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Resident CD8(+) and migratory CD103(+) dendritic cells control CD8 T cell immunity during acute influenza infection.

Waithman J, Zanker D, Xiao K, Oveissi S, Wylie B, Ng R, Tögel L, Chen W - PLoS ONE (2013)

Bottom Line: This transcription factor is required for the development of lymph node resident CD8(+) and migratory CD103(+)CD11b(-) DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+.We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes.Our results show that resident CD8(+) DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes.

View Article: PubMed Central - PubMed

Affiliation: Telethon Institute for Child Health Research and Centre for Child Health Research, University of Western Australia, Perth, Western Australia, Australia. jwaithman@ichr.uwa.edu.au

ABSTRACT
The identification of the specific DC subsets providing a critical role in presenting influenza antigens to naïve T cell precursors remains contentious and under considerable debate. Here we show that CD8(+) T lymphocyte (TCD8+) responses are severely hampered in C57BL/6 mice deficient in the transcription factor Batf3 after intranasal challenge with influenza A virus (IAV). This transcription factor is required for the development of lymph node resident CD8(+) and migratory CD103(+)CD11b(-) DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+. Using a similar ex vivo approach, many publications on this subject matter excluded a role for resident, non-migratory CD8(+) DC. We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes. Our results show that resident CD8(+) DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes.

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Antigen presentation by DC subsets after influenza infection.B6 mice were inoculated with PR8 and 3 days post infection the lung draining mediastinal lymph nodes were pooled and DC isolated. (A) Gating strategy for isolation of enriched DC subpopulations: CD8+ DC were purified on the basis of expression of CD11c and CD8 (upper left; right gate); CD11c+CD8− cells (upper left; left gate) were segregated into CD103+CD11b− (upper right; top gate) and CD103−CD11b+ (upper right; lower gate); and finally CD11c− cells were isolated (upper left; bottom gate). (B) The antigen-specific T cell activation for the T cell line specific for the H-2Db restricted influenza epitope NP366–374 was assessed using B6 bone-marrow derived DCs pulsed with NP366–374 peptide at indicated dilutions in a standard ICS assay for IFNγ. (C) Production of IFNγ by NP366–374 T cells (5×104) co-cultured for 6 hours with serially diluted DC subsets as identified in (A). Data are representative of two independent experiments, which showed a similar trend.
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pone-0066136-g003: Antigen presentation by DC subsets after influenza infection.B6 mice were inoculated with PR8 and 3 days post infection the lung draining mediastinal lymph nodes were pooled and DC isolated. (A) Gating strategy for isolation of enriched DC subpopulations: CD8+ DC were purified on the basis of expression of CD11c and CD8 (upper left; right gate); CD11c+CD8− cells (upper left; left gate) were segregated into CD103+CD11b− (upper right; top gate) and CD103−CD11b+ (upper right; lower gate); and finally CD11c− cells were isolated (upper left; bottom gate). (B) The antigen-specific T cell activation for the T cell line specific for the H-2Db restricted influenza epitope NP366–374 was assessed using B6 bone-marrow derived DCs pulsed with NP366–374 peptide at indicated dilutions in a standard ICS assay for IFNγ. (C) Production of IFNγ by NP366–374 T cells (5×104) co-cultured for 6 hours with serially diluted DC subsets as identified in (A). Data are representative of two independent experiments, which showed a similar trend.

Mentions: We next turned to an ex vivo antigenic presentation assay to determine the individual antigen presentation contributions of the different DC subsets, especially those implicated in the above in vivo experiments. This involves DC isolation from the lung draining lymph node of IAV-infected mice, segregation into defined subpopulations before co-culture with IAV-specific T cells. If the T cells respond, it is indicative that the co-cultured DC subtype is presenting antigen. We prepared DC at the reported peak of antigen presentation, D3 p.i. [6], by first depleting lymph node suspensions of other cell types and then staining for pan-DC marker CD11c as well as the markers CD8, CD103 and CD11b. Then, we sorted CD11c+ DC by flow cytometry on the basis of differences in expression of these markers to delineate the subsets. We collected CD11c− cells as a negative control as well as CD11c+CD8+ DC before fractionating the CD11c+CD8− population into the CD103+CD11b− DC subset and a CD11b+ subset (Fig 3A). It is imperative to isolate CD8+ DC first, as we have previously demonstrated they can express CD103 [20] and could potentially contaminate the migratory CD103+CD11b− population. Following sorting, we examined these populations for their presentation of an influenza-derived epitope by co-culturing independent subsets with a T cell line capable of responding to as little as 10−13 M of the immunodominant influenza-derived nucleoprotein peptide (NP366–374) (Fig 3B). Figure 3C shows that both CD8+ and CD103+ DC could stimulate NP366–374-specific T cells to generate the effector molecule IFNγ. These findings suggest that both these subpopulations have acquired influenza antigen and are capable of evoking anti-IAV TCD8+ immunity.


Resident CD8(+) and migratory CD103(+) dendritic cells control CD8 T cell immunity during acute influenza infection.

