LA JOLLA, CA – May 7, 2012 – Scientists at The Scripps Research Institute have made an important discovery about the internal programming of B cells, the immune cells that make antibodies against infections. The finding opens the way for the development of vaccines that can work more efficiently and hints at therapies for conditions in which B cells cause harm—such as the autoimmune disease lupus erythymatosus, severe allergies, and B-cell lymphomas.
The discovery reveals that B cells produce special proteins to maintain themselves in a particular functional “class,” even as they lie dormant in the memory-cell state, awaiting a new infection. The class of a B cell determines how its antibodies marshal other components of immunity, and thus how well they can remove a certain type of threat, say bacteria on the skin versus intestinal parasites.
“This is a real breakthrough, in the sense that we now have a much better understanding of how B cell class is regulated, and how we might target that regulatory process in vaccine and drug design,” said Michael McHeyzer-Williams, a Scripps Research professor who was the principal investigator for the study, published in Nature Immunology’s advance online edition on May 6, 2012.
Specialized Infection Fighters
Young, “naïve” B cells begin their careers as infection fighters when they are exposed, in the right way, to pieces of an invading microbe that happen to match their main receptor (the B cell receptor, or BCR). Some then become plasma B cells, and slowly ramp up the active production of antibodies. Others instead become memory B cells, which can lie in wait for years, primed to respond very rapidly and nip in the bud any reinfection.
Either way, as B cells move out of the naïve state, helper T cells secrete chemical signals that typically force the B cells into particular classes. IgG-class B cells are the most common in humans, and are broadly effective against viruses and bacteria. IgA-class B cells are predominantly found on mucosal surfaces such as in the throat and intestines. IgE-class cells and their antibodies protect against intestinal worms and other parasites. Some B cells stay in the default IgM class. The class of a B cell is marked by the type of “stem” it has on its Y-shaped antibodies; this stem, or effector, can mobilize other elements of the immune system, such as inflammatory chemicals, when the antibody binds to an invader.
It had been long assumed that the switching of a B cell to a particular class is the result of a one-time signaling event. “The idea was that the signals that produce this switch don’t persist in B memory cells, for example,” said Nathaniel Wang, a graduate student in the Scripps Research Kellogg School of Science and Technology working in the McHeyzer-Williams laboratory who was first author of the new study.
In the study, Wang, McHeyzer-Williams, and their colleagues tried to determine whether that assumption is true. They knew, for example, that when T cells cause naïve B cells to switch to the IgG2a class, a potent antiviral class, they do so by inducing the production in B cells of a particular protein called T-bet. To clarify T-bet’s role, the researchers engineered transgenic mice whose B cells lack the protein.
Without T-bet, they found, the mouse B cells could not be switched to the IgG2a class, even when presented with all the normal stimuli, and even though other IgG classes could be produced normally—or even in higher amounts. Even more surprisingly, in existing IgG2a memory B cells, the abrupt knockdown of T-bet levels caused the cells to lose their ability to respond to a new infection. In fact, most of the T-bet-deprived memory B cells became undetectable within a few days.
“T-bet turns out to be the central molecule that enforces the IgG2a class in B cells, and if its production stops in IgG2a memory cells, they become dysfunctional and die,” Wang said.
The finding that T-bet has this all-important, ongoing function in IgG2a memory cells suggested that other proteins play analogous roles in other classes of memory B cell. Wang therefore turned to memory B cells of the IgA class, and, with a similar set of experiments, showed that these memory B cells depend on the transcription factor RORα. “It essentially does for IgA memory cells what T-bet does for IgG2a memory cells,” said Wang.
Implications for Science and Medicine
Wang and McHeyzer-Williams and their colleagues are now searching for the proteins that keep other memory B cells healthy and in their classes. But already the work has clarified how memory B cells work. “Until now we haven’t really had a good conceptual framework for the development and maintenance of these cells,” McHeyzer-Williams said.
The findings clearly also have implications for medicine. By supplying a particular class-enforcement protein at the same time that it exposes B cells to microbial proteins, a vaccine could induce a long-term immunity that is heavily weighted towards a desired antibody class. “If you’re designing a vaccine for certain types of virus, for example, you would like to have lots of IgG2a and IgA memory cells,” said McHeyzer-Williams. “So the goal would be to design a chemical adjuvant for the vaccine that drives B cells into those classes.”
