Today, some basic basic science. The August 1 issue of the Journal of Infectious Diseases has two interesting articles which use mice to investigate the pathogenesis of TB, hence, the John Steinbeck title. Hopefully we’ll all learn something along the way!
The first article, written by researchers from Seattle, is entitled “Interferon γ and Tumor Necrosis Factor Are Not Essential Parameters of CD4+ T-Cell Responses for Vaccine Control of Tuberculosis.” Mice and cytokines, yikes (eyes glazing over)– I definitely didn’t take “Mouse Immunology 101″ during medical school.
So the world desperately needs a new TB vaccine. The BCG vaccine was developed by Albert Calmette and Camille Guérin almost 100 years ago. It has some efficacy and is still used widely but clearly isn’t good enough to stop TB transmission.
In addition, MDR-TB and XDR-TB are a growing threat, as I’ve described elsewhere on this blog. Unfortunately, a 2013 Lancet clinical trial of the MVA85A vaccine in South African infants did not show efficacy against TB. That MVA85A vaccine was a subunit boosting vaccine and was designed to enhance whatever protection is already provided by BCG, but it didn’t work.
Here’s the “big picture” as I understand it. The adaptive immune system evolved to protect us from microbes. CD4+ T cells mostly act by producing cytokines that communicate with other cells of the innate or adaptive immune systems. CD4+ T cells can be subdivided into several subgroups based on the cytokines they produce (i.e. TH1, TH2, and TH17).
CD4+ T cells, interferon γ (IFN-γ), and tumor necrosis factor (TNF) are thought to be essential for the control of TB. Recall that activated antigen presenting cells (APCs) make cytokines that influence the type of T helper cell that is produced. For example, in the figure below, an APC gets this CD4+ T cell jazzed up to become an effector T cell (Th1 cell) and that pumps out IFN-γ. Then, the macrophage becomes activated and gets better at killing the bacteria. IFN-γ also stimulates macrophages to produce more IL-12, which in turn potentiates TH1 cell development (setting up a positive feedback loop). But TB is one tough cookie. After infecting the macrophages, it is thought to kill them, leading to caseous necrosis (more on this later).
IFN-γ is essential to prevent progressive, fatal infection with TB. Clinically, we frequently use interferon gamma release assays (IGRAS), such as TSPOT and Quantiferon TB Gold, in the place of skin testing (PPD/TST). IGRAS measure T cell release of IFN-γ following stimulation by antigens unique to TB.
What about TNF? It’s another cytokine that is synthesized by activated macrophages and T cells and serves to activate other cells in the immune system. But TNF blockers (like infliximab) cause can cause people with latent TB to reactivate and develop active TB. I’ve seen it happen and it isn’t pretty.
Ok, back to the first JID article. The activity of TH1 cells that simultaneously produce IFN-γ and TNF has been proposed as a candidate mechanism of vaccine efficacy. But the failed MVA85A vaccine trial means we need to look more closely at this. So the authors used a mouse model of T-cell transfer and aerosolized TB infection to assess the contributions of TNF and IFN-γ to vaccine efficacy. To do that, they gave mice a vaccine called “ID93+GLA-SE” (who knew you could vaccinate mice for TB!) The ID93 vaccine apparently elicits a high frequency of multifunctional TH1 cells and reduces pulmonary TB by approximately 90% in vaccinated mice (i.e. it limits TB infection). After immunization with the ID93 vaccine, the researchers isolated T cells from the donor mice and transferred them intravenously into naive, uninfected recipients. Then, mice were infected with M. tuberculosis H37Rv and assessed for bacterial burdens.
But what is H37Rv? It is a strain of tuberculosis which was originally isolated from human lungs in 1905 by Dr. Edward R. Baldwin. H37 originally gained attention for its virulence in the guinea pig model. Nowadays, you can purchase H37Rv from an organization called ATCC which is located in Manassas, Virginia. 110 years later, it is still used widely in scientific experiments. More on H37Rv later.
Now on to results from the paper. As seen in this graph below, mice were not protected by vaccination with ID93, indicating that CD4+ T cells are necessary to transfer protection against aerosolized TB. However, neither CD4+ T cell–produced TNF nor host cell responsiveness to IFN-γ were necessary for protection. The authors’ conclusion is that induction of TH1 cells that coexpress IFN-γ and TNF is not a requirement for vaccine efficacy against TB. However, IFN-γ and TNF are essential for control of TB in nonvaccinated animals.
What did I take away from this first paper, besides that fact that I clearly need to take “Mouse Immunology 101″ and we aren’t getting an effective TB vaccine anytime soon? I learned that CD4+ T cells are necessary and sufficient for vaccine efficacy. But IFN-γ and TNF are not needed for vaccine-elicited control of aerosolized TB. Therefore, induction of TH1 cells is not needed for the generation of protective immunity against TB by vaccination. More basic science studies are needed before additional TB vaccine trials are launched.
Now, on to the second JID study, by researchers in the UK, entitled “The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis.” This study I found a lot easier to follow, perhaps because their article was full of color figures and photos (not just black and white bar graphs)! The British researchers are questioning a central tenet of TB pathogenesis, that caseous necrosis leads to extracellular matrix destruction (Figure A, below). When we say “caseous” necrosis, we mean cheeselike (human tissue, destroyed by TB, looks like cheese). In this set of experiments, the researchers infected mice with TB (the H37Rv strain used in the Seattle article and a more recently isolated strain of TB). They concluded that collagen / extracellular matrix destruction happens first, leading to cell death and caseous necrosis later (see Figure B).
Surprisingly, the British researchers found that the H37Rv strain of tuberculosis did not cause caseous necrosis or formation of multinucleate giant cells in infected mice. However, these pathologies were observed after infection with a recently isolated clinical strain of M. tuberculosis. The authors write:
“This implies that the prolonged laboratory culture of H37Rv since its isolation from a patient in 1905 has resulted in loss of currently unidentified factors that cause giant cell formation and caseous necrosis despite being able to proliferate rapidly.”
I’m certainly not a basic scientist, but I would like to ask the Seattle researchers what they think of this. Given that the H37Rv strain may have changed significantly since 1905, is it appropriate to have been used in the Seattle study?
Dr. Tim Lahey, an infectious diseases doctor at Dartmouth responded on Twitter, “lab adaptation is a valid concern. Even greater is that mice don’t develop latency so model applicability to us is uncertain.”