Plasmacytoid and CD141+ Myeloid Dendritic Cells Cooperation with CD8+ T Cells in Lymph Nodes is Associated with HIV Control

Abstract

Dendritic cells (DC) are known to modulate antiviral immune responses; however, the knowledge about the role of different DC subsets in antiviral T cell priming in human tissues remains uncompleted. In the context of HIV infection, we determined the phenotype and location of plasmacytoid and CD141+ myeloid DCs (pDCs and mDCs) in lymph nodes of people living with HIV (PLWH). We found an interaction between pDCs and CD141+ mDCs with CD8+ T cells, being associated with participants' viral levels in blood and tissue. Moreover, we demonstrated a higher and more polyfunctional superantigen- and HIV-specific CD8+ T cell response after the coculture with Toll-like receptor (TLR)-primed pDCs and CD141+ mDCs. Last, we showed the potential of programmed cell death-1 (PD-1) blocking using pembrolizumab to further increase antigen-specific CD8+ T cell response along with TLR agonists. Therefore, these results showed a cooperation between pDCs, CD141+ mDCs and CD8+ T cells in lymph nodes of PLWH, which is associated with higher HIV control, highlighting the importance of DC subsets crosstalk to achieve a more potent anti-HIV response and support the use of DC-based immunotherapies for HIV control.

Keywords: CD141 myeloid DC; CD8 T cell; HIV; dendritic cell (DC); lymph node; plasmacytoid DC.

FIGURE 1

pDCs and CD141+ mDCs are associated with viral load and follicular T cells. (A) Representative pseudocolor dot plots showing the gating strategy for pDC, CD141+ mDC, and fCD8+ T cell subsets and markers. (B) Correlations between pDC and CD141+ mDC percentages with viral load in lymph nodes (LN, green) and peripheral blood (blood, blue). (C) Correlation of PD‐L1+ pDC percentage with viral load in LN (green). (D) Correlation matrix representing the associations between pDCs, CD141+ mDCs, and follicular CD8+ (fCD8) and CD4+ (TFH) T cells in LN. Green and purple colors represent positive and negative correlations, respectively. The intensity of the color and the size of the squares indicate the R coefficient. Spearman test was used (n = 7) and ROUT method was utilized to identify and discard outliers (Q = 0.1%). *p < 0.05, **p < 0.01. p Values between 0.05 and 0.1 were considered as tendency and were shown as numbers.

FIGURE 2

pDC, CD141+ mDC, and CD8+ T cell interaction in LN of PLWH. (A) Dot plot graphs showing representative data of histocytometry analysis of a LN (LNP012), all cells are represented in blue and CD20+ cells (follicles) in red; follicles (F) and the closest T cell zones (T) were gated and pDC and CD141+ mDC percentages were determined (left). Bar graphs showing the percentages of pDCs and CD141+ mDCs of LNs from PLWH within the follicles (F) and T cell zones (T); each dot represents a follicle (right). (B) Representative confocal images showing the contact (white arrowheads) between pDCs (cyan) with CD8+ T cells (magenta) and CD141+ mDCs (yellow) with CD8+ T cells (magenta) (up panels). M1 and M2 Manders coefficients of CLEC9a (CD141)–CD8 and CD123 (pDC)–CD8 according to the colocalization patterns in confocal images (bottom panels). (C) Representative 3D microscopy image showing the contact between CD8+ T cells (magenta), CD141+ mDCs (yellow), and pDCs (cyan) in LN from PLWH (LNP012). (D) Representative images showing the analysis of the distance of DC–CD8+ T cell interaction to the follicles in LN; CD8+ T cells (magenta), CD141+ mDCs (yellow), pDCs (cyan), and follicles (red). (E) Bar graphs showing the distance (µm) of pDC–CD8+ T cell and CD141+ mDC–CD8+ T cell interaction and triple interaction to the follicle (up panel). Each dot represents an interaction. Correlation of the distance of pDC–CD8+ T cell interaction to the follicle with viral load (VL). All interactions are shown in black and the median of the interactions in each participant are represented in purple (bottom panel). PLWH are represented with different colors. Wilcoxon and Spearman tests were used (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

The interaction of primed pDCs and CD141+ mDCs with CD8+ T cells induces a higher SEB‐specific CD8+ T cell response in HD. (A) Representative pseudocolor dot plots and (B) bar graphs showing the percentages of IFNγ+, TNFα+, IFNγ+TNFα+, CD107+, and perforin (PRF)+ CD8+ T cells after prestimulated DC–CD8+ T cell coculture in the presence of enterotoxin type B (SEB) in HD. (C) Bar graphs showing the fold of increase of SEB‐specific IFNγ+, TNFα+, IFNγ+TNFα+, CD107+, and PRF+ CD8+ T cells after DC coculture, normalizing data with only CD8+ T cell condition. Each dot represents a participant. Friedman test was used (n = 13) and ROUT method was utilized to identify and discard outliers (Q = 0.1%). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 4

