Cytotoxic Non-T_H2 CD4⁺ T Cells are associated with Asthma Severity

Sara Herrera-De La Mata¹, Syed Hasan Arshad², Ramesh J Kurukulaaratchy² and Grégory Seumois^1*
*Correspondence: Grégory Seumois, Department of Vaccine Discovery, La Jolla Institute for Immunology, United States of America, Email:

Keywords

CD4⁺ tissue-resident memory T cells • Airway T cells • Asthma • Cytotoxic CD4⁺ T cells • Severe asthma • Single-cell RNA sequencing • Translation to patients

Introduction

Asthma is one of the most common chronic inflammatory diseases affecting both children and adults which is characterized by coughing, wheezing, shortness of breath, and chest tightness [1,2]. These classical symptoms are triggered by the inflammation of the airways which results in increased mucus production, remodeling of the airways, bronchial hyperresponsiveness, and airway obstruction [1]. This airway inflammation has been previously associated with CD4⁺ T Helper 2 (T_H2) cell responses and their cytokines (Interleukin) IL-4, IL-5, IL-9, and IL-13 which are involved in the recruitment and activation of high number of innate immune cells such as eosinophils, basophils, and mast cells. While current treatments aiming to suppress airway inflammation focus on the use of corticosteroids or/and, more recently, biological agents blocking type 2 cytokines or Immunoglobulin E (Ig E), many patients with severe forms of asthma fail to respond to these treatments and suffer from persistent symptoms and recurrent life-threatening exacerbations [1,3-5]. Recent studies performed in airway specimens from asthmatic patients have also implicated other effector CD4⁺ T cell subsets with asthma pathogenesis [3,6-11]. However, due to the difficulty in sample collection from severe asthmatics and limitations in the number of cells available for research, unbiased studies providing a precise characterization of CD4⁺ T cell subsets associated with asthma severity are limited [1,12-20].

Literature Review

In our study, we specifically focused on identifying differences in the properties of CD4⁺ T cells present in the airways of patients with severe asthma. Therefore, we collected Bronchoalveolar Lavage (BAL) samples from severe asthmatic patients (n=16, 50% male) from the Wessex AsThma CoHort of difficult asthma (WATCH) study [21], which has extensively characterized patients with severe asthma requiring ‘high dose’ and/or ‘continuous or frequent use of oral corticosteroid’, in accordance with Global Initiative of Asthma (GINA) management steps 4 and 5 [22,23]. We also collected BAL samples from mild asthmatic patients (n=14, 64% male) as controls.

Using an oil-droplet-based single-cell RNA-sequencing platform (10 x Genomics), we performed an unbiased clustering analysis of airway CD4⁺ T cells (n=27,771), which revealed eight transcriptionally distinct subsets. Among this heterogeneity, we identified two clusters that accounted for the majority (71%) of CD4⁺ T cells present in the airways of these asthmatic patients and that were significantly enriched for Tissue-Resident Memory (T_RM) signature genes [24,25], and thus were defined as T_RM cells. Even though both T_RM clusters expressed high levels of the T_RM marker gene CD69, they differed in the expression of another T_RM marker gene, ITGAE, encoding for the alpha chain of the integrin CD103, a transmembrane protein which is required for the adhesion of T cells to E-cadherin expressed by epithelial cells [26-27]. Hence, based on the expression of these T_RM markers, we referred to these clusters as CD103⁺ T_RM and CD103^– T_RM .

Interestingly, when we looked at the quantitative differences of these subsets between disease severity, we observed that the proportions of the CD103⁺ T_RM cluster were significantly increased in the airways of patients with severe asthma compared to mild asthma (46% versus 21%), while the proportions of the CD103^– T_RM subset were significantly decreased (22% versus 44%). Moreover, we observed that there was an influence of biological sex on the composition of airway CD4⁺ T cells in severe asthma, as the increase in the proportions of the CD103⁺ T_RM cluster was significant only when comparing male patients with severe and mild asthma (64% versus 13%), while the proportions of the CD103^– T_RM cluster were significantly lower in males with severe asthma. An unbiased Spearman correlation analysis also determined a positive correlation in male but not female asthmatics between the proportions of the CD103⁺ T_RM cluster and several clinical and physiological parameters related to asthma severity, such as a composite asthma severity score adapted from the Asthma Severity Scoring System (ASSESS) [28] ranging from 0-20 from the Severe Asthma Research Program (SARP) [29] (rS=0.8 and P<0.01), and severity of airflow obstruction (using post-bronchodilator FEV₁ and FEV₁/FVC) (rS=0.7 and P<0.01). Next, to determine the differences among the molecular properties between both T_RM subsets, we performed a Differential Gene Expression Analysis (DGEA) between cells in the CD103⁺ T_RM cluster and cells in the CD103^– T_RM cluster across all patient groups and using sex as a covariate. The CD103⁺ T_RM cluster showed a significant enrichment of genes involved in cytotoxic function (GZMB, GZMA, GZMH, FASLG) [30] together with an increased expression of transcripts encoding for transcription factors linked to cytotoxicity in T cells; HOBIT (ZNF683), which is linked to T_RM differentiation and persistence of cytotoxic effector T cells, [31,32] and HOPX, known to regulate GZMB expression and to increase in vivo persistence of T_H1 cells. We also observed a significant upregulation of genes associated with T Cell Receptor (TCR) signaling (HLA-DRB1, HLADRB5, HLA-DRA), suggesting that the CD103⁺ T_RM subset may be enriched for T cells that were recently activated in the asthmatic airways due to the association of HLA-DR expression with T cell activation following in vivo antigen-specific TCR engagement [33,34]. A number of transcripts encoding for several cytokines (IFN-γ, TNF, LIGHT/TNFSF14) and chemokines (CCL4/MIP-1β, CCL5/RANTES) linked to airway inflammation and remodeling were also significantly expressed in the CD103⁺ T_RM subset [35-39].

Furthermore, when we performed a DGEA of CD4⁺ T cells from severe versus mild asthmatics for each sex and per cluster, we observed that the most differentially expressed genes increased in severe asthma were significantly enriched in male patients, especially those linked to cytotoxicity (GZMB, GZMH, ZNF683, HOPX, CCL4) which were differentially expressed only in the CD103⁺ T_RM subset.

However, the CD103^– T_RM cluster showed a significant enrichment of several transcripts encoding for molecules known to dampen TCR signaling and effector functions in T cells (CREM, DUSP1, DUSP2, DUSP4, TNFAIP3). One of the most upregulated differentially expressed genes is CREM (Cyclic AMP Responsive Element Modulatory), which encodes for a transcription factor known to repress promoters of inflammatory cytokine genes like IL2, IL3, IL4 and to dampen type 2 inflammation [40-43].

Finally, we stimulated ex vivo a fraction of BAL cells with Phorbol 12-Myristate 13-Acetate (PMA) and Ionomycin for 2 hours, and performed single-cell RNA-sequencing on sorted CD4⁺ T cells to assess the effector potential of airway CD4⁺ T cells from patients with mild and severe asthma. We observed that transcripts encoding for chemokines known to be released by cytotoxic CD4⁺ T cells like CCL3, CCL4, and CCL5 that play key roles in the recruitment of several immune cell types, [30,44-46] and therefore have the potential to drive airway inflammation and remodeling, were significantly expressed in stimulated airway CD4⁺ T cells from patients with severe asthma compared to mild asthma. In addition, transcripts associated with TH1 effector (IFNG, TNF, FASLG, XCL1, XCL2), and pro-fibrotic (LIGHT, AREG, TGFB1) properties were also upregulated by stimulation.

Conclusion

Notably, we observed that several of these pro-inflammatory cytokines and chemokines contributing to airway inflammation, fibrosis, and remodeling co-expressed with canonical cytotoxicityassociated maker genes such as (Granzyme B) GZMB. However, only a relatively small fraction of cells expressed T_H2 and TH17 cytokine transcripts and they were significantly reduced in patients with severe asthma compared to mild asthma. Overall, these findings supported the existence of a cytotoxic non-T_H2 CD4⁺ T_RM population with effector potential present in the airways of severe asthmatics.

The findings from our study highlight the heterogeneity of the disease and the importance to look beyond the T2-paradigm when categorizing patients purely through a T2 High/Low lens to better stratify severe asthma diversity. The identification of airway cytotoxic CD4⁺ T cells as a potentially important driver of asthma pathogenesis in a subgroup of severe asthmatic patients also supports the need to explore other asthma biomarkers different than the current ones to develop effective therapies, mostly for patients that are unresponsive to the current available asthma treatments.

References

1. Dharmage, Shyamali C, Jennifer L. Perret, and Adnan Custovic. "Epidemiology of Asthma in Children and Adults." Front Pediatr 7(2019): 246.

   [Crossref] [Google Scholar] [Pubmed]

2. Leon, Beatriz, and Andre Ballesteros-Tato. "Modulating Th2 Cell Immunity for the Treatment of Asthma." Front Immunol 12 (2021): 637948.

