Access to self antigens during germinal center reaction improves self/nonself discrimination against mimicry antigens

This week journal Science published short paper from Chis Goodnow's lab that raises very interesting question about biological significance for existence of anergic self-reactive B cells. Ordinarily, developing B cells when encountering self-antigens undergo deletion, receptor editing or physiological receptor signaling down-regulation that makes such 'anergic' B cells refractory to presence of normal level of self antigens. However, anergic B cells could be re-energized if challenged with high density self antigens or antigens sharing epitope similarity with self antigen.

Now, new study indicates that rather than developing into full blown auto-reactive immune response, anergic B cells when challenged with mimicry antigens mutates its receptors in a such a way, during process of hypermutation, as to achieve a high degree of discrimination between mimicry antigen and actual self antigen.   

The experimental set up itself is quite simple, only complex aspect was to analyze single cell B cell receptor mutation and their binding affinity recovered after antigen challenge. Two type of hosts were used here. Both groups harbor small numbers of self-reactive B cells (CD45.1+ SW_HEL B cells)  but only one group also harbored a specific antigen detected by these transgenic SW_HEL B cells and expressed "as as an integral membrane protein, mHEL^3X, encoded by a transgene with a ubiquitin promoter".

As expected SW_HEL B cells in double transgenic hosts were anergic with decreased surface immunoglobulin M (IgM) expression. However, these anergic B cells could be re-activated in germinal centers when challenged with Sheep red blood cells (SRBCs) covalently coupled with self antigen, HEL^3X, at high density.

Next set of experiments however showed very unusual results. When challenged with mimicry antigen DEL which slightly differs from self HEL antigen anergic B cell receptors in double transgenic hosts rapidly accumulated mutations that decreased binding affinity to self HEL antigen.

In fact, single cell BCR receptor analysis clearly showed that presence of self antigens dramatically enhanced anergic B cell receptor mutations that allowed up to 5,000-fold better discrimination capacity between self and mimicry antigen (pre vs. post comparison). This is based on assumption that starting affinity to self are the same for both normal and anergic SW_HEL B cells population. 

In summary, this study suggests that during germinal center reaction where B cell receptors undergo hypermutation, anergic B cell repertoire, in presence of self antigen, could be salvaged (redeemed) by accelerated accumulation of mutations that modifies their original specificity away from self antigens and allowing more fine discrimination between self and mimicry, cross-reactive nonself antigen. In this scenario, self antigens serve as negative-feedback templates that hypermutating receptors interacts repeatedly in real time to achieve minimal level of binding.

In my view such negative-feedback loop to B cells can only delivered by specialized cell type in germinal center that maintains, keeps memory of host's unadulterated "self antigen collection'' visible to B cells, a task somewhat similar to Foxp3+ Tregs. So, it is possible that new cell type need to be discovered that does it or it is also possible that the same Foxp3+ Tregs localized in germinal centers, referred as follicular Foxp3+ T regs, do it too. 

What are the global implication for such mechanism: It could explain why anergic B cells hang around and how their repertoire could be salvaged without compromising tolerance. The authors also puts forward another intriguing idea that commensal mcrobes and their antigens could serve as negative-feedback loop 'self' templates for anergic B cells that allows them to discriminate between self and mimicking nonself during immune response. 

posted by David Usharauli       

Selective elimination of autoimmune B cells using novel CAR-T technology based on antigen decoy

Few days ago journal Science published very interesting study describing a novel approach of treating autoimmune [auto-antibody]-dependent diseases. Called chimeric auto-antibody receptor (CAAR) T cell technology, this method can selectively eliminate autoantigen specific B cells in autoimmune diseases such as Pemphigus vulgaris, lupus, Myasthenia Gravis, Graves diseases and so on.

This new technology is based on a simple idea: antigen decoy. For example, during pemphigus vulgaris autoimmune B cells secrete autoantibodies to the keratinocyte adhesion protein desmoglein (Dsg3) that causes severe skin inflammation. Short-term management of diseases is achieved by total B cell depletion, but disease returns. The authors reasoned that if engineered T cells would express Dsg3 as a CARs, such antigen decoy CAAR T cells would selectively engage disease-causing anti-Dsg3-specific auto-antibody producing B cells. Such interaction should eliminate only Dsg3 specific B cells, sparing normal, infectious-specific B cells.

