Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8+ memory T cells
Article first published online: 18 JAN 2013
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 43, Issue 3, pages 693–704, March 2013
How to Cite
Tartz, S., Deschermeier, C., Retzlaff, S., Heussler, V., Sebo, P., Fleischer, B. and Jacobs, T. (2013), Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8+ memory T cells. Eur. J. Immunol., 43: 693–704. doi: 10.1002/eji.201142262
- Issue published online: 11 MAR 2013
- Article first published online: 18 JAN 2013
- Accepted manuscript online: 10 DEC 2012 06:39PM EST
- Manuscript Accepted: 5 DEC 2012
- Manuscript Revised: 29 NOV 2012
- Manuscript Received: 18 NOV 2011
- collaborative research centre 841 (SFB 841). Grant Numbers: 310/08/0447, AV0Z50200510
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Figure S1. Gating strategy for the detection of H2-Kd/CSP245-253-binding CD8+ T cells. Total splenocytes were analysed by flow cytometry after staining with anti-CD8-eFluor®450 and APC-labelled H2-Kd/CSP245- 253-Pentamer. Cells were gated on lymphocytes in FSC/SSC (left dot plots, gate R1) and CD8+ afterwards (middle dot plots, gate R2). Percentage of H2-Kd/CSP245-253-binding cells within gates R1+R2 was determined as shown by the right dot plots.
Figure S2. Gating strategy for the detection of intracellular cytokines. Total splenocytes were analysed by flow cytometry after incubation with BD GolgiStop™ alone (A) or in the presence of CSP245-253 peptide and BD GolgiStop™ (B) and staining with anti-CD8-PerCP-Cy5.5, anti-IFNγ-PE, anti-TNFα-FITC and anti-IL-2-APC. Cells were gated on lymphocytes in FSC/SSC (left dot plot, gate R1) and afterwards on CD8+ (second dot plot, gate R2). Percentage of total IFNγ+ cells within gate R1+R2 was determined by gate R3 (third dot plot). Afterwards a quadrant was drawn within gate R3 to determine TNFα- and IL-2- expression of IFNγ+ cells (right dot plot).
Figure S3. In vivo cytotoxicity assay. CFSE highly labelled splenocytes loaded with CSP peptide and CFSE weakly labelled control splenocytes were adoptively transferred into naïve, immunised and challenged mice. Transferred cells were recovered from spleens three hours after transfer and analysed by flow cytometry. Cells were gated on lymphocytes (left dot plot, R1) and CFSE (middle dot plot, R2). The ratio of CFSEhigh and CFSElow cells was determined by histogram plots. Representative results of a naïve control mouse, one mouse only immunised and one mouse immunised and challenged is shown.
Figure S4. Phenotypic analysis of CSP-specific memory T cells. Splenocytes and liver lymphocytes from immunised and challenged mice were isolated 9 weeks after boost immunisation. Cells were analysed by flow cytometry after staining with anti-CD8-eFluor®450, APC-labelled H2-Kd/CSP245-253-Pentamer, anti- CD62L-FITC, anti-CD127-PE, anti-CD27-PE, anti-CD43-FITC, anti-CD122-biotin and anti-KLRG-1- biotin together with streptavidin-PE/Cy7. Cells were gated on H2-Kd/CSP245-253-specific cells as shown in Sup. Fig. 1 and the percentage of cells highly expressing the different surface molecules was determined by histogram plots (grey-shaded: isotype control). Representative stainings of liver lymphocytes are shown.
Figure S5. CD62L expression of non-CSP-specific CD8+ T cells. In order to exclude that a significant shedding of CD62L upon cell isolation has occurred, we looked for CD62L on non CSP-specific T cells. Splenocytes or liver cells from immunised and challenged BALB/c mice were analysed 9 weeks after boost immunisation by flow cytometry after staining with anti-CD8-eFluor®450, anti-CD44-V500, APClabelled H2-Kd/CSP245-253-Pentamer and anti-CD62L-FITC. (A) Cells were gated on lymphocytes in FSC/SSC (left dot plot, gate R1) and on CD8+ cells afterwards (middle dot plot, gate R2). Naïve and memory CD8+ T cells were distinguished by CD44 expression (right dot plot) and CD62L expression was analysed on CD44hi and CD44lo cells (histograms). Percentages of CD62Lhi of CD44hi and CD44lo cells within spleens (B) and livers (C) are summarised.
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