Similarly, therapies that knock down class-enforcement signals such as T-bet could usefully reduce or eliminate memory B cells in certain classes. “Some autoimmune, allergic and lymphoma conditions are driven by B cells of a particular class, for example IgE cells in allergies,” said McHeyzer-Williams. “Being able to target just that class of B cell would be an obvious advantage over existing therapies, such as steroids, that knock down large parts of the immune system.”
Other contributors to the paper, “Divergent Transcriptional Programming of Class-Specific B Cell Memory by T-bet and RORα,” were Louise J. McHeyzer-Williams and Shinji L. Okitsu of the McHeyzer-Williams lab; Thomas P. Burris of the Jupiter, Florida campus of Scripps Research, who provided crucial reagents for manipulating RORα levels; and Steven L. Reiner of Columbia University’s College of Physicians and Surgeons, who supplied transgenic mice.
Nathaniel Wang is a CTSA TL-1 scholar in association with the Scripps Translational Science Institute (STSI).
Professor McHeyzer-Williams’s research is funded in part by the National Institutes of Health.
About The Scripps Research Institute
The Scripps Research Institute (TSRI) is one of the world's largest independent, not-for-profit organizations focusing on research in the biomedical sciences. TSRI is internationally recognized for its contributions to science and health, including its role in laying the foundation for new treatments for cancer, rheumatoid arthritis, hemophilia, and other diseases. An institution that evolved from the Scripps Metabolic Clinic founded by philanthropist Ellen Browning Scripps in 1924, the institute now employs about 3,000 people on its campuses in La Jolla, CA, and Jupiter, FL, where its renowned scientists—including three Nobel laureates—work toward their next discoveries. The institute's graduate program, which awards PhD degrees in biology and chemistry, ranks among the top ten of its kind in the nation. For more information, see www.scripps.edu.
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Professor Michael McHeyzer-Williams
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Nathaniel Wang, a CTSA TL-1 scholar in association with the Scripps Translational Science Institute (STSI).
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Confirming Age-Associated B Cells as an Important Cause of Autoimmunity
Most of the better known and more common forms of autoimmune disease are not all that age-related, though incidence for many of them ticks upwards with age as the immune system becomes ever more dysfunctional in later life. There are many more autoimmunities that are age-related, however, mostly comparatively poorly understood, and new ones are discovered on a fairly regular basis. It is fair to say that autoimmunity as a whole is poorly understood, however. The immune system is enormously complex, and it remains to be established as to how exactly it falls into the malfunctioning states that cause it to attack specific tissues, cells, and proteins that it should normally leave alone. It is unlikely that there is any one root cause, but the hope in the research community is that the broad range of quite different autoimmunities do in fact have commonalities, as is the case for cancer. Just as in cancer research, meaningful progress in the medical control of autoimmunity will likely hinge on identification and targeting of mechanisms shared by many or a majority of the diseases in this category.
One of the most promising approaches to autoimmunity is to bypass the investigation of its mechanisms and just destroy the entire population of adult immune cells. The state-related data of the immune system, such as its memory, and including the errors that cause it to attack tissues rather than pathogens, is stored entirely in those cells. Wiping it clean and starting over has been shown to cure multiple sclerosis, for example. Unfortunately this is a fairly risky and damaging process at the moment, given the harsh nature of the high-dose immunosuppressants required, which makes it unsuitable for all but the most dangerous autoimmune conditions. One path forward is to produce better targeted cell-killing technologies, therapies that lack side-effects, and that is certainly a going concern in the biotechnology community. Look at the past decade of work emerging from the cancer research community, for example, or the programmable gene therapy cell destruction approach pioneered by Oisin Biotechnologies. Such a side-effect-free therapy would still leave the patient without a functioning immune system for a period of time, however, which would add considerably to the support needed to make such a treatment safe enough for widespread use, especially in older people.
What if a much smaller population of errant immune cells could be identified and selectively destroyed, however? The autoimmunity could be suppressed or removed without having to purge the entire immune system, and that could possibly be achieved to a good enough degree with existing technologies. That is the promise offered by research into age-associated B cells, a class of dysfunctional immune cell discovered not so many years ago. In the paper and publicity materials noted here, an important role for these cells appears to be confirmed for a range of classes of autoimmunity. This seems to me to be an noteworthy step forward in the field, and opens a number of paths towards forms of effective treatment for autoimmune conditions.
