Page 1 SUPPLEMENTARY FIGURE LEGENDS
Supplementary Figure 1 α4β7 is downregulated on CD8+ T cells upon coculture with live or inactivated bacteria. Expression of α4β7 on CD8+ T cells after total splenocytes were cocultured with live or heat-inactivated S. typhimurium or E. coli (MOI of 10). (A) Representative histograms are gated on CD8+ T cells. (B) Diagrams show the mean values of quadruplicates ± s.e.m. (C) α4β7 downregulation on CD8+ T cells upon TLR stimulation is dependent on MyD88 and results in reduced binding to MAdCAM-1. Expression of α4β7 on CD8+ T cells after total splenocytes from wild-type or MyD88-deficient (MyD88-/-) mice were cultured in the presence of TLR ligands for 36 h. (D) Adhesion of cultured splenic CD8+
T cells or TK-1 cells to plate-bound recombinant MAdCAM-Fc chimera. CD8+ T cells were purified from splenocytes that were cultured with or without CpG (3 µg/ml) for 24 h. Adhesion of untreated CD8+ T cells to non-coated wells was included as negative control (‘no MAdCAM’). Data show the mean values of at least triplicate samples ± s.e.m. All results are representative of at least two independent experiments. **, P < 0.01; ***, P < 0.001; ns, not significant. unstim, unstimulated.
Supplementary Figure 2 TLR-activated bystander CD8+
T cell retain a naïve phenotype and do not proliferate. Flow cytometry analysis of CD8+ T cells from mice that were treated once s.c. with CpG 24 h prior to the analysis. (A) Representative plots are gated on CD8+ T cells and show frequency of CD44lowCD62Lhigh (naïve) cells in secondary lymphoid organs. (B) Mean frequency of naïve, effector (CD44high
CD62Llow) and central (CD44highCD62Lhigh) memory T cells in secondary lymphoid organs of n = 5 untreated mice ± s.e.m. from two independent experiments. (C) naive (CD62L+ CD44low) Data give the mean values of at least n = 5 individual mice ± s.e.m from two independent experiments. An asterisk without brackets
Published in "7KH-RXUQDORI,PPXQRORJ\ ±" which should be cited to refer to this work.
Page 2 indicates comparison to the equivalent CD8+ T cell subset from unstimulated mice.
(D) Diagrams show CD69 MFI on naive CD8+ T cells in secondary lymphoid organs of individual mice. Horizontal bars represent the mean of individual mice (n = 5). (E) Purified splenic CD8+ T cells from wild-type or OT-I transgenic mice were labeled with Cell Proliferation Dye eFluor 670 and separately transferred into naïve recipient mice. Recipient mice were immunized with 100 µg CpG and 500 µg OVA i.p. and proliferation of transferred cells was determined by dye exclusion analyzed with flow cytometry. Representative plots are gated on CD8+ T cells and show cell proliferation dye staining and CD62L expression. The numbers indicate the percentage of proliferating and non-proliferating transferred CD8+ T cells. All results are representative of at least two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
Supplementary Figure 3 Cytokine production of CpG-stimulated splenocytes and their effect on α4β7 expression on different T-cell subsets. (A) Total splenocytes were cultured in the presence of CpG (3 µg/ml). After 48 h cytokine concentrations in the culture supernatant were determined by ELISA. (B) Expression of α4β7 on CD8+ T cells after total splenocytes were cultured in the presence of increasing concentrations of recombinant cytokines. The dashed line represents α4β7 expression of CpG-activated cells. (C) α4β7 expression is differentially regulated on effector and regulatory CD4 T-cell subsets following TLR activation. Complete splenocytes were cultured for 36h in the presence of either CpG or recombinant IL-6 and α4β7 expression on different T-cell subtypes was then analyzed by flow cytometry. Data is expressed as fold change of α4β7 expression of unstimulated T cells. All data show the mean values of at least triplicate samples ± s.e.m. All results are representative of two independent experiments. **, P < 0.01; ***, P < 0.001; ns, not significant.
