There is no question that the SARS-CoV-2 (COVID-19) pandemic has reversed many of the gains made in fighting the effort to control the infection and spread of Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), and has set back the fight against TB by years (1). Nevertheless, we cannot blame our lack of success in controlling TB on COVID-19 alone. We have lived with TB long enough not to have it as the leading cause of death from a single infectious disease prior to SARS-CoV-2, and by all accounts it is set to regain its number one spot again (2). The major challenges to realizing the global 2030 End TB reduction targets include the lack of efficient TB vaccines, diagnostic challenges, drug resistance, and inaccessibility to effective chemotherapy (2, 3).

The best intervention against TB, or in fact any infectious disease, is one that breaks the transmission cycle. Lessons from other infectious diseases that have now been eradicated, such as smallpox and rinderpest, have shown that vaccination is the best way to break transmission; polio is almost eradicated as well due to the WHO’s determined vaccination strategy (4). TB vaccination has been extensively taken up by most TB-endemic countries. However, it is well-known that BCG, currently the only licensed TB vaccine, is not effective against adult pulmonary TB, the most transmissible form of the disease in endemic countries (5). We need to develop more effective TB vaccines and develop shorter drug regimens, amongst a plethora of other interventions (2). The last few years have witnessed a reawakening of novel TB vaccine approaches. There is hope in some current trial vaccines (M72, BCG REVAC, VPM1002, H56/IC31), but tertiary trial results will not be out for several years (6, 7). In the meantime, we need vaccine strategies based on deeper insights into the immunity underlying TB, and not just in blood, but most importantly, at the site of infection in humans.

This is not to say that we have little idea of tissue-specific cell subsets in human tissue; tissue-resident immune cells have been successfully isolated from the lungs of organ donors (8, 9), and include subsets such as tissue-resident memory (TRM) T cells (10), macrophages (11), NK and innate lymphoid cell (ILC) subsets (12–14), mucosal-associated invariant T (MAIT) cells (15, 16) and γδ T cells (17). But only a few studies have looked at these subsets in the lungs of TB-diseased individuals (17–19), and none in otherwise healthy individuals with a latent TB infection (LTBI). Studies have been largely restricted to relatively accessible sites such as bronchoalveolar lavage (BAL) (20–22), the pleural fluid (23–25), or occasionally pleural or lymph node biopsies when clinically indicated (26). New techniques such as Positron Emission Tomography – Computed Tomography (PET-CT) can help us to understand the resolution or progression of disease (27–31), but cannot assess specific cellular functions. We therefore have only a superficial knowledge of how the immune system responds at the main site of TB infection and disease in humans – the lungs.

To address this, we conducted a postmortem study to determine whether we could use immune cells isolated from tissues taken postmortem for medical research. We show that we were able to get consent from the next of kin(s) (NoKs), perform a full postmortem, and collect and process tissues with little loss in cell viability or function, up to 14 hours post-death.

To determine whether tissue-specific immunology studies could be performed on tissues donated for medical research following death, we assessed two essential elements: 1) could we get consent from the NoK, and if so, 2) how viable would cells be following death and the postmortem process.

We found that we could get consent from the NoK with very high acceptability rates at the SEU (94% of those approached; Table 2 ), and with more modest rates on the TB and ID Wards (35% of those approached; Table 2 ). The latter consent rate was in line with a previous study assessing consent across all Wards at MNRH (33). The high rate of consent at the SEU was a surprise, as we had assumed a sudden, unexpected death might mean that the NoK would be less willing to consent, whereas the consent rate on the TB and ID Wards might be higher because the patient and family have usually built a rapport with hospital staff, and may therefore be more keen to know the actual cause of death of their relative. Most NoKs believed that understanding the reason for a loved one’s sudden death would help them to better come to terms with their loss; the low consent rate on the TB and ID Wards was mainly attributed to religious norms that do not allow a postmortem to be undertaken ( Table 2 ).

