by VISOR-CyTOF, highlighting dissimilarity of bystander cells to their productively infected counterparts. tSNE plots were generated using all markers in the CyTOF panel. (E and F) Productively infected cells express high levels of VISORs relative to their bystander counterparts. Shown are viral sensors and restriction factors that are significantly elevated in productively infected CD4 + T (E) and myeloid (F) cells relative to their bystander counterparts. Viral sensors significantly upregulated in productively infected CD4 + T and myeloid cells were capsid sensors (PQBP1 and MX2) and DNA sensors (TLR9, AIM2, IFI16, cGAS, and pIRF3). In productively infected CD4 + T but not myeloid cells, HIV RNA sensors RIGI and pSTING were upregulated. Restriction factors upregulated in both productively infected CD4 + T and myeloid cells were those blocking HIV reverse transcription (SAMHD1 and PAF1), nuclear import (MX2), integration (TRIM28), transcription (TRIM28, IFI16, and BRD4), and translation (IFITM1). Productively infected CD4 + T but not myeloid cells also upregulated inactive pSAMHD1. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, as assessed using Student’s two-sided paired t tests. Error bars correspond to SD.

Article Snippet: CD4 , Standard BioTools , Cat#3174004B; RRID:AB_3661864.

Techniques: Infection, Expressing, Generated, Blocking Assay, Reverse Transcription

Journal: Cell reports

Article Title: Anatomical, subset, and HIV-dependent expression of viral sensors and restriction factors

doi: 10.1016/j.celrep.2024.115202

Figure Lengend Snippet: (A) Experimental design for HIV infection of cGAMP-stimulated PBMCs. PHA-stimulated PBMCs ( N = 3) were incubated for 20 h with cGAMP-containing viral-like particles (VLPs), or control VLPs harboring catalytically inactive cGAS mutant, and then analyzed by VISOR-CyTOF. In parallel, VLP-treated cells from the same donor were mock-infected or exposed to HIV-F4.HSA, cultured for 3 days, and monitored for infection rates. Created with BioRender.com . (B) cGAMP-treated CD4 + T cells exhibited elevated expression of HIV DNA sensors (AIM2, cGAS, and pIRF3), as well as restriction factors targeting reverse transcription (SAMHD1) and translation (IFITM1). By contrast, inactive pSAMHD1 trended lower among cGAMP-treated cells. * p < 0.05, ** p < 0.01, as assessed using Student’s two-sided paired t tests. (C and D) cGAMP-treated CD4 + T cells restrict HIV infection. Representative CyTOF plots of uninfected and infected PBMC cultures treated with control or cGAMP VLPs (C) and cumulative results from 4 independent donors (D). Results were pre-gated on live, singlet, CD19 – CD3 + CD8 – cells. * p < 0.05, ** p < 0.01; n.s., non-significant, as assessed using one-way repeated measures ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: CD4 , Standard BioTools , Cat#3174004B; RRID:AB_3661864.

Techniques: Infection, Incubation, Control, Mutagenesis, Cell Culture, Expressing, Reverse Transcription

Journal: Cell reports

Article Title: Anatomical, subset, and HIV-dependent expression of viral sensors and restriction factors

doi: 10.1016/j.celrep.2024.115202

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CD4 , Standard BioTools , Cat#3174004B; RRID:AB_3661864.

Techniques: Virus, Recombinant, Electron Microscopy, Clinical Proteomics, Modification, Antibody Labeling, Staining, Software