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Supplemental Digital Content Supplementary Methods Study population The cohort was formed in 1990 as an open prospective cohort for whom regular yearly serological and clinical examinations were offered. The cohort included 4878 individuals at its closure in 2011. In 2013 participants of the current study were recalled for another followup visit. Antiretroviral treatment was available in Guinea-Bissau at the time of sampling, but blood samples from the individuals that, at any time, had received treatment were not analysed in this study. FACS staining Immunophenotyping of NK and NKT cells was performed on Cytochex (Streck) stabilized whole blood by multicolor flow cytometry. Before antibody labelling, red blood cells were lysed using lysis buffer (BD FACS™) and washed with PBS containing 2mM EDTA. All antibodies used were anti-human (see Table S1). The surface staining was carried out in two staining rounds. Cells were washed between staining procedures with PBS containing 2mM EDTA. Prior to intracellular staining, cells were permeabilized using Fixation/ Permeabilization solution and washed with permeabilization buffer (eBiosciences). Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV infections Measurement of CD38, PLZF and T-bet As CD38 is commonly expressed on T and NK cells the MFI of the CD38 expression was used as a proxy for activation rather than the percentage of cells expressing CD38. In our evaluation of flow cytometry data generated from blood samples collected in Cyto-Chex BCT tubes we noted that CD38 expression levels were not affected by the preservatives (data not shown). However, we were uncertain as to how the preservative in the tubes would affect the expression and staining of the transcription factors (TFs), PLZF and T-bet. We therefore decided to measure the MFI of the TFs in cell types determined to express these factors and relate these to cells from the same sample that did not express the TFs (internal negative control) (Fig. S1), allowing us to record a patient specific increase for the TFs and minimise any potential effect of the Cyto-Chex tubes. 2 Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV infections Supplementary Tables Table S1: Fluorochrome conjugated antibodies used in the study. Antibody Conjugate Clone Company Comment CD14 V500 M5E2 BD Horizon™ 1. surface staining Identification of invariant natural killer T cells (iNKT) CD3 Alexa Fluor® 700 UCHT1 BD Pharmingen™ 1. Surface staining CD4 R-PE-Cy®5.5 S3.5 Lifetechnologies TCR Vα24 biotin C15 Beckman Coulter™ TCR Vβ11 PE C21 Beckman Coulter™ Streptavidin Qdot® 655 Q10123MP Lifetechnologies 2. Surface staining Identification of natural killer cells (NK) CD56 (NCAM) Brilliant Violet 711™ HCD56 BioLegend 1. Surface staining HIT2 BD Horizon™ 1. Surface staining Intracell. staining Markers of immune activation CD38 PE-CF594 Transcription factors PLZF APC 6318100 R&D Systems™ T-bet Brilliant Violet 605™ 4B10 BioLegend 3 Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV infections Table S2: Results from the univariate general linear models Tests of Between-Subjects Effects Dependent Variable: CD38 MFI on iNKT Type III Sum of Source Squares df Mean Square F Sig. 12281120.639a 7 1754445.806 7.362 .000 Intercept 1408390.404 1 1408390.404 5.910 .019 Age 190194.644 1 190194.644 .798 .376 Log_VL 627054.329 1 627054.329 2.631 .111 Log_CD4% 6038228.503 1 6038228.503 25.338 .000 HIV 149532.340 2 74766.170 .314 .732 Gender 67962.295 1 67962.295 .285 .596 HIV * Gender 309275.479 1 309275.479 1.298 .260 Error 12153513.346 51 238304.183 Total 30021450.600 59 Corrected