Analysis of T cell subsets induced in response to Mycobacterium tuberculosis infection
Date
2009
Journal Title
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Abstract
Tuberculosis, an ancient disease, still kills more people each year than does any other bacterial infection. The global epidemic of tuberculosis (TB) results in eight million new tuberculosis cases per year and two million deaths; 98% of these occur in developing countries. At present the only available vaccine against tuberculosis, M. bovis Bacillus Calmette-Guerin (BCG), has proven unreliable and only minimally protects against pulmonary tuberculosis in adults. The reasons why the BCG vaccine is not fully protective are still very unclear, and even though novel vaccines are being developed, there is not a clear understanding of what kind of immune response they should elicit in order to provide maximum protection. The overall aim of these studies, therefore, was to investigate the T cell subsets generated by M. tuberculosis [including clinical strains] as well as further analysis of those generated by BCG, in order to explore their role in protection against M. tuberculosis infection.
I started my thesis work by studying a strain of M. tuberculosis lacking the 19kDa lipoprotein, an important mycobacterial antigen, in order to evaluate if the mutation could alter the capacity of this strain to infect mice in vivo. The results showed that the expression of the 19kDa lipoprotein is essential for the replication of the organism in the lungs of mice, both normal and immunocompromised, and that without it the bacilli persisted as a low grade chronic infection. In a second set of experiments the 19kDa mutant was used as a vaccine and compared to the gold standard BCG. Interestingly, when resistant C57BL/6 mice were vaccinated with the 19kDa mutant similar resistance to aerosol challenge infection was observed compared to the BCG control.
In an attempt to further understand the type of immune responses necessary to generate long lived protection against M. tuberculosis infection, I performed a series of experiments designed to evaluate the types of memory T cells involved in vaccination and challenge infection. C57BL/6 mice were infected by low dose aerosol with M. tuberculosis, and compared to animals BCG vaccinated but not challenged.
The rationale for these studies reflected current information that indicates that there are at least two subsets of memory T cells, which are generated to protect the organism against a second infection caused by the same organism. These two subsets have been designated as effector memory cells (TEM) and central memory cells (TCM). TEM are thought to provide a first line of defense and are localized in peripheral tissues, while TCM represent a second line of defense, and are localized in more central lymphoid tissues including the lymph nodes and the spleen.
My studies showed that BCG vaccinated, as well as chronically infected mice, both established significant populations of CD4 and CD8 T cells in the lungs that have characteristics of TEM cells. In contrast, cells with a phenotype characteristic of TCM represented a far smaller population. Based on these findings, our laboratory is now hypothesizing that the BCG vaccine is a poor inducer of central memory T cells and that defect may be the fundamental reason why the vaccine appears to lose efficacy in children over a period of several years.
I then addressed what might happen to the distribution of these memory T cell subsets if the chronic infection was resolved by chemotherapy. In my studies this experimental approach indicated that the numbers of central memory cells can be increased and that they respond very quickly in the lungs if the mouse is reinfected. This was then followed by a second wave of T cells expressing an effector memory T cell phenotype, and the combination of these two responses clearly rendered the animal highly resistant, in comparison to control mice undergoing primary infection in which the bacterial load by day 30 was 2-3 log higher. Quite unexpectedly however, in the second month of the reinfection the memory T cell subsets significantly declined in numbers, the lungs become highly consolidated by macrophages, and the animals died. These observations challenge the notion that successful chemotherapy renders the individual highly resistant to secondary infection in the longer term. In this model I thus showed that resistance is potent but it is also transient, and propose that this may potentially be explained by rapid transition of the two main memory T cell subsets into secondary effector cells that are short-lived and rapidly lose their protective capacity.
Clinically, the efficacy of BCG seems to be very poor against newly emerging W-Beijing strains such as the highly virulent strain HN878. In my final piece of work I obtained this strain and characterized it in the mouse model. The results of this study showed that the strain HN878 is clearly more virulent for mice than other strains of M. tuberculosis, growing faster and inducing severe lung damage. After 20 days or so the infection is contained and the lung pathology apparently slows, but animals still die more quickly than other virulent strains. Using flow cytometry I was able to demonstrate that mice infected with HN878 induce a potent TH1 [CD4, IFNγ-secreting] response but this is then followed by the emergence of regulatory T cells expressing a CD25hi Foxp3+ phenotype, with many of these cells also staining positive for IL-10. Whether this is a general characteristic of such highly virulent clinical strains is not known, but if so it could have a serious negative impact on the efficacy of new TB vaccines currently being developed.
