BLyS drives affinity maturation

I have found an interesting paper that describes in the very detailed way how the interactions between two specialized subsets of lymphocytes which are germinal center (GC) B cells and follicular helper (FH) T cells may influence the affinity maturation of antibodies. The cooperation of these two subsets is known for the long time. However, the novelty this publication brings about is that it looks specifically at the germinal center reaction and dissects the role that the molecule called BLyS (or interchangeably BAFF) may play in the affinity maturation of antibodies from other tasks fulfilled by the same protein. In other words, BLyS has important functions in many aspects of B cells life and the exact definition of its role in the affinity maturation process was not possible using relatively straightforward methods like studying mice with deletion of gene encoding BLyS.

The link:

Authors perform the study on mice that were immunized with nitrophenacethyl hapten conjugated with chicken gamma globulin which is a widely used method to elicit a strong germinal center reaction. What forms the basis of this paper is the observation that receptor-bound BLyS seems to be selectively excluded form B cells that are located inside germinal centers. Investigators follow with the demonstration that activated B cells downregulate TACI (one of three BLyS receptors), that IL-21 may be responsible for the observed TACI downregulation and finally that in the context of germinal center (and unlike systemically) the main source of BLyS comes from cells of hematopoietic origin, namely follicular helper T cells.

But what role the T cell-derived BLyS may have for the quality of the antibody response? To answer this question authors create an experimental setting (mixed bone marrow chimeras) where the T cell lineage lacks BLyS. In such circumstances germinal centers do form and are maintained mostly normally. However, the ability to form high-affinity antibodies is visibly impaired when germinal centers operate without T-cell derived BLyS.

Radhika Goenka, Andrew H. Matthews, Bochao Zhang, Patrick J. O’Neill, Jean L. Scholz, Thi-Sau Migone, Warren J. Leonard, William Stohl, Uri Hershberg, and Michael P. Cancro (2014). Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation Journal of Experimental Medicine DOI: 10.1084/jem.20130505

On the wild mice and autoimmunity

Only the relatively small part of mammalian genome is formed by protein-coding sequences. The long stretches between these protein-bearing fragments contain other components which could be either non-transcribed regulatory elements or the variety of non-translated RNAs many of which are also taking part in the regulation of gene expression. This intricate network influences the decision whether a given protein is present in the particular physiological situation and how much of it is available. Therefore the issue how proteins are regulated may in fact be equally or more important than the structural differences in coding parts of the protein in question. The problem of transcriptional regulation that affects the autoimmunity development composes the leading theme of the publication I am discussing today. Like the majority of experimental studies this work uses the cornerstone model organism – the mouse Mus musculus. However, unlike the most it provides also some glimpse into the natural populations of rodents.

The link:

The particular problem analyzed by this publication comprises the association between FcγRIIb receptor and the aptitude to develop autoimmune reactions. Authors analyze the populations of wild mice from various part of the globe and find out that the vast majority of them are in the possession of FcγRIIb haplotype that is also present in several laboratory mice known for their propensity to develop the autoimmunity. Such arrangement is of interest because it suggests that in the wild populations such autoimmunity-correlated variant of FcγRIIb may actually be positively selected.

FcγRIIb is the receptor that recognizes the constant portion of an antibody that has switched to the IgG isotype and unlike other receptors that also bind to IgG its ligation on B cells causes the modulation of immune responses. To gain an insight of how FcγRIIb haplotype which is predominantly present in the wild mice may influence the immune response of the classic laboratory strain C57BL/6 (which itself has FcγRIIb haplotype not associated with the autoimmunity) investigators exchange the copies of that receptor by the knock-in approach and study the immune parameters of the resulting strain.

