1/3/13. Conflict of Interest. Adaptive Immune Responses in Rhinovirus Infection. Learning Objectives

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1 Conflict of Interest Adaptive Immune Responses in Rhinovirus Infection None relevant to the current program (Grant Support: NIH, Dupont Consulting Work: Endo Speaker: Merck) Larry Borish, M.D. Carter Immunology Center Asthma and Allergic Disease Center University of Virginia Health System Charlottesville, VA Learning Objectives September (and May) Epidemics of Asthma Exacerbations in Children and Adolescents in North America To describe the utility of using autologous mdc to evaluate AND RV-specific immune responses To understand the phenotype of RV-specific and cells in: immune surveillance, and in response to RV infection Number of hospitalizations of children age 5 to 15 yo By week of the year in Ontario Johnston et al. J Allergy Clin Immunol 5 Feb; 115 : Children (age 3) and Adolescents Hospitalized for Asthma in Charlottesville, VA: Adenovirus Parainfluenza 1,, 3 Wheeze Asthma Control and this occurs despite compelling evidence that RV infections only occur in September (and May) RSV A, B Metapneumovirus Influenza A, B Rhinovirus p<.1 RV infections occur year round i.e., this cannot be explained by kids returning to school Enterovirus Coronavirus Percent of Patients Who Tested Positive for Each Virus Heymann et al JACI 4;114:

2 Seasonal incidence of respiratory viral infections in hospitalized patients. Johnston et al. Am J. Respir Crit Care Med. 1996;154: Asthma exacerbations link to a highly allergic phenotype Evidence of an allergic reaction: eosinophilia, high ECP, high leukotrienes High total IgE allergic sensitization (atopy) Hypothesis:? Autumn exacerbation requires: sensitivity to ragweed, alternaria, dermatophagoides Spring exacerbation requires: sensitivity to grass Zambrano, J., et al. J Allergy Clin. Immunol. 3;111:18-16 So, if this were true what would we observe in an environment where allergen exposure is perennial? Hypothesis: If the allergen exposure is perennial, and RV infections occur perennially then exacerbations will occur perennially without a seasonal spike Costa Rica ER Study Hospital Nacional de Niños, San José (Children Ages 7 to 1 years) February enrollment when children start school October enrollment Enrollment included: 96 children treated for acute asthma exacerba;ons, and 16 non- asthma;c controls 65 children who required treatment for asthma within the previous 1 months (i.e., stable asthma at the ;me of enrollment) JACI (1); 19 (6): e5 Children with Posi;ve Tests for Viral Infec;on (qpcr) % Children with Positive PCR for RV, HEV, or RSV 1% 8% 6% 4% % % * 8% 73% p<.1 p<.1 14% 14% 1% 9% February October February October February October Exacerbating Current Wheeze Stable Asthma Control asthmatics N N= = Rhinovirus RSV Enterovirus *p<.3 for the percentage of RV posi;ve tests in Feb and Oct

3 Total IgE levels in Costa Rica p=.1 p<.1 Costa Rica: Titers of allergen-specific IgE antibody in children and adolescents having an asthma exacerbation: 1 5 Total IgE p<.1 Titer of IgE (IU/ml) Wheeze Asthma Stable Stable Asthma Controls Exacerba;on Asthma (n=13) (n=95) (n=64) <.35 x 9 x 9 x 44 x 58 x 53 x 88 x 81 x 69 x 77 x 8.1 % Positive: D. pter Blomia B. germ P. amer Ascaris Alternaria *GM values (in blue) include only children whose ;ters were.35 IU/ml Aspergillus Dog Cat Bahia gr Probability of Asthma Exacerbation Based on Titers of IgE ab to D. pteronyssinus and Tests for Rhinovirus (Costa Rica) A RV Negative B RV Positive Conclusions, part I Probability of Current Wheeze Most asthma exacerbations in children and adolescents occur in association with RV infection This reflects concomitant presence of allergic sensitization to aerollergens expressed at the time of the infection IgE to Mite (IU/ml) IgE to Mite (IU/ml) Two Schools of Thought The seed= the virus Three virus clades A (ICAM/LDL), B (ICAM/LDL) and C (?) Asthmagenic Rhinovirus (Strain C?)* The soil= the asthmatic Innate immune deficiency in asthmatics Inability to control virusà increased viral load + exacerbations Robust immune response to viral infection Inflamed lungs of asthmatics + cytokine barrage from RV à exacerbations And this immune response can be directed to either: the virus itself bystander allergens Does the virus multiply better or worse in the asthmatic? *this could reflect either increased virulence or altered immune response RV qpcr for RV in Nasal Washes from Children with Asthma Exacerba;ons, Acute Rhini;s, and Controls in the ER however, this isn t comparing viral load on the same day post-infection therefore, need to do viral challenge approach Shaker, Kennedy, et al. Manuscript in progress. 3