Indeed, the authors showed that CAAR-T cells expressing Dsg3 as a CAR construct (EC1-4 CAAR) can selectively interact with Dsg3-specific autoimmune B cells in vitro.  

More importantly in vivo NSG mice experiments confirmed that Dsg3 CAAR T cells could selectively eliminate human Nalm6 CD19+ B cell line expressing autoimmune Dsg3-specific receptors (PVB28/F779). In addition the authors claim that Dsg3 CAAR-T cells did not interfere with normal human skin epithelial functioning expressing Dsg3's natural ligand desmocollins.

In summary, if the results of this study is confirmed by other groups it would open up a new path for treating auto-antibody dependent autoimmune diseases.

David Usharauli

Depletion of "hybrid" B cells could explain benefits of anti-CD20 immunotherapy in MS patients

Multiple Sclerosis (MS) is believed to be an autoimmune disease. During MS, the patient's own immune cells attack peripheral nerves' myelin sheath disrupting proper signal transmission. In mouse model of MS, called experimental autoimmune encephalomyelitis (EAE), several myelin proteins have been identified as such targets (such as MBP and MOG). 

While self-specific T cells are widely thought to play a pathogenic role in MS progression, clinical trials revealed surprising benefits of B cell depletion in MS. Mechanism is unknown.    

Now new paper is Science Translational Medicine provided initial data suggesting that depletion of pro-inflammatory GM-CSF secreting B cell subset could explain the clinical benefits of anti-CD20 antibody therapy in MS.

First, the authors showed that activated peripheral B cells from healthy donors express pro-inflammatory cytokine GM-CSF. Interestingly, this B cell population does not overlap with B cell population expressing anti-inflammatory cytokine IL-10.

Next, the authors showed that activated B cells from MS patients tend to express more GM-CSF compared to B cells from healthy donors.

In vitro co-culture of B cells and macrophages revealed that B cell-derived GM-CSF contributed to Th1-type macrophage polarization.

More complex co-culture experiments showed that macrophages derived from MS patients soon after B cell depletion produced less of pro-inflammatory cytokine IL-12p40.

And more importantly, this tendency was maintained even after 1 year of B cell depletion, even though new B cell came back by this time.

This points to a re-configuration of cytokine network in anti-CD20 antibody treated MS patients such as reversal of B cell-derived GM-CSF/IL-10 ratio.

In summary, this study provides additional clues why anti-CD20 antibody depletion might benefit MS patients. Antibody-independent function of B cells is a novel concept [and not widely known or accepted]. One of the reasons for this resistance is lack or inadequate knowledge of B cell receptor specificity of such specialized B cell subset. We need to go one step further and connect the dots between BCR specificity or other innate receptors and B cell's "hybrid" function. Only by developing this biological "framework" can we fully understand the role B cells play in health or diseases.

If you are interesting in science of MS, I will recommend a book by Susan Quinn's "Human Trials: Scientists, Investors, And Patients In The Quest For A Cure" (2001).

David Usharauli

B cells reduce effectiveness of platinum-based anti-cancer compounds

Some things cannot be explained by conventional thinking. Here is one example. Earlier I reviewed new Nature paper discussing the beneficial role of allo-IgG in cancer therapy. 

This time, however, another team of scientists reported in the same issue of Nature that B cells and IgA secreting cell in particular, suppress effectiveness of platinum-based anti-cancer compounds, such as oxaliplatin.

The authors, led by Michael Karin at the University of California, San Diego (UCSD), analyzed oxaliplatin's effect in tumor bearing mice. Surprisingly, B cell deficient mice showed significant synergy with oxaliplatin.   

This beneficial effect of B cell ablation on anti-cancer effect of oxaliplatin was CD8 T cell-dependent.

Next, the authors showed that inhibition of oxaliplatin-induced anti-cancer effect was related to TGF-beta signaling in B cells and their differentiation into IgA producing cells.  

Furthermore, the authors showed that B cell-specific expression of IL-10 and PD-L1/2 contributed to cancer resistance towards oxaliplatin.

Finally, the authors showed that adoptive co-transfer of B cells with T cells induced cancer resistance to oxaliplatin's treatment via TGF-beta signaling.