It turns out that the alterations between two haplotypes lie in their transcriptional regulation. In the non-autoimmune setting (as demonstrated by C57B/6 strain) FcγRIIb is upregulated on germinal center B cells whereas the knocked-in variant of this receptor cancels this up-regulation. Additionally, when compared to to C57BL/6 line the novel knock-in strain displays the reduced amount of FcγRIIb on activated B cells, splenic transitional B cells and bone marrow residing pre-B cells. Authors pin down the difference in the DNA sequence that underlies the disparate regulation of two variants and propose the transcription factor which may be responsible for the up-regulation seen when the non-autoimmune haplotype is present. Finally, investigators also show that the two haplotypes in questions differ in qualitative terms that comprise the number of germinal center B cells, affinity maturation and autoimmunity development.

Why do I think that this report is interesting? When you look at the paper conclusions from the broader perspective they seem to confirm the notion that in the natural circumstances the autoimmunity is not a problem. The widespread presence of that particular FcγRIIb haplotype among the wild mice suggests that at least for germinal center B cells the natural selection may have favored the situation where the efficiency of immune reaction is maximized even at the cost of potential collateral damage. Maybe it looks like the obvious notion but it is good to have some hard data that confirm it.

Espeli, M., Clatworthy, M., Bokers, S., Lawlor, K., Cutler, A., Kontgen, F., Lyons, P., & Smith, K. (2012). Analysis of a wild mouse promoter variant reveals a novel role for Fc RIIb in the control of the germinal center and autoimmunity Journal of Experimental Medicine, 209 (12), 2307-2319 DOI: 10.1084/jem.20121752

PAX5 ability to repress BLIMP1 is phosphorylation-dependent

When a B cell starts releasing antibodies on the large scale it undergoes the sequence of deep morphological and physiological changes. It expands its cytoplasm and switches on the machinery that lets it cope with the enhanced protein production in preparation to professional antibody secretion. On the molecular level this transition it governed by the onset in expression of certain transcription factors whereas other transcription factors are being switched off. The main players in the transformation of a B cell to a plasma cell are transcription factors PAX5 and BLIMP1. PAX5 is expressed in all B cells except the plasma cell stage while BLIMP1 holds the position of chief regulator that opens the way to antibody secreting cell phenotype. Additionally, it is known that PAX5 represses BLIMP1 and that this repression can be relieved by signaling through BCR. I have read the publication which examines details of the interactions between PAX5, BLIMP1 and the signaling relay activated by BCR stimulation.

The link:

The report’s leading theme is the analysis how the phosphorylation status of PAX5 may influence its ability to repress BLIMP1 promoter. Initially authors identify two residues in PAX5 sequence that can be phosphorylated by ERK kinase which is a part of cascade that transmits the signal originating from BCR. Since the repression competence of PAX5 may be dictated by its phosphorylation state investigators test whether it is possible to activate Blimp-1 promoter when PAX5 is modified to become unresponsive to ERK-driven phosphorylation. To this end they demonstrate that the BCR activation starts off the signaling cascade which terminates with PAX5 phosphorylation by ERK and that the phosphorylated PAX5 is unable to maintain the repression of BLIMP1 promoter. However, the mutated form of PAX5 with its phosphorylation sites replaced by alanine substitutions continues the transcriptional repression of Blimp-1 regardless of BCR activation. Thus the signal at BCR may initiate the plasmacytic differentiation because it changes PAX5 phosphorylation status.

The discussed publication studies the molecular events only and due to a number of technical limitations (see the discussion chapter in the original paper) it does not attempt to translate its findings to the actual immune response (e.g. whether the same PAX5 modifications may influence plasma cell counts). Why do I think it is interesting, though? The answer is I have just read another paper published by Mark Shlomchik’s group (B Cell Receptor Signal Transduction in the GC Is Short-Circuited by High Phosphatase Activity. Science; 2012. 336: 1178-81). That paper examines the extend of BCR signaling during the germinal center reaction and finds it very limited.

According to authors in GC B cells the phosphorylation level of several components belonging to BCR signaling cascade is diminished compared to non-GC B cells. Such reduction is due to the enhanced phosphatase activity which abolishes signals that could be potentially transmitted downstream when BCR is activated during germinal center reaction. Moreover, the specific to B cells inactivation of SHP-1 phosphatase decreases the GC B cell frequency which indicates that the temporal inhibition of BCR signaling may be actually vital for the proper GC maintenance.

