Lung Tissue Resident Memory T-Cells in the Immune Response to Mycobacterium tuberculosis

Despite widespread BCG vaccination and effective anti-TB drugs, Tuberculosis (TB) remains the leading cause of death from an infectious agent worldwide. Several recent publications give reasons to be optimistic about the possibility of a more effective vaccine, but the only full-scale clinical trial conducted failed to show protection above BCG. The immunogenicity of vaccines in humans is primarily evaluated by the systemic immune responses they generate, despite the fact that a correlation between these responses and protection from TB disease has not been demonstrated. A novel approach to tackling this problem is to study the local immune responses that occur at the site of TB infection in the human lung, rather than those detectable in blood. There is a growing understanding that pathogen specific T-cell immunity can be highly localized at the site of infection, due to the existence of tissue resident memory T-cells (Trm). Notably, these cells do not recirculate in the blood and thus may not be represented in studies of the systemic immune response. Here, we review the potential role of Trms as a component of the TB immune response and discuss how a better understanding of this response could be harnessed to improve TB vaccine efficacy

Lung tissue resident memory T and B cells in the immune response to Mycobacterium tuberculosis in humans.

Doctoral Degree. University of KwaZulu-Natal, Durban.Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) is the main cause of death from a single infectious agent. Although a quarter of the world population is estimated to be infected with TB, only 5–10% of these individuals will develop active disease during their lifetime, suggesting that the immune system is highly effective at containing Mtb infection in most people. Extensive analysis of T-cells in blood has, however, failed to identify correlates of protection in humans, which is a prerequisite for rationale vaccine design. A likely explanation for this failure is that most memory T-cells in lung tissue do not recirculate in blood. Indeed, adoptive transfer of lung T-cells from infected mice protects recipients from Mtb challenge whereas transfer of blood T-cells does not. While the crucial role of T-cells in controlling Mtb infection is well appreciated, the role of B cells and humoral immunity in TB disease is unclear. Furthermore, human studies that directly compare the B-cell involvement in TB between infected lung tissue and matched peripheral blood are lacking. Therefore, this study was based on the premise that studying the human adaptive immune responses to Mtb at the site of infection would provide new findings that are not present in the systemic circulation. Thus, we hypothesised that the lung T and B-cell responses to TB in humans are fundamentally different from those in the blood, because they involve cells that do not recirculate (particularly T-cells). The overall aim was to determine the phenotype, function and TB specificity of adaptive immune responses (T and B-cells) at the site of TB disease and to understand how they correlate with the responses detectable in circulation. To achieve this, a cohort of study participants undergoing medical lung resection for tuberculosis related lung pathologies was established The results presented in this thesis show that lung T-cells are different from peripheral blood T-cells in terms of phenotype, function, and specificity. Lung tissue contains a distinct population of effector memory T-cells expressing canonical markers of Tissue Resident Memory (Trm) T-cells, including CD69 and CD103, which are expanded during active TB disease. These Trm are highly functional, are enriched for IL-17 and IFN-γ secreting cells, and are depleted by HIV co-infection, compared to matched blood samples. In addition, they are highly enriched for TB-specific T-cells, including TB-specific Th17 and regulatory T-cell subsets. Th17 TB-specific T-cells in the lung negatively correlate with plasma cytokine markers of inflammation, suggesting an important role for this subset in human lung in limiting disease severity. Interestingly, although CD103 expressing T-cells produce the highest levels of cytokine by non-specific stimulation, TB-specific T-cells in the lung lack CD103 expression. Finally, detailed phenotyping of lung Trms was conducted using Mass cytometry and a panel of 40 T-cell markers. This confirmed patterns of expression of markers that are consistent with published data, including CXCR3, PD1, KLRG1 and other integrins. The existence of Trms in TB infected lung tissue was also investigated by global TCR sequencing. This revealed a significant clonal expansion of T-cells in the lung tissue compared to peripheral blood, consistent with a non-recirculating tissue resident memory population. When T-cells from blood and lung tissue were pre-sorted by flow cytometry before TCR sequencing, the results clearly demonstrated that the lung tissue is made of up to 20% clonally expanded T-cell clones that do not recirculate. This approach was also used to investigate the potential contribution of non-classical, donor unrestricted T-cells to lung immunity. By flow cytometry and TCR sequencing, we show that unconventional T-cells such as mucosal associated invariant T-cells (MAIT), germline-encoded mycolyl-reactive (GEM) T-cells and γδ T-cells are all depleted in peripheral blood of TB infected individuals compared to healthy controls while iNKT cells are not. However, this is unlikely to be due to recruitment to the site of disease, as no subset was consistently expanded in the lung, GEMs were rarely detected and γδ T-cells were present at a much lower frequency than in matched blood. Indeed, this study demonstrates the presence of unique and expanded γδ T-cells in TB infected lung that are absent in circulation, suggesting some DURTs may also exist as nonrecirculating lung resident memory cells. Analysis of B-cells in individuals with TB revealed skewing of blood B-cell phenotypes in both active and latent TB infection and an enrichment of total B-cells in the lung compared to matched blood. Lung B-cells were mainly of memory B-cell phenotype including plasmablasts, and, like T-cells, were enriched for the expression of the Trm marker CD69, suggesting that B-cells in the lung probably do not recirculate. This potential Trm B-cell population was enriched for B-cell activation markers, CD95 and HLA-DR, suggesting involvement at the site of disease. However, using TB-specific BAITs, the frequency of TB specific B-cells in the lung tissue was found to be lower compared to TB specific B-cells in blood. The functional role of B-cells in TB disease warrants further investigation. These results provide clear evidence of compartmentalization of the adaptive immune responses to Mtb in the human lung, which maybe important when interpreting data from human PBMC for correlates of protection or vaccine response