Total 24434633.986 58 Corrected Model a. R Squared = .503 (Adjusted R Squared = .434) 4 Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV infections Tests of Between-Subjects Effects Dependent Variable: CD38 MFI on CD4 iNKT Type III Sum of Source df Mean Square F Sig. 1498617.757a 7 214088.251 2.810 .020 14856.163 1 14856.163 .195 .662 149.676 1 149.676 .002 .965 Log_VL 359869.177 1 359869.177 4.723 .037 Log_CD4% 130460.106 1 130460.106 1.712 .199 HIV 355244.876 2 177622.438 2.331 .113 .714 1 .714 .000 .998 396.071 1 396.071 .005 .943 Error 2590655.184 34 76195.741 Total 7264858.575 42 Corrected Total 4089272.941 41 Corrected Model Intercept Age Gender HIV * Gender Squares a. R Squared = .366 (Adjusted R Squared = .236) 5 Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV infections Tests of Between-Subjects Effects Dependent Variable: CD38 MFI on CD56 dim NK Type III Sum of Source df Mean Square F Sig. 132081629.060a 8 16510203.632 5.253 .000 Intercept 12558699.228 1 12558699.228 3.996 .050 Age 1773616.578 1 1773616.578 .564 .455 48474.267 1 48474.267 .015 .902 Log_CD4% 39229823.555 1 39229823.555 12.483 .001 HIV 9492717.945 2 4746358.972 1.510 .229 Gender 14880660.472 1 14880660.472 4.735 .034 HIV * Gender 12224946.991 2 6112473.495 1.945 .152 Error 185419987.176 59 3142711.647 Total 822902020.000 68 Corrected Total 317501616.235 67 Corrected Model Log_VL Squares a. R Squared = .416 (Adjusted R Squared = .337) 6 Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV infections Tests of Between-Subjects Effects Dependent Variable: CD38 MFI on CD56 bright NK Type III Sum of Source df Mean Square F Sig. 112223198.456a 8 14027899.807 8.853 .000 13079012.776 1 13079012.776 8.254 .006 Age 660866.287 1 660866.287 .417 .521 Log_VL 363600.400 1 363600.400 .229 .634 Log_CD4% 22767783.748 1 22767783.748 14.369 .000 HIV 3079391.111 2 1539695.556 .972 .385 Gender 1783878.616 1 1783878.616 1.126 .293 HIV * Gender 9050416.465 2 4525208.232 2.856 .066 Error 91901702.708 58 1584512.116 Total 1288343521.000 67 Corrected Total 204124901.164 66 Corrected Model Intercept Squares a. R Squared = .550 (Adjusted R Squared = .488) 7 51.4 0.268 81.7 3.66 CD4 Va24 98.1 CD56 SSC-A Supplementary Figures 49.9 37.7 FSC-A CD14 CD3 Vb11 Internal negative % of Max SSC-A 0.282 Bulk CD4 T cells CD56dim NK cells CD38 PLZF T-bet Figure S1) Measurement of CD38, PLZF and T-bet This figure illustrates an example of histograms generated from the acquisition of CD38, PLZF and T-bet data of Bulk CD4+ T cells and CD56dim NK cells. CD8 Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV infections CD3+ cells (a) NK cells (CD56dim/bright) iNKT cells 250K 10 5 10 5 200K 10 4 10 4 150K 10 3 10 3 10 2 10 2 3.9 0.423 100K 63.8 0 0 (b) 10 2 10 3 10 4 10 5 CD3+ cells 250K 200K 150K 0 9.52 0 10 3 10 4 10 5 CD3 SSC 0 TCR Valpha24 50K 0 10 10 4 10 3 10 2 3 10 4 10 5 NK cells (CD56dim/bright) iNKT cells 5 10 10 5 10 4 10 3 10 2 3.47 0.514 100K 57.9 0 0 50K 7.54 0 0 10 2 10 3 10 4 10 5 0 CD3 (c) 3 10 4 10 5 0 TCR Vbeta11 CD56dim NK cells % of Max 10 CD56bright NK cells 100 100 80 80 80 60 60 60 40 40 40 20 20 20 0 0 10 3 10 4 10 5 3 10 4 10 5 iNKT cells 100 0 10 CD56 0 0 10 3 10 4 10 5 0 10 3 10 4 10 5 T-bet (d) CD56dim NK cells CD56bright NK cells iNKT cells 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 0 0 0 10 3 10 4 10 5 0 0 10 3 10 4 10 5 0 10 3 10 4 10 5 T-bet Figure S2) Comparison of NK, iNKT and intracellular transcription factor T-bet staining using fresh and Cyto-chex stabilized whole blood. This figure illustrates examples on dot-plots generated during pre-testing of protocols for staining and flow cytometry analysis of