The interaction of primed pDCs and CD141+ mDCs with CD8+ T cells induces a higher HIV‐specific CD8+ T cell response in PLWH. (A) Representative pseudocolor dot plots and (B) bar graphs showing the percentages of IFNγ+, TNFα+, IFNγ+TNFα+, CD107+, and perforin (PRF)+ CD8+ T cells after prestimulated DC–CD8+ T cell coculture in the presence of HIV Gag peptides in PLWH. (C) Bar graphs showing the fold of increase of Gag‐specific IFNγ+, TNFα+, IFNγ+TNFα+, CD107+, and PRF+ CD8+ T cells after DC coculture, normalized with only CD8+ T cell condition. Red triangles represent PLWH under ART and red circles, naïve for treatment. Friedman and Wilcoxon tests were used (n = 6) and ROUT method was utilized to identify and discard outliers (Q = 0.1%). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. p Values between 0.05 and 0.1 were considered as tendency and were shown as numbers.

FIGURE 5

The interaction of primed pDCs and CD141+ mDCs with CD8+ T cells induces a more polyfunctional antigen‐specific CD8+ T cell response. (A) Pie charts representing SEB‐specific CD8+ T cell polyfunctionality after DC–CD8+ T cell coculture in HD. Each sector represents the proportion of CD8+ T cells expressing 4 (red), 3 (green), 2 (pink), and 1 (blue) function. Arcs represent the type of function (CD107a, IFNγ, PRF, and TNFα) expressed in each sector. (B) Bar graphs showing the percentage of CD8+ T cells expressing different combinations of studied functions in HD. (C) Pie charts representing HIV‐specific CD8+ T cell polyfunctionality and (D) bar graphs showing the percentage of CD8+ T cells expressing different combinations of studied functions after DC–CD8+ T cell coculture in PLWH. Each dot represents a participant; black circles represent HD, red circles ART naïve PLWH and red triangles PLWH on ART. Permutation and Friedman tests were used (HD, n = 13; PLWH, n = 6) and ROUT method was utilized to identify and discard outliers (Q = 0.1%). *p < 0.05, **p < 0.01, ****p < 0.0001. p Values between 0.05 and 0.1 were considered as tendency and were shown as numbers.

FIGURE 6

PD‐1 blockade‐mediated the increase of CD8+ T cell response after TLR stimulation in peripheral blood of PLWH. (A) Representative pseudocolor dot plots and (B) bar graphs showing the percentages of HIV Gag‐specific IFNγ+, TNFα+, IFNγ+TNFα+, CD107a+, and perforin (PRF)+ CD8+ T cells in ART naïve PLWH, after unfractionated cells stimulation with TLR agonists, in the presence and absence of pembrolizumab (Pembro). (C) Bar graphs showing the percentage of CD8+ T cells expressing different combinations of studied functions (CD107a, IFNγ, PRF, and TNFα) in ART naïve PLWH, after unfractionated cells stimulation with TLR agonists, in the presence and absence of pembrolizumab. Correlations between the percentages of HIV Gag‐specific IFNγ+, perforin (PRF)+ and CD107a+ CD8+ T cells after PD‐1 blockade in vitro, with ex vivo CD86+CD83+ (D) and CD86+ (E) CD141+ mDC percentages, in ART naïve PLWH. Each dot represents a participant. Friedman and Spearman tests were used (n = 7) and ROUT method was utilized to identify and discard outliers (Q = 0.1%). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

PD‐1 blockade‐mediated increase of CD8+ T cell response after TLR stimulation in HD's tonsils. (A) Representative pseudocolor dot plots and (B) bar graphs showing the percentages of SEB‐specific IFNγ+, TNFα+, IFNγ+TNFα+, CD107a+, and perforin (PRF)+ CD8+ T cells in HD tonsils, after unfractionated cells stimulation with TLR agonists, in the presence and absence of pembrolizumab (Pembro). (C) Bar graphs showing the percentage of CD8+ T cells expressing different combinations of studied functions (CD107a, IFNγ, PRF, and TNFα) in HD tonsils, after unfractionated cells stimulation with TLR agonists, in the presence and absence of pembrolizumab. Each dot represents a participant. Friedman test was used (n = 8) and ROUT method was utilized to identify and discard outliers (Q = 0.1%). *p < 0.05, **p < 0.01, ***p < 0.001.