4 Nasal wash viral titers in asthmatics and controls after experimental inoculation with RV16 Virus titer in nasal wash fluid log (TCID 5 / ml +1) Non-asthmatic controls (n=11) High IgE asthmatics (384-8 IU/mL; n=6) Low IgE asthmatics (9-15 IU/mL; n=11) Peak viral load in nasal wash fluid log (TCID 5 / ml) Peak and Cumulative (Days 1-4) Nasal Wash Viral Titers in Asthmatics and Controls after Experimental Inoculation with RV16 Peak Viral Load in Nasal Wash (usually days 4-7) in asthmatics and controls. Controls p=.998 Asthmatics Cumulative (days 1-4) viral load in nasal wash fluid log (TCID 5 / ml+1) Cumulative (days 1-4) Viral Load in Nasal Wash in asthmatics and controls. Controls p=.978 Asthmatics Study Day Shaker, Kennedy, et al. Manuscript in progress. Shaker, Kennedy, et al. Manuscript in progress. Conclusions, part II RV-induced asthma exacerbations cannot be ascribed to differences in viral load or, by extension, to differences in innate immune responses If the viral load is the same, why do asthma;cs exacerbate? i.e., it s the soil (there may be asthmagenic viruses but it is not their inherent virulence but the nature of the immune response they generate that is the problem) Immune Response to RV Viral titer consistently begins to decline 4-7 dpi This can NOT represent a de novo adaptive immune response by naïve T cells which is never seen prior to day 7 post infection Therefore these adaptive responses MUST be being mediated by effector memory cells None of our subjects demonstrated evidence of recent infection with RV16 (or RV39) All were serotype negative Antibodies precludes ability to infect volunteers These effector memory cells must have developed in response to a structurally related RV (± other picornaviruses) and be recognizing a shared epitope (heterologous immunity) Non-asthmatic controls (n=11) High IgE asthmatics (384-8 IU/mL; n=6) Low IgE asthmatics (9-15 IU/mL; n=11) Tetramer Tetramer-guided epitope mapping (TGEM) of RV39 capsid proteins PBMCs were stimulated with pooled HRV39 peptides (P11-15) for 14 days before staining with tetramers containing each of the peptides P11 Tetramer P1 P13 P14 P15 Peptide 14: AQVRRKFEMFTYVRFDSEIT Peptide 15: MFTYVRFDSEITLVPCIAGR Fig. 1. Detection of + T Cells Specific for HRV- 39 by Tetramer Staining. PBMCs from a DRB1 *41+ individual were stimulated VP1 epitope: MFTYRFDSEIT (overlapping peptides ) maps to a region that is highly conserved among RV groups A, B, and C implies memory + T cells induced by one strain can respond to a different strain; thereby allowing a rapid T cell recall response could facilitate recruitment of RV-specific + T cells to the respiratory tract soon after RV infection Kwok, W and Woodfolk, J unpublished 4

