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: http://jem.rupress.org/content/211/1/45.abstract

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

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Memory CD4 T cells and the neonatal gut

I have found a short paper on the potential mechanism of how HIV virus may be transmitted between mother and child. I think it is interesting because it not only provides the information which may be useful for a given pathology but it also poses some questions as to the basic immunology processes. The main theme of the paper is the quest for HIV targets among neonatal CD4 T cells. As it is known the virus tends to infect memory CD4 T cells but these cells are practically absent in the cord blood. Thus authors inspect neonatal CD4 T populations from various anatomical compartments and find that CD4 T cells bearing a memory marker and HIV co-receptor abound at the intestinal mucosa.

The link: http://bloodjournal.hematologylibrary.org/content/120/22/4383.abstract

CD4 T cells collected for this study derive from children born to healthy mothers therefore this report asks only about the potential mechanism of mother to child transmission. Authors follow CD4 T cells that bear also CD45RO (which is a marker of memory state) and CD5 (HIV uses this molecule as a co-receptor to infect an individual cell – only CD5-tropic strains tend to become transmitted form mother to child). The main conclusion of this publication is that the population of CD4+CD45RO+CD5+cells (the potential HIV target according to the current state of knowledge) exists at the neonatal gut mucosa but not in the lymph nodes, spleen or blood. Additionally, around half of this intestinal memory CD4+CD45RO+CD5population appears to be differentiated into Th17 phenotype since these cells express RORγt transcription factor and CCR6.  In an in vitro experiment investigators also show that neonatal CD4 T cells from the gut are more susceptible to HIV infection than CD4 T cells from the lymph nodes or blood.

Based on obtained data authors propose a model of how HIV gets transmitted from mother to child. According to them the virus may take the oral route of transmition by the ingestion of infected body fluids during the delivery or milk shortly afterwards. I lack the clinical knowledge to critically evaluate such proposal. But I have more basic question instead. This paper not only shows the presence of memory CD4 T cells population at the neonatal gut mucosa but it also provides the evidence that these memory cells underwent substantial clonal expansion that must have happened in utero. I would like to know more details on the nature of antigenic challenge that underlies such prenatal activation of the adaptive immune system.

Bunders MJ, van der Loos CM, Klarenbeek PL, van Hamme JL, Boer K, Wilde JC, de Vries N, van Lier RA, Kootstra N, Pals ST, & Kuijpers TW (2012). Memory CD4+CCR5+ T cells are abundantly present in the gut of newborn infants to facilitate mother-to-child transmission of HIV-1. Blood, 120 (22), 4383-90 PMID: 23033270

First prime and then pull – the novel immunization approach

Some areas of our body enjoy a special status as far as the immune reaction is concerned. Anatomical entities like the gut or female genital tract as well as other mucosal surfaces do not support the same extend of protective response compared to many non-mucosal tissues. This exclusion is crucial to avoid the unwanted inflammation in places that are regularly exposed to the outer environment but sometimes it may present a problem when there is the need to elicit the strong protective response at such privileged site. I have found an interesting report which applies the novel vaccination strategy aimed to enhance the protection against herpes simplex virus 2 which being the virus transmitted through the contact with infected body fluids often enters the body through the genital organs. The innovation that this report introduces consists of double treatment (“prime and pull”) which bypasses the restrictive entry of memory T cells into the vaginal mucosa.

The link: http://www.nature.com/nature/journal/v491/n7424/full/nature11522.html

The mentioned “prime and pull” strategy is the subcutaneous immunization with an attenuated strain of HSV-2 (prime) which is followed by the topical application of chemokines CXCL9 and CXCL10 to the vaginal mucosa (pull). Authors follow the localization of CD8 T cells that recognize an epitope within one of HSV-2 glycoproteins and activated CD4 T cells to show that the distal immunization event plus the localized chemokine treatment provokes the significant recruitment of activated lymphocytes to the vagina whereas the immunization alone has much weaker effect. Interestingly, this recruitment is specific to CD4 and CD8 lymphocytes and does not encompass other cell types that express the relevant chemokine receptor CXCR3.