The key findings of these two papers (1) The signaling at BCR may open the way to plasma cell phenotype through phosphorylation cascade which completes with PAX5-mediated relieve of BLIMP1 repression. (2) In GC B cells BCR signaling is limited because of high phosphatase activity make a lot sense when combined together. Germinal centers are anatomical sites where B cells undergo antigen-driven affinity maturation and the GC reaction which is terminated too early would compromise the quality of immune response. It looks like the stimulation at BCR may be able to set off the change in the transcription factor architecture (through the phosphorylation-dependent mechanism) that could promote the immediate plasmacytic differentiation. Therefore the temporal modulation of BCR signaling during GC reaction could be crucial because it would ensure enough time to generate high affinity antibody response. Is this the case? Maybe it is.

Yasuda T, Hayakawa F, Kurahashi S, Sugimoto K, Minami Y, Tomita A, & Naoe T (2012). B cell receptor-ERK1/2 signal cancels PAX5-dependent repression of BLIMP1 through PAX5 phosphorylation: a mechanism of antigen-triggering plasma cell differentiation. Journal of immunology (Baltimore, Md. : 1950), 188 (12), 6127-34 PMID: 22593617

Borrelia burgdorferi – the master manipulator

Who are the most accomplished immunologists in the world? The title may go to several pathogenic organisms that are apparently able to manipulate immune responses and do it in the way that puzzles many researchers. Bacterium Borrelia burgdorferi (the causative agent of Lyme disease) definitely belongs to the elite club. I have learnt that during infection it does not even try to hide away and assumes distinctively bold tactics as it migrates to the very hub of protective action – the draining lymph node. And there it does not sit quietly either since it can cue B cells to what it looks like the unusual (plus yet unexplained) proliferation which probably hinders the quality of ensuing protective response.

The link:

The discussed paper is a continuation of the report which was published by the same group last year (Lymphoadenopathy during lyme borreliosis is caused by spirochete migration-induced specific B cell activation. PLoS Pathog. 7: e1002066). Since I think it is important to combine the information from both papers I am going to summarize shortly the findings of that first publication before moving on to more recent results. Authors observed that when they infected mice with Borrelia using the natural route (tick’s bite) sick animals displayed the substantial enlargement of lymph nodes that were most adjacent to bite locations. In order to control the actual site of infection (ticks are living animals and they can move freely before starting their blood meal) as well as to avoid the direct use of culture-grown bacteria (which may stimulate the different type of immune response than bacteria from infested ticks) investigators have devised a modified infection procedure. Shortly, they injected immunocompromised mice (SCID) with culture-grown Borrelia and transplanted biopsies from such infected animals into the right tarsal joint of naive mice. This innovation has allowed focusing on the single draining lymph node while it exposed animals to host-adapted bacteria.

The particular problem that authors have tackled was how the Borrelia infection altered the right inguinal lymph node and whether there were any further modifications to the lymphatic architecture as the disease progressed. Investigators confirmed the rapid and intense accumulation of B cells in the draining lymph node and also noticed that this accumulation subsequently spread to more distant lymph nodes but not to the spleen. Such ensuing B cell response was critically dependent on the presence of live bacteria inside the lymph node yet quite surprisingly it occurred without any perturbations in the absence of MyD88. Apart from that, authors demonstrated that the immune reaction going on in affected lymph nodes was at least partially specific to Borrelia antigens.

In the follow-up paper researchers attempt to answer the question what is the role of CD4 T cells in the B cell accumulation prompted by Borrelia infection. They find out that CD4 T cells from affected lymph nodes do not increase their numbers as it happens to B cells yet they become activated along the course of disease. Nevertheless, the B cell buildup takes place without CD4 T cells as it did without MyD88. The anti-Borrelia antibody response, however, is weaker when there are no CD4 T cells around.