5 Tetramer-guided epitope mapping (TGEM) of RV39 capsid proteins Identified DR4-restricted T cell epitopes within RV capsid proteins: VP1 and VP Present at frequency of ~11.9/1 6 + T cells. + T cells P6 Tetramer VP (P6) epitope (P6) CCR7+ suggestive of a central memory phenotype (preferentially home to secondary lymphoid organs) CCR7 Kwok, W and Woodfolk, J unpublished Adaptive Immune Response to RV Adaptive Immune Responses + T cells engaging antigens presented by APCs on MHC class II + cytotoxic T cells engaging MHC class I on infected epithelium RV-associated asthma exacerbations are linked to the presence of an increased type cytokine signature (IL-4, IL-5, and IL-13) Hypothesis: RV-specific adaptive T cells in asthmatics who develop an asthma exacerbation demonstrate a Th cytokine bias HOWEVER, we cannot rule out the possibility that RV infection merely acts to induce/exacerbate an adaptive Th effector immune response to bystander allergens Autologous Dendritic Cells as RV Antigen-Presenting Cells In order to test this we needed to develop DC model of RV antigen presentation PBMCs from volunteers are cultured to generate an expanded population of autologous DC CD14 + monocytes (>95%) enriched using magnetic affinity purification CD14 + cells cultured in medium with 1% autologous serum containing hgm-csf (1 U/ml) and hil- 4 (1 U/ml) for 5 days with the addition of fresh media and cytokines at 3-day intervals <PE-A> <PE-A>: Isotype <PE-A>: CD DC Phenotyping DC identified by flow cytometry morphologic criteria: DC markers -MHC I hi -MHC II hi -CD11c hi -CD14 low -CD1c + Activation markers <PE-A> <APC-A> Sample_No Staining Cells.fcs Event Count: <PE-A>: <APC-A> 1 5 Sample_No FSC-A, Staining SSC-A subset Cells.fcs Event Sample_.fcs Count: 1955 Event Count: 155 <PE-A>: Sample_.fcs Event Count: 155 PE-isotype <PE-A>: Isotype <PE-A> <PE-A>: <PE-A> <PE-A>: <PE-A>: 3 <PE-A>: <PE-A> <PE-A>: Sample_CD14.fcs DC Phenotyping Event Count: <PE-A>: Isotype <APC-A> <APC-A>: <APC-A>: Isotype Isotype 51.1 FSC-A, SSC-A 1 subset APC-isotype 1 3 <APC-A> FSC-A, FSC-A, SSC-A SSC-A subset <APC-A>: Isotype FSC-A, SSC-A 1subset subset Sample_No Staining 1 Cells.fcs Sample_Mouse IgG IgG APC,f,PE.fcs Sample_No Staining Cells.fcs Sample_Mouse IgG APC,f,PE.fcs Event FSC-A, Count: 1955 Event Event Count: Count: <APC-A>: Event SSC-A Count: subset 1955 Event Count: <APC-A>: Isotype FSC-A, SSC-A subset Sample_Mouse IgG APC,f,PE.fcs Sample_CD14.fcs Sample_3.fcs Sample_6.fcs Event Count: Event Count: 157 Event Count: 1591 Event Count: <APC-A>: Isotype Sample_Mouse IgG APC,f,PE.fcs Event Count: <PE-A>: 3 <PE-A>: <PE-A>: CD14 Isotype <PE-A>: Isotype <PE-A>: 6 CD <PE-A>: CD FSC-A, FSC-A, SSC-A SSC-A subset FSC-A, SSC-A subset subset 1 Sample_CD14.fcs Sample_CD14.fcs Event Event Count: 157 Event FSC-A, Count: SSC-A subset 1 5 Sample_CD11c.fcs Event Count: <PE-A>: <PE-A>: 6 3 <PE-A>: subset <APC-A>: 1 1 HLA-DR SSC-A subset subset <APC-A>: subset subset <APC-A>: subset FSC-A, SSC-A FSC-A, SSC-A FSC-A, FSC-A, SSC-A 1 5 FSC-A, SSC-A Sample_6.fcs SSC-A FSC-A, Sample_3.fcs Sample_.fcs Sample_6.fcs Sample_3.fcs Sample_.fcs Event FSC-A, Count: SSC-A 159 subset Event FSC-A, Count: SSC-A 1591 subset Event FSC-A, Count: SSC-A 155 subset Event Count: 159 Event Event Count: 1591 Event Event Count: Count: Sample_CD11c.fcs Sample_6.fcs Sample_3.fcs Event Count: Event Count: 159 Event Count: 1591 <PE-A>: CD14 <PE-A>: <PE-A>: CD11c CD14 CD11c <PE-A>: 6 <PE-A>: CD11c <PE-A>: 6 <PE-A>: <PE-A>: CD11c <PE-A>: CD11c <APC-A>: 1 3 HLA-DR Sample_CD11c.fcs Sample_CD11c.fcs Event Count: Event Count:

