Understanding Tuberculosis and the Immune Response

 

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a significant global health challenge. In response to infection with intracellular bacteria, such as Mtb, both innate and adaptive immune cells elicit a coordinated response dependent on cytokines to facilitate host bacterial clearance. Upon Mtb inhalation, alveolar macrophages and other innate cells surveying the upper respiratory tract serve as the first line of defense, phagocytose Mtb bacteria, and stimulate the production of IL-1β, IL-6, TNF-α, and additional chemokines¹.

 

 

CD4⁺ Th1 cells are a key factor in the adaptive immune response to intracellular bacteria. Importantly, Th1 cells secrete IFN-γ, IL-2, and TNF-α to further recruit inflammatory monocytes and neutrophils to the site of infection and directly enhance the anti-microbial function of macrophages. While CD4⁺ T helper cells have been highlighted as a critical cell type in the host response, the function of cytotoxic CD8⁺ T cells in controlling Mtb infection is not well understood. However, emerging evidence suggests that cytokines such as IFN-γ, granulysin, and granzymes may be important in CD8⁺ T cell responses against Mtb¹.

 

 

Flow cytometry-based LEGENDplex™ assays provide a solution for simultaneous quantification of up to 14 analytes in only 25 µL of sample. Our pre-defined panels offer ready-to-go solutions for monitoring immune responses and cytokine secretion in the context of multiple cell types, diseases, and pathogens in a variety of different biological samples. Whether you're characterizing in vitro immune cell activation or investigating systemic or local tissue immune responses, LEGENDplex panels offer a high-sensitivity, streamlined approach to quantify multiple biomarkers for research applications.

 

 

Role of CD8⁺ T cells in tuberculosis and the immune response

 

In a recent study, researchers used our LEGENDplex assays to profile cytokine release among subsets of CD8⁺ T cells to define their role in controlling Mtb infection and elucidate new targets for the development of host-directed therapies. The authors highlight two populations of polycytotoxic CD8⁺ T cells expressing the natural killer receptors NKG2A or NKG2C, which exhibited unique cytokine expression profiles upon co-culture with Mtb-infected macrophages, suggesting distinct cytotoxic capabilities among CD8⁺ T cell subsets exist in the context of Mtb infection².

 

Measuring cytotoxic molecules and cytokines using LEGENDplex

 

Cytokines and cytotoxic molecule secretion between NKG2A⁺ and NKG2C⁺ expressing CD8⁺ T cell populations was compared using LEGENDplex Human CD8/NK Panel (13-plex). Sorted NKG2A⁺ or NKG2G⁺ expressing CD8⁺ T cells from 14 donors were co-cultured with Mtb-infected macrophages that had been previously incubated with anti-CD3 antibodies. After 24 hours of co-culture, the supernatant was collected and loaded into LEGENDplex plates, and analyte concentrations were calculated and analyzed using our complimentary Data Analysis Software Suite for LEGENDplex.

 

The authors determined that NKG2A⁺ cells released significantly higher levels of IFN-γ, IL-10, granzyme A, granzyme B, and granulysin compared to other subsets while NKG2C⁺ cells exhibited increased production of IL-2 relative to NKG2A^- NKG2C^- CD8⁺ T cells, demonstrating the unique expression profiles across T cell populations. IL-6, IL-4, and soluble Fas concentrations were below the level of detection in all donors².

 

NKG2C⁺ and NKG2A⁺ cytotoxic T cell subsets exhibit distinct cytokine profiles in response to Mtb infection. Mtb-infected macrophages coated with anti-CD3 antibodies were co-cultured with sorted T cells for 24 hours at a 1:1 ratio. The mean of triplicate samples is presented (n=14 donors). LEGENDplex Human CD8/NK Panel (13-plex) was used to assess cytokine production in cell culture supernatant. Creative Commons CC BY 4.0.

