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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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DC subset upregulation of MHC II during acute influenza infection.B6 mice were inoculated i.n. with PKH26 or carrier 16 hours before i.n. infection with influenza virus. One to three days post infection, individual lung draining mediastinal lymph nodes were harvested and DC isolated. Enriched DC were stained with anti-CD11c, anti-CD8, anti-CD103, anti-CD11b, and anti-MHC II and analyzed on a flow cytometer. (A) Representative FACS plots of – Dye (left) and + Dye (middle) treated animals are shown. In addition, a representative histogram overlay from day 1 post infection (right) shows expression of MHC II on the CD11c+CD8+ DC (Gate I-filled) and PKH26+ DC (Gate II-open) subsets. (B) Representative dot plot of pKH26+ cells expression of CD103 and CD11b. (C–D) Upper histograms are an overlay showing expression of MHC II on PKH26+ cells from naïve (dotted line) or PR8 infected (open) mice day 2 (C) or day 3 (D) post infection. Lower histograms are identical with exception they represent CD8+ DC from naïve (dotted line) or PR8 infected (filled) mice. (E–F) Similar to C–D, with the exception expression of CD86 is shown. (G) Dot plot shows CD8+ DC (red dots) from naïve (left) or D3 PR8 infected (right) mice as compared to total CD11c+ DC from naïve mice. The numbers in each plot represent the frequency of CD8+ DC in the MHC IIhigh population. Representative histograms from 1 of 5 mice from two independent experiments are shown.
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pone-0066136-g004: DC subset upregulation of MHC II during acute influenza infection.B6 mice were inoculated i.n. with PKH26 or carrier 16 hours before i.n. infection with influenza virus. One to three days post infection, individual lung draining mediastinal lymph nodes were harvested and DC isolated. Enriched DC were stained with anti-CD11c, anti-CD8, anti-CD103, anti-CD11b, and anti-MHC II and analyzed on a flow cytometer. (A) Representative FACS plots of – Dye (left) and + Dye (middle) treated animals are shown. In addition, a representative histogram overlay from day 1 post infection (right) shows expression of MHC II on the CD11c+CD8+ DC (Gate I-filled) and PKH26+ DC (Gate II-open) subsets. (B) Representative dot plot of pKH26+ cells expression of CD103 and CD11b. (C–D) Upper histograms are an overlay showing expression of MHC II on PKH26+ cells from naïve (dotted line) or PR8 infected (open) mice day 2 (C) or day 3 (D) post infection. Lower histograms are identical with exception they represent CD8+ DC from naïve (dotted line) or PR8 infected (filled) mice. (E–F) Similar to C–D, with the exception expression of CD86 is shown. (G) Dot plot shows CD8+ DC (red dots) from naïve (left) or D3 PR8 infected (right) mice as compared to total CD11c+ DC from naïve mice. The numbers in each plot represent the frequency of CD8+ DC in the MHC IIhigh population. Representative histograms from 1 of 5 mice from two independent experiments are shown.

Mentions: To examine if phenotypic changes occur in migratory DCs trafficking from the infectious site to the lung draining mediastinal lymph node, mice were instilled i.n. with a solution containing the dye PKH26 16 hours prior to influenza infection. Dye-labeled DCs were readily identifiable in the mediastinal lymph node 1–3 days post infection (Fig 4A). CD11c+CD8+ DC did not stain for the dye, consistent with the notion they are a non-migratory, lymphoid resident cell population [25] (Fig 4A GI). Dye-labeled cells (Fig 4A GII) were a mixture of the migratory CD103+CD11b− DC and CD103−CD11b+ DC populations previously described [5] (Fig 4B). Consistent with previously published results, a higher proportion of migrated pKH26+CD103−CD11b+ DCs was observed (data not shown). Day 1 p.i., the expression of MHC II surface molecules on CD8+ DC were at intermediate levels as compared to a high level of expression on their migratory PKH26+ counterparts (Fig 4A histogram). However, a clear shift in MHC II expression within the resident CD8+ DC subpopulation was readily observed on day 2 (Fig 4C bottom). Upregulation of this marker by the majority of this DC subset was most evident on day 3 (Fig 4D bottom), a time point previously reported as the peak of antigen presentation [6]. In addition, the kinetics of expression of the co-stimulatory marker CD86 in this DC subset mirrored MHC II expression (Fig 4E–F bottom). No alteration in MHC II expression was observed in the PKH26+ migratory populations (Fig 4C–D top) between D1-3 p.i.. Interestingly, no alteration in CD86 expression was observed on pKH26+ migratory DCs on D2 p.i. (Fig 4E top). Increased expression of this accessory molecule was only observed D3 p.i. (Fig 4F top) in both the pKH26+CD11b+CD103− and pKH26+CD11b−CD103+ subpopulations (data not shown). These results clearly show that DC subpopulations are responsive to a peripheral pathogenic encounter, progressing from an immature phenotype to a more mature state.