Is the “prime and pull” approach able to provide the longstanding and reliable protection? Data demonstrate that CD8 T cells (but not CD4 T cells) are retained at vaginal mucosa after the primary response period is over. Most importantly the “prime and pull” treatment may be indeed superior in enforcing the better protective immunity to HSV-2 challenge than the immunization alone. Investigators also ask about the mechanism by which the protection is delivered by the “prime and pull” strategy. It appears that this application can prevent the virus from entering the nervous system where HSV-2 propagates past the mucosal stage of infection.

What will be the future of “prime and pull”, though? The pros are obvious – there is the simple method to enhance the mucosal migration of protective lymphocytes without the “ugly face” of immunity which in this case would be the excessive inflammation at the sensitive anatomical location. Authors speculate about the future applications ranging from HIV protection to solid tumors treatment. The method itself may also be developed as in the discussed paper it provides the optimal protection only in conjunction with the adoptive transfer of virus-specific lymphocytes. The “pull” works as well with the endogenous population of CD8 T cells; however, the protection is suboptimal in such scenario. I will follow this story.

Shin H, & Iwasaki A (2012). A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature, 491 (7424), 463-467 PMID: 23075848

The own versus foreign microbiota

It was long known that the absence of the gut microbiota impairs the full functionality of mammalian immune system. However, it appears that the immune system may require the species-specific microbiota not just any microbiota to develop its proper responses as the recent publication indicates. I think that this report has important implications both for the better understanding of principal immune events as well as for predicted and much expected innovative research applications like the advent of experimental animals with humanized microbiota.

The link: http://www.cell.com/abstract/S0092-8674(12)00629-0

In the course of this research authors colonize germ-free mice (it means – without any microbiota) with intestinal bacteria that are derived from several sources. Two main of these sources are murine or human fecal samples. Additionally, some experimental mice are also provided with rat microbiota. Summarizing the applied methodology, the starting fecal material (murine, human or rat) serves to prepare a respective probe that provides the formerly germ-free mice with commensal bacteria which differ by the species origin.  Using such model the publication answers two outstanding questions. The first analyzes how different species microbiotas are accommodated inside the murine intestinal tract by looking what is the difference between the original colonization sample and the established microbiota. The second attempts to find out what could be the influence of incongruent microbiota (in this case human or rat-derived) for the development of murine immune responses that are known to be affected by commensal bacteria.

Obviously, humans and mice harbor different microbiotas and data obtained by investigators reflect this simple fact as species identification among two different experimental microbiotas (murine or human-derived) reveals quite dissimilar results. But what is really interesting involves how, or maybe rather to what extend human-derived commensal bacteria could be maintained inside the murine gastrointestinal tract. It appears that recipients of human microbiota demonstrate a period of instability to their intestinal bacterial community after which a constant state is achieved. However, the final microbiota of such mice differs remarkably from the original sample. This is not the case for animals that received murine microbiota. Thus human intestinal commensals cannot be maintained in mice in their entirety. Additional and important piece of information is that Firmicutes may contain the bulk of bacterial species that are specific to humans and unstable in mice.

What is even more striking – mice colonized with human microbiota resemble germ-free mice in many immune parameters that are normally influenced by the presence of commensal bacteria. Studying such mice investigators document many changes in the immune structures of the small intestine such as smaller number of T cells in the lamina propria, less αβ CD4 T cells in the intraepithelial compartment, smaller Peyer’s patches and less T cells inside Peyer’s patches. The large intestine of mice with human microbiota is also affected but in quite contrasting way since it holds less γδ T cells in the intraepithelium but there are no other changes. Peripheral immune organs like spleen or brachial lymph nodes seem to be not altered by the change in microbiota origin. As an additional argument for the need of species-specific microbiota in the proper development of mucosal immune responses authors provide germ-free mice with rat-derived microbiota and observe similar disfuntionalities of the intestinal immune system as in the case of human microbiome transfer.

When it comes to the mechanism responsible for the impaired accumulation of lymphocytes at intestinal sites in mice with humanized microbiota it looks like it is the proliferation in Peyer’s patches and mesenteric lymph nodes that may be hold accountable. On the other hand the gut homing ability seems to not be affected by the heterologous microbiota transfer. Among other results that this publication contains the observation that the colonization with different species microbiota causes different bias in T cell effector phenotypes compared to the colonization with homologous microbiota definitely merits the further attention. Authors come to such conclusion after performing the detailed transcriptional analysis of CD4 T cells from lamina propria isolated from mice that were given the transfer of either murine or human commensal bacteria.

