The Costimulatory Molecule CD27 Maintains Clonally

Transcription

1 Immunity, Volume 35 Supplemental Information The Costimulatory Molecule CD7 Maintains Clonally Diverse CD8 + T Cell Responses of Low Antigen Affinity to Protect against Viral Variants Klaas P.J.M. van Gisbergen, Paul L. Klarenbeek, Natasja A.M. Kragten, Peter-Paul A. Unger, Marieke B.B. Nieuwenhuis, Felix M. Wensveen, Anja ten Brinke, Paul P. Tak, Eric Eldering, Martijn A. Nolte, and Rene A.W. van Lier 1

2 (CFSE, Log 1 fluorescence) Cell division (CFSE, Log 1 fluorescence) Cell division A Control isotype anti-cd7 CD7 Tg B Control CD7 Tg CD7 MHC I CD8 CD86 C pg/ml control CD7 D Cell death (PI, Log 1 fluorescence) SIIQFEKL SAINFEKL SIINFEKL control CD Cell death (PI, Log 1 fluorescence) 6.7 Figure S1: Antigen density and avidity determine the outcome of CD7-driven costimulation. (A) Histograms display the expression of an isotype control and CD7 on B cells within spleen preparations of (control) and x CD7 Tg (CD7 Tg)

3 mice that were used as APCs. (B) Similarly, expression of MHC class I, CD8, and CD86 is shown for control and CD7 Tg APCs. (C-D) OTI cells were stimulated with peptide for 3 days in the presence of irradiated splenocytes of mice or x CD7 Tg mice. (C) Representative dotplots display proliferation by CFSE dilution and cell death by PI staining of OTI cells stimulated with the indicated concentrations of SIINFEKL peptide. (D) Similarly, representative dotplots display proliferation and cell death of OTI cells that were stimulated with identical concentrations (3 pg/ml) of SIIQFEKL, SAINFEKL and SIINFEKL peptide. Representative data is shown of three separate experiments. 3

4 EM/Effector CD8 + T cells (x 1 6 ) 5 A/PR8/34 Day mln Spleen Lungs Figure S: CD7 lowers the affinity of the virus-specific CD8 + T cell population. Total number of CD8 + T cells with effector memory or effector phenotype (CD44 + CD6L - ) were enumerated in mln, spleen and lungs of and mice at day 1 after infection with A/PR8/34. Error bars denote standard error of the mean (SEM). 4

5 Viral titer (log RNA copies) Expression CD43 (%) Expression CD5 (%) Tetramer + CD8 + T cells (x 1 6 ) Tetramer + CD8 + T cells (x 1 6 ) (Log 1 fluorescence) CD17 A 8.5 +/- 1.3 EMtype MPEC CMtype MPEC.8 +/ / /-.5 SLEC B / /- 1.7 CD6L (Log 1 fluorescence) mln.5 Spleen C Time (days) 1 8 D Time (days) 4 3 E Time (days) Time (days) 3 1 Day 5 Day 8 Day 1 Figure S3: CD7 restricts effector differentiation of low but not high affinity CD8 + T cells. (A) The phenotype of influenza-specific CD8 + T cells was analyzed in spleen of 1-5

6 day A/PR8/34-infected and mice using CD17 and CD6L. (B) The total number of influenza-specific CD8 + T cells was determined at the indicated timepoints after infection with A/PR8/34 in mln (left panel) and spleen (right panel) of and mice. (C-D) The expression of the activation markers (C) CD43 and (D) CD5 was followed in time on tetramer + CD8 + T cells within the spleen of and after influenza infection with the A/PR8/34 virus. (E) Viral loads were determined within the lungs at day 5, 8, and 1 after infection with the A/PR8/34 virus using qpcr. Error bars denote standard error of the mean (SEM). 6

