1. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraMathematical models of CD8 T cell responses:measuring CD8 T cell mediated killing in vivoVitaly V. GanusovLos Alamos National Laboratory Los Alamos, NM, USA1 / 55

2. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraOutline 1Immunology 101 Immune system 2Killing in acute viral infections Experimental details Model 3Killing in chronic viral infections Problem Experiments and Analysis Conclusions 4Appendix I LCMV infection 5Appendix II PyV infection 6Extra Pictures2 / 55 3. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraOutline 1Immunology 101 Immune system 2Killing in acute viral infections Experimental details Model 3Killing in chronic viral infections Problem Experiments and Analysis Conclusions 4Appendix I LCMV infection 5Appendix II PyV infection 6Extra Pictures3 / 55 4. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraFocus of this talk: CD8 T cells CD8+ T cells (T-killers, Cytotoxic T Lymphocytes (CTLs))have the major function to kill virus-infected cells.CD8 T cells recognize viral antigens (epitopes) that arepresented on the surface of infected cells.4 / 55 5. Immunology 101 Killing in acute viral infectionsKilling in chronic viral infections Appendix I Appendix IIExtraKilling by CD8 T cells in vivoKilling in vivoMempel et al Immunity 2006 5 / 55 6. Immunology 101Killing in acute viral infections Killing in chronic viral infectionsAppendix I Appendix II ExtraCD8 T cells and viral clearanceLCMV ArmstrongLCMV clone 13 107 107 NP396NP396 106 106 populations populations 105 105104CD8 cells spleen 104viral load, PFU ml 103 103 2 CD8 cells spleen 10102 viral load, PFU ml 1 1 1010 0 204060800 20 406080 time, days time, days6 / 55 7. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraTopics to discussAcute viral infections:quantifying cytotoxic efcacy of CD8 T cells in vivoChronic viral infections:is failure to clear chronic infection due to inefcientCD8 T cell response?7 / 55 8. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraOutline 1Immunology 101 Immune system 2Killing in acute viral infections Experimental details Model 3Killing in chronic viral infections Problem Experiments and Analysis Conclusions 4Appendix I LCMV infection 5Appendix II PyV infection 6Extra Pictures8 / 55 9. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraMeasuring effector functions of CD8+ T cellsMost of effector functions of T cells are currently measuredex vivo or after cell culturing in vitro.Effector functions in vivo and ex vivo can differ.We propose a quantitative framework for estimation ofkilling efcacy of T cells based on the in vivo cytotoxicityassay. Barber et al. JI 20039 / 55 10. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraSpleenSome details Spleen is a secondary lymphoid organ. Lymphocytes enter and leave spleen via blood. T cell responses take place in the white pulp of the spleen (which is only 10 to 20% of whole spleen).10 / 55 11. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraSpleenSome details Spleen is a secondary lymphoid organ. Lymphocytes enter and leave spleen via blood. T cell responses take place in the white pulp of the spleen (which is only 10 to 20% of whole spleen).10 / 55 12. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix IIExtraMeasuring killing in vivo Target cells (splenocytes) arelabeled with different concentrationsof CFSE.A subpopulation of target cells ispulsed with a peptide (to which CD8T cells are specic).Pulsed and unpulsed target cells aretransferred into new syngenic host(generally i.v.) harboringepitope-specic CD8 T cells.Killing is measured by the reductionin the ratio of peptide-pulsed tounpulsed percent of pulsed targets(generally in the spleen). Barber et al. JI 2003; Regoes et al. PNAS 2007 11 / 55 13. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraMeasuring killing in vivo: FACS Ratio of pulsed to unpulsed R = 0.15/0.84 : 44.7/54.5 0.22 Percent cells killed L = 1 R = 78%Ingulli Meth Mol Biol 200712 / 55 14. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraMeasuring killing in mice infected with LCMV SetupThree target cell populations were transferred into miceinfected 8 days (or 30 to 100 days) previously withLCMV-Arm: unpulsed, cells pulsed with NP396 and GP276epitopes of LCMV.Killing was measured in the spleen in several time points.Barber et al. JI 200313 / 55 15. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraMeasuring killing in mice infected with LCMV SetupThree target cell populations were transferred into miceinfected 8 days (or 30 to 100 days) previously withLCMV-Arm: unpulsed, cells pulsed with NP396 and GP276epitopes of LCMV.Killing was measured in the spleen in several time points.Barber et al. JI 200313 / 55 16. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix IIExtraResearch questions For the data of Barber et al. 2003What are the half-life times of peptide-pulsed targets inacutely infected and LCMV-immune mice?How does the death rate of targets scale with themagnitude of the CD8 T cell response?What are the per capita efcacy of memory and effectorCD8 T cells at killing peptide-pulsed targets? Ganusov and De Boer J Virol (2008); Ganusov et al. (in prep)14 / 55 17. Immunology 101Killing in acute viral infections Killing in chronic viral infectionsAppendix IAppendix IIExtraComplied Data 106 NP396 pulsed unpulsed acute 100 memory unpulsed spleen1 10 5 102 acute 10memory3 10 104 050100150 200 250050 100 150 200250 minminBarber et al. JI 2003; Regoes et al. PNAS 2007 15 / 55 18. Immunology 101Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraRecruitment of unpulsed targets into spleen where SB (0) = 5 106 cells and S(0) = 0.16 / 55 19. Immunology 101Killing in acute viral infectionsKilling in chronic viral infections Appendix I Appendix II ExtraRecruitment of unpulsed targets into spleen dSB (t) = ( + + )SB (t), dtdS(t) = SB (t) S(t).dt where SB (0) = 5 106 cells and S(0) = 0. 16 / 55 20. Immunology 101Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraKilling of peptide-pulsed targets in the spleen where similarly, TB (0) = 5 106 cells and T (0) = 0, and K is the death rate of peptide-pulsed targets.17 / 55 21. Immunology 101Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraKilling of peptide-pulsed targets in the spleendTB (t)= ( + + )TB (t),dt dT (t)= TB (t) ( + K)T (t).dt where similarly, TB (0) = 5 106 cells and T (0) = 0, and K is the death rate of peptide-pulsed targets.17 / 55 22. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix IIExtraPredictions on dynamics of target cells c S(t) = e t edt , d T (t) (d ) edt e( +K)tR(t) = =, S(t)( + K d) e t edt where d = ++ is the rate of cell removal from the blood, and c = SB (0) is the initial rate of migration ofcells into the spleen.18 / 55 23. Immunology 101Killing in acute viral infectionsKilling in chronic viral infections Appendix I Appendix II ExtraPredictions on dynamics of target cellsc S(t) =e t edt ,dT (t) (d ) edt e( +K)t R(t) = =,S(t)( + K d) e t edt where d = + + is the rate of cell removal from the blood, and c = SB (0) is the initial rate of migration ofcells into the spleen.2.5 d 0.02100 d0.02 c 5000 cells spleen, 105 0pulsed unpulsed2 1 10 1.52 101 0K0.01 0.0053 K0.08 0.5 10 0.01 K0.2 0 4K1 10 050 100150 200 0 50100150200 time, mintime, min 18 / 55 24. Immunology 101Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraFits of the model to data: killing killing of NP396-pulsed targets100 acute NP396 100memory NP396 pulsed unpulsedpulsed unpulsed 110 210 1 10 310 0 50100 150 200250 0 50 100150200 250minmin killing of GP276-pulsed targets100 acute GP276 100memory GP276pulsed unpulsedpulsed unpulsed 110 210 1 10 310 0 50100 150 200250 0 50 100150200 250minmin19 / 55 25. Immunology 101Killing in acute viral infectionsKilling in chronic viral infections Appendix I Appendix II ExtraFits of the model to data: recruitmentacute memory acute memory 106 106 unpulsed spleen unpulsed spleen 105 105 104 104 0 501001502002500 50 100 150200 250min min 20 / 55 26. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix IIExtraRobust quantitative resultsHalf-life times of pulsed targetsT1/2 = 2 and 14 minutes for NP396 and GP276-pulsedtargets in acutely infected mice.T1/2 = 48 minutes and 2.9 hour for NP396 andGP276-pulsed targets in memory mice. Ganusov and De Boer J Virol (2008)21 / 55 27. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix IIExtraRobust quantitative resultsHalf-life times of pulsed targetsT1/2 = 2 and 14 minutes for NP396 and GP276-pulsedtargets in acutely infected mice.T1/2 = 48 minutes and 2.9 hour for NP396 andGP276-pulsed targets in memory mice.Recruitment rate of recruitment of targets into the spleen is proportional to the spleen size.Ganusov and De Boer J Virol (2008)21 / 55 28. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix IAppendix IIExtraSpecic (testable) predictions of the modelThe model predicts a relatively slow removal of cells fromthe blood (T1/2 2 h). This allows for the biphasic declinein the ratio of pulsed to unpulsed targets.In the model, due to a rapid preparation-induced cell death,only 2 105 targets (or less than 5% of injected cells) areable to migrate to other tissues (beside spleen). About 9%of injected splenocytes are predicted to home to the spleenin 24 hours.Ganusov and De Boer J Virol 2008 22 / 55 29. Immunology 101Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraEstimating the per capita killing efcacy Death rate K and CD8 T cell numbersK = kEi(mass-action)kEi K = (saturation in E) 1 + cE EikEi K =(decrease with T) 1 + cT T i kEi K =(E/T ratio) E i + cT T i where Ei and Ti is the frequency of effector and target cells in the spleen, respectively.23 / 55 30. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraAdditional (adoptive transfer) experiments PredictionIf killing follows the law of mass-action, estimated death rate of targetsshould be proportional to the density of antigen-specic CD8 T cells. 24 / 55 31. Immunology 101 Killing in acute viral infections Killing in chronic viral infectionsAppendix I Appendix II ExtraDeath rate vs. CD8 T cell density AB death rate K, 1 min death rate K, 1 minNP396 NP396GP276 GP2761 GP331 GP33 10102 1010 2 3 10 3 1010 1 100105 106107% of epitope specific CD8 T cells # of epitope specific CD8 T cells Results:Linear regression suggests that K = kE 1 .Ganusov et al. (in prep)25 / 55 32. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraPer capita killing vs CD8 T cell densityA6 B5 NP396NP396 cell min GP276GP276 5 GP33 4 GP33K E, 1 min 43 3 82 K E, 10 2 1100 10 1 100 105 106107 % of epitope specific CD8 T cells# of epitope specific CD8 T cellsResults: Killing is simply proportional to the frequency (or number) of antigen-specic CD8 T cells in the spleen. The average killing efcacy of CD8+ T cells is 2.33 min1 (panel A) or 2.62 108 cell1 min1 (panel B). Ganusov et al. (in prep) 26 / 55 33. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraDoes this all make sense? Results:We found that LCMV-specic CD8 T cells rapidly clearpeptide-pulsed targets from the spleen. NP396-specic effectorskill half of the targets in 2 minutes.The death rate of targets is roughly independent of the T cellspecicity and simply proportional to the magnitude of theantigen-specic CD8 T cell response. 27 / 55 34. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraDoes this all make sense? Results:We found that LCMV-specic CD8 T cells rapidly clearpeptide-pulsed targets from the spleen. NP396-specic effectorskill half of the targets in 2 minutes.The death rate of targets is roughly independent of the T cellspecicity and simply proportional to the magnitude of theantigen-specic CD8 T cell response.Problems with results:In vitro and in vivo studies found that it takes 10-60 minutes forCD8 T cell to kill a target cell which seems to be incompartiblewith the 2 min half-life estimated.Given 10-15 min handeling time for the kill, how can theestimated death rate of targets be simply proportional to thedensity of T cells? 27 / 55 35. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraOutline 1Immunology 101 Immune system 2Killing in acute viral infections Experimental details Model 3Killing in chronic viral infections Problem Experiments and Analysis Conclusions 4Appendix I LCMV infection 5Appendix II PyV infection 6Extra Pictures 28 / 55 36. Immunology 101Killing in acute viral infections Killing in chronic viral infectionsAppendix I Appendix II ExtraChronic viral infections LCMV ArmstrongLCMV Clone 13 107 107 NP396NP396 106 106 populations populations 105 105 4 10 CD8 cells spleen 104viral load, PFU ml 3 10103 CD8 cells spleen 102 102 viral load, PFU ml101 101 0 20406080020406080 time, days time, days Problems: How can viruses establish a state of persistent infection? 