After obtaining consent, the body was taken for a full postmortem. Tissues were taken to assess the viability of cells over time, from the time of death through sample processing to cell counting in the laboratory. We found that cells were viable up to 14 hours post-death, but that there was considerable variability in viability depending on the tissue sample ( Figure 3 ). Arterial blood maintained a high level of viability out to 14 hours post-death, but other tissues examined were considerably lower; around 40% of the cells isolated were viable. A regression analysis showed no effect of time from death on viability for any tissue, with the exception of PBMCs. For PBMCs, we found a significant decrease of 1.4% viability for every hour following death that the samples were processed. We did not see a difference in cell viability in any tissue between TB and non-TB groups.

Having shown that we could isolate viable cells from the organs of deceased subjects, we next tested whether the cells were functional using the T-SPOT^®.TB assay to assess exposure to M.tb antigens (ESAT-6 and CFP-10) ( Figure 4 ). We found that PBMCs isolated postmortem were just as functional as PBMCs isolated from the venous blood of a living subject ( Figure 4A ). Furthermore, the T-SPOT^®.TB assay worked on spleen and bronchoalveolar lavage (BAL) samples, showing that cells isolated from tissues are not just viable, but are functional as well. In undertaking the T-SPOT^®.TB assay, we were able to identify both T-SPOT^®.TB positive and negative subjects ( Figure 4B ). PBMCs isolated postmortem from HIV-positive blood did not give reliable responses, so HIV-positive participants were excluded from this analysis. We found that 61% ( Figure 4B ) of the subjects tested were T-SPOT^®.TB positive – a finding similar to other studies conducted in the same population (34, 35), and suggesting no overt bias in our non-TB postmortem population.

Lastly, we asked whether T cells isolated from different organs showed evidence of classical immunological naïve and memory T cell subsets ( Figures 5A, B ; representative data). In all organs analyzed, both CD4 ( Figure 5A ) and CD8 ( Figure 5B ) T cells had naïve (CCR7⁺CD45RA⁺; T_N), central memory (CCR7⁺CD45RA^-; T_CM), effector memory (CCR7^-CD45RA^-; T_EM) and effector memory RA (CCR7^-CD45RA⁺; T_EMRA) subsets, albeit at different frequencies. Additionally, we asked whether Tissue Resident Memory (TRM) T cells, defined by co-expression of CD69 and CD103, were present in the different organs, for both CD4 ( Figure 5C ) and CD8 ( Figure 5D ) T cells. As expected, TRM T cells (CD69⁺CD103⁺) could be identified in the lung tissue (LUNG), bronchoalveolar lavage (BAL), and the lung draining hilar lymph nodes (HLN), but were not found in the blood.

Development of better TB vaccine strategies needs to be based on deeper insights into the immunity underlying TB, and not just in blood, which is commonly undertaken, but perhaps more importantly, at the site of infection. Our knowledge of TB immune responses at the main site of infection, the lungs, is largely derived from animal models and whilst clearly important, few animal models exist that recapitulate the complex natural history of disease progression and reactivation as it exists in humans. Non-human primate (NHP) models are our closest model to human infection and disease, but are limited by cost and exposure to concurrent (or prior) unrelated infections and vaccine responses. Current human lung studies are sampled from patients with severe lung complications at the extreme end of the TB disease spectrum, and often lack normal lung tissue from non-diseased individuals for comparison. Another approach to tackle the burden of disease is to undertake postmortem studies. Postmortem studies allow us to not only compare the immunology within the tissues of people who have died from TB disease from those who have died of other causes, but also allow us to study the immunology of disease at other, non-involved tissues within the same individual. Postmortem studies also allow us to sample the entire population, and study tissue-specific responses in otherwise healthy people at the time of death – road traffic accident victims, for example.

