Kappler and Philippa Marrack designed and produced essential tools; Paul Garside designed experiments; Megan K

Kappler and Philippa Marrack designed and produced essential tools; Paul Garside designed experiments; Megan K. producing CD4 T cells. Table S1. The top 33 gene. IMM-162-68-s001.pdf (925K) GUID:?52E25146-FB4A-49DB-BE60-6F32864F756D File S1. Panther over\representation test of GO terms for DEGs expressed at lower levels in tolerised memory CD4 T cells compared to control CD4 T cells. IMM-162-68-s002.xlsx (14K) GUID:?00088116-1341-4E1D-A3C5-3E940320A1D4 Data Availability StatementTranscriptomics data have been deposited on GEO, accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE145310″,”term_id”:”145310″GSE145310. Abstract We have examined the response of memory CD4 T cells to tolerogenic signals. Although memory CD4 T cells could respond to antigen delivered without adjuvant, they undergo cell death upon further restimulation. Our data suggest that the Tcells die as a consequence of mitotic catastrophe that occurs when cells are unable to complete cell division. value of <0.05; **cytokine production; cytokine responses are limited in antigen/adjuvant models. 42 Thirty days after mice were infected with IAV, they were injected with immunogenic or tolerogenic NP311C325 i.n. Anitrazafen and then rested for 30?days (Figure ?(Figure6A).6A). Bone marrow dendritic cells loaded with NP311C325 were used to examine the cytokine potential of the memory CD4 T cells and activated CD4 T cells from mice given a tertiary immunisation with NP311C325\OVA and alum delivered i.p. (Figure S7). Open in a separate window Figure 6 Activation of CD4 T cells with peptide in the absence of adjuvant does not affect CD4 Tcell cytokine production but does prevent them providing accelerated help to B cells. C57BL/6 mice were infected with influenza A virus (IAV) on day ?30. On day 0, mice received NP311C325 +/? PolyIC and some of these mice were immunized i.p with NP\OVA with alum on day 30 (A). On day 35, cells from the spleen, MedLN and lung were co\cultured with bmDCs loaded with NP311C325 for 6 hours in the presence of Golgi Plug and the number of interferon\gamma\ (IFN\), tumour necrosis factor\ (TNF) and interleukin\2 (IL\2)\producing CD44hi CD4+ T cells examined (B). The levels of IgG, IgG1 and IgG2c anti\OVA antibodies UPA in the serum were determined on day 5 (C). Each symbol represents one mouse, and error bars are SD. In B, the grey dashed line represents the background staining in na?ve animals. Data in B are combined from 2C3 experiments (3C5 mice/experiment). Data in C are combined from 3 experiments with 4 mice/experiment. All statistics calculated using a one\way ANOVA with multiple comparisons; ns?=?not significant, *<0.05, **<0.01, ***<0.001, ****<0.0001. The numbers of interferon\gamma\ (IFN\), tumour necrosis factor\ (TNF) or IL\2\producing antigen\specific memory CD4 T cells were similar in mice exposed to immunogenic or tolerogenic NP311C325 peptide 35?days previously (Figure ?(Figure6B)6B) with no consistent changes found across the three organs and three cytokines. Five days after reactivation with NP\OVA+alum, there was an increase of TNF and IL\2 producing cells in the spleen and the MedLN in mice previously exposed to NP311C325 and PolyIC. In contrast, there was no increase in the number of cytokine\producing cells in mice previously exposed to tolerogenic NP311C325. In neither Anitrazafen group did we see an increase in IFN\ producing CD4 T cells. Together, these data suggest that, while exposure to tolerogenic signals affected accumulation of T cells, it did not prevent their ability to produce cytokines. To investigate the functional responses of the T cells further, we examined their ability to provide accelerated help to primary responding OVA\specific B cells. 18 , 43 We measured the levels of class\switched, OVA\specific antibody 5?days after the tertiary reactivation. As expected, primary responding mice had very little class\switched, OVA\specific antibody and IAV\infected mice previously exposed to immunogenic signals had clearly detectable levels of OVA\specific immunoglobulin. 43 Anitrazafen In contrast, IAV\infected mice that had previously received tolerogenic signals failed to produce these antibodies at levels above primary immunised animals, demonstrating an impaired functional response (Figure ?(Figure6C6C). Memory CD4 T cells exposed to tolerogenic signals expand following reactivation with influenza virus Finally, we wanted to test whether the failure of CD4 T cells exposed to tolerogenic signals to accumulate could be rescued by reactivation with a more inflammatory stimulus. We therefore challenged IAV\infected mice given immunogenic or tolerogenic signals with an heterosubtypic form of IAV, X31 (Figure ?(Figure7A7A). Open in a separate window Figure 7 CD4 T cells exposed to peptide in the absence of adjuvant expand following re\infection with influenza A virus (IAV). C57BL/6 mice were.