Protective Potential of Antioxidant Enzymes as Vaccines for Schistosomiasis in a Non-Human Primate Model

Schistosomiasis remains a major cause of morbidity in the world. The challenge today is not so much in the clinical management of individual patients, but rather in population-based control of transmission in endemic areas. Recent large-scale efforts aimed at limiting schistosomiasis have produced limited success. There is an urgent need for complementary approaches, such as vaccines. We demonstrated previously that anti-oxidant enzymes such as Cu-Zn superoxide dismutase (SOD) and glutathione S peroxidase (GPX), when administered as DNA-based vaccines induced significant levels of protection in inbred mice, greater than the target 40% reduction in worm burden compared to controls set as a minimum by the WHO. These results led us to investigate if immunization of non-human primates with antioxidants would stimulate an immune response that could confer protection, as a prelude for human trials. Issues of vaccine toxicity and safety that were difficult to address in mice were also investigated. All baboons in the study were examined clinically throughout the study and no adverse reactions occurred to the immunization. When our outbred baboons were vaccinated with two different formulations of SOD (SmCT-SOD and SmEC-SOD) or one of GPX (SmGPX), they showed a reduction in worm number to varying degrees, when compared with the control group. More pronounced, vaccinated animals showed decreased bloody diarrhea, days of diarrhea and egg excretion (transmission), as well as reduction of eggs in the liver tissue and in the large intestine (pathology) compared to controls. Specific IgG antibodies were present in sera after immunizations and 10 weeks after challenge infection compared to controls. PBMC, mesenteric and inguinal node cells from vaccinated animals proliferated and produced high levels of cytokines and chemokines in response to crude and recombinant antigens compared with controls. These data demonstrate the potential of antioxidants as vaccine candidates

Major TCR Repertoire Perturbation by Immunodominant HLA-B*44:03-Restricted CMV-Specific T Cells

Lack of disease during chronic human cytomegalovirus (CMV) infection depends on the maintenance of a high-frequency CMV-specific T cell response. The composition of the T cell receptor (TCR) repertoire underlying this response remains poorly characterised, especially within African populations in which CMV is endemic from infancy. Here we focus on the immunodominant CD8+ T cell response to the immediate-early 2 (IE-2)-derived epitope NEGVKAAW (NW8) restricted by HLA-B*44:03, a highly prevalent response in African populations, which in some subjects represents >10% of the circulating CD8+ T cells. Using pMHC multimer staining and sorting of NW8-specific T cells, the TCR repertoire raised against NW8 was characterised here using high-throughput sequencing in 20 HLA-B*44:03 subjects. We found that the CD8+ T cell repertoire raised in response to NW8 was highly skewed and featured preferential use of a restricted set of V and J gene segments. Furthermore, as often seen in immunity against ancient viruses like CMV and Epstein-Barr virus (EBV), the response was strongly dominated by identical TCR sequences shared by multiple individuals, or “public” TCRs. Finally, we describe a pair “superdominant” TCR clonotypes, which were germline or nearly germline-encoded and produced at remarkably high frequencies in certain individuals, with a single CMV-specific clonotype representing up to 17% of all CD8+ T cells. Given the magnitude of the NW8 response, we propose that this major skewing of CMV-specific immunity leads to massive perturbations in the overall TCR repertoire in HLA-B*44:03 individuals

