Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Representation of HIV pMorpheus-V5 genome, with the LTR-dependent (HSA-mCherry-IRES-Nef) and LTR-independent (PGK-V5-NGFR) cassettes highlighted. (b) Representation of peripheral blood CD4 + T naïve and memory subsets (T memory stem cells [T SCM ], central memory [T CM ], transitional memory [T TM ] and effector memory [T EM ]). (c) Representative Uniform Manifold Approximation and Projection (UMAP) of 211,934 cells overlaying five CD4 + T cell subsets (naïve, T SCM , T CM , T TM , T EM ) within total CD4 + T cell population (gated on live cells, CD4 + T cell subsets are color-coded), at five days post-infection (dpi). (d) Bar graph showing the frequencies for each CD4 + T cell subset within total live CD4 + population (n=8), at five dpi. (e) Representative contour plot showing FACS gating strategy to define non-productively infected (NP), productively infected (P) and negative-exposed (NE) CD4 + T cells (cells gated on total CD4 + cells), defined by NGFR and mCherry expression. (f) Frequencies of the NP, P and NE populations in total CD4 + T cells, at the time of FACS analysis (five days post-infection, n=8). (g) Percentages of non-productively and productively infected cells in each CD4 + T cell subset considered (n=8) with the ratio of productive over non-productive (P/NP ratio) highlighted for each subset. Each symbol represents a different healthy blood donor, with the four donors used for subsequent CD4 + T SCM sorting highlighted with different symbols. Donors used for analysis but not sorted are represented by paler dots. Primary CD4 + T cells were stimulated with αCD3/CD28 antibodies for 72 hours before infection with HIV pMorpheus-V5 single-round reporter virus for five days before analysis. Data presented as the mean with SD.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: Infection, Expressing, Virus

Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Representative gating strategy of purified peripheral blood CD4 + T cells infected with HIV pMorpheus-V5 reporter virus, at the time of sorting (five days post-infection). Total cultured CD4 + T cells were gated using physical parameters, then gated for Live Dead NIR and subsequently for CD45RA/CD45RO (“1” and “2”). CD45RA + cells (“1”) were gated for CD27/CCR7. CD27 + CCR7 + cells were gated for CD95/CCR7: CD95 - CCR7 + cells were considered CD4 + naïve cells, CD95 + CCR7 + cells were considered CD4 + T SCM . NP CD4 + T SCM cells were NGFR + mCherry - (Q1), P cells were NGFR + mCherry + (Q2), NE cells were NGFR - mCherry - (Q4). CD45RO cells (“2”) were gated for CD27/CCR7: CD27 + CCR7 + cells were considered T CM , CD27 + CCR7 - cells were considered T TM , CD27 - CCR7 - cells were considered T EM . (b) Dot plot representing total infection levels (NGFR + mCherry - + NGFR + mCherry + ) in CD4 + T naïve and memory subsets (n=8). Significance was calculated using one-way ANOVA (Dunnett’s multiple comparisons test, *, P < 0.05: **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, ns, not significant). Each symbol represents a different healthy blood donor, with the four donors used for subsequent CD4 + T SCM sorting highlighted with different symbols. Donors used for analysis but not sorted are represented by dots. (c) Percentages of non-productively (NP) and productively (P) infected cells in total CD4 + T cells and in (d) each CD4 + T cell subset considered, in the four donors sorted (each donor is represented by a different symbol, n=4). (e) Frequencies of CD4 + T SCM NP, P and NE (negative-exposed) sorted populations in the four sorted donors (each donor is represented by a different symbol, n=4). (f) Dot plot representing the ratio between productively and non-productively infected cells in total CD4 + T cells and in the subsets considered (n=7). Primary CD4 + T cells from four different healthy donors (donors 1, 2, 3, 4) were stimulated with αCD3/CD28 antibodies for 72 hours before infection with HIV pMorpheus-V5 single-round reporter virus for five days before sorting. Data presented as the mean with SD.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: Purification, Infection, Virus, Cell Culture

Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Experimental strategy. Freshly isolated CD4 + T cells from peripheral blood were spinoculated with the pMorpheus-V5 dual reporter virus. Five days post-infection, cells were stained with a panel of antibodies to discriminate between CD4 + T cell subsets, and CD4 + T SCM were sorted based on the infection outcome (NP, P, NE or mock). RNA and DNA were extracted from lysed cells and bulk RNAseq was performed (n=4). (b) Principal component analysis (PCA) of gene expression pattern in sorted populations (NP, P, NE, mock) of four donors (donor 1-2-3-4). (c) Unsupervised clustering heatmap with dendrograms from bulk RNA sequencing (RNAseq) showing the expression levels (Z-score) of differentially expressed genes across sorted populations and donors. (d) Heatmap of selected differentially expressed genes (Z-score is shown, row normalized considering only the displayed genes) across all sorted CD4 + T SCM populations. (e) Volcano plots representing the relative expression of all genes, with highlighted genes encoding for CCR4-binding chemokines, tryptophan catabolism enzymes, cytoskeletal rearrangement and other relevant genes in NP vs P (left) and NP vs mock (right) comparisons. 131 genes upregulated in NP vs P, 185 upregulated in NP vs mock, (log 2 FoldChange (FC) >= 2, FDR <= 0.01). (f) Gene ontology (GO) molecular function (MF) of significantly upregulated genes in NP CD4 + T SCM , compared to P (left) and mock (right). (g) Pre-ranked gene set enrichment analysis (GSEA) for genes differentially expressed between NP and P sorted populations, using a molecular signature of chemokine signaling (left) and tryptophan metabolism (right). Normalized enrichment score (NES) is shown.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: Isolation, Virus, Infection, Staining, RNA sequencing, Gene Expression, RNA Sequencing, Expressing, Binding Assay

Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Schematic of pMorpheus-V5 reporter virus with arrows denoting the location of 4 probes, Gag-FAM, 5’Pol-ABY, 3’Pol-VIC, and Tat-Cy5. (b) Representative data from dPCR with single and double positive wells of each probe pair. (c) dPCR-PIA was run on DNA isolated from infected and sorted for non-productive and productive proviruses, from either sorted CD4 + T SCM or total CD4 + T cells. Schematic to the left depicts the permutations of probe combinations while the bar graphs to the right show the % of positive wells that correspond to each combination averaged across four donors (or three donors for total CD4 + NP). (d) Pie charts summarize the percentages of proviruses which are called as intact (++++) or positive for any 3, 2, or 1 probe. The brackets above denote the significance of paired t-test comparing the fraction of intact in NP vs P across paired donors (ns, Not Significant, *, p<0.05, **, p<0.01).

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: Virus, Isolation, Infection

Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Mean-difference (MD) plot showing that no differentially expressed genes were found in CD4 + T SCM NE compared to Mock (|log 2 FC| >= 2 and FDR <= 0.01). (b) Volcano plot representing the relative expression of all genes in the CD4 + T SCM P vs mock comparison, with upregulated genes in red and downregulated genes in blue. (c) Venn diagram showing significantly upregulated genes in CD4 + T SCM NP compared to P, NE and mock (log 2 FC >= 2 and FDR <= 0.01). (d) Heatmap of CD4 + T SCM selected differentially expressed genes (Z-score is shown, row normalized considering only the displayed genes) across all sorted populations. (e) Boxplots representing transcript counts per million (CPM) detected by RNAseq in CD4 + T SCM for genes encoding for CCL22 , CCL17 , KYNU, IDO1 , BASP1 and TNFAIP2 in all sorted conditions (n=4). (f) Venn diagram of significantly upregulated genes in CD4 + T SCM harboring an integrated pMorpheus-V5 provirus (either NP or P compared to NE and mock, log 2 FC >= 2 and FDR <= 0.01). The six shared significantly upregulated genes are highlighted in the box below the Venn diagram. (g) Gene Ontology (GO) of Biological Processes (BP) of significantly upregulated genes in NP CD4 + T SCM , compared to P (left) and mock (right).

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: Expressing, Comparison, RNA sequencing

Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Representative gating strategy of purified peripheral blood CD4 + T cells infected with HIV pMorpheus-V5 reporter virus, at the time of sorting (five days post-infection). Total cultured CD4 + T cells were gated using physical parameters, then negative for Live Dead NIR, then gated for NGFR and mCherry. NGFR + mCherry - cells were gated for HSA: NGFR + mCherry - HSA - cells were considered CD4 + T cells non-productively infected with HIV pMorpheus-V5. NGFR + mCherry + were gated for HSA: NGFR + mCherry + HSA + were considered CD4 + T cells productively infected with HIV pMorpheus-V5. (b) Percentages of non-productive, productive infections and negative-exposed in total sorted CD4 + T cells, in the five sorted donors (donors 5-6-7-8-9, n=5). Each sorted donor is represented by a different symbol. Primary CD4 + T cells were stimulated with αCD3/CD28 antibodies for 72 hours before infection with HIV pMorpheus-V5 single-round reporter virus. Data presented as the mean with SD.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: Purification, Infection, Virus, Cell Culture

Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Expression of CCL22 and CCL17, (b) KYNU and IDO1, (c) BASP1 and TNFAIP2 mRNA in FACS-sorted total CD4 + T cells (Productively infected P, negative-exposed NE, mock unexposed mock, non-productively infected NP, compared to P) infected with HIV pMorpheus-V5 reporter virus (n=3). Significance was calculated on log-transformed fold-change values (normalized to P fold-change values, set as 1) using both one-way ANOVA with multiple comparisons (Dunnett’s multiple comparisons test, between NP, NE and mock) and one-sample t and Wilcoxon test (between single populations compared to P, only NP vs P t test significance is shown). *, P < 0.05: **, P < 0.01, ***, P < 0.001, ****, P < 0.0001, ns, not significant. Each dot represents a different donor.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Virus, Transformation Assay

Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Uniform manifold approximation and projection (UMAP) of 43,112 naïve (TN) and memory (TM) CD4 + T cells from Cano-Gamez et al. (2020) . Cell type populations in the UMAP are colored according to the legend: naïve, central (T CM ) and effector (T EM ) memory cells, effector memory cells re-expressing CD45RA (T EMRA ), natural T regulatory (nTreg). (b) Dot plot showing the average gene expression per cell type and respective percentage of cells expressing each of 16 NP-upregulated genes, including chemokines ( CCL22, CCL17, CCL19, CXCL9, CXCL10, EBI3 ), tryptophan catabolic enzymes ( IDO1, IDO2, KYNU, IL4I1 ), and cytoskeletal regulators ( TNFAIP2, BASP1, FSCN1, MARCKS, PLEK, CYRIA ). (c) Heatmap displaying the expression levels of the 16 genes and corresponding UCell enrichment scores at the single-cell level in 2,756 CD4 + T cells (6.4%) in cells with detectable levels of CCL22 , CCL17 or CCL19 (marked by *). (d) UMAP of 1,821,725 human immune cells within the Human Immune Health Atlas (T cells, B cells, monocytes, natural killer (NK) cells, and 12 other subsets including dendritic cells (DC) and hematopoietic precursors) . Cell type populations in the UMAP are colored according to the legend. (e) Same as panel (b) but for the Human Immune Health Atlas . (f) Same as panel (c) but for the 448 cells (0.025%) with detectable CCL22 , CCL17 or CCL19 expression in the Human Immune Health Atlas (marked by *) .

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: Expressing, Gene Expression, Single Cell

Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Experimental outline of CD4 + T cells treated with either CCL22, Tryptophan or IL-2 (media control). CD4 + T cells were TCR activated for three days in the presence of IL-2. Six hours before spinoculation, CCL22 or tryptophan was added to the cells. After spinoculation, cells were kept in either CCL22, tryptophan or control media until FACS staining and analysis. (b through g) Bar graphs showing the percentages of NP and P cells in the conditions treated with CCL22, tryptophan or IL-2 (media control), in total CD4 + T cells (b), naïve (c), T SCM (d), T CM (e), T TM (f) and T EM (g, n=3), with the ratio between P and NP highlighted on each condition (P/NP).

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: Control, Staining

Journal: bioRxiv

Article Title: Three immunoregulatory signatures define non-productive HIV infection in CD4 + T memory stem cells

doi: 10.64898/2026.03.20.713012

Figure Lengend Snippet: (a) Representative contour plots gated on CD4 + T SCM population showing the percentage of CCL22 + IDO1 + cells in NP, P, NE and mock populations. (b) Representative contour plots gated on CD4 + T SCM population showing the percentage of single positive populations for either IDO1 or CCL22, in CD4 + T SCM NP, P, NE and Mock. (c through h) Bar graphs displaying the percentages of CCL22 + IDO1 + cells in every subset considered, in naïve (c), T SCM (d), T CM (e), T TM (f), T EM (g) and total CD4 + T cells (h, n=3). (i) Bar graph showing the percentage of CCL22 + IDO1 + in NP population across the CD4 + T cell subsets considered (n=3). Significance was calculated using Repeated Measures one-way ANOVA with multiple comparisons (Dunnett’s multiple comparisons test, between NP, P, NE and Mock). *, P < 0.05: **, P< 0.01, ***, P < 0.001, ****, P < 0.0001, ns, not significant. Data presented as the mean with SD.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified through density gradient centrifugation (Lymphoprep, Stemcell Technologies # 07851) and CD4 + T cells were isolated through negative selection (Miltenyi, CD4 + T cell Isolation Kit human # 130-096-533) according to the manufacturer’s instructions.

Techniques: