Comprehensive Tissue Cytometry Analysis of CD3+ T cells in Lungs of COVID-19 Patients


The appearance of a novel highly infectious human coronavirus (SARS-CoV-2) in 2019 resulted in a global pandemic that caused social and economic devastation worldwide. Infection with SARS-CoV-2 has a highly heterogenous presentation but can lead to life-threatening respiratory failure, even in otherwise healthy individuals. Across the world, SARS-CoV-2 infections has lead to the disease COVID-19, which has been associated with more than six million deaths.

Initially, the spread of the virus was controlled through strict lockdowns, but later the development of effective vaccines enabled limitations to be safely relaxed. Intensive research to further understand the SARS-CoV-2 virus, and human responses to infection and vaccination, are ongoing.

Interestingly, humoral immune responses mediated by antibodies to SARS-CoV-2 are often relatively small and of limited durability after natural infection1,2. To better understand why this is the case, alterations to the humoral immune system components were investigated in lymphoid tissue and the spleen, revealing dysregulated COVID-19-specific humoral immunity during early COVID-19 infection3. In particular, there were no lymph node and splenic germinal centers or Bcl-6-expressing B cells.

In addition, the generation and differentiation of Bcl-6+ T follicular helper cells, which are important for the differentiation of B cells into antibody-producing plasma cells, were found to be defective; this could explain the limited durability of humoral immunity to COVID-19, and the failure to achieve herd immunity through natural infection. Even among acutely ill patients with extremely high viral loads, germinal centers do not form despite a robust T-cell-mediated activation of B cells, precluding the creation of long-lived memory B cells3. The poor humoral response may also explain findings suggesting that the COVID-19 virus persists in various organs for up to 7 months after infection, possibly contributing to the development of long-covid4,5.

To further unravel the human immune response to SARS-CoV-2 infection, early and late T-cell subset alterations were studied in the lungs and draining lymph nodes of patients diagnosed with severe COVID-19 disease6.

T-cell Response in COVID-19 patients

The T-cell response to SARS-CoV-2 infection has been shown to be highly variable in analyses of peripheral blood of COVID-19 patients, and specific patterns of T-cell response have been linked to distinct clinical outcomes7. Blood analyses have shown that expansion of antigen-specific T helper cells and cytotoxic CD4+ T cells occurs following  SARS-CoV-2 infection8. However, there is no evidence of antigen-specific CD8+ T cell clonal expansion in acute COVID-19 cases9.

Post-mortem lung evaluation using spatial transcriptomic imaging detected a limited number of T cells, but no details about individual T-cell subsets have been obtained10,11. Using a rapid autopsy protocol and tissue cytometry, the T-cell subsets in the lung parenchyma and draining thoracic lymph nodes of patients with severe COVID-19 disease, have now been directly quantified6.

Quantifying T-cell Subset Alterations in COVID-19

This study provides the first quantitative in-depth analysis of T-cell subset alterations in the lung parenchyma that occur during the acute stages of severe COVID-19 6.


Thoracic lymph nodes and lung samples were collected from 22 patients undergoing autopsy at the Brigham and Women's Hospital Department of Pathology, 16 of whom had tested positive for the SARS-CoV-2 infection upon hospital admission.

The tissue specimens were stained with various markers to perform high-content T cell phenotyping.

The IF stained tissue sections were acquired using the TissueFAXS PLUS12. Cells of a given phenotype were identified and quantitated using the TissueQuest software and cell-to-cell contact was quantified using the StrataQuest IF Cellular Contact App (TissueGnostics)13. Applied together, these enabled in-depth spatial phenotyping of single cells within tissue sections.


A comparison of samples taken during early and later stages of the disease revealed that there was no increase in CD8+ T cells or of re-activated Granzyme B + CD8+ T cells in the lung. Indeed, the only T-cell subset found to undergo significant expansion in the lungs during severe COVID-19 disease, was the cytotoxic CD4+ T cells.

The CD8+ T cells present in the lung, however, were shown to express high levels of PD-1.

This data suggests that CD8+ T cells in severe COVID-19 cases may be dysfunctional. Furthermore, it appears that the number of CD4+ T cells are increased, which alters functional CD8 + T cells and leads to a decreased anti-viral immune reaction.


Quantitative analysis of T-cell subsets in a disease-affected organ is required to associate specific T cells/T-cell subsets to the disease pathology. A greater understanding of the immune response can be obtained when tissue and peripheral blood data are interpreted in parallel with data from draining lymph nodes.

The quantitative data on T-cell subsets in the lung parenchyma and lymph nodes acquired using rapid autopsy protocols, as followed in this study, can be evaluated in conjunction with other non-quantitative data; such as antigen-specific T-cell subsets in the blood of living patients, to build a more accurate picture of the adaptive T-cell response in the lungs of patients infected with SARS-CoV-2.

Further research is required to discern the underlying mechanisms through which COVID-19 alters the lymph node milieu resulting in sub-optimal immune pressure in the lungs3,6.


TissueFAXS is a versatile multifluorescence imaging platform12. Its upright configuration includes an 8-slide stage that can accommodate over-sized slides. TissueFAXS PLUS provides automated multi-channel fluorescence and brightfield whole slide scanning, as well as sophisticated quantitative image analysis. The contextual image analysis software from TissueGnostics, StrataQuest, enables the user to run streamlined analysis solutions, such as the IF Cellular Contact App, or to create self-designed analysis algorithms. 


The StrataQuest software13 allows the analysis solution to identify the cellular phenotype of specific fluorescence-stained cell populations, and to further establish the cellular contacts to their neighboring cells. It readily provides the staining intensity per marker and other morphometric parameters for each segmented cell/cell compartment, as well as the percentage of cells of different phenotypes in direct contact.

The analysis is conducted using a IF Cellular Contact App, which is available online in the TissueGnostics App Center.

References and Further Reading

  1. Long QX, et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat. Med. 2020;26:845-848.
  2. Robbiani CF, et al. Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature2020;584:437-442.
  3. Kaneko N, et al. Loss of Bcl-6-Expressing T Follicular Helper Cells and Germinal Centers in COVID-19. Cell 202;183(1):143-157.
  4. Daniel C., et al. 2022. Nature Portfolio.
  5. Crook H., et al. Long covid-mechanisms, risk factors, and management. BMJ. 2021;374:n1648.
  6. Kaneko N, et al. Temporal changes in T cell subsets and expansion of cytotoxic CD4+ T cells in the lungs in severe COVID-19. Clin Immunol. 2022;237. Epub Mar 29.
  7. Mathew D, et al. Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications. Science. 2020;369(6508).
  8. Meckiff BJ, et al. Imbalance of regulatory and cytotoxic SARS-CoV-2-reactive CD4(+) T cells in COVID-19. Cell. 2020;183(5):1340–1353.e16.
  9. Zhao Y, et al. Clonal expansion and activation of tissue-resident memory-like Th17 cells expressing GM-CSF in the lungs of severe COVID-19 patients. Sci. Immunol. 2021;6(56).
  10. Desai N, et al. Temporal and spatial heterogeneity of host response to SARS-CoV-2 pulmonary infection. Nat.Commun. 2020;11:6319.
  11. Delorey TM, et al. COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets. Nature. 2021;595(7865):107–113.
  12. TissueGnostics. TissueFAXS Plus Upright Brightfield And Fluorescence System Available at
  13. TissueGnostics. StratQuest. Available at

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