I started my thesis work by studying a strain of M. tuberculosis lacking the 19kDa lipoprotein, an important mycobacterial antigen, in order to evaluate if the mutation could alter the capacity of this strain to infect mice in vivo. The results showed that the expression of the 19kDa lipoprotein is essential for the replication of the organism in the lungs of mice, both normal and immunocompromised, and that without it the bacilli persisted as a low grade chronic infection. In a second set of experiments the 19kDa mutant was used as a vaccine and compared to the gold standard BCG. Interestingly, when resistant C57BL/6 mice were vaccinated with the 19kDa mutant similar resistance to aerosol challenge infection was observed compared to the BCG control.
In an attempt to further understand the type of immune responses necessary to generate long lived protection against M. tuberculosis infection, I performed a series of experiments designed to evaluate the types of memory T cells involved in vaccination and challenge infection. C57BL/6 mice were infected by low dose aerosol with M. tuberculosis, and compared to animals BCG vaccinated but not challenged.
The rationale for these studies reflected current information that indicates that there are at least two subsets of memory T cells, which are generated to protect the organism against a second infection caused by the same organism. These two subsets have been designated as effector memory cells (TEM) and central memory cells (TCM). TEM are thought to provide a first line of defense and are localized in peripheral tissues, while TCM represent a second line of defense, and are localized in more central lymphoid tissues including the lymph nodes and the spleen.
My studies showed that BCG vaccinated, as well as chronically infected mice, both established significant populations of CD4 and CD8 T cells in the lungs that have characteristics of TEM cells. In contrast, cells with a phenotype characteristic of TCM represented a far smaller population. Based on these findings, our laboratory is now hypothesizing that the BCG vaccine is a poor inducer of central memory T cells and that defect may be the fundamental reason why the vaccine appears to lose efficacy in children over a period of several years.
I then addressed what might happen to the distribution of these memory T cell subsets if the chronic infection was resolved by chemotherapy. In my studies this experimental approach indicated that the numbers of central memory cells can be increased and that they respond very quickly in the lungs if the mouse is reinfected. This was then followed by a second wave of T cells expressing an effector memory T cell phenotype, and the combination of these two responses clearly rendered the animal highly resistant, in comparison to control mice undergoing primary infection in which the bacterial load by day 30 was 2-3 log higher. Quite unexpectedly however, in the second month of the reinfection the memory T cell subsets significantly declined in numbers, the lungs become highly consolidated by macrophages, and the animals died. These observations challenge the notion that successful chemotherapy renders the individual highly resistant to secondary infection in the longer term. In this model I thus showed that resistance is potent but it is also transient, and propose that this may potentially be explained by rapid transition of the two main memory T cell subsets into secondary effector cells that are short-lived and rapidly lose their protective capacity.
Clinically, the efficacy of BCG seems to be very poor against newly emerging W-Beijing strains such as the highly virulent strain HN878. In my final piece of work I obtained this strain and characterized it in the mouse model. The results of this study showed that the strain HN878 is clearly more virulent for mice than other strains of M. tuberculosis, growing faster and inducing severe lung damage. After 20 days or so the infection is contained and the lung pathology apparently slows, but animals still die more quickly than other virulent strains. Using flow cytometry I was able to demonstrate that mice infected with HN878 induce a potent TH1 [CD4, IFNγ-secreting] response but this is then followed by the emergence of regulatory T cells expressing a CD25hi Foxp3+ phenotype, with many of these cells also staining positive for IL-10. Whether this is a general characteristic of such highly virulent clinical strains is not known, but if so it could have a serious negative impact on the efficacy of new TB vaccines currently being developed.
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Subject
Mycobacterium tuberculosis
T cell subsets
vaccines
microbiology
immunology