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Transcript
Salmonella Typhi-specific effector/memory CD8+ T cell responses elicited in a wild-type S. Typhi controlled human infection model Stephanie Fresnay1, Monica A. McArthur1, Thomas C. Darton2, Claire Jones2, Claire S. Waddington2, Christoph J. Blohmke2, Gordon Dougan3, Brian Angus2, Myron M. Levine1, Andrew J. Pollard2, Marcelo B. Sztein1 1University of Maryland, Baltimore, MD, United States, 2University of Oxford, Oxford, United Kingdom, 3Wellcome Trust Sanger Institute, Hinxton, United Kingdom Infection Prevention and Control, November 28 2016 CVD Typhoid fever More than 20 millions cases of enteric fever and 200,000 death / year Salmonella enterica serovar Typhi: human-restricted pathogen Current limited knowledge on correlates of protection due to the lack of appropriate clinical and preclinical models Cellular immune responses are likely to play a crucial role in protection Limited data on immune status before infection and its effects on clinical outcome http://www.infectionlandscapes.org The Oxford challenge model Challenge with wild-type S. Typhi to establish a 60-75% attack rate Typhoid Diagnosis (TD), determined by fever >38C for 12 hours or bacteremia Effector functions tested at different time points for: - Volunteers with positive typhoid diagnosis: TD (n=13) - Volunteers not diagnosed with typhoid: NoTD (n=7) Experimental Design: in vitro stimulation of T effectors with S. Typhi-infected targets Target Cells Autologous B-EBV cells, HLA-E-restricted AEH cells, autologous blasts TD (n=13) NoTD (n=7) PBMC S. Typhi infection + g-irradiation 14-18 hrs Flow cytometry Experimental Design: Flow cytometry gating 2500 R2: 95.28% 800 1000 2000 1000 500 0 200 400 600 800 104 CD62-L CD8 TEM IFN-g 103 104 102 CD45RA Qdot 800-A 103 101 102 TNFa Alexa700-A 103 TNF- 104 103 104 100 101 CD69 ECD-A 102 103 104 CD69 ECD-A 102 101 100 100 104 TEMRA CD45-RA 103 104 CD69 ECD-A 102 102 IFNg PE-Cy 7-A 101 104 100 104 101 101 R9: 8.56% TCM TN CD69 ECD-A 102 103 100 100 R7: 56.02% 101 102 CD3 BV650-A 103 104 CD69 ECD-A 102 101 100 CD107a 104 R6: 13.17% R8: 15.53% 100 101 100 101 103 104 103 CD69 ECD-A 102 CD69 101 100 103 10 4 CD8+ subsets CD62L APC-EF780-A 102 103 104 CD8 PerCP-Cy5-5-A 102 103 R4: 23.47% CD3 102 CD107a/b FITC-A 10 1 10 2 10 3 YEVID + CD14/19/45 BV570-A Yevid-CD14-CD19-CD45 CD8+ T cells 100 101 10 0 1000 FSC-H FSC-H 100 101 102 IL-2 BV605-A IL-2 103 104 100 600 FSC-A FSC-A 101 400 0 0 200 R201: 75.70% Number 1500 FSC-A 600 400 200 400 0 200 SSC-A SSC-A 600 FSC-A R1: 43.89% 0 100 Live T cells 800 800 1000 Singlets 1000 Lymphocytes 100 101 102 IL-17 BV421-A 103 IL-17A 104 100 101 102 MIP1b PE-A 103 MIP-1 104 S. Typhi-specific CD8+ TEM responses: Baseline CD107a IFN-g TNF- TD EBV TD AEH TD blast NoTD EBV % positive cells NoTD AEH NoTD blast median MIP-1 IL-17A IL-2 p<0.05 p<0.01 Higher baseline responses at baseline are associated with disease development Relationship between S. Typhi-specific baseline responses and time to diagnosis CD107a IFN-g C D 107a 2 0 TNF- T N F - IF N -g p = 0.09 3 2 0 1 0 1 0 5 5 2 % positive cells 1 0 1 0 0 1 5 0 2 0 0 2 5 0 0 1 0 0 MIP-1 2 0 0 2 5 0 1 0 0 1 5 0 IL-17A M IP -1 1 0 2 0 0 2 5 0 IL-2 IL -2 IL -1 7 A p = 0.09 2 0 0 1 5 0 2 0 .8 0 .6 5 1 0 .4 0 .2 0 1 0 0 0 0 .0 1 5 0 2 0 0 2 5 0 1 0 0 1 5 0 2 0 0 2 5 0 1 0 0 1 5 0 2 0 0 High S. Typhi-specific baseline responses are associated with delayed time to diagnosis 