   [Crossref] [Google Scholar] [Pubmed]

3. Lambrecht, Bart N, Hamida Hammad, and John V. Fahy. "The Cytokines of Asthma." Immunity 50(2019): 975-991.

   [Crossref] [Google Scholar] [Pubmed]

4. Seumois, Gregory, Jose Zapardiel-Gonzalo, Brandie White and Divya Singh, et al. "Transcriptional Profiling of Th2 Cells Identifies Pathogenic Features Associated with Asthma." J Immuno Res 197(2016): 655-664.

   [Crossref] [Google Scholar] [Pubmed]

5. Wadhwa, Ridhima, Kamal Dua, Ian M. Adcock and Jay C. Horvat, et al. "Cellular Mechanisms Underlying Steroid-Resistant Asthma." ERR 28(2019).

   [Crossref] [Google Scholar] [Pubmed]

6. Chung, Kian Fan, Sally E. Wenzel, Jan L. Brozek and Andrew Bush, et al. "International ERS/ATS Guidelines on Definition, Evaluation and Treatment of Severe Asthma." Eur Respir J 43(2014): 343-373.

   [Crossref] [Google Scholar] [Pubmed]

7. Castro, Mario, Jonathan Corren, Ian D. Pavord and Jorge Maspero, et al. "Dupilumab Efficacy and Safety in Moderate-To-Severe Uncontrolled Asthma." NEJM 378(2018): 2486-2496.

   [Crossref] [Google Scholar] [Pubmed]

8. Ortega, Hector G, Mark C. Liu, Ian D. Pavord and Guy G. Brusselle, et al. "Mepolizumab Treatment in Patients with Severe Eosinophilic Asthma." NEJM 371 (2014): 1198-1207.

   [Crossref] [Google Scholar] [Pubmed]

9. Humbert, Marc, Camille Taillé, Laurence Malan and Vincent Le Gros, et al. "Omalizumab Effectiveness in Patients with Severe Allergic Asthma According to Blood Eosinophil Count: The STELLAIR Study." Eur Respir J 51(2018): 1702523.

   [Crossref] [Google Scholar] [Pubmed]

10. Rupani, Hitasha, Mohammed Aref Kyyaly, Adnan Azim and Rana Abadalkareen, et al. "Comprehensive Characterization of Difficult-To-Treat Asthma Reveals Near Absence of T2-Low Status." JACI: In Practice 11(2023): 2812-2821.

    [Crossref] [Google Scholar] [Pubmed]

11. Liu, Weimin, Sucai Liu, Mukesh Verma and Iram Zafar, et al. "Mechanism of TH2/TH17-Predominant and Neutrophilic TH2/TH17-Low Subtypes of Asthma." JACI: In Practice 139(2017): 1548-1558.

    [Crossref] [Google Scholar] [Pubmed]

12. Koch, Sonja, Nina Sopel, and Susetta Finotto. "Th9 and Other IL-9-Producing Cells in Allergic Asthma." Semin Immunopathol 39(2017): 55-68.

    [Crossref] [Google Scholar] [Pubmed]

13. Zhou, Ting, Xiji Huang, Yun Zhou and Jixuan Ma, et al. "Associations between Th17-Related Inflammatory Cytokines and Asthma in Adults: A Case-Control Study." Sci Rep 7(2017): 15502.

    [Crossref] [Google Scholar] [Pubmed]

14. Cui, Junqing, Stephen Pazdziorko, Joy S. Miyashiro and Paresh Thakker, et al. "TH1-Mediated Airway Hyperresponsiveness Independent of Neutrophilic Inflammation." JACI: In Practice 115(2005): 309-315.

    [Crossref] [Google Scholar] [Pubmed]

15. Bošnjak, Berislav, Sahar Kazemi, Lukas M. Altenburger and Gordana Mokrović, et al. "Th2-Trms Maintain Life-Long Allergic Memory in Experimental Asthma in Mice." Front Immunol 10 (2019): 453185.

    [Crossref] [Google Scholar] [Pubmed]

16. Lloyd, Clare M, and Edith M. Hessel. "Functions of T Cells in Asthma: More than Just TH2 Cells." Nat Rev Immunol 10(2010): 838-848.

    [Crossref] [Google Scholar] [Pubmed]

17. Seumois, Grégory, Ciro Ramírez-Suástegui, Benjamin J. Schmiedel and Shu Liang, et al. "Single-Cell Transcriptomic Analysis of Allergen-Specific T Cells in Allergy and Asthma." Sci Immunol 5(2020): eaba6087.

