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. 2024 Feb 20;25(5):2476.
doi: 10.3390/ijms25052476.

Immunological Landscapes in Lung Transplantation: Insights from T Cell Profiling in BAL and PBMC

Tharushi Ayanthika de Silva  1   2 , Simon Apte  2   3 , Joanne Voisey  1 , Kirsten Spann  4 , Maxine Tan  2   3 , Daniel Chambers  1   2   3 , Brendan O'Sullivan  1   2   3
Affiliations

Immunological Landscapes in Lung Transplantation: Insights from T Cell Profiling in BAL and PBMC

Tharushi Ayanthika de Silva et al. Int J Mol Sci. .

Abstract

Lung transplant recipients frequently encounter immune-related complications, including chronic lung allograft dysfunction (CLAD). Monitoring immune cells within the lung microenvironment is pivotal for optimizing post-transplant outcomes. This study examined the proportion of T cell subsets in paired bronchoalveolar lavage (BAL) and peripheral PBMC comparing healthy (n = 4) and lung transplantation patients (n = 6, no CLAD and n = 14 CLAD) using 14-color flow cytometry. CD4+ T cell proportions were reduced in CD3 cells in both PBMC and BAL, and positive correlations were discerned between T cell populations in peripheral PBMC and BAL, suggesting the prospect of employing less invasive PBMC sampling as a means of monitoring lung T cells. Furthermore, regulatory T cells (Tregs) were enriched in BAL when compared to peripheral PBMC for transplant recipients. A parallel positive correlation emerged between Treg proportions in BAL and peripheral PBMC, underscoring potential avenues for monitoring lung Tregs. Finally, the most promising biomarker was the Teff (CD8+Granzyme B+)-Treg ratio, which was higher in both the PBMC and BAL of transplant recipients compared to healthy individuals, and increased in the patients with CLAD compared to no CLAD and healthy patients. Conclusions: Distinct T cell profiles in BAL and peripheral PBMC underscore the significance of localized immune monitoring in lung transplantation. The Teff (CD8+granzyme B+)-Treg ratio, particularly within the context of CLAD, emerges as a promising blood and BAL biomarker reflective of inflammation and transplant-related complications. These findings emphasize the imperative need for personalized immune monitoring strategies that tailored to address the unique immunological milieu in post-transplant lungs.

Keywords: BAL; CLAD; PBMC; Tregs; granzyme B; lung transplant.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
T cell populations in BAL and blood. (a) Flow cytometry plots depicting identified T cell populations in peripheral blood and bronchoalveolar lavage. (b) (i) CD4+ T cells (ii) CD8+ T cells. (b) (iii) Cell proportions of CD4+ T cells between blood and BAL. (b) (iv) Cell proportions of CD8+ T cells between blood and BAL * p ≤ 0.05; ** p ≤ 0.01.
Figure 2
Figure 2
Effector T cells in BAL and peripheral blood. (a) Flow cytometry plots depicting effector T cell populations (expressing granzyme B) in (i) peripheral blood and (ii) bronchoalveolar lavage. (b) (i) CD4+GZMB+ T cells; (ii) CD8+GZMB+ T cells; (iii) cell proportions of CD4+GZMB+ T cells between blood and BAL; (iv) cell proportions of CD8+GZMB+ T cells between blood and BAL. ** p ≤ 0.01.
Figure 3
Figure 3
Memory T cells in BAL and peripheral blood. (a) Flow cytometry plots depicting memory T cell populations (expressing CD45RO) in (i) peripheral blood and (ii) bronchoalveolar lavage. (b) (i) CD4+CD45RO+ T cells (ii) CD8+CD45RO+ T cells. (iii) Cell proportions of CD4+CD45RO+ T cells between blood and BAL; (iv) cell proportions of CD8+CD45RO+ T cells between blood and BAL. * p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001.
Figure 4
Figure 4
Non-Tregs expressing CD39 in BAL and peripheral blood. (a) Flow cytometry plots depicting non-Treg population expressing CD39 cell populations in (i) peripheral blood and (ii) bronchoalveolar lavage. (b) (i) CD4+CD39+ T cells (ii) CD8+CD39+ T cells; (iii) cell proportions of CD4+CD39+ T cells between blood and BAL; (iv) cell proportions of CD8+CD39+ T cells between blood and BAL. ** p ≤ 0.01.
Figure 5
Figure 5
Regulatory T cells in BAL and peripheral blood. (a) Flow cytometry plots depicting conventional Tregs (CD25+CD127−) and Tregs expressing CD39 cell populations in (i) peripheral blood and (ii) bronchoalveolar lavage. (b) (i) CD4+CD25+CD127− Tregs (ii) CD4+CD25+CD127−CD39+ T cells; (iii) cell proportions of CD4+CD25+CD127− T cells between blood and BAL; (iv) cell proportions of CD4+CD25+CD127−CD39+ T cells between blood and BAL.* p ≤ 0.05; *** p ≤ 0.001.
Figure 6
Figure 6
Subpopulation of Tregs based on CD45RO, HLA-DR, and CD194. (a) Flow cytometry plots depicting Treg subpopulations in peripheral blood; (b) flow cytometry plots depicting Treg subpopulations in bronchoalveolar lavage. (c) (i) naïve Tregs (CD45RO−HLA-DR−); (ii) memory Tregs (CD45RO+HLA-DR−); (iii) memory activated Tregs (CD45RO+HLA-DR+); (iv) non-migratory naïve Tregs (CD45RO−CD194−); (v) non-migratory memory Tregs (CD45RO+CD194−); (vi) migratory memory Tregs (CD45RO+CD194+). *** p ≤ 0.001; **** p ≤ 0.0001.
Figure 6
Figure 6
Subpopulation of Tregs based on CD45RO, HLA-DR, and CD194. (a) Flow cytometry plots depicting Treg subpopulations in peripheral blood; (b) flow cytometry plots depicting Treg subpopulations in bronchoalveolar lavage. (c) (i) naïve Tregs (CD45RO−HLA-DR−); (ii) memory Tregs (CD45RO+HLA-DR−); (iii) memory activated Tregs (CD45RO+HLA-DR+); (iv) non-migratory naïve Tregs (CD45RO−CD194−); (v) non-migratory memory Tregs (CD45RO+CD194−); (vi) migratory memory Tregs (CD45RO+CD194+). *** p ≤ 0.001; **** p ≤ 0.0001.
Figure 7
Figure 7
Granzyme B–Treg Ratio Between Healthy and Transplant (GZMB- Granzyme B). The ratio of CD8+GZMB+ T cell proportion to CD4+CD25+CD127− Treg proportion between healthy and transplant cohorts in BAL and PBMC. * p ≤ 0.05; ** p ≤ 0.01.
Figure 8
Figure 8
T cell subtypes comparing groups. (a) Effector T cells across groups (i) of CD4+GZMB+ cells; (ii) of CD8+GZMB+ cells. (b) Memory T cells across groups (i) of CD4+CD45RO+ cells; (ii) of CD8+CD45RO+ cells. (c) CD39 expressing cells across groups (i) of CD4+CD39+ cells; (ii) of CD8+CD39+ cells. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
Figure 9
Figure 9
Tregs comparing groups. (a) Conventional Tregs (CD4+CD25+CD127−); (b) CD39+ Tregs; (c) naïve Tregs (CD45RO+HLA-DR+); (d) non-migratory naïve Tregs (CD45RO−CD194−); (e) memory Tregs (CD45RO+HLA-DR−); (f) non-migratory memory Tregs (CD45RO+CD194−); (g) memory activated Tregs (CD45RO+HLA-DR+); (h) migratory memory Tregs (CD45RO+CD194+). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
Figure 10
Figure 10
Granzyme B–Treg ratio between healthy and transplant (GZMB- granzyme B). Ratio of CD8+GZMB+ T cell proportion: CD4+CD25+CD127− Treg Proportion across groups in PBMC and BAL. * p ≤ 0.05; ** p ≤ 0.01.
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