Page 3 Supplementary Figure 4 Loss of CD8+ T-cell numbers in Peyer’s patches following
repetitive CpG treatments is self-limiting. (A) Mice were treated four times at 2-day intervals with 100 µg CpG s.c. Absolute numbers of CD8+ T cells in secondary lymphoid organs were counted and analyzed by flow cytometry. The diagram shows CD8+ T cell numbers of individual mice one or five days after the last CpG injection. Horizontal bars represent the mean of individual mice where applicable. (B) Comparison of TLR-induced downregulation of α4β7 on T cells in vitro and in vivo. Mice were injected s.c. with 100 µg CpG. 24 h later, spleens were prepared and lymphocytes were analyzed by flow cytometry. Representative dot plots show α4β7 expression on CD8+ T cells from untreated and CpG-injected mice. For in
vitro stimulation complete splenocytes from untreated animals were cultured in the presence
of the indicated stimulants and α4β7 expression on CD8+ T cells was analyzed 48 h later. Horizontal bars indicate α4β7 mean fluorescence intensity. Numbers give percentage of α4β7high
6 6
47 is downregulated on CD8+ T cells upon coculture with live or
inactivated bacteria. 0 20 40 60 80 100
Live Inactivated Unstimulated
S. typhimurium Events (% of max.) 4 7 on CD8+ T cells 0 102 103 104 105 0 102 103 104 105 Unstimulated E. coli 0 20 40 60 80 100 Events (% of max.) 4 7on CD8+ T cells 0 103 104 105 0 103 104 105 Live Inactivated Salmonella typhimurium Escherichia coli Unstim. Live Inactivated Live Inactivated 0 500 1000 ** *** ** *** Unstimulated E. coli S. typhimurium on CD8 + T cells (MFI)
A
B
TK-1 T cells no MAdCAM 100 75 50 25 Untreated CpG # c ells bound t o M A d C A M (x 10 3)D
4 7 Anti-0 nsUnstim.LPSR848 CpG PMA LPS R848 CpG PMA
0 1000 2000 3000 ** ** ** *** *** Wild-type MyD88 -/-Unstim. ns ns on CD8 + T cells (MFI) ** PAM 3 CSK4 PAM 3 CSK4 ND Flagellin ns Flagellin ND
C
Supplementary Figure 2: TLR-activated bystander CD8+ T cells retain a naïve phenotype and do not proliferate.
A
D
CpG s.c. Untreated CD44 on CD8+ T cells 76.4 83.4 80.5 48.3 53.6 77.9 83.6 75.5 CD62L Spleen PLN MLN PP 0 20 40 60 0 20 40 60 0 20 40 60 0 20 40 60 Spleen PLN MLN PP Untreated CpG s.c. P = 0.003 P = 0.011 P < 0.001 P < 0.021 CD69 on naïve CD8 + T cells (% positive)C
0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 102 103 105 104 0 102 103 105 104 0 CD69 on naïve CD8 + T cells (% positive) CD69 on naïve CD8 + T cells (% positive) CD69 on naïve CD8 + T cells (% positive)E
PLN MLNWild-type OT-I transgenic
Cell Proliferation Dye eFluor 670
CD62L
Wild-type OT-I transgenic
OVA+CpG i.p. Unstimulated 2.1 97.9 93.8 6.2 93.6 6.4 95.3 4.7 3.7 96.3 3.2 96.8 2.8 97.2 95.2 4.8
gated on CD8+ T cells gated on CD8+ T cells
0 103 104 105 0 103 104 105
Cell Proliferation Dye eFluor 670
0 103 104 105 0 103 104 105 103 105 104 0 103 105 104 0 CD62L 103 105 104 0 103 105 104 0 Spleen Total Naive 0 500 1000 Untreated CpG s.c. on CD8 + T ce ll s (MF I) PLN Tota l Naiv e 0 500 1000 Tota l Naive 0 500 1000 PP Total Naiv e 0 500 1000 MLN ** *** ns ** ** ns * * ns ** *** ns
B
NaiveCentral memory Effe
ctor memory 0 20 40 60 80 100 Spleen PLN MLN PP % of total C D 8 + T c ells 0 50K 100K150K200K250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K life gate 91.9 0 103 104 105 0 103 104 105 0 103 104 105 B cells 9.97 T cells 84 0 103 104 105 CD4+ 52 CD8+ 43.1 FSC CD3 CD8 CD4 CD19 SSC
Supplementary Figure 3: Cytokine production of CpG-stimulated splenocytes and their effect 47 expression on different T-cell subsets.
IL-4 IL-6 IL-10
IL-12p70 IFN-0 500 1000 1500 2000 2000 6000 10000 Unstimulated CpG pg/ ml *** *** ** ns ns
A
B
on CD8 + T cells (MFI) 0 0,01 0,1 1 10 0 500 1000 1500 2000 IL-1ß [ng/ml] 0 0,01 0,1 1 10 0 500 1000 1500 2000 IL-2 [ng/ml] 0 0.01 0.1 1 10 0 500 1000 IL-15 [ng/ml] 0 0.01 0.1 1 10 0 500 1000 TNF- ng/ml] CD4 + TH cells CD4 + TReg cells CD8 + T cells 0.0 0.5 1.0 Unstim. CpG IL-6 *** *** *** *** ns ns 4 7 on T c ells (M F I, fo ld c h a n g e o f u n s tim .)C
6 6
Supplementary Figure 4: Loss of CD8+ T-cell numbers in Peyer’s patches following repetitive CpG
treatments is self-limiting.
B
In vivo CpG stimulation: In vitro CpG stimulation: 4 7 CD8 Untreated CpG s.c. 0 102 103 104 105 0 103 104 105 0102 103 104 105 0 103 104 105 0102 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 4 7 CD8 Unstimulated CpG anti-CD3/CD28 + Retinoic acid 2.6 0.0 15.1 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105A
PLN Untreated1 day post CpG s.c.5 days post CpG s.c. 0 2 4 6 8 Abs olut e CD8 + T ce ll s (x1 0 6 Untreated
1 day post CpG s.c.5 days post CpG s.c. 0.00 0.05 0.10 0.15 Abs olut e CD8 + T c e ll s ( x 1 0 6 ) ) Peyer’s patches