7 IFN- + CD8 + T cells (x 1 6 ) IFN- and IL- + CD8 + T cells (x 1 6 ) (Log 1 fluorescence) IFN- CD8 + T cells (x 1 6 ) A B Host Donor /-.3 +/ / /-. C.4.3 IL- (Log 1 fluorescence) Day 1 D.1.8 Day 1 * medium peptide medium peptide Figure S4: CD7 regulates CD8 + T cells in a cell-intrinsic manner. (A) The total number of splenic CD8 + T cells originating of the recipient mice, and of the and donor mice were determined in mixed BM chimeras. (B-D) Splenocytes of and mice that had been infected with the influenza virus HK/68 1 days previously were re-stimulated for 4 hrs with the influenza-specific peptide ASNENMDAM. (B) Representative dotplots display intracellular staining of CD8 + T 7

8 cells of (left) and mice (right) for IFN- and IL-. Insets denote the mean +/- SEM of the percentage of CD8 + T cells within the respective quadrants. The percentage of CD8 + T cells within upper right quadrants is significantly different between and mice (P <.5). (C-D) The absolute numbers of (C) IFN- -producing CD8 + T cells and (D) IFN- and IL- co-producing CD8 + T cells of and mice were determined after mock and peptide re-stimulation. Error bars denote standard error of the mean (SEM). 8

9 CD17 CD17 A / / / /- 1.8 B / /- 1.1 CD6L / / / / /-.8 CD /- 1.1 Figure S5: CD7 ensures the presence of low affinity clones in the memory CD8 + T cell pool. and mice were infected with the influenza virus A/PR8/34 and 4 days later the phenotype of influenza-specific CD8 + T cells was analyzed. Influenzaspecific CD8 + T cells were analyzed for expression of (A) CD17 and CD6L or (B) CD17 and CD43. Insets represent mean +/- SEM of the percentage of cells within the quadrant. 9

10 Dominant clone of total reads (%) Number of clones > 1% Public clones of total reads (%) Expression V (%) Tetramer binding (geo MFI) Tetramer Expression V 8.3 (%) Expression V 8.3 (%) Expression V 8.3 (%) A 6 4 B Day 1 * * C * 4 4 Thymus Control Infected CM EM SLEC Spleen MPEC Donor D E F HK/68 6 ** 4 HK/68 3 ** V 8.3 V 11 Total V G * H * V 11 I ** Figure S6: CD7 favors subdominant clones within the virus-specific CD8 + T cell pool. (A-B) The usage of Vβ8.3 was determined within (A) the indicated non-tetramer + populations and (B) the indicated tetramer + populations of and mice at day 1 of infection with A/PR8/34. (C) Similarly, the percentage of Vβ8.3 + CD8 + T cells within 1

11 the spleen of mixed BM chimeras was determined for tetramer + CD8 + T cells of and donor origin. (D-F) Vβ usage was examined in and mice 1 days after infection with HK/68. (D) The percentage of Vβ8.3 + and Vβ11 + clones within the tetramer + population was analyzed in spleen of and mice. (E) The geo MFI of tetramer binding was determined of the indicated influenza-specific populations of and mice. (F) Representative dotplots display tetramer binding and Vβ11 expression of influenza-specific CD8 + T cells of (left panel) and mice (right panel). (G-I) and mice were infected with the influenza virus A/PR8/34 and 8 days later influenza-specific CD8 + T cells were isolated and analyzed for the TCR repertoire within the Vβ8.3 segment using deep sequencing. (G) The frequency of the dominant clone within the tetramer + and Vβ8.3 restricted CD8 + T cell population was determined for and mice. (H) As an arbitrary measure for subdominant clones within the influenza-specific CD8 + T cell population, the number of clones with a relative frequency of more than 1% was determined for and mice. (I) The combined frequency of the public clones was examined in and mice. Only clones with a minimal frequency of.1 % in all of the mice were considered public. Error bars denote standard error of the mean (SEM). 11

