Immune Netw.  2017 Jun;17(3):163-170. 10.4110/in.2017.17.3.163.

Clonal Expansion of Allergen-specific CD4⁺ T Cell in the Lung in the Absence of Lymph Nodes

• Choi G
• Kim BS
• Park YJ
• Shim I
• Chung Y

• KMID: 2383354
• DOI: http://doi.org/10.4110/in.2017.17.3.163

Abstract

The expansion of allergen-specific CD4⁺ T cells is a critical step in inducing airway inflammation during allergic asthma. Such clonal expansion of T cells is initiated through the interaction between allergen-bearing dendritic cells and allergen-specific naïve T cells in the draining lymph nodes. Whether such T cell clonal expansion also occurs in the lung, the primary organ encountering inhaled allergens, remains unclear. Compared with wild-type mice, we found similar frequencies of CD4⁺ T cells in the lung of lymph node-deficient Rorgt(gfp/gfp) mice after repeated exposure to inhaled allergens. In addition, we observed an evident population of CD4⁺ T cells that underwent clonal expansion in the lung of allergen-challenged mice treated with an S1P antagonist FTY720 in an in vivo proliferation study with CFSE-labeled OT-II T cells. Moreover, the expansion of allergen-specific CD4⁺ T cells was significantly enhanced in the lungs of Rorgt(gfp/gfp) mice in comparison to that of wild-type mice. These results together demonstrate that the clonal expansion of allergen-specific CD4⁺ T cells occurs in the absence of the lymph nodes, indicating that the lung can act as a primary site of the clonal expansion of CD4⁺ T cells in response to inhaled allergens.

Keyword

Allergens; Lung; RORγt; CD4⁺ T cell; Proliferation

MeSH Terms

Allergens
Animals
Asthma
Dendritic Cells
Fingolimod Hydrochloride
Inflammation
Lung*
Lymph Nodes*
Mice
T-Lymphocytes
Allergens
Fingolimod Hydrochloride

Figure

• 

  Figure 1 CD4+ T cell population in the lung of Rorgtgfp/gfp mice in steady-state. (A) Representative pictures of the superficial cervical lymph nodes, the mLN, the mesenteric lymph nodes, the inguinal lymph nodes and the Peyer's patches of wild-type mice and Rorgtgfp/gfp mice. (B) The number of CD45.2+ immune cells in the lungs of Rorgt+/gtp and Rorgtgfp/gfp mice and (C) the proportion of CD4+ cells among CD45.2+ cells in the lung of Rorgt+/gtp and Rorgtgfp/gfp mice before intranasal challenge. n=3 mice per group (B and C).

• 

  Figure 2 CD4+ T cells are major immune cells in the lung of RORγt-deficient mice after intranasal challenges with allergens. (A) Experimental scheme. Wild-type mice and Rorgtgfp/gfp mice were intranasally injected with PAO/Ova every other day for 4 times and analyzed one day after the last challenge. (B) The proportion of CD4+ cells among CD45.2+ cells in the lung of wild-type mice and Rorgtgfp/gfp mice after intranasal challenges with PAO/Ova. Data are means±SEM. n=4 mice per group. Data are representative of two independent experiments.

• 

  Figure 3 Kinetics of allergen-specific CD4+ T cell proliferation in the BALT in response to intranasal allergen. (A) Experimental scheme. (B-D) Proliferation of OT-II T cells after intranasal PAO/Ova in the indicated tissue. CD45.1+ donor T cells were gated and analyzed for CFSE dilution by flow cytometry. Data are means±SEM. *p<0.05 and **p<0.01 in comparison with the mLN. Data are representative of three independent experiments.