The commentary that I would like to make concerns the differential ability of murine or human microbiotas to stimulate the proliferation of CD4 T cells at mucosal sites. CD4 T cells proliferate as the response to the antigen stimulation and since they bear anticipatory receptors (the true cornerstone of adaptive immunity) the obvious question that comes to mind is why the origin of microbiota matters that much. I do not have explanation for this unexpected result and authors also do not offer a definitive answer, although they make some intelligent guesses as to the potential reason why heterologous microbiota fail to stimulate CD4 T cells proliferation to the same extend as murine commensals (impaired antigen uptake, decreased ability to penetrate mucus layer). An interesting observation is that the host epithelium seems to be more proficient at detecting host-specific than foreign bacteria. Perhaps the stratification by mucus layer is not as stringent in the case of certain host-specific bacterial species.

My last remark touches more practical thing. I have read recently a number of eloquently written review articles that postulated the need to engineer experimental mice with humanized microbiota as the exciting models to study microbiota-influenced diseases like inflammatory bowel disease or metabolic syndrome. But in the light of data that this publication presents the generation of such models looks more complicated than it was thought before. Obviously it is pertinent now that these findings be revisited by other laboratories. The following studies may confirm, widen or even contradict the conclusion presented in the discussed paper. However, maybe we assumed just too much and did not take into account the deep symbiotic relationship between host and its specific commensal bacteria that may be very difficult to recapitulate in a heterologous model.

Chung H, Pamp SJ, Hill JA, Surana NK, Edelman SM, Troy EB, Reading NC, Villablanca EJ, Wang S, Mora JR, Umesaki Y, Mathis D, Benoist C, Relman DA, & Kasper DL (2012). Gut immune maturation depends on colonization with a host-specific microbiota. Cell, 149 (7), 1578-93 PMID: 22726443

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: http://www.jimmunol.org/content/188/11/5612.abstract

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

The developmental diversion of thymocytes

I like reports that make me learn something new and appreciate novel developments leading to more integral view of immunological concepts. My understanding of the thymic central tolerance process was that thymocytes receiving strong signals from tissue antigens through TCR undergo invariably the clonal deletion. But it looks like the clonal deletion of cells that can develop into potentially autoreactive T lymphocytes is not the only way which exists for such population in the thymus. I have read the paper that suggests that there may be actually two outcomes for thymocytes with self-reactive TCR – first is the clonal deletion whereas second the developmental diversion.

The link: http://www.nature.com/ni/journal/v13/n6/abs/ni.2292.html

What is the developmental diversion, though? According to authors it is a process that happens when a thymocyte gets the signal through its autoreactive TCR but is not able to receive the costimulation with CD28 molecule. In such case it can enter a pool of DN cells (double negative for CD4 and CD8) and turn up in the intestinal epithelium where it re-expresses CD8 (in its αα form). Cells derived from the developmental diversion are anergic and when the clonal deletion is impaired (as for example in CD28 knockout mice) the efficiency of central tolerance is not reduced because autoreactive thymocytes have an substitute pathway that sequesters them from harmful and self-reactive mature population.

How the the developmental diversion was detected? The publication contains a lot of data, so I will focus on most crucial evidence. The initial observation made by investigators was that CD28 knockout mice (and also B7 double knockout with no CD80 and CD86 which are CD28 ligands) has unusually numerous population of DN thymocytes that express TCRαβ. In normal mice DN thymocytes are in their majority TCRαβ-negative. The DN population from mice deleted for CD28 contains also the similar proportion of autoreactive TCRs as pre-selection DP (double positive) thymocytes but mature C4 or CD8 T cells from the same strain are mostly deprived of self-reactive rearrangement. Therefore authors conclude that the clonal deletion of autoreactive thymocytes requires CD28 costimulation and in its absence such cells are diverted into the alternative developmental way.