Researchers have identified a trigger for autoimmune diseases such as lupus, Crohn's disease and multiple sclerosis. The findings help explain why women suffer autoimmune disease more frequently than men, and suggest a therapeutic target to prevent autoimmune disease in humans. "Our findings confirm that Age-associated B Cells (ABCs) drive autoimmune disease. We demonstrated that the transcription factor T-bet inside B cells causes ABCs to develop. When we deleted T-bet inside B cells, mice prone to develop autoimmune disease remained healthy. We believe the same process occurs in humans with autoimmune disease, more often in elderly women."
B cells are important players in autoimmune disease. The research team previously identified a subset of B cells that accumulate in autoimmune patients, autoimmune and elderly female mice. They named the cells Age-associated B cells, or ABCs. Subsequent research showed that the transcription factor T-bet plays a crucial role in the appearance of ABC. Transcription factors bind to DNA inside cells and drive the expression of one or several genes. Researchers believe that T-bet appears inside cells when a combination of receptors on B-cell surfaces - TLR7, Interferon-gamma and the B-cell receptor - are stimulated.
Through breeding and genetic techniques the research team eliminated the ability of autoimmune-prone mice to express T-bet inside their B cells. As a result, ABCs did not appear and the mice remained healthy. Kidney damage appeared in 80 percent of mice with T-bet in the B cells and in only 20 percent of T-bet-deficient mice. Seventy-five percent of mice with T-bet in their B cells died by 12 months, while 90 percent of T-bet-deficient mice survived 12 months. "Our findings for the first time show that ABCs are not only associated with autoimmune disease, but actually drive it."
B cells are known to be involved in different aspects of autoimmune diseases and may contribute in a number of ways including the secretion of autoantibodies, processing and presentation of autoantigen to T cells, and production of inflammatory cytokines. Therefore, B cells are promising targets for treatment of autoimmune diseases. Indeed, this idea has been put into practice and B cell depletion therapy has been tested for multiple autoimmune diseases. It is not yet known why B cell depletion is effective for some but not all diseases and for some but not all patients with a particular malady. One possibility is that the depletion therapies might not affect all B cell subsets equally well and different diseases, or different patients, might have involvements of different B cell subsets.
A novel subset of B cells named age-associated B cells (ABCs) has recently been identified by others and ourselves. Unlike other B cells, ABCs express high levels of CD11c and the transcription factor T-bet. T-bet was subsequently demonstrated to be necessary and sufficient for the appearance of this subset, and triggering of the B cell antigen receptor (BCR), IFN-γ receptor (IFN-γR), and TLR7 on B cells induces high levels of T-bet expression. Our previous data demonstrated that T-bet+ ABCs appear in autoimmune patients and in autoimmune-prone mice. These cells produce high amounts of autoantibodies upon stimulation in vitro, suggesting that they are major precursors of autoantibody-secreting cells.
Moreover, our recent findings indicate that ABCs are very potent antigen-presenting cells and therefore might participate in autoimmune responses by presenting self-antigen to autoreactive T cells. In agreement with our findings, a recent study demonstrated elevated levels of T-bet expression in B cells obtained from peripheral blood mononuclear cells of lupus patients when compared with healthy donors, suggesting that T-bet expression in B cells may be critical for the development of lupus in humans. Others have reported that T-bet-expressing B cells are associated with Crohn's disease activity, and an increased expression of T-bet in B cells was found in a patient with MS and celiac disease, altogether suggesting an important role for T-bet-expressing B cells in human autoimmunity.
Therefore, we hypothesized that ablation of ABCs will prevent or delay the development of lupus-like autoimmunity. We tested this hypothesis by conditionally deleting T-bet from B cells in a mouse model of lupus. Our data demonstrate that this deletion leads to reduced kidney pathology, prolonged survival, and delayed appearance of autoantibodies in these mice. Moreover, our data suggest that T-bet expression in B cells is required for the rapid formation of spontaneous germinal centers that develop without purposeful immunization or infection during such autoimmune responses. The results indicate a critical role for T-bet expression in B cells for the generation of efficient autoimmune responses and the development of lupus-like autoimmunity, and suggest that specific targeting of T-bet+ B cells might be a useful therapy for some autoimmune diseases.