• 

  Figure 4 Expansion of allergen-specific CD4+ T cell in the lung in mice treated with FTY720. (A) Experimental scheme. CFSE-labeled CD45.1+OT-II T cells were transferred into wild-type mice (CD45.2+/+) and the recipients were intranasally challenged with PAO/Ova. Six hours after the intranasal challenge, the mice were intraperitoneally injected with 1 mg/kg FTY720 or vehicle (n=3). (BD) Forty-eight hours later, the frequency and the dilution of CFSE of donor T cells were analyzed in the indicated lymphoid tissues or blood. Data are means±SEM. **p<0.01 in comparison with vehicletreated group. Data are representative of two independent experiments.

• 

  Figure 5 Expansion of allergen-specific CD4+ T cell in the lung of Rorgtgfp/gfp mice. (A) Experimental scheme (n=3). (B) Forty-eight hours after the challenge, the proportion of divided cells among CFSE-labeled donor T cells was analyzed by flow cytometry. Numbers on the line indicate the percentage of divided cells. (C) Seventy-two hours after co-culture of the indicated T cells with BMDC in the presence of anti-CD3, CFSE-dilution was measured to determine the proliferation of the T cells. Data are means±SEM. **p<0.01 in comparison with wild-type mice. Data are representative of two independent experiments.

Reference

1. Mueller DL, Jenkins MK, Schwartz RH. Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu Rev Immunol. 1989; 7:445–480.

2. Mittler JN, Lee WT. Antigen-specific CD4 T cell clonal expansion and differentiation in the aged lymphoid microenvironment. II. The memory T cell response is diminished. Mech Ageing Dev. 2004; 125:59–68.

3. Bousso P. T-cell activation by dendritic cells in the lymph node: lessons from the movies. Nat Rev Immunol. 2008; 8:675–684.

4. Gunzer M, Weishaupt C, Hillmer A, Basoglu Y, Friedl P, Dittmar KE, Kolanus W, Varga G, Grabbe S. A spectrum of biophysical interaction modes between T cells and different antigen-presenting cells during priming in 3-D collagen and in vivo. Blood. 2004; 104:2801–2809.

5. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol. 2012; 12:180–190.

6. Chen L, Flies DB. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol. 2013; 13:227–242.

7. Corthay A. A three-cell model for activation of naive T helper cells. Scand J Immunol. 2006; 64:93–96.

8. Britschgi MR, Link A, Lissandrin TK, Luther SA. Dynamic modulation of CCR7 expression and function on naive T lymphocytes in vivo. J Immunol. 2008; 181:7681–7688.

9. MartIn-Fontecha A, Sebastiani S, Hopken UE, Uguccioni M, Lipp M, Lanzavecchia A, Sallusto F. Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J Exp Med. 2003; 198:615–621.

10. GeurtsvanKessel CH, Willart MA, Bergen IM, van Rijt LS, Muskens F, Elewaut D, Osterhaus AD, Hendriks R, Rimmelzwaan GF, Lambrecht BN. Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus-infected mice. J Exp Med. 2009; 206:2339–2349.

11. Halle S, Dujardin HC, Bakocevic N, Fleige H, Danzer H, Willenzon S, Suezer Y, Hammerling G, Garbi N, Sutter G, Worbs T, Forster R. Induced bronchus-associated lymphoid tissue serves as a general priming site for T cells and is maintained by dendritic cells. J Exp Med. 2009; 206:2593–2601.

12. De Togni P, Goellner J, Ruddle NH, Streeter PR, Fick A, Mariathasan S, Smith SC, Carlson R, Shornick LP, Strauss-Schoenberger J. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science. 1994; 264:703–707.

13. Koni PA, Sacca R, Lawton P, Browning JL, Ruddle NH, Flavell RA. Distinct roles in lymphoid organogenesis for lymphotoxins alpha and beta revealed in lymphotoxin beta-deficient mice. Immunity. 1997; 6:491–500.

14. Kuprash DV, Alimzhanov MB, Tumanov AV, Grivennikov SI, Shakhov AN, Drutskaya LN, Marino MW, Turetskaya RL, Anderson AO, Rajewsky K, Pfeffer K, Nedospasov SA. Redundancy in tumor necrosis factor (TNF) and lymphotoxin (LT) signaling in vivo: mice with inactivation of the entire TNF/LT locus versus single-knockout mice. Mol Cell Biol. 2002; 22:8626–8634.