Investigators follow this phenomenon by studying at what exact stage of thymocyte development the diversion may occur and what happens with diverted thymocytes once they leave the thymus (they end up in the intestinal epithelium as already has been remarked). The most interesting thing, however, is that the developmental diversion seems to take place in normal mice as well as TCRαβ+CD8ααintraepithelial lymphocytes from the wild type strain are enriched for autoreactive specificities. I definitely need to start following this story.

Pobezinsky LA, Angelov GS, Tai X, Jeurling S, Van Laethem F, Feigenbaum L, Park JH, & Singer A (2012). Clonal deletion and the fate of autoreactive thymocytes that survive negative selection. Nature immunology, 13 (6), 569-78 PMID: 22544394

B cells can secrete IL-6 and drive Th17 response in autoimmunity

The main task of B cells is to release protective immunoglobulins. Yet it is not their only role since they are apparently capable to take on the diverse array of activities that do not directly form a part of effector humoral responses. Instead of just secreting antibodies B cells can influence the outcome of an immune response by dictating the behavior of other cell types. It appears that such mechanism may underlie the development of autoimmunity in the central nervous system. I have found the publication that by combining the work on a mouse experimental model and the analysis of human patients presents the compelling evidence pointing to how B cells stimulate CD4 T cells into the pathogenic phenotype through the antibody-independent action.

The link: http://jem.rupress.org/content/209/5/1001.abstract

The aim of the paper is to seek a clarification for the unexplained outcomes of various B cell depletion treatments during the course of multiple sclerosis or its mouse model – EAE. For example, the targeted reduction of antibody-secreting plasma cells results in the worsening of disease symptoms. Similarly, the broad B cell depletion leads to the improvement that precedes the drop in the level of autoantibodies. To understand whether B cells may have an additional antibody-independent role in the pathogenesis of multiple sclerosis or EAE investigators focus on IL-6 which is a pro-inflammatory cytokine and the essential factor in autoimmune conditions that afflict the central nervous system. They analyze how B cells can contribute to IL-6 secretion in the context of autoimmunity and assess if B cell-derived IL-6 may influence the conduct of CD4 T cells which are the main factor in multiple sclerosis/EAE development.

Authors show that B cells have the inherent ability to secrete IL-6 when they are stimulated with ligands engaging innate receptors (LPS or CpG plus anti-CD40 antibody) and attempt to prove that such B cells’ capacity may have the physiological importance.  To this end they demonstrate that B cells collected from mice that received EAE-driving immunizations are enriched for IL-6 mRNA compared to B cells from healthy mice. Additionally, the abrogation of IL-6 expression in B cells blunts the severity of neurological symptoms typical to EAE. The final proof indicating for the antibody-independent role of B cells in murine EAE pathogenesis comes from BCDT (B cell depletion therapy) experiments as such treatment is effective in alleviating the EAE-resulting damage only when B cells are competent to release IL-6. On the other hand, knocking out IL-6 expression in B cells does not influence their capacity to secrete antibodies. Investigators also pinpoint that marginal zone B cells are the subset which is most proficient in IL-6 release.

IL-6 is a cytokine involved in the formation of Th17 subset which is known for its participation in pathogenic responses pertinent to the autoimmunity driven by CD4 T cells. Because EAE stands as a representative such disorder, authors ask whether B cell-derived IL-6 could impact the development of Th17 population in the course of this disease. They demonstrate that it is indeed the case since the ablation of IL-6 expression in B cells is able to diminish the propagation of Th17 response during EAE. Remarkably, the B cell-derived IL-6/Th17 axis is operative regardless of antigen specificity as the Th17 population is also less numerous following immunizations with EAE-irrelevant OVA peptide. In the last part of paper devoted to human patients investigators show that the ability of B cells to control Th17 development through IL-6 release may be conserved across mammalian species.

