Peripheral T follicular Helper Cells (pTfh)

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Peripheral T follicular Helper Cells (pTfh) - A biomarker for vaccine induced responses? Suresh Pallikkuth & Savita Pahwa University of Miami

Gating strategy for pTfh cells Singlets Exclusion-CD3+ Scatter CD4 and CD8 T cells Memory CD4 pTfh FSC-H FSC-W Exclusion CD3 SSC-A FSC-A CD4 CD45RO CD4 CXCR5 CD4 CD8 Non-pTfh pTfh cells show higher expression of PD1, ICOS, CCR7, HLA-DR and Ki67 than non-pTfh cells Isotype pTfh 23.3 (141) 22.8 (280) 77.2 (420) 2.1 (122) 2.1 (2368) 2.2 (338) Non-pTfh 11.1 (86) 15.6 (180) 1.8 (304) 58.8 (102) 1.8 (106) 1.7 (1451) Clone of CCR5? Titered mAb? Ctrl for CCR5 expression? Cryopreserved PBMC obtained at day 28 post-influenza vaccination from a healthy donor were thawed, rested overnight and cultured at 1x106 cells/ml for 12 hrs at 37°C in the presence of 5 µg/ml H1N1 vaccine Ag in the presence of 10µg/ml Brefeldin A. After incubation, cells were stained with markers specific for Tfh cells along with intracellular staining for cytokines IL-21, IL-2, IFNg and TNFa. Flow cytometric dot plots showing the gating strategy and expression of intracellular cytokines in pTfh and non-pTfh cells CD3+CD4+CD45RO+ memory CD4 T cells were gated from live (ViViD-) CD3+ cells. pTfh (CXCR5+) and non-pTfh (CXCR5-) were identified in the memory cells and analyzed for the intracellular expression of IL-21, IL-2, TNFa and IFNg. 23.3 (701) 20.9 (130) 14,2 (560) 93 (29252) 3.9 (286) 0.63 (51) 81.7 (21727) 2.8 (162) 32.1(7851) 28.3 (320) 8.6 (290) 0.32 (33)

pTfh function by ICC: intracellular IL-21 and IL-2 production by pTfh cells after 12 hrs of stimulation Intracellular IL-2 by total CD4 T cells and pTfh cells Intracellular IL-21 by total CD4 T cells and pTfh cells H1N1 Medium H1N1 SEB SEB Medium Total CD4 Total CD4 pTfh cells pTfh cells Cryopreserved PBMC obtained 4 weeks after H1N1/09 vaccination were thawed and rested overnight and stimulated with H1N1 antigen (5 µg/ml), SEB (positive control) and medium (negative control) for 12hr. After incubation cells were surface stained for pTfh markers, fixed, permeabilized and stained intracellularly for cytokines and analyzed by flow cytometry. Total CD4, pTfh (ViViD-CD3+CD4+CD45RO+CXCR5+) and non-pTfh (ViViD-CD3+CD4+CD45RO+CXCR5-) cells were Gating strategy for the identification of pTfh cells. Intracellular expression of IL-21 and IL-2 in total CD4 T cells, pTfh and non-pTfh cells Non-pTfh cells Non-pTfh cells

Literature review: delineation of pTfh Markers used for pTfh identification Reference pTfh: CD3+CD4+CD45RAneg CXCR5+ Subsets within pTfh: Th1: CXCR3+CCR6negCCR4neg Th2: CXCR3negCCR4+CCR6neg Th17: CXCR3negCCR6+CCR4neg Morita et al, Immunity 2011 pTfh: CD3+CD4+CD4+CXCR5+ and CD3+CD4+CD45RAnegCXCR5+ Pallikkuth et al, Blood 2012 pTfh: CD3+CD4+CD45RAnegCXCR5+ Th1: CXCR3+CCR6neg Th2: CXCR3negCCR6neg Th17: CXCR3negCCR6+ Bentebibel et al. Science translational Med 2013 pTfh: CD3+CD4+CD45RAnegPD1+CXCR3neg Locci et al., Immunity 2013 pTfh: CD3+CD4+CD45RAnegCCR7loPD1hi He et al, Immunity 2013

pTfh cells can be used to monitor the immune response against vaccination: expansion of pTfh post-vaccination occurs in vaccine responders Day 0 Day 7 Day 28 CD4+CD45RO+ Isotype Vaccine responder Vaccine non-responder Vaccine responder Vaccine non-responder P=0.013 P=0.001

pTfh cell changes in VR (not seen in Non- pTfh or VNR) Signature Immunological Changes in pTfh and B cells in Vaccine Responders (VR) following H1N1/09 Influenza Vaccine, at T1 (7d), T2 (4 wk) pTfh cell changes in VR (not seen in Non- pTfh or VNR) Expansion of pTfh at T1, T2 Ag-stimulated IL-21 production in pTfh (ICC) “Help” to autologous B cells for Ag-specific IgG production and B cell differentiation in purified pTh plus B cell co-cultures at T2 B cell changes in VR (deficient in VNR) Increase in plasmablasts at T1 Spontaneous H1N1 influenza Ab secreting cells (ASC) at T1 Increase in total memory B cells and switch memory at T2 Upregulation of IL-21R on total B and memory B cells at T2 Increase in plasma cells at T2 Increase in TACI expression on total B memory B cells at T2 Downregulation of BAFF-R expression on total B and memory B cells at T2 PBMC / plasma findings in VR (deficient in VNR) Production of IL-21 and CXCL13 in Ag stimulated culture supernatants with increases in plasma IL-21 Increase in plasma BAFF and APRIL levels Pallikkuth et al, J Immunol 2011, JACI 2011, Blood 2012