Anti-PD-1 immunotherapy leads to tuberculosis reactivation via dysregulation of TNF-alpha

Previously, we developed a 3-dimensional cell culture model of human tuberculosis (TB) and demonstrated its potential to interrogate the host-pathogen interaction (Tezera et al., 2017a). Here, we use the model to investigate mechanisms whereby immune checkpoint therapy for cancer paradoxically activates TB infection. In patients, PD-1 is expressed in Mycobacterium tuberculosis (Mtb)-infected lung tissue but is absent in areas of immunopathology. In the microsphere model, PD-1 ligands are up-regulated by infection, and the PD-1/PD-L1 axis is further induced by hypoxia. Inhibition of PD-1 signalling increases Mtb growth, and augments cytokine secretion. TNF-a is responsible for accelerated Mtb growth, and TNF-a neutralisation reverses augmented Mtb growth caused by anti-PD-1 treatment. In human TB, pulmonary TNF-a immunoreactivity is increased and circulating PD-1 expression negatively correlates with sputum TNF-a concentrations. Together, our findings demonstrate that PD-1 regulates the immune response in TB, and inhibition of PD-1 accelerates Mtb growth via excessive TNF-a secretion.</p

Finding antigens for TB vaccines: the good, the bad and the useless

Prospective, longitudinal clinical studies incorporating high-throughput, single-cell analyses could identify which bacterial antigens to include in TB vaccines — and which to avoid

Recognition and control of Mycobacterium tuberculosis-infected cells: from basics to the clinic: a NIAID/WGNV workshop report 2023

Vaccination is crucial for the control of tuberculosis (TB), and safe, more effective, and accessible vaccines against Mycobacterium tuberculosis (Mtb) infection are critically needed to achieve TB control milestones envisioned in the End TB Strategy. TB vaccine research and development faces numerous challenges including, but not limited to, insufficient knowledge of the most informative antigens to prioritize as potential vaccine candidates, lack of defined correlates of protection, and incomplete knowledge of anatomical and cellular locations of the Mtb-infected cell in vivo, among others. To take stock of the progress, challenges, and opportunities in TB vaccine R&amp;D, the Stop TB Partnership Working Group on New TB Vaccines (WGNV), in partnership with the National Institute of Allergy and Infectious Diseases (NIAID) cohosted a two-day virtual workshop on 13–14 June 2023 with experts from all over the world. In this report, we summarize key themes and discussions from the meeting, highlighting progress and gaps in the TB vaccine research

Performance Evaluation of Lateral Flow Assays for Coronavirus Disease-19 Serology.

The coronavirus disease of 2019 (COVID-19) pandemic, caused by infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has undoubtedly resulted in significant morbidities, mortalities, and economic disruptions across the globe. Affordable and scalable tools to monitor the transmission dynamics of the SARS-CoV-2 virus and the longevity of induced antibodies will be paramount to monitor and control the pandemic as multiple waves continue to rage in many countries. Serologic assays detect humoral responses to the virus, to determine seroprevalence in target populations, or induction of antibodies at the individual level following either natural infection or vaccination. With multiple vaccines rolling out globally, serologic assays to detect anti-SARS-CoV-2 antibodies will be important tools to monitor the development of herd immunity. To address this need, serologic lateral flow assays (LFAs), which can be easily implemented for both population surveillance and home use, will be vital to monitor the evolution of the pandemic and inform containment measures. Such assays are particularly important for monitoring the transmission dynamics and durability of immunity generated by natural infections and vaccination, particularly in resource-limited settings. In this review, we discuss considerations for evaluating the accuracy of these LFAs, their suitability for different use cases, and implementation opportunities