The information that this report contains is obviously important because of its practical value but it also stimulates to ask broader questions. Why we are equipped with signaling pathways like the one described in the discussed report? To what end the apparently pathogenic (in the context of autoimmunity) B cell-derived IL-6/Th17 axis has evolved? Why B cells are so prone to act as autoimmunity mediators when stimulated with TLR ligands (especially nucleic acid-recognizing ligands)? The case of lupus and now that of multiple sclerosis have provided enough evidence for such ostensibly rebellious nature of B cells. Could potentially pathogenic pathways starting with the recognition of TLR ligands by B cells represent an evolutionary trade-off with the better control of gut commensal bacteria as an asset and the danger of autoimmunity as a liability? As a matter of fact MyD88 signaling in B cells has been shown to take part in accommodating the intestinal microbiota by preventing their systemic spread when colonic injury occurs (B Cell-Intrinsic MyD88 Signaling Prevents the Lethal Dissemination of Commensal Bacteria during Colonic Damage: Immunity. 2012; 36 (2): 228-38).

I have one more remark concerning the data that this paper presents. Authors show that CpG (TLR9 ligand) is more efficient that LPS (TLR4 ligand) in driving IL-6 secretion by B cells. Nucleic acid-recognizing TLRs are unusual because they are hidden from the cell surface to endolysosomal compartments. Apart from that, recent publications by Gregory Barton’s group reveal that TLR9, TLR7 and TLR3 (expressed by a macrophage cell line) require proteolytic processing prior to becoming functional detectors. Such feature is interpreted as an additional safety measure since ligands for these receptors are expressed both by host cells and pathogens. I am not really sure if it makes sense but nobody checked if the similar requirement for proteolytic cleavage exists in B cells.

Barr TA, Shen P, Brown S, Lampropoulou V, Roch T, Lawrie S, Fan B, O’Connor RA, Anderton SM, Bar-Or A, Fillatreau S, & Gray D (2012). B cell depletion therapy ameliorates autoimmune disease through ablation of IL-6-producing B cells. The Journal of experimental medicine, 209 (5), 1001-10 PMID: 22547654

The effect of MyD88 deletion on autoimmunity driven by Foxp3 inactivation

Over last 10 years few subjects in immunology have received more attention than regulatory CD4 T cells called in abbreviation Tregs. Tregs are considered to have the potent suppression activity over adaptive immune responses and their lack may result in the autoimmunity development. The central trait pertinent to Tregs is the expression of the transcription factor Foxp3.  The essential role of Foxp3 in Tregs’ life is underscored by the fact that Foxp3-deficient mice or human patients with mutations in the respective gene acquire massive systemic autoimmunity due to the absence of Tregs and generally do not fare well. The publication I have found adds yet another twist to Tregs and Foxp3 story. It turns out that the concurrent to Foxp3 deletion of MyD88 (the crucial adaptor protein linking the innate recognition of microbial signature patterns to the expression of genes involved in defense mechanisms) imparts effects that are not identical between major environmental surfaces (skin, gastrointestinal tract or lungs) and the systemic compartments.

The link: http://www.jci.org/articles/view/40591

Foxp3-deficient mice suffer from the advanced inflammatory skin condition and as a result have grossly increased skin pathology indicators like dryness, loss of hair and bleeding. Apart from that their ears and tails are seriously necrotized. However, animals deleted for both Foxp3 and MyD88 show many substantial improvements. Authors demonstrate that the removal of MyD88 from Foxp3-deficient background diminishes immune infiltration to the epidermis and locally deactivates molecular pathways involved in the amplification of inflammatory signals and cellular trafficking (NF-κB translocation to the nucleus, the expression of ICAM-1 on keratinocytes). Additionally, the skin level of numerous cytokines is reduced in doubly deficient animals compared to mice with the single Foxp3 deletion.

What is remarkable, MyD88 deletion on Foxp3-deficient background has also significant systemic effects because such mice grow to much bigger size than visibly runted Foxp3 single mutants. Therefore investigators analyze the extend of immune infiltration in multiple organs of double Foxp3/MyD88 mutants and find out that they have decreased inflammation scores and the expression of pro-inflammatory cytokines not only in the skin but in the small intestine and lungs as well. However, the alleviating consequences of MyD88 removal are restricted to environmental surfaces as the symptoms characteristic for Foxp3 deletion continue unabated in the liver and the pancreas of Foxp3/MyD88-deficient animals (and are even enhanced in their salivary glands). Moreover, the detailed examination of spleen and lymph nodes (authors indicate that they focus on skin draining lymph nodes and mesenteric lymph nodes) shows that cellular counts, proliferation indicators and the expression of various cytokines are elevated in Foxp3/MyD88-deficient mice compared to animals with Foxp3 deletion.