12 # of mice containing clone CDR3 Sequence # of reads % of total reads CDR3 Sequence # of reads % of total reads 3 CDR3 Sequence # of reads % of total reads CD7 KO 1 CDR3 Sequence # of reads % of total reads CD7 KO CDR3 Sequence # of reads % of total reads CD7 KO 3 CDR3 Sequence # of reads % of total reads 1 CASSGGGNTGQLYF % 1 CASSARTANTEVFF % 1 CASSDAANTEVFF % 1 CASSGGSNTGQLYF % 1 CASSGGGNTGQLYF % 1 CASSGGSNTGQLYF % CASSEWGTGQLYF % CASSGGSNTGQLYF % CASSGGANTGQLYF % CASSGGGNTGQLYF % CASSGGSNTGQLYF % CASSGGGNTGQLYF % 3 CASSGGSNTGQLYF % 3 CASSDAAATEVFF % 3 CASRGGGNTGQLYF % 3 CASSGGANTGQLYF % 3 CASSGGANTGQLYF % 3 CASSGGANTGQLYF % 4 CASSGGANTGQLYF % 4 CASSGGANTGQLYF % 4 CASKGGGNTGQLYF % 4 CASRWGGNTGQLYF % 4 CASRWGGNTGQLYF 61.93% 4 CASRGGGNTGQPYF 6.8% 5 CASKGGANTGQLYF % 5 CASSGGGNTGQLYF 183.5% 5 CASSGGSNTGQLYF % 5 CASSGGANTGQLYL % 5 CASSGRGNTGQLYF 4.61% 5 CASSDGGDTEVFF 35.46% 6 CASSDHRNTEVFF % 6 CASRGGSNTGQLYF % 6 CASSGGSNTGQLCF % 6 CASSVGQNTEVFF 55.86% 6 CASKGGGNTGQRYF 3.46% 6 CASSGGPNTGQLYF 9.38% 7 CASSDARNTEVFF 9 1.% 7 CASSDNYNSPLYF 59.66% 7 CASSGGANTGQPYF % 7 CASSGGGVTGQLYF 9.46% 7 CASSGGGTTGQLYF 7.41% 7 CASSGRGNTGQLYF.9% 8 CASSDDERLFF 58.76% 8 CASSDNGAGNTLYF 39.44% 8 CASSGTVQNTLYF % 8 CASSGGGTTGQLYF 8.44% 8 CASSEGGNSDYTF 1.3% 8 CASSDGGSQNTLYF 19.5% 9 CASSDGMYEQYF 56.73% 9 CASSDAPGTDTEVFF 31.35% 9 CASSGGGNTGQLYF 51.66% 9 CASSGGRNTGQLYF 7.4% 9 CASSGEGNTGQLYF 13.% 9 CASSPWGGEDYAEQFF 18.4% 1 CASRDSANTEVFF 5.65% 1 CASSEALGSQNTLYF 6.9% 1 CASSDGGANSDYTF 6.33% 1 CASGGGANTGQLYF 3.36% 1 CASSGGPNTGQLYF 1.18% 1 CASSGGSLAETLYF 17.% 11 CASSDGDRDGYAEQFF 48.63% 11 CASSGGHLTEVFF 3.6% 11 CASSEGSNTGQLYF.6% 11 CASSDPGQGAEAPLF 3.36% 11 CASSGGRNTGQLYF 1.18% 11 CASSNLSNERLFF 17.% 1 CASSDTGTGQDTQYF 45.59% 1 CASSDAWGNTLYF.5% 1 CASSPGLGGYEQYF 16.1% 1 CASSGNTLYF 3.36% 1 CASSEAGEDTQYF 1.18% 1 CASSGGRNTGQLYF 15.% 13 CASSDYRNSDYTF 44.57% 13 CASSGGRNTGQLYF 1.4% 13 CASSDATNTEVFF 16.1% 13 CASSDRGGQDTQYF 17.7% 13 CASRGGANTGQLYF 11.17% 13 CASSDARGPSSQNTLYF 13.17% 14 CASSDGQGPEQFF 4.5% 14 CASSDGGETLYF 18.% 14 CASSGWGSTETLYF 15.19% 14 CASSQWYAEQFF 16.5% 14 CASSVGTGYNSPLYF 9.14% 14 CASSGRWGGAETLYF 13.17% 15 CASSDNANTEVFF 39.51% 15 CASSDATGQLYF 18.