Another recent potential option for MS is selective suppression of the T cells that attack Myelin via T regs produced in the lymph nodes.
Just directly inject into a lymph node myelin, some peptide to hold it in the lyphm node, and some rapamycin, and bobs your uncle.
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Win $100,000 to host your own conference. Correction ARTICLE Corrigendum: Characterization of T-bet and Eomes in Peripheral Human Immune Cells
Characterization of T-bet and Eomes in Peripheral Human Immune Cells
by Knox JJ, Cosma GL, Betts MR, McLane LM. Front Immunol (2014) 5:217. doi: 10.3389/fimmu.2014.00217
The corrigendum regards data and text for the final figure of the manuscript, Figure 7:
Subsequent analysis of T-bet levels in human lymphocytes comparing different permeabilization procedures (eBioscience FoxP3 transcription factor kit, BD Pharmingen Cytofix/Cytoperm) has revealed variable findings in the level of T-bet expression detected within certain lymphocyte populations. While this does not change our conclusions for the majority of the populations assessed in this study, B cells in particular show differences under these conditions. Specifically, permeabilization via the eBioscience FoxP3 transcription factor staining buffer set indicates that subpopulations of memory B cells express significantly higher levels of T-bet (MFI) compared to plasmablasts, and that plasmablasts express T-bet only at low levels. Subsequent RNA transcript analysis confirms that plasmablasts express T-bet RNA at a level comparable to naïve B cells. Together, in combination with fluorescence-minus-one and isotype control studies, these new findings suggest that subsets memory B cells, not plasmablasts, express the highest levels of T-bet in the B cell compartment and plasmablasts express T-bet at a lower frequency than is reported in Figure 7.
Figure 7 Legend should read:
(C) Histograms depicting T-bet expression levels in B-cells and NK cells from a representative donor. Histograms represent the following subsets: naïve B-cells (thick black line), memory B-cells (shaded gray), plasmablasts (thin black line), CD56 bright NK cells (gray line), and CD56 dim NK cells (shaded black).
B-cell results section should be titled “T-bet is predominantly expressed in mature memory B-cells” and should read:
While Eomes was undetectable in B-cells (data not shown), we found T-bet in ~10% of B-cells (Figure 7B). This T-bet expression was largely relegated to memory B-cells, with significantly lower amounts observed in transitional/immature B-cells, naïve B-cells, and plasmablasts (Figure 7B). Greater than 15% of memory B-cells expressed T-bet, a significantly higher frequency than that of all other B-cell populations, suggesting that T-bet may play a particularly important role in memory B-cell function.
The discussion related to T-bet expression in plasmablasts should be reconsidered as follows:
Figure 7. T-bet expression in antigen-experienced B-cells. (A) T-bet gating strategy for B-cell populations is shown. Transitional, naïve, memory B-cells, and plasmablasts populations are depicted as a contour plot overlaying a density plot of total B-cells. T-bet + events are gated from a representative donor. (B) The frequency of T-bet + B-cells within B-cell subpopulations is shown. Each symbol represents an individual subject. Statistical differences of interest, as measured using non-parametric Wilcoxon matched, paired two-tailed t tests, are described in the text. *p < 0.04. (C) Histograms depicting T-bet expression levels in B-cells and NK cells from a representative donor. Histograms represent the following subsets: naïve B-cells (thick black line), memory B-cells (shaded gray), plasmablasts (thin black line), CD56 bright NK cells (gray line), and CD56 dim NK cells (shaded black).
We found that T-bet is not significantly expressed in transitional/immature B-cells, naïve B-cells, and plasmablasts, but is highly expressed in subsets of memory-B cells. Reduced frequencies of T-bet expression in plasmablasts indicate a specific role for T-bet at the memory B-cell stage of development, which may no longer be necessary after further differentiation to the plasmablast stage.Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Keywords: T-box transcription factors, T-cells, NK cells, B-cells, T-bet
Citation: Knox JJ, Cosma GL, Betts MR and McLane LM (2016) Corrigendum: Characterization of T-bet and Eomes in Peripheral Human Immune Cells. Front. Immunol. 7:337. doi: 10.3389/fimmu.2016.00337
Received: 10 August 2016; Accepted: 22 August 2016;
Published: 08 September 2016
Copyright: © 2016 Knox, Cosma, Betts and McLane. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
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