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. 2023 Feb 21;13:1041552. doi: 10.3389/fimmu.2022.1041552

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Copyright © 2023 Kozlovski, Regev, Sapoznikov, Kizner, Achdout, Petrovich-Kopitman, Elkahal, Addadi, Silva Castanheira, Feigelson, Kubes, Erez, Garbi and Alon

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Defective homing of early CD4⁺ T cells post influenza infection is counterbalanced by augmented cDC2 accumulation and activation inside ICAM-1/2^-/- mediastinal LNs. (A) The total numbers of CD45⁺ cells, CD4⁺ T cells, CD19⁺ and B220⁺ B cells in resting MedLNs of either WT or ICAM-1/2^-/- mice determined by flow cytometry. The subset totals were recovered from the same mice, except for the B220⁺ totals, which were analyzed from a separate experiment. (B) Paraffin sections of resting MedLNs of naïve WT (left panel) and ICAM-1/2^-/- (right panel) mice stained with anti PNAd (green) mAb. Scale bar represents 50µm. (C) Experimental design and timeline of B220⁺ B cell accumulation in the MedLNs of ICAM-1/2^-/- mice after 1 or 3 days post I.N. infection (dpi) with a sublethal dose of PR8-OVAII influenza. I.N. PBS administration was performed in control experiments. Results shown (bottom panel) are the mean +- SEM. (D) Experimental design and timeline of B1-a cell accumulation in the MedLNs of both WT and ICAM-1/2^-/- following PR8 infection (top panel). Results shown (bottom panel) are the mean +- SEM. (E) Experimental design, timeline and results of accumulation of extrafollicular B cell (EFBC, CD138⁺, B220⁺) in the MedLNs of either WT or ICAM-1/2^-/- infected mice. Mice were I.N. administered with a sublethal dose of PR8-OVAII influenza 1 or 3 days post I.N. infection (dpi) (top panel). Results shown (bottom panel) are the mean +- SEM. (F) Experimental design and timeline of flow cytometry analysis of OT-II CD45.1 CD4⁺ T cell accumulation at steady state or 1 day following OVA-II PR8 infection (top panel). Results shown are the mean +- SEM (bottom panel). (G) Experimental design and timeline of flow cytometric analysis of total OT-II CD45.1 cells and their differentiated derivatives. Numbers of total OT-II CD45.1 cells (H), early Tfh cells (CD45.1⁺PD-1⁺CXCR5⁺) (I) and the BCL6 expression levels of these early Tfh cells (J) determined in the MedLNs of WT or ICAM-1/2^-/- 4 day post OVA-II PR8 infection. (K) Experimental design and timeline protocol for the flow cytometry analysis of cDC2s (CD45⁺MHC-II⁺CD11b⁺CD103^-) accumulated in the MedLNs of WT and ICAM-1/2^-/- at steady state or 3 days post infection with OVA-II PR8 (top panel). (L) Results shown (bottom left panel) are the mean +- SEM. (M) cDC2 activation profiles assessed by expression levels of CD86, CD80, CD40 and IL-6. Statistical significance was determined by two-tailed, unpaired Student’s t tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. The numbers of each experimental group are indicated in the graphs. The error bars indicate the SEM of each measurement.