Functional and transcriptional properties of DC distinguish a subset of HIV-1 controller neutralizers Enrique Martin-Gayo Ph.D Ragon Institute of MGH, MIT and Harvard Laboratory Dr. Xu G. Yu Mo B CD4 DC
Evolution of bnabs against HIV-1 Functional and transcriptional properties of DC distinguish a subset of HIV-1 controller neutralizers The bnab responses in HIV-1 infected individuals are mainly driven by high level viremia and immune activation (Sajadi MM et al, J Acquir Immune Defic Syndr 211; Cortez V et al, Plos Pathog 212 ) A small proportion of HIV controllers are capable to produce bnabs in the presence of extremely low viremia. Mechanisms of bnab development in these HIV controller neutralizer patients will be informative for the design of an HIV vaccine.
Proportion of PD1lo TFH (%) Background 1 1 How homogeneous is DC Function in Neutralizers?.1 T+B + NNDC T+B + NeutDC Martin-Gayo E. et al. JCI insight 217
Study design HIV-1 Controller cohort 46 Neutralizers 15 Non Neutralizers Sorting primary cell populations: cdc, Monocytes, CD4T cells and B cells Full genome RNAseq CD4 T cell counts Plasma Viral Load Ab Breadth Nt NN Nt NN Nt NN
Neutralizers can be subdivided in two different groups based on DC transcriptional patterns : Nt2 (n=21; different from Non Neuts) and Nt1 (n=25; overlapping with Non Neut) NT2 NT1 NN VL Nt1 vs Nt2 vs NN Genes Std>2 >1 genes 61 samples Orange:Nt2 (21) Yelow:Nt1 (25) Green:NN (15) CD4 Ab. Breadth
Pathway analysis of DC transcriptional patterns suggest increased maturation and activation G DEG Nt2 vs NN : 189 genes(fdr<1e-5) 15 <.1.9 I D.793 15 CD86 MFI S 1.21 DC Maturation 2 IL12 Sig. ICOS-ICOSL Sig. 5 NFKB Sig. 1 5 TREM1 CD28 Sig. in T Helpercells A NT1 NN LPS NT2 NT1 NN CD4 Sig. CD4L 8 CD4 MFI NT2 2 FOXO1 IL6.85 25.1949 6 4 of PRR in Role Path Recog. STAT4 1 2 NT2 NT1.632.367 NN JAK/STAT Sig. TGFB1. PDL1+L2 MFI E Upstream Reg. CD83 MFI Cannonical Pathways 7 2 15 1 TLR Sig. 5 NT2 NT1 NO and ROS prod. by Mo NN Increased expression of costimulatory molecules In cdc from Nt2 compared to NN and Nt1 Cytokines/TLR pathways are upregulated in DC Nt2 vs NN
Number of differentially expressed genes NT2 vs NN What is the relationship between cdc responses and other cell types and their association with Ab. Production? 8 6 B Correlation Significance Mo CD4 DC 4 2 B Mo DC CD4 Mo B DC CD4 Mo B CD4 p<1e-5 FDR-adjusted p<5e-2 DC Differences between Nt2 and NN Are dominated by DC and CD4T cell Transcriptional signatures Top 1 DEG Nt2 and NN from DC and CD4T cell are highly correlated
Proportion of CD4 T cells (%) cells Total ptfh Nt (%) Transcriptional patterns of CD4 T cells from Nt2 Patients are enriched in Tfh/TH1 signatures Total CXCR5+ PD1+ ptfh p=.1 6.224.152 6 r=.5875 Nt2 4 4 Nt1 2 2 Proportions of ptfh are higher In Nt2 than NN and Nt1. Higher contribution of the CXCR3+ ptfh compartment. Transcriptional differential patters of CD4 T cells from Nt2 are enriched In Tfh and Th1 signatures NT2 NT1 NN 5 1 15 2 CD86 MFI on mdc
DC from Nt2 are more effective than Nt1 inducing polarization of naïve CD4+ T cells into Tfh-like cells Nt2 vs Nt1 + Nt1 + Nt2
Bcl-6 CXCR5 Mechanisms reponsible for function of cdc from Nt2 Upstream Reg. Do cytokines or TLR stimulation can boost 1 5 2.19 2.7 Tfh priming function in cdc towards an Nt2 1 4 1 3 Functional phenotype? 1 2 84.9 1.8 1 2 1 3 1 4 1 5 PD-1 1 5 2.2 1 4 1 3.421 2.29 1 2 87.4 9.9 1 2 1 3 1 4 1 5 PD-1 Pre-conditionning of cdc with cytokines (IL-12), but not TLR ligands enhance their Tfh-priming function
Impact of IL-12 signaling on Tfh priming function of cdc 48h cdc From Nt2 STAT4 inhibitor Naïve T and B cells IL-12 efficiently induce Costimulatory molecule expression on cdc Differences in CXCR5+ PD-1+ Bcl-6+ Tfh-like cells?? Tfh priming function of cdc from Nt2 Depends on STAT4 activation
Number of differentially expressed genes NT2 vs NN Which cells are potentially responsible for more active cytokine induction in Nt2 patients? Cannonical Pathways 8 6 4 2 Function of macrophages Phagocytosis IL-6 Sig. Inflammatory Response DC CD4 Mo B DC CD4 Mo B TLR Sig. Activation of cells IL-12 Sig. p<1e-5 p<5e-2 FDR-adjusted NFKB Sig.