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)

DC subset upregulation of MHC II during acute influenza infection.B6 mice were inoculated i.n. with PKH26 or carrier 16 hours before i.n. infection with influenza virus. One to three days post infection, individual lung draining mediastinal lymph nodes were harvested and DC isolated. Enriched DC were stained with anti-CD11c, anti-CD8, anti-CD103, anti-CD11b, and anti-MHC II and analyzed on a flow cytometer. (A) Representative FACS plots of – Dye (left) and + Dye (middle) treated animals are shown. In addition, a representative histogram overlay from day 1 post infection (right) shows expression of MHC II on the CD11c+CD8+ DC (Gate I-filled) and PKH26+ DC (Gate II-open) subsets. (B) Representative dot plot of pKH26+ cells expression of CD103 and CD11b. (C–D) Upper histograms are an overlay showing expression of MHC II on PKH26+ cells from naïve (dotted line) or PR8 infected (open) mice day 2 (C) or day 3 (D) post infection. Lower histograms are identical with exception they represent CD8+ DC from naïve (dotted line) or PR8 infected (filled) mice. (E–F) Similar to C–D, with the exception expression of CD86 is shown. (G) Dot plot shows CD8+ DC (red dots) from naïve (left) or D3 PR8 infected (right) mice as compared to total CD11c+ DC from naïve mice. The numbers in each plot represent the frequency of CD8+ DC in the MHC IIhigh population. Representative histograms from 1 of 5 mice from two independent experiments are shown.
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pone-0066136-g004: DC subset upregulation of MHC II during acute influenza infection.B6 mice were inoculated i.n. with PKH26 or carrier 16 hours before i.n. infection with influenza virus. One to three days post infection, individual lung draining mediastinal lymph nodes were harvested and DC isolated. Enriched DC were stained with anti-CD11c, anti-CD8, anti-CD103, anti-CD11b, and anti-MHC II and analyzed on a flow cytometer. (A) Representative FACS plots of – Dye (left) and + Dye (middle) treated animals are shown. In addition, a representative histogram overlay from day 1 post infection (right) shows expression of MHC II on the CD11c+CD8+ DC (Gate I-filled) and PKH26+ DC (Gate II-open) subsets. (B) Representative dot plot of pKH26+ cells expression of CD103 and CD11b. (C–D) Upper histograms are an overlay showing expression of MHC II on PKH26+ cells from naïve (dotted line) or PR8 infected (open) mice day 2 (C) or day 3 (D) post infection. Lower histograms are identical with exception they represent CD8+ DC from naïve (dotted line) or PR8 infected (filled) mice. (E–F) Similar to C–D, with the exception expression of CD86 is shown. (G) Dot plot shows CD8+ DC (red dots) from naïve (left) or D3 PR8 infected (right) mice as compared to total CD11c+ DC from naïve mice. The numbers in each plot represent the frequency of CD8+ DC in the MHC IIhigh population. Representative histograms from 1 of 5 mice from two independent experiments are shown.
Mentions: To examine if phenotypic changes occur in migratory DCs trafficking from the infectious site to the lung draining mediastinal lymph node, mice were instilled i.n. with a solution containing the dye PKH26 16 hours prior to influenza infection. Dye-labeled DCs were readily identifiable in the mediastinal lymph node 1–3 days post infection (Fig 4A). CD11c+CD8+ DC did not stain for the dye, consistent with the notion they are a non-migratory, lymphoid resident cell population [25] (Fig 4A GI). Dye-labeled cells (Fig 4A GII) were a mixture of the migratory CD103+CD11b− DC and CD103−CD11b+ DC populations previously described [5] (Fig 4B). Consistent with previously published results, a higher proportion of migrated pKH26+CD103−CD11b+ DCs was observed (data not shown). Day 1 p.i., the expression of MHC II surface molecules on CD8+ DC were at intermediate levels as compared to a high level of expression on their migratory PKH26+ counterparts (Fig 4A histogram). However, a clear shift in MHC II expression within the resident CD8+ DC subpopulation was readily observed on day 2 (Fig 4C bottom). Upregulation of this marker by the majority of this DC subset was most evident on day 3 (Fig 4D bottom), a time point previously reported as the peak of antigen presentation [6]. In addition, the kinetics of expression of the co-stimulatory marker CD86 in this DC subset mirrored MHC II expression (Fig 4E–F bottom). No alteration in MHC II expression was observed in the PKH26+ migratory populations (Fig 4C–D top) between D1-3 p.i.. Interestingly, no alteration in CD86 expression was observed on pKH26+ migratory DCs on D2 p.i. (Fig 4E top). Increased expression of this accessory molecule was only observed D3 p.i. (Fig 4F top) in both the pKH26+CD11b+CD103− and pKH26+CD11b−CD103+ subpopulations (data not shown). These results clearly show that DC subpopulations are responsive to a peripheral pathogenic encounter, progressing from an immature phenotype to a more mature state.

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