T, iNKT and NK cell surface markers comparing (a) fresh blood and (b) Cytochex stabilized blood. The histograms are illustrating T-bet stainings of NK cells and iNKT cells comparing (c) fresh blood and (d) Cyto-chex stabilized blood. Grey filled histograms represent control samples without intracellular staining. Black lines indicate fully stained samples 9 Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV infections Figure S2 %CD56bright NK cells %CD56dim NK cells % iNKT cells %CD4+ iNKT of iNKT (a) %CD56bright NK cells %CD56dim NK cells %CD4+ iNKT of iNKT % iNKT cells (b) Figure S3: iNKT and NK cell subsets as percentage of lymphocytes. Percentages of iNKT and NK cell subsets assessed in blood samples of HIV-1, HIV-2, HIVD and non-infected control individuals. (a) Percentage of iNKT cells (filled squares), CD4+ iNKT cells (open squares) CD56dim NK cells (open circles), and CD56bright NK cells (filled circles) of peripheral blood lymphocytes. Comparisons of percentages of (b) total iNKT, CD4+ iNKT, CD56dim NK cell and CD56bright NK cell subsets (labelled as described above) in HIV-2 aviremic (viral load 75 RNA copies/mL blood) and viremic individuals (viral load > 75 RNA copies/mL). The dashed line represents the median absolute number of the respective cell type in HIV negative individuals. Median is depicted as solid bars. KruskalWallis test with Dunn´s post test, and Mann-Whitney test were used where appropriate. *p<0.05. 10 Bächle SM, Malone DFG, et al iNKT and NK cell activation in HIV infections iNKT 800 CD4+ iNKT 600 CD38 MFI CD38 MFI 600 400 200 0 400 200 0 -200 -200 non-HIV HIV-2 aviremic non-HIV CD56dim NK ** CD38 MFI 6000 4000 2000 0 HIV-2 aviremic CD56bright NK 6000 CD38 MFI ** 800 ** 4000 2000 0 non-HIV HIV-2 aviremic non-HIV HIV-2 aviremic Figure S4: Subsets of iNKT and NK cells in aviremic HIV-2 infection display enhanced activation compared with HIV seronegative individuals. Degree of activation represented by the median fluorescence intensity (MFI) level of CD38 surface expression was assessed. Comparison of CD38 MFI expression levels on total iNKT (filled squares), CD4+ iNKT (open squares), CD56dim (open circles) and CD56bright NK cells (filled circles) between HIV seronegative (non-HIV) and aviremic HIV-2 infected individuals. The dashed line represents the median CD38 MFI level of non-infected individuals. Mann-Whitney U-test was used for the comparison.**p<0.01. 11 Bächle SM, Malone DFG, et al T-bet in iNKT PLZF in CD4+iNKT *** 3 15 2 10 1 5 0 0 HIV-1 HIV-2 HIV-D HIV-1 HIV-2 HIV-D * 15 3 10 2 1 5 0 non-HIV T-bet in CD56bright NK 20 4 20 non-HIV PLZF in CD56bright NK 25 4 relative MFI T-bet in CD56dim NK relative relativeMFI MFI relative MFI 5 T-bet in CD4+iNKT relative MFI PLZF in CD56dim NK (b) relative MFI relative MFI (b) relative MFI relative MFI relative MFI PLZF in iNKT relative MFI relative MFI (a) (a) iNKT and NK cell activation in HIV infections 0 non-HIV HIV-1 HIV-2 HIV-D non-HIV HIV-1 HIV-2 HIV-D Figure S5: PLZF and T-bet expression in HIV infection Expression of transcription factors PLZF and T-bet in iNKT and NK cells subsets of HIV-1, HIV-2, HIV-D and controls were assessed by intracellular flow cytometry. All transcription factors are expressed as a relative expression, MFI ratio, to an internal baseline control as previously described. The dashed line at 1 represents the baseline level. A) Expression of PLZF and T-bet in total iNKT (filled squares) and CD4+ iNKT (open squares) cell subsets in HIV-infected and HIV-negative individuals. B) Expression of PLZF and T-bet in CD56dim (open circles) and CD56bright (closed circles) NK cell subsets. *p<0.05, ***p<0.001 12