Waithman J, Zanker D, Xiao K, Oveissi S, Wylie B, Ng R, Tögel L, Chen W - PLoS ONE (2013)

Antigen presentation by DC subsets after influenza infection.B6 mice were inoculated with PR8 and 3 days post infection the lung draining mediastinal lymph nodes were pooled and DC isolated. (A) Gating strategy for isolation of enriched DC subpopulations: CD8+ DC were purified on the basis of expression of CD11c and CD8 (upper left; right gate); CD11c+CD8− cells (upper left; left gate) were segregated into CD103+CD11b− (upper right; top gate) and CD103−CD11b+ (upper right; lower gate); and finally CD11c− cells were isolated (upper left; bottom gate). (B) The antigen-specific T cell activation for the T cell line specific for the H-2Db restricted influenza epitope NP366–374 was assessed using B6 bone-marrow derived DCs pulsed with NP366–374 peptide at indicated dilutions in a standard ICS assay for IFNγ. (C) Production of IFNγ by NP366–374 T cells (5×104) co-cultured for 6 hours with serially diluted DC subsets as identified in (A). Data are representative of two independent experiments, which showed a similar trend.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672151&req=5

pone-0066136-g003: Antigen presentation by DC subsets after influenza infection.B6 mice were inoculated with PR8 and 3 days post infection the lung draining mediastinal lymph nodes were pooled and DC isolated. (A) Gating strategy for isolation of enriched DC subpopulations: CD8+ DC were purified on the basis of expression of CD11c and CD8 (upper left; right gate); CD11c+CD8− cells (upper left; left gate) were segregated into CD103+CD11b− (upper right; top gate) and CD103−CD11b+ (upper right; lower gate); and finally CD11c− cells were isolated (upper left; bottom gate). (B) The antigen-specific T cell activation for the T cell line specific for the H-2Db restricted influenza epitope NP366–374 was assessed using B6 bone-marrow derived DCs pulsed with NP366–374 peptide at indicated dilutions in a standard ICS assay for IFNγ. (C) Production of IFNγ by NP366–374 T cells (5×104) co-cultured for 6 hours with serially diluted DC subsets as identified in (A). Data are representative of two independent experiments, which showed a similar trend.
Mentions: We next turned to an ex vivo antigenic presentation assay to determine the individual antigen presentation contributions of the different DC subsets, especially those implicated in the above in vivo experiments. This involves DC isolation from the lung draining lymph node of IAV-infected mice, segregation into defined subpopulations before co-culture with IAV-specific T cells. If the T cells respond, it is indicative that the co-cultured DC subtype is presenting antigen. We prepared DC at the reported peak of antigen presentation, D3 p.i. [6], by first depleting lymph node suspensions of other cell types and then staining for pan-DC marker CD11c as well as the markers CD8, CD103 and CD11b. Then, we sorted CD11c+ DC by flow cytometry on the basis of differences in expression of these markers to delineate the subsets. We collected CD11c− cells as a negative control as well as CD11c+CD8+ DC before fractionating the CD11c+CD8− population into the CD103+CD11b− DC subset and a CD11b+ subset (Fig 3A). It is imperative to isolate CD8+ DC first, as we have previously demonstrated they can express CD103 [20] and could potentially contaminate the migratory CD103+CD11b− population. Following sorting, we examined these populations for their presentation of an influenza-derived epitope by co-culturing independent subsets with a T cell line capable of responding to as little as 10−13 M of the immunodominant influenza-derived nucleoprotein peptide (NP366–374) (Fig 3B). Figure 3C shows that both CD8+ and CD103+ DC could stimulate NP366–374-specific T cells to generate the effector molecule IFNγ. These findings suggest that both these subpopulations have acquired influenza antigen and are capable of evoking anti-IAV TCD8+ immunity.

Bottom Line: This transcription factor is required for the development of lymph node resident CD8(+) and migratory CD103(+)CD11b(-) DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+.We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes.Our results show that resident CD8(+) DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes.

View Article: PubMed Central - PubMed

Affiliation: Telethon Institute for Child Health Research and Centre for Child Health Research, University of Western Australia, Perth, Western Australia, Australia. jwaithman@ichr.uwa.edu.au

ABSTRACT
The identification of the specific DC subsets providing a critical role in presenting influenza antigens to naïve T cell precursors remains contentious and under considerable debate. Here we show that CD8(+) T lymphocyte (TCD8+) responses are severely hampered in C57BL/6 mice deficient in the transcription factor Batf3 after intranasal challenge with influenza A virus (IAV). This transcription factor is required for the development of lymph node resident CD8(+) and migratory CD103(+)CD11b(-) DCs and we found both of these subtypes could efficiently stimulate anti-IAV TCD8+. Using a similar ex vivo approach, many publications on this subject matter excluded a role for resident, non-migratory CD8(+) DC. We postulate the differences reported can partially be explained by how DC are phenotyped, namely the use of MHC class II to segregate subtypes. Our results show that resident CD8(+) DC upregulate this marker during IAV infection and we advise against its use when isolating DC subtypes.

Show MeSH
Related in: MedlinePlus