Such difference between the systemic compartments and environmental surfaces could be explained by several factors. Authors show that introducing MyD88 deletion to Foxp3-deficient background disrupts the chemokine gradient between lymph nodes and effector tissues. They also demonstrate that homing ability of CD4 T cells to lungs is incapacitated and as a consequence lymphocytes may accumulate in draining lymph nodes. Finally, in a series of adoptive transfer experiments it is established that the protective effect of MyD88 deletion acts at the level of target tissue and is independent on whether CD4 T cells express MyD88.

In an interesting, although not entirely conclusive part of the paper authors follow the hypothesis that the protective influence of MyD88 deletion in this model may be due to the removal of capability to process activation signals derived from microbiota. To prove such concept they attempt to mimic the effect of MyD88 ablation by purging commensal bacteria from gastrointestinal tracts of Foxp3-deficient animals with two different antibiotic treatments. The first such treatment includes two antibiotics (doxycycline and cotrimoxazole) and indeed relieves some symptoms of Foxp3 inactivation in the skin and lungs. However, the second regimen comprising four antibiotics (kanamycin, vancomycin, metronidizol, and amphotercin-B) actually worsens the state of Foxp3-deficient animals and accelerates their death. I would be interesting to know what part of commensal microflora can be hold responsible for either such protective or detrimental effects in the context of ongoing autoimmunity.

Rivas MN, Koh YT, Chen A, Nguyen A, Lee YH, Lawson G, & Chatila TA (2012). MyD88 is critically involved in immune tolerance breakdown at environmental interfaces of Foxp3-deficient mice. The Journal of clinical investigation, 122 (5), 1933-47 PMID: 22466646

IL-21 reporter mouse shines more light on follicular helpers

Follicular helpers are the special population of CD4 T cells that localize to germinal centers and are involved in enhancing the humoral response. They are distinguishable from other CD4 T cell subsets by their joint expression of CXCR5, ICOS, PD-1 and the transcription factor Bcl-6 as well as the capacity to secrete IL-21 cytokine. The recent paper describes a novel model to study the biology of TFH – the IL-21 reporter mouse. Such mouse has a GFP encoding cassette knocked-in into IL-21 locus to create the animal that expresses simultaneously IL-21 and GFP. IL-21 is not a cytokine characteristic exclusively for TFH but in conditions applied by this study the GFP presence in secondary lymphoid organs is restricted to activated CD4 T cells that are positive for CXCR5 and PD-1 thus being follicular helpers according to the accepted definition. The ability to track TFH population in vivo allows answering questions concerning their fate after the primary immune response is over and germinal centers undergo resolution.

The link: http://www.nature.com/ni/journal/v13/n5/full/ni.2261.html

There are two main contributions that this paper adds to the existing knowledge of TFH. First, it dissects follicular helpers into IL-21 secreting and IL-21 negative populations and characterizes both subsets. Second, it analyzes the ability of TFH to form the memory compartment and participate in recall responses. In order to track IL-21 secreting cells among TFH authors follow general follicular helper population (defined as CD4+CD44+CD62LCXCR5+PD-1+cells) after the immunization with NP-KLH (a T cell-dependent antigen). They discover that GFP-positive cells constitute a stable proportion of TFH (~30-40%) at every time points during the course of primary response. Both IL-21 secreting (GFP+) and IL-21 negative (GFP) subpopulations are mostly equivalent in terms of transcription factors (Bcl-6, IRF4, Blimp-1, T-bet and GATA3 tested) and cytokine expression (IFN-γ, IL-4 and IL-10 examined), proliferation and functional abilities (NP-specific GC B cell response analyzed). The only difference between two subsets was an increase in transcripts encoding T-bet, IFN-γ and IL-10 among GFPcells.