    [Crossref] [Google Scholar] [Pubmed]

18. Alladina, Jehan, Neal P. Smith, Tristan Kooistra and Kamil Slowikowski, et al. "A Human Model of Asthma Exacerbation Reveals Transcriptional Programs and Cell Circuits Specific to Allergic Asthma." Sci Immunol 8(2023): eabq6352.

    [Crossref] [Google Scholar] [Pubmed]

19. Vieira, Braga Felipe A, Gozde Kar, Marijn Berg and Orestes A. Carpaij, et al. "A Cellular Census of Human Lungs Identifies Novel Cell States in Health and in Asthma." Nat Med 25(2019): 1153-1163.

    [Crossref] [Google Scholar] [Pubmed]

20. Azim, Adnan, Heena Mistry, Anna Freeman and Clair Barber, et al. "Protocol for the Wessex AsThma CoHort of Difficult Asthma (WATCH): A Pragmatic Real-Life Longitudinal Study of Difficult Asthma in the Clinic." BMC Pulm. Med 19(2019): 1-11.

    [Crossref] [Google Scholar] [Pubmed]

21. Larsson, Kjell, Hannu Kankaanranta, Christer Janson and Lauri Lehtimäki, et al. "Bringing Asthma Care into the Twenty-First Century." NPJ Prim Care Respir Med 30(2020): 25.

    [Crossref] [Google Scholar] [Pubmed]

22. Global Initiative for Asthma. Global Strategy for Asthma Management and Prevention. (2021).
23. Anna, E Oja, Berber Piet, Christina Helbig and Regina Stark, et al. "Trigger-Happy Resident Memory CD^+; T Cells Inhabit the Human Lungs." Mucosal Immunol 11(2018): 654-667.

    [Crossref] [Google Scholar] [Pubmed]

24. Kumar, Brahma V, Wenji Ma, Michelle Miron and Tomer Granot, et al. "Human Tissue-Resident Memory T Cells are Defined by Core Transcriptional and Functional Signatures in Lymphoid and Mucosal Sites." Cell Rep 20(2017): 2921-2934.

    [Crossref] [Google Scholar] [Pubmed]

25. Casey, Kerry A, Kathryn A. Fraser, Jason M. Schenkel and Amy Moran, et al. "Antigen-Independent Differentiation and Maintenance of Effector-Like Resident Memory T Cells in Tissues." J Immunol Res 188: 4866-4875.

    [Crossref] [Google Scholar] [Pubmed]

26. Cepek, Karyn L, Sunil K. Shaw, Christina M. Parker and Gary J. Russell, et al. "Adhesion Between Epithelial Cells and T Lymphocytes Mediated by E-Cadherin and the Αeβ7 Integrin." Nature 372(1994): 190-193.

    [Crossref] [Google Scholar] [Pubmed]

27. Fitzpatrick, Anne M, Stanley J. Szefler, David T. Mauger and Brenda R. Phillips, et al. "Development and Initial Validation of the Asthma Severity Scoring System (ASSESS)." JACI: In Practice 145(2020): 127-139.

    [Crossref] [Google Scholar] [Pubmed]

28. Teague, W. Gerald, Brenda R. Phillips, John V. Fahy and Sally E. Wenzel, et al. "Baseline Features of the Severe Asthma Research Program (SARP III) Cohort: Differences with Age." JACI: In Practice 6(2018): 545-554.

    [Crossref] [Google Scholar] [Pubmed]

29. Patil, Veena S, Ariel Madrigal, Benjamin J. Schmiedel and James Clarke, et al. "Precursors of Human CD^+; Cytotoxic T Lymphocytes Identified by Single-Cell Transcriptome Analysis." Sci Immunol 3(2018): eaan8664.

    [Crossref] [Google Scholar] [Pubmed]

30. Mackay, Laura K, Martina Minnich, Natasja AM Kragten and Yang Liao, et al. "Hobit and Blimp1 Instruct a Universal Transcriptional Program of Tissue Residency in Lymphocytes." Science 352(2016): 459-463.

    [Crossref] [Google Scholar] [Pubmed]

31. Oja, Anna E, Felipe A. Vieira Braga, Ester BM Remmerswaal and  Natasja AM Kragten, et al. "The Transcription Factor Hobit Identifies Human Cytotoxic CD^+; T Cells." Fron Immunol 8(2017): 325.