The overall picture of the immune response to Borrelia in the model that uses host-adapted bacteria (which mimics the natural infection) looks somehow paradoxical and misshapen. First pathogens invade the closest lymph node and seem to provoke there the massive B cell proliferation which disperses later to other lymph nodes. This proliferation is independent of mitogenic cues imparted by TLR signaling and it happens without CD4 T cell-driven costimulation as well. The specific anti-Borrelia antibody response (partially dependent on CD4 T cells) is then switched on but it gives the impression of being not completely normal, too. Authors show that the germinal center induction in lymph nodes is delayed and all germinal centers tend to decline very rapidly. However, plasma cells (which are thought to derive from the germinal center reaction) accumulate with kinetics suggesting that they are not generated in germinal centers located in lymph nodes. Investigators postulate that these plasma cells may originate from ectopic lymphoid tissues.

But it is the initial B cell accumulation that probably distorts the quality of anti-Borrelia immune response. Authors present data showing that this accumulation is indeed able to destroy the inherent organization of an affected lymph node. The question that I have is whether it happens because of sheer number of B cells or maybe through some defined B cell-specific antibody-independent effector mechanism like for example the release of a chemokine that interferes with the layout of a lymph node. Another interesting enigma is how Borrelia targets B cells and what receptor on B cell surface intercepts the signal.

Hastey CJ, Elsner RA, Barthold SW, & Baumgarth N (2012). Delays and diversions mark the development of B cell responses to Borrelia burgdorferi infection. Journal of immunology (Baltimore, Md. : 1950), 188 (11), 5612-22 PMID: 22547698

Germinal centers in pathogenic SIV infection

The characteristic feature of HIV and some SIV infections is the chronic immune activation that probably drives the continuous CD4 T cell depletion and progression towards AIDS. What exactly causes this aberrant activation is not clear; however, traits inherent to the immune response seem to be more responsible than the virus itself. For example, there are data showing that the same strain of virus (SIV) can induce lasting but non-pathogenic infection in one primate species (sooty mangabeys) and devastating disease which resembles AIDS in the other (rhesus macaques). It may be therefore instructive to study details of immune response against SIV in pathogenic conditions. B lymphocytes are not spared from overall activation during HIV/SIV infection – in fact, sick individuals display hypergammaglobulinemia, or elevated levels of IgG antibodies. Switched antibodies are generally produced by cells that derive from the germinal center reaction where B lymphocytes interact with the unique subset of CD4 T cells called follicular helpers (TFH). Follicular helpers can be distinguished from other CD4 T cell subsets by their expression of CXCR5, ICOS, PD-1 and the transcription factor Bcl-6 as well as the high level of IL-21 cytokine secretion. TFH have been shown to be crucial for the successful generation of plasma cell and memory compartments. The recent paper takes a look at germinal centers during SIV infection in monkeys that develop AIDS-like disease.

The link:

Authors use immunohistological staining and flow cytometry to prove that PD-1 positive CD4 T cells (according to what we know TFH population) accumulate in germinal centers along disease progression in rhesus macaques infected with SIVmac239. They also correlate the enhanced PD-1 expression on CD4 T cells with remodeling of B cell populations that reside in lymph nodes (the trend is towards reducing naïve subset and enriching memory subsets) and the increased IgG secretion. Additionally, it is demonstrated that CD8 T cells are excluded from germinal centers (both in naïve and infected monkeys). Investigators conclude that the buildup of TFH population during pathogenic SIV infection may play the important role in shifting B cell activation status. Apart from that they suggest that germinal centers could form anatomical sites of potential viral escape due to the lack of CD8 T cells presence (and alleged insufficient control of infected CD4 T cells there).

The experimental setting of this publication is restricted to recording several immune parameters at time points that represent either acute or chronic SIV infection. However, it is interesting because it attempts the systematic documentation of changes in the immune response that may underlie the pathologic immune activation distinctive for some primate lentiviruses. The obvious question for the future is whether there may be any significant changes in germinal center reaction between species that develop pathogenic or non-pathogenic SIV infection.