The potential of TFH to participate in recall responses is tested by adoptive transfer approach. Follicular helpers generated in the course of immune response to influenza virus (flu infection similarly to NP-KLH immunization leads to the formation of GFP+ and GFP– subsets of TFH) were injected into congenic recipients challenged subsequently with the same virus. It is observed that antigen-experienced  TFH have the ability to expand on re-stimulation in vivo. Transferred follicular helpers are then analyzed by their surface marker expression and cytokine profile.  In secondary lymphoid organs TFH have a definite propensity to continue as TFH. However, at effector sites (lungs) the vast majority of former follicular helpers become conventional effectors CD4 T cells. Follicular helpers retain also the substantial ability to release cytokines on antigen re-exposure. It is interesting to know that TFH have some plasticity and may end up doing different tasks after germinal centers of primary immune response are disbanded.

Lüthje, K., Kallies, A., Shimohakamada, Y., Belz, G., Light, A., Tarlinton, D., & Nutt, S. (2012). The development and fate of follicular helper T cells defined by an IL-21 reporter mouse Nature Immunology, 13 (5), 491-498 DOI: 10.1038/ni.2261

Memory compartment regeneration in SIV infection does not rely on naive CD4 T cells

Among mechanisms hold responsible for the severe CD4 T cell depletion in AIDS are those that contribute to increased apoptosis rates for CD4 T cells (either infected or non-infected) and decreased CD4 T cells regeneration capacity. Pathogenic HIV/SIV clones are in their majority CCR5-tropic. CCR5 is expressed on tissue-resident effector memory CD4 T cell populations and not surprisingly these subsets are decimated during the primary HIV/SIV infection. However, the CCR5-negative central memory subset which resides in secondary lymphoid organs is relatively spared and may form the reservoir for the subsequent effector memory subsets regeneration. The publication I have found investigates details of memory CD4 T cells renewal in non-human primate model infected with SIV and makes an intriguing point about what is not needed to bring back depleted memory compartments.

The link: http://jem.rupress.org/content/209/4/641.abstract

The chief aim of this paper is to assess whether naïve CD4 T cells presence is needed for the memory compartment replenishment during pathogenic SIV infection. Authors employed rhesus macaques and subjected them to a number of manipulations before proper infection. Briefly, experimental groups have been either thymectomized or left intact and subsequently CD4 T cells in each group were depleted with anti-CD4 antibody following which animals were left to regenerate CD4 T cell counts. Such approach allows comparing regeneration of memory subsets and general disease outcome between two different conditions – complete lack vs. normal level of naïve CD4 T cells. If you have read my previous post, note that this report assumes that the thymus is the only place of significant naïve CD4 T cell development.

After complete rebuilding of CD4 T cells counts took place animals from each group were infected with SIVmac239. Then they underwent the long data collection period which included initial untreated infection, anti-retroviral therapy phase and treatment discontinuation in order for animals to develop AIDS-like symptoms. Investigators amassed data relating to multiple parameters – most notably they followed plasma viral load, memory CD4 T cells depletion levels (in blood and at mucosal lung surfaces) and the presence of anti-SIV adaptive immunity over the entire span of experiment. They also observed the disease progression and appearance of opportunistic infections in both groups. The summary conclusion of this study is that in the course of pathogenic SIV infection naïve CD4 cells presence is not necessary for the regeneration of memory CD4 T cells subsets. Additionally, the depletion of naive CD4 T cells do not seem to influence the disease advancement.

Major findings of this publication are very convincing and well supported with data. However, I would like to pose one question – it looks like the depletion of naive CD4 T cells in thymectomized group impairs the scope of adaptive anti-SIV response. For example, anti-SIV CD4 T cell responses (defined as the percentage of memory compartment specific to gag+pol+env+nef) never really start working. Thymectomized animals show much slower seroconversion rate and blunted anti-SIV CD8 T cell reactions as well. Yet the general disease outcome between both groups is remarkably similar at all studied stages. How to explain it?

Okoye, A., Rohankhedkar, M., Abana, C., Pattenn, A., Reyes, M., Pexton, C., Lum, R., Sylwester, A., Planer, S., Legasse, A., Park, B., Piatak, M., Lifson, J., Axthelm, M., & Picker, L. (2012). Naive T cells are dispensable for memory CD4+ T cell homeostasis in progressive simian immunodeficiency virus infection Journal of Experimental Medicine, 209 (4), 641-651 DOI: 10.1084/jem.20112071