% 15 CASSDKGSEVFF 15.19% 15 CASNDRGRGNTLYF 15.4% 15 CASSGGGSTGQLYF 9.14% 15 CASSDWQFSAETLYF 1.13% 16 CASSERRVNSDYTF 37.48% 16 CASSEGGAQNTLYF 17.19% 16 CASSGGRNTGQLYF 1.15% 16 CASSGGPNTGQLYF 11.17% 16 CASSDEGDEQYF 9.14% 16 CASRGGANTGQLYF 9.1% 17 CASRGGANTGQLYF 3.4% 17 CASSGPGQGYAEQFF 17.19% 17 CASSGLNQDTQYF 9.1% 17 CASSGRGNTGQLYF 1.16% 17 CASSDAWDTQYF 8.1% 17 CASSVRVTEVFF 9.1% 18 CASSDPPANTGQLYF 7.35% 18 CASSASGQNTGQLYF 17.19% 18 CASSHTGEDTGQLYF 8.1% 18 CASSERGRGNTLYF 1.16% 18 CASRPGQPNSDYTF 8.1% 18 CASSPGQGYSDYTF 8.11% 19 CASSEEGNNQDTQYF 5.33% 19 CASSWGDYEQYF 14.16% 19 CASSGGPNTGQLYF 7.9% 19 CASSDADRVYEQYF 9.14% 19 CASSPQGAGNTLYF 8.1% 19 CASSDATEGQNTLYF 8.11% CASSDTGGGAETLYF 4.31% CASSEAGGNERLFF 13.15% CASSDAGGQNTLYF 6.8% CASSEPRLGDAEQFF 8.13% CASSDAGPYQAPLF 7.11% CASNGRWGGAETLYF 6.8% 1 CASSGRGGTGQLYF 4.31% 1 CASSAQTANTEVFF 11.1% 1 CASSDQLGGREQYF 5.6% 1 CASSSGGRNTLYF 7.11% 1 CASSEVYAEQFF 6.9% 1 CASRWRGNTGQLYF 6.8% CASSLDKANTEVFF 3.3% CASSGTAEVFF 11.1% CASSGRSNTGQLYF 5.6% CASSGEGNTGQLYF 6.9% CASSGGGNTGRLYF 6.9% CASSQGWNNQAPLF 6.8% 3 CASSGRGLEQYF.9% 3 CASGAETLYF 1.11% 3 CASSGGGTTGQLYF 4.5% 3 CASRAGTGGAEQYF 6.9% 3 CASGGGGNTGQLYF 6.9% 3 CASSEAYTGNYAEQFF 6.8% 4 CASSDYLGDYAEQFF.9% 4 CASSPGQGALDTQYF 9.1% 4 CASRWGGNTGQLYF 4.5% 4 CASRVGGNTGQLYF 6.9% 4 CASSGAGNTGQLYF 5.8% 4 CASSGESNTGQLYF 4.5% 5 CASRLGGDTQYF 18.3% 5 CASSARAANTEVFF 8.9% 5 CASSDEGTGNEQYF 3.4% 5 CASSGGDNQAPLF 6.9% 5 CASSDEDTQYF 4.6% 5 CASGGGSNTGQLYF 4.5% 6 CASSDSGSDYTF 16.1% 6 CASSAANTEVFF 8.9% 6 CASKEGGNTGQLYF 3.4% 6 CASSGSSNTGQLYF 5.8% 6 CASSEWVQDTQYF 4.6% 6 CASSEMGDTEVFF 3.4% 7 CASSGTDSAETLYF 15.% 7 CASKGGAGTGQLYF 8.9% 7 CASSGRANTGQLYF 3.4% 7 CASSDAWGGAETLYF 5.8% 7 CASSDWDSANTEVFF 4.6% 7 CASSGGGSTGQLYF 3.4% 8 CASKGRDASAETLYF 15.% 8 CASSDRLGQDTQYF 8.9% 8 CAGKGGGNTGQLYF 3.4% 8 CASSDRTGGEFYAEQFF 4.6% 8 CASSVDRGLGEQYF 4.6% 8 CASSGGGTTGQLYF 3.4% 9 CASSDRDSQNTLYF 14.18% 9 CASKGGGNTGQLYF 8.9% 9 CASSGDSAETLYF 3.4% 9 CARGTGGLEVFF 4.6% 9 CASSGTGGEDEQYF 4.6% 9 CASSGGGDTGQLYF.3% 3 CASSDHRGTEVFF 1.16% 3 CASGGGSNTGQLYF 7.8% 3 CASSGESNTGQLYF 3.4% 3 CASNGGSNTGQLYF 4.6% 3 CASGGGSNTGQLYF 3.5% 3 CASSGGGNAGQLYF.3% 31 CASSGWGTGQLYF 11.14% 31 CASSGRGNTGQLYF 7.8% 31 LCQQRGGSKHRGSSTF.3% 31 CASSGRSNTGQLYF 4.6% 31 CASSGGLNTGQLYF 3.5% 31 CASSDSGGAETLYF.3% 3 CASSVRQGWDTEVFF 11.14% 3 CASSARTVNTEVFF 6.7% 3 CACSGGSNTGQLYF.3% 3 CASNRGANTGQLYF 4.6% 3 CASSRGLGGYEQYF 3.5% 3 CASSNPSNERLFF.3% 33 CASSGRGNTGQLYF 1.13% 33 CASSGGTGGGEQYF 6.7% 33 CASRGGGNAGQLYF.3% 33 CASSGVGANTEVFF 4.6% 33 CASSARLGGQEQYF 3.5% 33 CASGGGGNTGQLYF.3% 34 CASSGGGSTGQLYF 9.1% 34 CASSTRGQNTLYF 6.7% 34 CASKGRGNTGQLYF.3% 34 CASSDRLGGREQYF 4.6% 34 CASSASQGGEQYF 3.5% 34 CASGGGANTGQLYF 1.1% 35 CASKGGGNTGQLYF 9.1% 35 CASSGGPNTGQLYF 5.6% 35 CASSDAVNTEVFF.3% 35 CASSDAGEAPLF 4.6% 35 CASSGLGGNYAEQFF 3.5% 35 CASSEGEDNQAPLF 1.1% 36 CASSDGGGGTEVFF 8.1% 36 