29 / 55 37. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraHow can viruses establish a persistent infection?Immune response is efcient (on per cell basis) but small.Immune response is large but inefcientOn per cell basis, killing is impaired (e.g., IL-10 productionby virus-infected cells)Killing of virus-infected cells is inefcient (e.g., latency)? 30 / 55 38. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraAcute vs. chronic LCMVConclusion: During LCMV infection, effector to target ratio determines the outcome of infection (clearance vs. persistence). It is unknown, however, if the functionality of LCMV-specic CD8 T cells is also impaired during the acute phase of LCMV clone 13 infection. 31 / 55 39. Immunology 101 Killing in acute viral infectionsKilling in chronic viral infectionsAppendix I Appendix II ExtraChronic infection with Polyoma virus (PyV) 107 106 cells,virus 105 104CD8 T cells spleen 103PFU mg 2 10 101 100 0 10 2030 4050days since infection Biology:Polyoma virus is a natural pathogen of mice causingpersistent infection.CD8 T cell response is important in the early control of thevirus dynamics.How PyV establishes a persistent infection is notcompletely understood. 32 / 55 40. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix IAppendix II ExtraKilling efcacy of PyV-specic CD8 T cellsTransfer targets (peptide-pulsed and unpulsed) intoPyV-infected mice at different times after the infection.Measure percent of targets killed at different times aftercell transfer.Estimate the death rate of targets due to CD8 T cellmediated killing. Byers et al. JI 200333 / 55 41. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix IIExtraMathematical modelc S(t) = e t edt , d T (t) (d ) edt e( +K)tR(t) = =, S(t)( + K d) e t edtwhere d = ++ is the rate of cell removal from the blood, and c = SB (0) is the initial rate of migration ofcells into the spleen.34 / 55 42. Immunology 101Killing in acute viral infectionsKilling in chronic viral infections Appendix I Appendix II ExtraModel ts to dataA 106B100pulsed unpulsed unpulsed spleen 105 110 104acuteacutechronicchronic 2 10310 050 100 150200250 050 100 150 200 250 min minResults: The half-life time of peptide-pulsed targets at the peak of the MT389-specic CD8+ T cell response is 15 minutes. During the chronic phase, half of MT389-expressing targets were eliminated in 47 minutes. Per capita killing efcacy of PyV-specic effectors (k = 4 min1 ) is similar to that of LCMV-specic effectors. 35 / 55 43. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix IAppendix II ExtraNo change in the killing efcacy over time 101 acutechronic1 kmax , min100 0 2550 75 100 125 150175 days after infectionKmax t where kmax = Kmax /E, Kmax = (1e Rt) and E is frequency of PyV-specic CD8 T cells in the spleen. Ganusov et al. (in prep)36 / 55 44. Immunology 101Killing in acute viral infectionsKilling in chronic viral infections Appendix I Appendix II ExtraKilling efcacy at lower peptide concentrations1 M0.1 M100100pulsed unpulsed pulsed unpulsed 110acute acutechronic 1 chronic 10 0 50 100 150 200250050 100 150200250 min min 0.01 M 0.001 M100pulsed unpulsed pulsed unpulsed0 10 acuteacute chronicchronic 050 100 150 200 250 050 100 150200250 min min 37 / 55 45. Immunology 101Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraKilling efcacy at lower peptide concentrations kpn101acutek= chronichn + pn 100 k, 1 minwhere k is the maximal killing efcacyof MT389-specic CD8+ T cells, p is 110the peptide concentration used to 2pulse target cells, h is the peptide103 1010 210 1100 101peptide concentration, M concentration at which killing ishalf-maximal, and n is the power ofthe Hill function.38 / 55 46. Immunology 101Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraKilling efcacy at lower peptide concentrations kpn101acutek= chronichn + pn 100 k, 1 minwhere k is the maximal killing efcacyof MT389-specic CD8+ T cells, p is 110the peptide concentration used to 2pulse target cells, h is the peptide103 1010 210 1100 101peptide concentration, M concentration at which killing ishalf-maximal, and n is the power ofthe Hill function.Conclusion: Persistence of PyV could arise due to low level expression of viral proteins but not due to low killing efcacy of T cells.Ganusov et al. (in prep) 38 / 55 47. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraConclusionsThe developmed framework allows estimation of the killingefcacy of the antiviral CD8 T cell response in the spleen.Killing in the spleen by effector CD8 T cells is rapid with most oftargets being killed in minutes after transfer.Killing of targets in the spleen follows the law of mass-actionwhereby the death rate of targets is simply proportional to thedensity of epitope-specic CD8 T cells.Persistence of chronic PyV infection could arise due to a lowlevel expression of viral proteins, and as the consequence, to alow killing efcacy by PyV-specic CD8 T cells.PyV-specic CD8 T cells are effective killers at high peptideconcentrations and do not loose their ability to kill over the time. 39 / 55 48. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraAcknowledgments and Contributions Acute LCMV infectionRob De Boer (Utrecht) and Daniel Barber (NIH)Chronic PyV infectionTony Byers (VaxDesign) and Aron Luckacher (Emory)FundingMarie Curie Incoming International Fellowship (FP6), HFSPgrant RGP0010/2004 (Rob De Boer), and Directorspost-doctoral fellowship (LANL). 40 / 55 49. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraI hope that youre not completely bored Post-doc position available:At the Department of Microbiology of the University of Tennessee in Knoxville starting March-April 2010 for at least 2 years. 41 / 55 50. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraOutline 1Immunology 101 Immune system 2Killing in acute viral infections Experimental details Model 3Killing in chronic viral infections Problem Experiments and Analysis Conclusions 4Appendix I LCMV infection 5Appendix II PyV infection 6Extra Pictures 42 / 55 51. Immunology 101 Killing in acute viral infectionsKilling in chronic viral infections Appendix I Appendix II ExtraRecruitment of targets into the spleenA B106 acute106 memoryunpulsed spleen unpulsed spleen56015 9030 12060 240105 120105 2702402701041040 1 108 2 108 3 108 4 10805 107 1 108 1.5 108 2 108splenocytes splenocytes43 / 55 52. Immunology 101Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraKilling of targets in the spleen A B 0.25eff NP396 eff GP276 log predicted R 0.2 log predicted R0.5 0.750.410.6 1.25 0.81.5t 5 t 5 t 15 1t 15 1.75t 30t 30 t 60t 60 t 120 1.2 t 1202t 240 t 240 t 270 t 27032.5 2 1.5 1 0.5 0 1.5 1.25 1 0.75 0.5 0.25 log observed Rlog observed RC Dmem NP396mem GP276 0.2 log predicted R log predicted R 0.1 0.4 0.2 0.6 t t 60 90 0.3t60t90 0.8 t 120t120 t 240t240 t 270t270 0.41 10.8 0.6 0.4 0.20 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0log observed Rlog observed R44 / 55 53. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraParameter estimates for the in vivo killingParameterMean Low 95% CIHigh 95% CI, min1 4.71 1033.34 103 6.17 103 A , min1 7.17 1012 5.58 10129.22 1012 M , min1 1.30 1011 0.92 10111.89 1011 KN P , min1a3.45 1012.59 101 4.56 101 KGP , min1a 5.0 1023.80 102 6.41 102 KN P , min1m1.44 1021.10 102 1.82 102 KGP , min1m4.15 1033.21 103 5.52 103 45 / 55 54. Immunology 101 Killing in acute viral infectionsKilling in chronic viral infections Appendix I Appendix II ExtraPoint estimates of the half-life time By assuming slow migration of targets into the spleen (i.e., at the limit d, e 0), we obtain the maximum estimate of the death rate of pulsed targets Kmax 1 eKmax t0 R0 = Kmax t0 By assuming very rapid migration of targets into the spleen (i.e., at the limit d ), we obtain the minimal estimate of the death rate of pulsed targets Kmin ln R0Kmin = t0 Half-life times are calculated aslog(2)T1/2 =K46 / 55 55. Immunology 101Killing in acute viral infectionsKilling in chronic viral infections Appendix IAppendix II ExtraPoint estimates of the half-life time104Eff NP396104 Eff GP276 103 103T1 2 , minT1 2 , min 102 102 101 101 100 100 050 100150 200 250050 100150 200 250 time after cell transfer, min time after cell transfer, min 104Mem NP396104 Mem GP2763 3 1010T1 2 , min 2T1 2 , min 10102 101 101 100 100 050 100150 200 250050 100150 200 250 time after cell transfer, min time after cell transfer, min46 / 55 56. Immunology 101 Killing in acute viral infections Killing in chronic viral infectionsAppendix IAppendix II ExtraParameter estimates from the adoptive transfer experimentsParameterMean95% CIsE/T E, % E, 106 cells Transferred , 1011 min1 2.141.886.16, 103 min11.1 0.41.6 , 102 min11.0 0.74.3 k1 , min1 1.770.912.12 0.14 0.06 0.05106 1 1.841.472.78 0.09 0.04 0.03 k2 , min1 1.770.912.12 0.34 0.18 0.102 106 2 0.550.360.81 0.25 0.15 0.09 k3 , min1 3.081.63.9 23.7 0.87 0.68107 3 0.3 0.260.39 9.48 1.25 1.05 k4 , min1 1.770.912.12 44.4 1.54 1.432 107 47 / 55 57. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraAlternative models explaining the dataEfux of targets migrated to the spleen, back to the blood.long retention of cells in the blood (T1/2 1.9 h);short average residence time in the spleen (T1/2 2 h);60-70% of injected targets migrate to other organs.Loss of recognition of peptide-pulsed targets by CTLsrapid removal of cells from the blood (T1/2 = 40 min);rapid loss of recognition of peptide-pulsed targets by CTLs(T1/2 4 h). 48 / 55 58. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraAlternative models explaining the dataEfux of targets migrated to the spleen, back to the blood.long retention of cells in the blood (T1/2 1.9 h);short average residence time in the spleen (T1/2 2 h);60-70% of injected targets migrate to other organs.Loss of recognition of peptide-pulsed targets by CTLsrapid removal of cells from the blood (T1/2 = 40 min);rapid loss of recognition of peptide-pulsed targets by CTLs(T1/2 4 h).More experimental data are need to discriminate betweenalternative models!48 / 55 59. Immunology 101 Killing in acute viral infectionsKilling in chronic viral infectionsAppendix I Appendix II ExtraEstimated killing efcacyA 4 B3effectormemory2.5kGP33 , per min 3 2GP332 1.5 110.500106 2 106107 2 1071062 106 107P14 cells transferredP14 cells transferredResults:These results suggest that there is a minimal change in the killingefcacy of GP33-specic effector CD8 T cells (with an average of2.10 0.17 per minute) with the number of effector cells transferred.In contrast, the per capita efcacy of memory CD8 T cells (average1.72 0.44 per minute) declines at high numbers of transferred memorycells.Ganusov et al. (in prep) 49 / 55 60. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraOutline 1Immunology 101 Immune system 2Killing in acute viral infections Experimental details Model 3Killing in chronic viral infections Problem Experiments and Analysis Conclusions 4Appendix I LCMV infection 5Appendix II PyV infection 6Extra Pictures 50 / 55 61. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix IAppendix II ExtraStatistical modeln (log Si log S(ti ))2 n (log Ri log R(ti ))212s21 2s2 L(S, R|p) =e1 e2i=12s2 1 i=12s2 2where Si and S(ti ) are the measured and predicted number of unpulsed targets in the spleen at time ti after cell transfer, respectively, and Ri and R(ti ) are the measured and predicted ratio of peptide-pulsed to unpulsed targets in the spleen at time ti after cell transfer, respectively, s1 and s2 are the standard deviation of the errors in the recruitment and killing data, respectively, n is the number of measurements, and p is the vector of model parameters to be estimated from the data. Ganusov et al. (in prep)51 / 55 62. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraParameter estimates: high peptide concentration parameter acute chronic , 104 min1 3.96 (2.905.37) , 103 min1 4.92 (2.957.04) k, min14.16 (3.424.84) 1.90 (1.652.18) 52 / 55 63. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraParameter estimates: k vs. peptide concentration parameter acute chronic d, 103 min1 5.83 (3.618.78) k, min13.50 (2.934.04) 1.89 (1.582.16) h, M 0.19 (0.100.37) 0.08 (0.040.14) 53 / 55 64. Immunology 101 Killing in acute viral infections Killing in chronic viral infections Appendix I Appendix II ExtraOutline 1Immunology 101 Immune system 2Killing in acute viral infections Experimental details Model 3Killing in chronic viral infections Problem Experiments and Analysis Conclusions 4Appendix I LCMV infection 5Appendix II PyV infection 6Extra Pictures 54 / 55 65. Immunology 101 Killing in acute viral infectionsKilling in chronic viral infections Appendix I Appendix II ExtraKilling by CD8 T cells in vitroKilling in vitro55 / 55