2 5 0 Post-challenge kinetics of responses IFN-g TD AEH NoTD 5 8 MIP-1 TD 5 8 5 5 0 0 0 0 -5 -5 -1 2 -8 -4 8 4 T D 12 % positive cells -5 -5 28 21 14 7 0 20 16 -1 2 D a y s p o s t c h a lle n g e T im e r e la tiv e to d ia g n o s is -8 -4 T D 4 8 12 16 D a y s p o s t c h a lle n g e IL17A 5 5 1 5 0 0 0 0 -1 2 -8 -4 T D 4 12 8 16 -1 2 28 21 14 7 0 20 -5 -1 -5 -5 -8 -4 T D 4 8 12 16 D a y s p o s t c h a lle n g e IL-2 TNF- 5 5 2 5 0 0 0 0 -5 -1 2 -8 -4 T D 4 8 12 16 -5 -2 28 21 14 7 0 20 -1 2 D a y s p o s t c h a lle n g e T im e r e la tiv e to d ia g n o s is 28 21 14 7 0 20 T im e r e la tiv e to d ia g n o s is D a y s p o s t c h a lle n g e -5 28 21 14 7 0 20 T im e r e la tiv e to d ia g n o s is CD107a T im e r e la tiv e to d ia g n o s is IFN-g+ cells NoTD -8 -4 T D 4 8 12 16 28 21 14 7 0 20 D a y s p o s t c h a lle n g e T im e r e la tiv e to d ia g n o s is Blasts EBV Early decreases in CD107a expression and cytokine production are associated 5 5 0 0 2 with disease development -5 -5 -1 2 -8 - 4 T D 4 8 1 2 1 6 2 0 0 7 1 4 2 1 2 8 2 0 0 -2 -2 0 7 1 4 2 1 2 8 Multifunctional (MF) CD8+ TEM responses: Baseline NoTD EBV median TD AEH NoTD AEH p<0.05 TD blast NoTD blast p<0.01 % positive cells TD EBV MF S. Typhi-specific responses are dominant in TD volunteers Summary / Discussion Development of typhoid disease is associated with: Higher baseline S. Typhi-specific responses Early decreases in blood of S. Typhi-specific TEM cells after challenge These results suggest that S. Typhi-specific TEM cells migrate to the site(s) of infection (e.g., gut, RES), resulting in an inflammatory process that favors S. Typhi to effectively infiltrate the mucosa and spread systemically. Delayed time to diagnosis among TD volunteers is associated with higher baseline S. Typhi-specific responses. These results suggest that high numbers of S. Typhi-specific T cells at baseline are sufficient to delay, but ultimately can not prevent, disease development. Summary / Discussion Protection against typhoid fever is associated with: Very low or no baseline S. Typhi-specific responses No changes observed in circulation after challenge These results suggest that in protected volunteers innate and/or adaptive immune responses in the gut microenvironment are able to control S. Typhi infection, precluding systemic dissemination. Take home messages This study provides unique insights into the human immune response during the development of typhoid fever Uncovered, for the first time, that S. Typhi-specific CD8 T cell baseline responses correlate significantly with clinical outcome after infection Revealed some of the immunological mechanisms responsible for delayed time to disease onset Demonstrated that multifunctional T cells appear to play a critical role in the development of typhoid disease These results argue for the measurement of defined immunological responses during the development of new generation typhoid vaccines Acknowledgements Center Vaccine Development, UMB Cellular Immunology Group Regina Harley Cathy Storrer CVD Paula Bernal Franklin Toapanta Rezwan Wahid Jeffrey Floyd Rosangela Mezghanni Haiyan Chen Kirsten Lyke Jay Booth Mark Rudolph Sarah Boudova UK Collaborators Jeremy Farrar Paul Langford Derrick Crook Stephen Lockhart Statistical support Larry Magder Supported by grants U19 AI082655 (UM-CCHI) & R01 AI-36525 from the NIH, the Passano Foundation Clinical Investigator Award, and by the Wellcome Trust