In summary, these results suggest that initial anti-cancer action of oxaliplatin primes B cells to develop into IgA secreting cells and to inhibit CD8 T cell's anti-cancer effector function via IL-10 and PD-L1/2 signalling.

It is not clear whether IgA secretion per se has any inhibitory role. It appears that oxaliplatin activates or amplifies pathways towards IgA differentiation that is inhibitory to CTL functions. Does oxaliplatin action mimics gut immune environment? This fact requires further investigation to understand its mode of action. It could have a separate usefulness for oral vaccine formulations. 

David Usharauli

  

Reverse engineering the T-independent type 2 (TI-2) antibody responses

A very interesting and thought-provoking research in humoral immunity was published in journal Science this week. It came from Bruce Beutler's lab at the UT Southwestern Medical Center. Professor Beutler, as many may know, is a 2011 recipient of Nobel Prize in Physiology or Medicine for his discovery of endotoxin receptor (TLR4).

Actually, before I review this paper I would like to highlight that since 2005 when Ruslan Medzhitov's lab has published the first paper in Nature that suggested the role of TLR in B cell responses and then later in 2006, David Nemazee's lab has published new study in Science questioning the validity of 2005 paper, there was a controversy in this field. In fact, in 2009, Ruslan Medzhitov's lab has published another study that confirmed that haptenated proteins used in 2006 study could promote antibody response independent of TLR signaling. However the mechanism remained elusive.

It appears that this new study in Science may be a first glimpse in the darkness to reconcile the differences.

I also would like to point out that Professor Beutler's lab is famous for studying ENU-induced random mutations in mice to generate unbiased molecular signature of immune deficiencies.

Humoral immunity is divided into T-dependent and T-independent (TI) antibody response. TI responses, are in turn segregated into two categories: TI-1 and TI-2. This new study focus on TI-2 response.

Initially, using NP-Ficoll immunization, as a TI-2 model antigen, the authors observed that NP-specific IgM response was diminished in single STING-KO, cGAS-KO, MAVS-KO mice but not in several TLR signaling KO mice. 

Mice, double deficient in cGAS/MAVS, had almost complete absence of NP-specific IgM response.

This was strange observation since STING, cGAS and MAVS are known to detect the presence of DNAs or RNAs and NP-Ficoll contained none of them.

Using adoptive B cell transfer from these three KO mice into RAG-KO hosts, the authors showed that this effect of NP-Ficoll was B cell intrinsic (and the number of B1 or MZ B cells were normal in non-responding KOs).

The authors concluded that DNAs and RNAs were generated as a result of B cell stimulation by NP-Ficoll. Indeed, several endogenous retroviruses were up-regulated in NP+ B cells as compared to NP- B cells.

Furthermore, NP-ficoll immunization induced Reverse Transcriptase (RT) activity in NP+ B cells.

Parallel experiments revealed that mice deficient in NF-kB signaling also lacked the ability to respond to NP-Ficoll immunization or express endogenous retroviruses.

The authors observed that B cells from MAVS-KO, but not from cGAS KO mice, showed reduced phosphorylation of NF-kB proteins after anti-IgM stimulation, implying that MAVS played more critical role in sustained activation of NF-kB.

In summary, the authors proposed the new model of TI-2 antibody response. According to this model, when antigens with repetitive epitopes engage specific B cells, this activates initial wave of NF-kB activity leading to expression of endogenous retroviruses as a RNA that are subsequently converted into cDNA by RT activity. These newly generated RNAs and cDNAs are recognized by MAVS and cGAS/STING pathways, respectively. In turn, MAVS activation induces second wave of NF-kB activation to sustain TI-2 antibody response.

As the authors correctly pointed out, at this stage, it is not clear whether expression of endogenous retroviruses in activated B cells indeed play a critical role in TI-2 response. Their presence could be coincidental to this process. It would require B cell devoid of endogenous retroviruses to definitely test their precise role (since both TI-1 and T-dependent antibody response were shown to be STING, MAVS and cGAS independent).

For me this results represent new concept how we should assess the role of endogenous viruses present among our genes. Not everyone carries them, even among laboratory mice strains there is significant differences. Why would nature develop such system where TI-2 response would depend on endogenous viruses? It is just a fascinating idea.  

David Usharauli