CASSSLSYEQYF 5.6% 36 CASGGGSNTGQLYF.3% 36 CASSAPDNGGGAQDTQYF 3.5% 36 CASSDHRANERLFF 3.5% 36 CASERRGNTGQLYF 1.1% 37 CASQGRDAEQFF 8.1% 37 YASSDAASSNERLFF 5.6% 37 CASSGERNTGQLYF.3% 37 CASSDGGRTEVFF 3.5% 37 CASSDWDRGNTEVFF.3% 37 CASSEGSNTGQLYF 1.1% 38 CASSDAGEQYF 6.8% 38 CASGARTANTEVFF 5.6% 38 CANSGGSNTGQLYF.3% 38 CASLRSNTEVFF 3.5% 38 CASSDGTSQDTQYF.3% 38 CASSGAGNTGQLYF 1.1% 39 CASSEKAGQLYF 6.8% 39 CASSDTGGEDTQYF 4.4% 39 CASRGWGNTGQLYF.3% 39 CASSGGGEQYF 3.5% 39 CASSDEGNEQYF.3% 39 CASGRGSNTGQLYF 1.1% 4 CASKGGRNTGQLYF 6.8% 4 CASSDVAATEVFF 4.4% 4 CAGSGGSNTGQLCF.3% 4 CASSEGSNTGQLYF 3.5% 4 CASSEGGRDEQYF.3% 4 CASRGRGNTGQLYF 1.1% 41 CASSEKANTGQLYF 6.8% 41 CASSPDRSAETLYF 4.4% 41 CASGGGANTGQLYF.3% 41 CASSEREYNNQAPLF 3.5% 41 CASSSGGNTGQLYF.3% 41 CASSENRGLQNTLYF 1.1% 4 CASSGGTNTGQLYF 6.8% 4 CASNARTANTEVFF 3.3% 4 CASKGGGDTGQLYF 1.1% 4 CASSLGGYAEQFF.3% 4 CASSGGGRTGQLYF.3% 4 CAGSGGANTGQLYF 1.1% 43 CASSGGGGTGQLYF 6.8% 43 CASSARIANTEVFF 3.3% 43 CAIRGGGNTGQLYF 1.1% 43 CASSGVGNTGQLYF.3% 43 CASSETVVYAEQFF.3% 43 CASRGGGNTGQLYF 1.1% 44 CASSRDRGTGQLYF 5.7% 44 CASSVRTANTEVFF 3.3% 44 CASRGDGNTGQPYF 1.1% 44 CASRINTEVFF.3% 44 CASNGGANTGQLYF.3% 44 CASGAGGNTGQLYF 1.1% 45 CASSEWGAGQLYF 5.7% 45 CASSARTTNTEVFF.% 45 CASEGGGNTGQLYF 1.1% 45 CASSEGQGALEQYF.3% 45 CAGRWGGTTGQLYF.3% 45 CASSGRANTGQLYF 1.1% 46 CASSGGENTGQLYF 5.7% 46 CASSDAAAAEVFF.% 46 CASRGGGDTGQLYF 1.1% 46 CASNGGGNTGQLYF.3% 46 CASSGGGDTGQLYF.3% 46 CASVGGSNTGQLYF 1.1% 47 CASSGXGNTGQLYF 4.5% 47 CASSDAGGTSAETLYF.% 47 CARKGGGNTGQLYF 1.1% 47 CASSGGGNAGQLYF.3% 47 CACSGGSNTGQLYF 1.% 47 CASTPWGGEDYAEQFF 1.1% 48 CASSGWGNTGQLYF 4.5% 48 CASSGEANTGQLYF.% 48 CASKGGGNAGQLYF 1.1% 48 CASSPRGRNTEVFF.3% 48 CASRVGGNTGQLYF 1.% 48 CASSRRGNTGQLYF 1.1% 49 CASGGGSNTGQLYF 4.5% 49 CASSEGANTGQLYF.% 49 CASSGVRNTGQLYF 1.1% 49 CASSDAGRGEQYF.3% 49 CAGSGGGSTGQLYF 1.% 49 CASSRGSNTGQLYF 1.1% 5 CASSTGGEHTQYF 3.4% 5 CASSDAGGGAYAEQFF.% 5 CASSGGRQTDRQPYF 1.1% 5 CASSRGGNTGQLHF.3% 5 CASRRGGNTGQLYF 1.% 5 CASSRGGNTGQLYF 1.1% 51 CASSGHRNTEVFF 3.4% 51 CASSTRTANTEVFF 1.1% 51 CASSGEGNTGQLYF 1.1% 51 CASSGASNTGQLHF.3% 51 CARPGGGNTGQLYF 1.% 51 CASSGWGNTGQLYF 1.1% 5 CASSGGRNTGQLYF 3.4% 5 CASSRGSNTGQLYF 1.1% 5 CASSGEANTGQLYF 1.1% 5 CASSSGGGNTLYF.3% 5 CASNLSGTGENTLYF 1.% 5 CASSGVGNTGQLYF 1.1% 53 CASSGGPNTGQLYF 3.4% 53 CASGGGANTGQLYF 1.1% 53 CASSGDRNTGQLYF 1.1% 53 CASSGAANTGQLYF.3% 53 CASGWGGNTGQLYF 1.% 53 CASSGSANTGQLYF 1.1% 54 CASSGGGTTGQLYF 3.4% 54 CASNGGSNTGQLYF 1.1% 54 CASSGARNTGQLYF 1.1% 54 CASSPTVNTGQLYF.3% 54 CASSXGGNTGQLYF 1.