15. Eberl G, Marmon S, Sunshine MJ, Rennert PD, Choi Y, Littman DR. An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol. 2004; 5:64–73.

16. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, Cua DJ, Littman DR. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell. 2006; 126:1121–1133.

17. Sun Z, Unutmaz D, Zou YR, Sunshine MJ, Pierani A, Brenner-Morton S, Mebius RE, Littman DR. Requirement for RORgamma in thymocyte survival and lymphoid organ development. Science. 2000; 288:2369–2373.

18. Choi G, Chung Y. Blockade of STAT3 in T cells inhibits germinal center reactions against intranasal allergens. Biomol Ther (Seoul). 2016; 24:244–251.

19. Lim H, Cho M, Choi G, Na H, Chung Y. Dynamic control of Th2 cell responses by STAT3 during allergic lung inflammation in mice. Int Immunopharmacol. 2015; 28:846–853.

20. Kheradmand F, Kiss A, Xu J, Lee SH, Kolattukudy PE, Corry DB. A protease-activated pathway underlying Th cell type 2 activation and allergic lung disease. J Immunol. 2002; 169:5904–5911.

21. Matloubian M, Lo CG, Cinamon G, Lesneski MJ, Xu Y, Brinkmann V, Allende ML, Proia RL, Cyster JG. Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature. 2004; 427:355–360.

22. Brinkmann V, Billich A, Baumruker T, Heining P, Schmouder R, Francis G, Aradhye S, Burtin P. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat Rev Drug Discov. 2010; 9:883–897.

23. Ciabattini A, Pettini E, Fiorino F, Prota G, Pozzi G, Medaglini D. Distribution of primed T cells and antigen-loaded antigen presenting cells following intranasal immunization in mice. PLoS One. 2011; e19346.

24. Reines I, Kietzmann M, Mischke R, Tschernig T, Luth A, Kleuser B, Baumer W. Topical application of sphingosine-1-phosphate and FTY720 attenuate allergic contact dermatitis reaction through inhibition of dendritic cell migration. J Invest Dermatol. 2009; 129:1954–1962.

25. Lan YY, De Creus A, Colvin BL, Abe M, Brinkmann V, Coates PT, Thomson AW. The sphingosine-1-phosphate receptor agonist FTY720 modulates dendritic cell trafficking in vivo. Am J Transplant. 2005; 5:2649–2659.

26. Krummel MF, Bartumeus F, Gérard A. T cell migration, search strategies and mechanisms. Nat Rev Immunol. 2016; 16:193–201.

27. Pitzalis C, Jones GW, Bombardieri M, Jones SA. Ectopic lymphoid-like structures in infection, cancer and autoimmunity. Nat Rev Immunol. 2014; 14:447–462.

28. Germain C, Gnjatic S, Dieu-Nosjean MC. Tertiary lymphoid structure-associated B cells are key players in antitumor immunity. Front Immunol. 2015; 6:67.

29. Sawicka E, Zuany-Amorim C, Manlius C, Trifilieff A, Brinkmann V, Kemeny DM, Walker C. Inhibition of Th1- and Th2-mediated airway inflammation by the sphingosine 1-phosphate receptor agonist FTY720. J Immunol. 2003; 171:6206–6214.

30. Idzko M, Hammad H, van Nimwegen M, Kool M, Müller T, Soullié T, Willart MA, Hijdra D, Hoogsteden HC, Lambrecht BN. Local application of FTY720 to the lung abrogates experimental asthma by altering dendritic cell function. J Clin Invest. 2006; 116:2935–2944.

31. Lochner M, Ohnmacht C, Presley L, Bruhns P, Si-Tahar M, Sawa S, Eberl G. Microbiota-induced tertiary lymphoid tissues aggravate inflammatory disease in the absence of RORgamma t and LTi cells. J Exp Med. 2011; 208:125–134.