Hong, J., Amancha, P., Rogers, K., Ansari, A., & Villinger, F. (2012). Spatial Alterations between CD4+ T Follicular Helper, B, and CD8+ T Cells during Simian Immunodeficiency Virus Infection: T/B Cell Homeostasis, Activation, and Potential Mechanism for Viral Escape The Journal of Immunology, 188 (7), 3247-3256 DOI: 10.4049/jimmunol.1103138

IgE class switching occurs in germinal centers

IgE antibody response is known to accompany infections with helminths. Organisms like Schistosoma mansoni, Nippostrongylus brasiliensis, Heligmosomoides polygyrus or Brugia malayi are able to induce the substantial class switching to IgE and increased levels of IgE serum antibody. IgE antibodies are also associated with allergies, asthma and generally Th2 effector responses. The other murine antibody class connected to Th2 is IgG1. Despite the importance of IgE response not all facts linked to basic IgE biology are entirely clear.  For example, it is not sure whether class-switching to IgE takes place in or outside germinal centers. Another elusive issue is the hypothetical presence of separate IgE memory population. There were some suggestions that IgE class switching needs to go through IgG1 intermediates and that IgE memory is maintained in IgG1 compartment. The recent publication uses IgE-reporter mouse strain to challenge the notion that IgE response occurs exclusively in tight connection with IgG1.

The link:

Authors use the fact that the secreted and membrane forms of antibody are translated from different mRNA splice variants. The IgE reporter strain they employ has the GFP sequence inserted downstream of exon that encodes the cytoplasmic domain of membrane IgE thus connecting the fluorescent signal with the membrane antibody version. Additionally, the IgE locus of reporter mice contains 52-amino acid region (called M1) derived from human antibody. Such approach allows detecting GFP signal as approximation of IgE membrane variant synthesis and subsequently verifying the presence of IgE with antibody against M1.

To confirm the validity of this strain for in vivo studies authors employ in vitro cultures with IgE response promoting factors (anti-CD40 and IL-4). It is established that GFP-positive population can be divided into GFPint and GFPhi subsets. The presence of soluble IgE correlates with appearance of GFPhi cells and only GFPhi fraction stains positively with anti-M1 antibody. Additionally, GFPhi cells express much more of IgE mature transcript than GFPint population which contains numerous cells positive for IgG1 presence. It is therefore concluded that only GFPhi fraction encloses IgE-switched cells.

To monitor how IgE-switched population comes into existence authors use the infection with Nippostrongylus brasiliensis. They aim to detect the germinal center (B220+IgDGL7+CD95+) and memory (B220+IgD+GL7CD38hi) compartments inside GFPhi (and thus IgE-switched) subset by flow cytometry. At the initial phase of infection all GFPhi cells bear features characteristic for germinal centers. At later stages some of them transform into memory cells. The appearance of IgE germinal center and memory populations occurs at the same time as analogous IgG1 subsets. Authors interpret these data as ruling against the possibility of IgG1 transitional stage for IgE-switched cells. Investigators also confirm the functionality of IgE memory population by adoptive transfer strategies. Additionally, the publication contains data about in vivo generation of IgE-secreting plasma cells and visualizations of germinal centers with IgE-switched cells (plus IgE memory and plasma cells) in lymph nodes of infected mice.

My first reaction after reading this paper was that of astonishment. Did they know so little about IgE response not to be sure whether there are IgE-switched cells inside germinal centers? The included data do not show anything very new or unexpected. They just extend basic facts from B cell biology for the IgE population.  Investigators show in the convincing way that IgE memory and plasma compartments derive from germinal center reaction and that memory IgE cells contribute to faster antibody response on re-infection. On the whole this is very interesting stuff which illustrates how many gaps are still in our knowledge.

Talay, O., Yan, D., Brightbill, H., Straney, E., Zhou, M., Ladi, E., Lee, W., Egen, J., Austin, C., Xu, M., & Wu, L. (2012). IgE+ memory B cells and plasma cells generated through a germinal-center pathway Nature Immunology, 13 (4), 396-404 DOI: 10.1038/ni.2256