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Ramses Ilarraza, PhD, Yingqi Wu, Christopher D

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1 Rhinovirus has the unique ability to directly activate human T cells in vitro 
Ramses Ilarraza, PhD, Yingqi Wu, Christopher D. Skappak, BSc, Farnam Ajamian, MD, David Proud, PhD, Darryl J. Adamko, MD, FRCP(C)  Journal of Allergy and Clinical Immunology  Volume 131, Issue 2, Pages (February 2013) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Rhinovirus-induced T-cell proliferation does not depend on MHC-II–restricted antigen presentation. A and B, moDCs incubated with RSV (Fig 1, A) or rhinovirus (Fig 1, B), with or without chloroquine (Chl), were cocultured with T cells (>90% pure). Proliferation was measured in relative light units. Chloroquine inhibited RSV- but not rhinovirus-induced T-cell proliferation. Unlike RSV, UV inactivation inhibited rhinovirus-induced proliferation. C, In the absence of moDCs, rhinovirus induced T-cell proliferation compared with RSV or the LPS and PHA controls. D, The rhinovirus effect is strain dependent. Shown is the mean value; error bars represent SEMs. Fig 1, A and B, n = 5; Fig 1, C, n = 4; Fig 1, D, n = 3. *P < .05, **P < .01, and ***P < n.s., No significant difference. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Rhinovirus induced CD4+ and CD8+ T-cell proliferation in a time- and dose-dependent manner. A and B, Freshly isolated, greater than 99% pure CD4+ (Fig 2, A) or CD8+ (Fig 2, B) T cells proliferated when exposed to rhinovirus but not to RSV. C and D, Time-course (Fig 2, C) and dose-response (Fig 2, D) effects on day 7 of rhinovirus infection (r = +0.79). E, Proliferation persisted unchanged, even after washing rhinovirus away (Tw), as measured on day 7. Fig 2, A, n = 5; Fig 2, B, n = 3-5; Fig 2, D and C, n = 3. Shown is the mean value; error bars represent SEMs. ***P < .001 and **P < .01 compared with respective controls: Fig 2, A and B, T cells alone; Fig 2, C, T cells for the particular time point; and Fig 2, E, as indicated. T-cell purity for Fig 2, C-E, is greater than 90%. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Rhinovirus VP2 protein is transiently expressed in T cells exposed to rhinovirus. T cells (>90% pure) exposed to rhinovirus were collected on days 1, 2, 3, 5, or 7 and analyzed by using flow cytometry for rhinovirus VP2 protein expression. There was transient VP2 expression in both the CD4 (A) and CD8 (B) subsets with rhinovirus but not RV-UV. Evidence suggests de novo synthesis of rhinovirus VP2. Shown is the mean value of the VP2+ cell percentage; error bars represent SEMs. Fig 3, A and B, n = 3. ***P < .001 compared with uninfected cells at each time point. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Rhinovirus induces death of CD8+ T cells, mainly through cytolysis. A, Apoptosis and cytolysis were measured by means of flow cytometry (Annexin-V and TO-PRO-3). Total T-cell death was determined as both Annexin-V+ and To-PRO-3+ cells. Rhinovirus induced T-cell death. B, Differentiating cytolytic (Cytolysis; Annexin-V+To-PRO-3+) from apoptotic (Apoptosis; Annexin-V+To-Pro-3−) cell death, we found that both increased with rhinovirus. C and D, Gating on CD3+CD4+ (Fig 4, C) or CD3+CD8+ (Fig 4, D) subpopulations showed cell death occurred in CD8+ but not CD4+ T cells. Shown is the mean of the percentage of dead cells; error bars represent SEMs. Fig 4, A-D, n = 4. **P < .01 and ***P < T-cell purity was greater than 90%. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Rhinovirus-induced T-cell proliferation is inhibited by Jnk and NF-κB antagonists. T cells (>99% pure) were incubated with rhinovirus (or RV-UV) in the presence of antagonists for Jnk (A) and NF-κB (B); both antagonists completely inhibited proliferation by rhinovirus. Shown is the mean value; error bars represent SEMs. Fig 5, A and B, n = 3. ***P < n.s., No significant differences. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 T cells release cytokines and induce eosinophil activation on exposure to rhinovirus but not RSV. Supernatants from T cells exposed to virus were analyzed for the following cytokines: A, IFN-γ; B, IL-13; C, IL-10; D, GM-CSF; and E, TNF. F, Eosinophil CysLT release was measured when incubated with T cells alone or in coculture with moDCs and exposed to rhinovirus. Shown is the mean value; error bars represent SEMs. Fig 6, A-E, n = 4-10, and Fig 6, F, n = 5-6. *P < .05, **P < .01, and ***P < Because cytokine release was comparable, data were pooled from experiments using greater than 90% or greater than 99% pure cells. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 VP2 protein is expressed in T cells from human nasal tissue exposed to rhinovirus. T cells from tissue exposed to rhinovirus were analyzed by means of flow cytometry for expression of RV-VP2. A-C, Representative histograms showing VP2 expression in CD3-gated cells. Overlay histograms of isotype control (dot-lined, clear histogram) along with VP2 staining of sham-treated (Fig 7, A), rhinovirus-treated (Fig 7, B) and RV-UV–treated (Fig 7, C) nasal tissue (shaded histogram). D, Shown is the percentage of VP2+ cells within the CD3+ gate in all 3 groups. Error bars represent SEMs. Fig 7, n = 3. *P < .05 and **P < .01. n.s., No significant difference. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E1 T cell proliferation in absence or presence of APC. To test the purity of the T cell isolates (i.e, the presence of contaminating APC), T cell incubated in the presence or absence of moDC. When APC are present, LPS induces proliferation of T cells (TD LPS). The absence of proliferation in the absence of DC provides evidence for the purity of the T cell isolates. n = 3; ***p < .001; n.s., No significant differences. T cell purity, >90%. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E2 Rhinovirus induced T cell proliferation in monocultures, as measured by Ki-67. T cells (T, >99% pure) were cultured with RV, either infectious (T RV) or UV-inactivated (T RV-UV), and proliferation was measured by flow cytometry staining for Ki-67 antigen (FITC channel). A. Representative histogram showing the isotype (I, hairline), T cells alone (T, continuous line), T cells with RV (T RV, shaded fill) and T cells incubated with UV-inactivated RV (T RV-UV, dashed line). B. Quantitative analysis of flow cytometry data using Ki-67+ gate (n = 2). PHA (T PHA) was used as a positive control for T cell proliferation. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E3 An anti-ICAM-1 antibody inhibits RV-induced T cell proliferation. T cells (>99% pure) were cultured for 24h in the presence, or absence, of an anti-ICAM-1 antibody (Clone HA58, BD Pharmigen, 20μg/ml), or a matching isotype control. Then, RV was added for 7 days. Proliferation was measured using BrdU. n = 5; *p < .05; ***, p < .001; n.s., No significant differences. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E4 RV-induced T cell proliferation is neither induced by TLR agonists nor impaired by inhibition of TLR3, 7-8 or 9. A. Agonists for TLR2 (Pam3CSK4), TLR3 (Poly(I:C)), TLR4 (LPS) and TLR7-8 (R848) were added to T cell monocultures (>99% pure) and proliferation was measured (day 7). None of the TLR agonists tested induced T cell proliferation. B. Chloroquine treatment to impair endosomal TLR did not alter RV-induced proliferation. Shown is the mean value; error bars represent SEM; n = 4. Statistical comparison groups: A, all groups compared to T; B, as indicated.***, p < .001, n.s., no significant differences. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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