Which cells are potentially responsible for more active cytokine induction in Nt2 patients? Just media (basal levels) cdc and Mo Mo and are cdc more from predisposed Cytokine secretion Nt2, Nt1, NN to produce IL-12 and IL-6 upon TLR stimulation than NN and Nt1 TLR stimulation (TLR8 and TLR2) after 24h IL-12p7/p4 IL-6 Mo cdc Med TLR8 TLR2 Med TLR8 TLR2 Med TLR8 TLR2 Nt2 Nt1 NN Med TLR8 TLR2 Med TLR8 TLR2 Med TLR8 TLR2 Nt2 Nt1 NN
Proportion of B cells (%) Number of differentially expressed genes NT2 vs NN Transcriptional analysis of B cells from Nt2 Disease and Function DEG Nt2 vs NN B cells Naïve Act. Unswitched MZ-like/B1 8 Bacterial infection 6 Death of B lymphocytes 4 B cell malignant tumor Rheumatoid Arthritis Abnormal morphology of B1a cells Lack of B cells Resting Effector Mem Activated Mem Arrest in differentiation of B cells 2 Antibacterial response Quantity of B cells Quantity of IgG NA IgD+ CD27- DC CD4 Mo B DC CD4 Mo B Quantity of MZ zone B cells Proliferation of B lymphocytes Production of Ab Quantity of IgM 1 8.455.5427 p<1e-5 FDR-adjusted p<5e-2 Quantity of follicular B cells Inflammatory Resp Quantity of IgA Differentiation of B cells Cell movement of B cells 5 1 -Log p value 6 4 2 NT2 NT1 NN
Proportion of B cells (%) Proportion from B cells (%) Phenotypical analysis of B cells from Nt2 Ag experienced Resting Effector Mem IgM+ DN-effector memory IgM+ 25 2 15 1 Ag experienced Activated Mem IgM+ Switched memory and PB <.1.2 2 15 1.2453.616 5 5 NT2 NT1 NN NT2 NT1 NN Increased proportions of Ag-experienced IgM+ CD27- B cells in Nt2 patients Are associated with ptfh proportions
Hypothetical model Bacterial translocation TLR Sensing HIV-1 Stimulation Trans IL-6 IL-12 DC IL-12 Tfh cell B cell Plasmablast Mo Naïve T cell Memory B cell Ab. Breadth and Potency
Conclusions 1. Neutralizers can be subdivided in two different patient groups based on transcriptional patterns of DC: Nt2 (Different fro NN) and Nt1 (Overlapping with NN). 2. Transcriptional profiles of cdc from are characterized by cytokine/tlr activation 3. DC from Nt2 are more functional inducing Tfh than DC from Nt1 patients. Such functionality can be boosted by inflammatory cytokine conditioning of cdc 4. Tfh-priming function of cdc from Nt2 is dependent on STAT4 signaling. 5. Transcriptional signatures of Monocytes from Nt2 reflect altered expression of genes related to cytokine and endocytosis. Indeed Mo and cdc from Nt2 patients are more predisposed to secrete cytokines upon TLR stimulation 6. B cells from Nt2 seem to be enriched in resting IgM+ memory cells, which could have implications for Ab. evolution
Acknowledgements Ragon Institute Xu Yu Hsiao Rong Chen Ce Gao Zhengyu Ouyang Stephane Hua Samanttha Chen Other members Flow core Michael Waring Nathalie Bonheur Processing lab Ildiko Toth Jamie Flynn Michael Baladiang Bruce Walker Facundo Batista Mathias Lichterfeld Guinevere Lee Hsiao Hsuan Kuo Pilar Garcia Broncano Collaborators Ragon-IMES UC Berkeley Alex K Shalek Nir Yosef Kellie E Kolb Michael Cole Funding HU CFAR Scholar award Tosteson and Fund ECOR developmental award NIH/NIAID Gates Foundation Ragon Institute