% 54 CASSGDANTGQLYF 1.1% 55 CASSGDSAETLYF 3.4% 55 CASSRGGNTGQLYF 1.1% 55 CASRWGANTGQLYF 1.1% 55 CASSGGVNTGQLYF 1.% 55 CASRGGGESQNTLYF 1.% 55 CASSGPGTGGNQDTQYF 1.1% 56 CASSGGVNTGQLYF.3% 56 CAGSWGDYEQYF 1.1% 56 CASSGVANTGQLYF 1.1% 56 CASNGGANTGQLYF 1.% 56 CASMGGSNTGQLYF 1.% 56 CASSGGSSAGQLYF 1.1% 57 CAGKGGANTGQLYF.3% 57 CASSGGGTTGQLYF 1.1% 57 CASRGGGSTGQLYF 1.1% 57 CASSVGSNTGQLYF 1.% 57 CASRWGANTGQLYF 1.% 57 CASSGGSDTGQLYF 1.1% 58 CACKGGANTGQLYF.3% 58 CASSDTDSANTEVFF 1.1% 58 CASSDGRANSDYTF 1.1% 58 ASSGGPGTGQLYF 1.% 58 CASRGLGGSAETLYF 1.% 58 CASSGGPLAETLYF 1.1% 59 CASSRGGNTGQLYF.3% 59 CASSDTAATEVFF 1.1% 59 CASSDGGTGYTEVFF 1.1% 59 CASNGGRNTGQLYF 1.% 59 CASSGSSNTGQLYF 1.% 59 CASSGGLNTGQLYF 1.1% 6 CASRGGSNTGQLYF.3% 6 CASSEGQGAYEQYF 1.1% 6 CASSPGLGGCEQYF 1.1% 6 CASRWGGNAGQLYF 1.% 6 CASSGGTNTGQLYF 1.% 6 CASSGGGRTGQLYF 1.1% 61 CASEGGANTGQLYF.3% 61 CASSGEGNTGQLYF 1.1% 61 CASTGGGNTGQLYF 1.1% 61 CASSAPDNGGGGQDTQYF 1.% 61 CASSGGENTGQLYF 1.% 61 CASSGGERTGQLYF 1.1% 6 CASSGGGATGQLYF.3% 6 CASSGGGDTGQLYF 1.1% 6 CASXGGSNTGQLYF 1.1% 6 CASSGGAGTGQLYF 1.% 6 CASSGEGSTGQLYF 1.% 6 CASSGGENTGQLYF 1.1% 63 CASSGTGLEQYF.3% 63 CASSARKANTEVFF 1.1% 63 CTSVPAGGVRTPGSSTF 1.1% 63 CASSDALGVNYAEQFF 1.% 63 CASSGEANTGQLYF 1.% 63 CASSGEGNTGQLYF 1.1% 64 CASSARTANTEVFF.3% 64 CASSGGGSTGQLYF 1.1% 64 CASRWGSNTGQLYF 1.1% 64 CASSDENERLFF 1.% 64 CASSGARNTGQLYF 1.% 64 CASSGSSNTGQLYF 1.1% 65 CASRWRGNTGQLYF.3% 65 CASRWRGNTGQLYF 1.1% 65 CASRRGGNTGQLYF 1.1% 65 CASSDGLGGPTGQLYF 1.% 65 CASSRGGNTGQLYF 1.% 66 CASSGEGNTGQLYF.3% 66 CASSGRANTGQLYF 1.1% 66 CASSWGGRAEQFF 1.% 66 CASSGSRENTLYF 1.% 67 CASRGGRNTGQLYF 1.1% 67 CASSGRNTGQLYF 1.1% 67 CASSEGNYNSPLYF 1.% 67 CASRWGGDTGQLYF 1.% 68 CASRGGGNTGQLYF 1.1% 68 CASSGRSNTGQLYF 1.1% 68 CASSGRGGQDTQYF 1.% 68 CASSGTGNTGQLYF 1.% 69 CASNGGSNTGQLYF 1.1% 69 CASSGTDSAETLYF 1.1% 69 CASSGWANTGQLYF 1.% 7 CASNGGGNTGQLYF 1.1% 7 CASSGVANTGQLYF 1.1% 7 CASSDLGDNYAEQFF 1.% 71 CASEVGANTGQLYF 1.1% 71 CASSGGGNAGQLYF 1.1% 71 CASSRGANTGQLYF 1.% 7 CASGGGGNTGQLYF 1.1% 7 CASSGAGPYRAPLF 1.% 73 CASKGGRTTEQLYF 1.1% 73 CASRWGRRTGQLYF 1.% 74 CASRGAGNTGQLYF 1.1% 74 CASRWGGNAGQLYF 1.% 75 CASSGEANTGQLYF 1.1% 75 CASSGSGNTGQLYF 1.% 76 CASSGAGAEVFF 1.1% 77 CASSEWGIGQLYF 1.1% 78 CASSGGGDTGQLYF 1.1% 79 CASSEGETTEVFF 1.1% 8 CASSGGGNAGQLYF 1.1% 81 CASSEEGNDQDTQYF 1.1% 8 CASSGVGNTGQLYF 1.1% 83 CASSDYLGGYAEQFF 1.1% 84 CASSGRANTGQLYF 1.1% 85 CASSGRGDTGQLYF 1.1% 86 CASSDHRDTEVFF 1.1% 87 CASSDGTYEQYF 1.1% 88 CASSGRSNTGQLYF 1.1% 89 CASSGGANAGQLYF 1.1% 9 CASSGVDTGQLYF 1.1% 91 CASSRGANTGQLYF 1.1% 1