    [Crossref] [Google Scholar] [Pubmed]

32. Serroukh, Yasmina, Chunyan Gu-Trantien, Baharak Hooshiar Kashani and Matthieu Defrance, et al. "The Transcription Factors Runx3 and Thpok Cross-Regulate Acquisition of Cytotoxic Function by Human Th1 Lymphocytes." Elife 7(2018): e30496.

    [Crossref] [Google Scholar] [Pubmed]

33. Albrecht, Inka, Uwe Niesner, Marko Janke and Astrid Menning, et al. "Persistence of Effector Memory Th1 Cells is Regulated by Hopx." Eur J Immunol 40(2010): 2993-3006.

    [Crossref] [Google Scholar] [Pubmed]

34. Tippalagama, Rashmi, Akul Singhania, Paige Dubelko and Cecilia S. Lindestam Arlehamn, et al. "HLA-DR Marks Recently Divided Antigen-Specific Effector CD4 T Cells in Active Tuberculosis Patients." J Immunol Res 207(2021): 523-533.

    [Crossref] [Google Scholar] [Pubmed]

35. Berbers, Roos-Marijn, M. Marlot van der Wal, Joris M. van Montfrans and Pauline M. Ellerbroek, et al. "Chronically Activated T-Cells Retain their Inflammatory Properties in Common Variable Immunodeficiency." J Clin Immunol 41(2021): 1621-1632.

    [Crossref] [Google Scholar] [Pubmed]

36. Makris, Spyridon, Michelle Paulsen and Cecilia Johansson. "Type I Interferons as Regulators of Lung Inflammation." Front Immunol 8(2017): 249453.

    [Crossref] [Google Scholar] [Pubmed]

37. Choi, Il-Whan, Young-Suk Kim, Hyun-Mi Ko and Suhn-Young Im, et al. "TNF-Α Induces the Late-Phase Airway Hyperresponsiveness and Airway Inflammation through Cytosolic Phospholipase A2 Activation." JACI: In Practice 116(2005): 537-543.

    [Crossref] [Google Scholar] [Pubmed]

38. Doherty, Taylor A, Pejman Soroosh, Naseem Khorram and Satoshi Fukuyama, et al. "The Tumor Necrosis Factor Family Member LIGHT is a Target for Asthmatic Airway Remodeling." Nat Med 17: 596-603.

    [Crossref] [Google Scholar] [Pubmed]

39. Lang, Roland and Faizal AM Raffi. "Dual-Specificity Phosphatases in Immunity and Infection: An Update." Int J Mol Sci 20(2019): 2710.

    [Crossref] [Google Scholar] [Pubmed]

40. Verjans, Eva, Kim Ohl, Lucy K. Reiss and Femke van Wijk, et al. "The Camp Response Element Modulator (CREM) Regulates TH2 Mediated Inflammation." Oncotarget 6(2015): 38538.

    [Crossref] [Google Scholar] [Pubmed]

41. Koga, Tomohiro, Christian M. Hedrich, Masayuki Mizui and Nobuya Yoshida, et al. "Camk4-Dependent Activation of AKT/Mtor and CREM-Α Underlies Autoimmunity-Associated Th17 Imbalance." JCI 124(2014): 2234-2245.

    [Crossref] [Google Scholar] [Pubmed]

42. Giordano, Marilyn, Romain Roncagalli, Pierre Bourdely and Lionel Chasson, et al. "The Tumor Necrosis Factor Alpha-Induced Protein 3 (TNFAIP3, A20) Imposes A Brake on Antitumor Activity of CD8 T Cells." PNAS 111(2014): 11115-11120.

    [Crossref] [Google Scholar] [Pubmed]

43. Meckiff, Benjamin J, Ciro Ramírez-Suástegui, Vicente Fajardo and Serena J. Chee, et al. "Imbalance of Regulatory and Cytotoxic SARS-Cov-2-Reactive CD^+; T Cells in COVID-19." Cell 183(2020): 1340-1353.

    [Crossref] [Google Scholar] [Pubmed]

44. Wang, Yue, Ziyi Chen, Tingjie Wang and Hui Guo, et al. "A Novel CD^+; CTL Subtype Characterized by Chemotaxis and Inflammation is Involved in the Pathogenesis of Graves’ Orbitopathy." Cell Mol Immunol 18(2021): 735-745.

    [Crossref] [Google Scholar] [Pubmed]

45. Juno, Jennifer A, David Van Bockel, Stephen J. Kent and Anthony D. Kelleher, et al. "Cytotoxic CD4 T Cells—Friend or Foe During Viral Infection?." Front Immunol 8(2017): 19.

    [Crossref] [Google Scholar] [Pubmed]