We report that hospital-driven postmortems are feasible in Uganda. Aligned with a previous study (36), we found that consent rates were highly dependent on whether death was sudden or not. The consent rate on the TB and ID Wards was more than two-fold lower than at the SEU, but comparable to what has previously been reported at Mulago National Referral Hospital (33). The reason for this may be that on Wards other than the SEU, patients are admitted and stay for longer periods of time, during which a clinical diagnosis is established, and relatives therefore have no need for a postmortem to be conducted to determine the cause of death. Indeed, the major reason for consent given by the NoKs at the SEU, where the average length of a hospital stay for RTA victims was 10 hours, was that knowing the cause of death helped in coming to terms with their relative’s death.

In this paper, we show that lymphocytes remain viable and functional up to 14 hours post-death. Previous studies have reported good cell viability up to several days following death (37–39), far longer than the time frame of our study. However, few studies have looked at the functionality of cells isolated postmortem (8, 9), even fewer in tissues, and even fewer that are focussed on M.tb (17–19). Of note, the T-SPOT^®.TB test that we used for M.tb-specific functionality required a change of incubation time from the manufacturer’s recommendation of 24 hours to 36 hours. In addition, changing the media to RPMI from the recommended media (AIMV) yielded more consistent results. It is unclear why these two changes improved the consistency of the T-SPOT^®.TB test in our case.

Finally, we presented a snapshot of T cell subsets in the blood and tissues of a subject from the SEU, showing that 1) classical naïve and memory T cell subsets were present in all organs, but at different frequencies, and 2) TRM T cells could be detected within the tissues and were absent from the blood, as would be expected. This work presents the platform under which we can undertake further research to understand the immune response to TB within different human tissues, and between those individuals who died from TB and those who were otherwise healthy at the time of death, but died from other causes, such as RTA victims.

This study presented the feasibility of undertaking postmortem studies for medical research. A clear limitation in this kind of hospital-based study is that patient demographics are skewed; this is particularly the case for TB subjects and is a well-recognized epidemiological quirk. In addition, our non-TB subjects were skewed in terms of gender, limiting the overall generalisability of this study; ongoing recruitment of further subjects may help resolve this issue.

These limitations notwithstanding, we have shown that postmortem studies are a feasible approach to studying tissue-specific immune responses to disease in humans. Lymphocytes can be isolated from tissues up to 14 hours after death with no loss of viability. These cells are functional and show tissue-specific surface markers. Different tissues show clear differences in classical T cell memory and TRM subsets. Human postmortem procedures remain a valuable and essential tool to advance our medical and scientific understanding, not just for understanding TB disease and its progression, but for any disease with a tissue-specific tropism. Although largely fallen out of favor in first-world settings, postmortem studies are clearly a valuable tool in our race to understand immune responses at the site of infection, and in the rational design of future tissue-specific vaccines.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by School of Biological Sciences Research Ethics Committee (SBS REC), Makerere University, Kampala, Uganda Mulago National Referral Hospital Research Ethics Committee (MNRH REC), MNRH, Kampala, Uganda Kiruddu National Referral Hospital Research Ethics Committee (KNRH REC), KNRH, Kampala, Uganda Uganda National Council for Science and Technology (UNCST), Kampala, Uganda London School of Hygiene and Tropical Medicine Research Ethics Committee (LSHTM REC), London, UK. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

GA: Investigation, Methodology, Writing – original draft, Writing – review & editing, Data curation, Project administration. MN: Data curation, Investigation, Methodology, Writing – review & editing. MM: Investigation, Methodology, Writing – review & editing. DO: Investigation, Writing – review & editing. RM: Investigation, Writing – review & editing. JN: Investigation, Writing – review & editing. FB: Investigation, Writing – review & editing. MK: Investigation, Writing – review & editing. HL: Investigation, Writing – review & editing. AA: Investigation, Writing – review & editing. DN: Investigation, Writing – review & editing. SM: Investigation, Writing – review & editing. RL: Investigation, Writing – review & editing. AK: Investigation, Writing – review & editing. JB: Investigation, Writing – review & editing. IA: Investigation, Writing – review & editing, Conceptualization. SC: Conceptualization, Investigation, Writing – review & editing, Funding acquisition, Methodology, Resources, Supervision, Writing – original draft.