13 Table S1: and mice were infected with the influenza virus A/PR8/34 and 8 days later influenza-specific CD8 + T cells were isolated and analyzed for the TCR repertoire within the Vβ8.3 segment using deep sequencing. The CDR3 sequence, abundance and frequency of the influenza-specific CD8 + T cell clones are provided of 3 and 3 mice. The number of recaptures of each clone in different mice has been indicated by color code. 13

14 Clone of total reads (%) HK/68 Tetramer binding (%) V 8.3 expression (%) Tetramer HK/68 A HKx31 A/PR8/ HK/68 A/PR8/ B * 9 of * 3 of 3 V 8.3 * 3 of of 3 6 of 9 C HKx31HK/68 HKx31 V V HK/68 CASSGGSNTGQLYF CASSGGANTGQLYF CASSGGGNTGQLYF CASKGGGNTGQLYF CASSSGGKNTLYF CASSARTANTEVFF CASRGGANTGQLYF Clones at frequency < 1% HKx31 A/PR8/34 HK/68 A/PR8/34 Figure S7: CD8 + T cell responses upon re-challenge with heterologous but not homologous virus require CD7. mice were infected with either HK/68 or HKx31, re-challenged 51 days later with A/PR8/34, and analyzed 7 days after rechallenge. (A) Dotplots show the expression of Vβ8.3 and the binding of HK/68- specific tetramers to A/PR8/34-specific CD8 + T cells of mice with the indicated infection history. Left panel is representative of 3 of 3 mice, center panel of 6 of 9 mice and right panel of 3 of 9 mice. (B) The percentage of binding of HK/68 tetramers (left 14