6 Antigen Loading DCs cannot be infected by RV Used RV itself as the means of DC maturation not requiring maturation cocktails that would influence T-cell immune deviation RV (5 µg/ml (1 5.6 pfu/ml)) for 48 hrs at 37 o C Measured RV-induced DC maturation by: cell surface staining mrna expression of activation markers ability to support RV-specific T cell proliferation RV-Induced DC HLA-DR Expression HLA- DR posieve cell % %HLA- DR+ - RV %HLA- DR+ + RV 1 RV-Induced DC Expression p<.7" 1 RV-Induced DC 6 Expression p<.3" 8 8 % positive 6 4 increased in 13/14 subjects % positive increased in 1/14 subjects Media" RV" Media" RV" % positive RV-DC 3 Expression Media" p<." RV" 3 decreased in 11/14 subjects RV-induced DC maturation in control subjects: mrna Cytokine ΔCT Medium ΔCT RV Fold change p value Homeostatic ckit 9.1± ±1.8.7 N.S. SCF 9.73± ± Innate TNF-α 4.76± ± N.S. IL-1β 7.67± ± N.S. IL ± ± Th1 IL-1p ± ± IL-1p4 6.63± ±.7.85 N.S. IL ± ± N.S. p8 1.± ± N.S. Th TSLP 9.59± ±1.5.5 N.S. Th17 IL-6 8.7± ± N.S. IL ± ±.98.7 N.S. Treg TGF-ß 3.47±.49.44± IL-1 7.4± ± EBI ± ± N.S. N.S.= not significant 6

7 Event Count: Event Count: Event Count: Event Count: Event Count: Event Count: Event Count: /3/13 Conclusions, part III Even though RV does not productively infect DC RV does provide DC maturation signals and via expression of RV-associated peptides these newly activated DC can be used to induce both MHC class I activation of s and MHC class II activation of s RV-Specific Memory T cells in Healthy Subjects PBMCs isolated from healthy subjects to generate mature DCs and collect T cells T cells labeled with the dilution sensitive dye CFSE prior to co-culture RV-loaded or control DC CFSE low/- cells represent the + and + T cells which have proliferated in response to the RV-matured DC DC presenting RV antigen co-cultured at a T cell: DC ratio of 1:1 using x1 6 cells per culture for 7 days CD3 + T cells co-cultured with DCs 7 days: s ICCS after stimulation after PMA/ionomycin/monensin stimulation -RV CFSE low IFNγ IL-4 IL <APC-A>: IL RV CD3 3 + T cells co-cultured with DCs 7 days: s ICCS after stimulation after PMA/ionomycin/monensin stimulation CFSE low IFNγ IL-4 IL IFNy <PE-A>: IL <APC-A>: IL-1 <APC-A>: <APC-A>: IL-1 IL RV <APC-A>: IL-1 <APC-A>: IL RV <APC-A>: IL <APC-A>: IL % CFSE low in DC:T cell co-cultures: healthy subjects % CFSE low and intracellular cytokines in DC:T cell co-cultures: healthy subjects 7

8 Secreted cytokines from DC:T cell cocultures: healthy subjects Activation and Phenotyping of RV- Specific T-Lymphocytes: RV Challenge PBMCs isolated from subjects undergoing RV challenge prior to infection to generate mature DCs At day 5 of infection T cells isolated from blood ICCS staining on CFSE low population % CFSE low and intracellular cytokines in DC:T cell co-cultures: asthmatic subjects Conclusions, part IV Circulating RV-specific effector memory s and to a lesser extent s can be identified in the circulation of healthy subjects where they are presumably involved in immune surveillance These cells recognize epitopes shared across RV-A, -B, and -C clades and via heterologous immunity this will allow rapid (day 5-7) immune responses to RV strains without need for previous exposure Effector memory cells in healthy subjects primarily express Th1 (IFN-γ) signature Increased numbers of circulating RV-specific effector T cells are observed during infection in healthy subjects these cells display a mixed Th1>Th cytokine signature a Th bias could be present in asthmatics who exacerbate Acknowledgements Borish/Steinke Lab Braciale Lab John W Steinke Tom Braciale Julie Negri Lixia Liu Heymann Lab Peter Heymann Joshua L Kennedy Holly Carper Deb Murphy Woodfolk Lab Judith A Woodfolk Rachana Agrawal Turner Lab Ron Turner Deborah Thacker Benaroya Institute: Bill Kwok 8