 

The authors highlight that polycytotoxic T cells (P-CTL), expressing three cytotoxic effector molecules perforin, granzyme B, and granulysin, significantly inhibited intracellular Mtb growth, suggesting that these cytotoxic molecules are key mediators of Mtb immunity. Therapies aimed at promoting the activation and expansion of P-CTLs and the anti-microbial molecules they secrete show promise as a novel strategy for the treatment of Mtb infection.

 

Immunoassays for Infectious Disease Research

 

We offer several LEGENDplex panels aimed at deciphering human immune responses to M. tuberculosis such as Human Tuberculosis Panel 1 and Human Tuberculosis Panel 2. Investigate cytokine release in response to viral infection with Human Anti-Virus Response Panel V02 and Mouse Anti-Virus Response Panel. We also offer panels focused on relevant cytokines and chemokines for key immune cell types such as Human Macrophage/Microglia Panels and Human T Helper Cytokine Panels Version 2. For individual analytes, we offer a wide range of ELISAs to focus on specific targets involved in the immune response to bacterial and viral pathogens, like IFN-γ, IL-6, and granzyme B. Or dive into single-cell level cytokine secretion and antigen specific T cell responses with ELISpot.

 

ELISpot MAX™ Deluxe IFN-γ assay is an advanced solution for TB research, designed to quantify interferon-gamma secretion at the single-cell level. This high-sensitivity assay empowers researchers to precisely identify and measure antigen-specific T cells, facilitating deeper insights into immune responses against Mycobacterium tuberculosis.

 

 

Assessment of single-cell secretion of Human IFN- γ using ELISpot. Human peripheral blood mononuclear cells (PBMC) at 10⁴ cells/well were incubated for 20 hours in the absence or presence of 1X Cell Activator without Brefeldin A (Cat. No. 423301), 1X Cell Activator with Brefeldin A (Cat. No. 423303), or Ultra-LEAF™ purified anti-human CD3 (Cat. No. 300331). Our Cell Activator cocktail contains PMA and ionomycin at optimized concentrations. Brefeldin A functions to inhibit intracellular protein transport. As a result, it inhibits secretion of cytokines such as IFN-γ.

 

Our reagents and solutions are designed for all aspects of your infectious disease research. For high-throughput detection of cytokines, learn more about Revvity's no-wash immunoassay technologies.

 

Featured LEGENDplex Panel

 

The LEGENDplex Human CD8/NK Panel is a bead-based multiplex assay, utilizing fluorescence-encoded beads intended for use on various flow cytometers. This panel allows simultaneous quantification of 13 human proteins, including IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ, TNF-α, soluble Fas, soluble FasL, Granzyme A, Granzyme B, Perforin, and Granulysin. This panel provides high sensitivities and broad dynamic range. The panel has been validated for use on serum, plasma, and cell culture supernatant samples.

 

This standard curve was generated using the LEGENDplex Human CD8/NK Panel for demonstration purposes only. A standard curve must be run with each assay.

 

Implications for Tuberculosis Research and Treatment

 

The study highlights the importance of P-CTLs, characterized by the co-expression of granzyme B, perforin, and granulysin, in controlling tuberculosis infection. These findings open up exciting possibilities for future tuberculosis research and treatment strategies. The frequency of P-CTL could potentially serve as a biomarker for disease progression, while targeting CD8⁺ NKG2⁺ T lymphocytes, particularly P-CTL, may lead to novel host-directed therapies for severe tuberculosis infections. As we continue to unravel the complex immune response against tuberculosis, this research paves the way for more effective diagnostic tools, treatments, and vaccines.

 

References

 

1. Zhuang, L., Yang, L., Li, L., Ye, Z., Gong, W., 2024. Mycobacterium tuberculosis: immune response, biomarkers, and therapeutic intervention, MedComm, 5, 1. doi: 10.1002/mco2.419.
2. Zumwinkel, M., Chirambo, A., Zähnle, M., Bürger, M., Grieshober, M., Romahn, V., Mwandumba, H., Stenger, S., 2024. Polycytotoxic T cells mediate antimicrobial activity against intracellular Mycobacterium tuberculosis, Infection and Immunity, 93, 1. doi: 10.1128/iai.00297-24.