15 panel) and the percentage of Vβ8.3 usage (right panel) is shown of A/PR8/34 tetramer + CD8 + T cells of mice re-challenged with A/PR8/34 that had previously been infected with either HKx31 or HK/68. As expected, the high degree of cross-reactivity in the influenza-specific CD8 + T cell population after heterologous re-challenge was not observed in homologous recall responses. Analysis of Vβ usage showed that influenzaspecific CD8 + T cells in homologous secondary responses displayed similar bias for Vβ8.3 as in primary responses. In contrast, heterologous secondary responses had either almost exclusive usage of Vβ8.3 (3 of 9 mice) or almost no usage of Vβ8.3 (6 of 9 mice). (C) Using deep-sequencing, the relative frequency of influenza-specific CD8 + T cell clones with a frequency of higher than 1% of the total pool was analyzed within 3 individual mice that had been sequentially infected with either HKx31 and A/PR8/34 or HK/68 and A/PR8/34. For the latter group the 3 mice that contained Vβ8.3 + influenza-specific CD8 + T cells were selected. Sequencing of the Vβ8.3-restricted CD8 + T cell responses after A/PR8/34 re-challenge revealed striking differences at the clonal level between heterologous and homologous recall responses. The Vβ8.3-restricted repertoire of influenza-specific CD8 + T cells during homologous recall responses was diverse, although less than that of primary memory responses, and contained both public and non-public clones. In contrast, the Vβ8.3 + heterologous recall responses were largely monoclonal and consisted of one identical non-public clone in each of the mice. It should be noted that the differences between homologous and heterologous CD8 + T cell recall responses are further enlarged, when usage of Vβ8.3 is taken into account. Error bars denote standard error of the mean (SEM). 15