Can microbial innate signaling generate peripheral FOXP3+ regulatory T cells?

In the past several years few papers have been published suggesting that some commensal microbiota species acting via their conserved molecules [i.e. polysaccharide A (PSA) from Bacteroides fragilis (Bf)] and metabolic products [i.e. short-chain fatty acids (SCFAs)] could induce peripheral FOXP3+ Tregs. The mechanisms are still controversial due to fact that there is no consensus whether thymus-derived and peripherally generated FOXP3+ Tregs (pTregs) represent two distinct lineages or distinct differentiation phases of unique thymus-derived FOXP3+ Tregs.

In this regard, a new paper in Science Immunology is of interest. In this study the authors tried to produce evidence that microbial cell surface β-glucan/galactan (CSGG) polysaccharides of Bifidobacterium bifidum [B. bifidum], a probiotic strain PRI1, could induce generation of FOXP3+ pTregs specific for diverse set of antigens. Lets review it.

First, the authors showed that monocolonization of germ-free mice with B. bifidum dramatically increased frequency of colonic Tregs compared to control species. Of note, Bacteroides fragilis (Bf) showed no effect thus putting some doubt on original observation.

Second, to nominally distinguish between conversion vs. expansion, the authors transferred naive CD4+Foxp3− T cells sorted from Foxp3^GFP reporter mice into germ-free mice and analyzed for GFP expression 3 weeks later. Around 5% of donor T cells up-regulated FOXP3 in colon tissue.

Of note, monocolonization of germ-free mice with B. bifidum induced quite robust IFN-γ response too.

In addition, transfer of OT-II TCR transgenic T cells [on RAG1 KO background] recognizing OVA into OVA-fed B. bifidum treated mice or transfer of CBir TCR transgenic T cells that recognize bacterial flagellin into B. bifidum treated mice on SPF RAG1 KO background could augment their differentiation into FOXP3+ Tregs. Of note, first, OT-II were transferred into germ-free mice that harbor natural, thymus-derived Tregs that could direct naive OT-II conversion into FOXP3+ Tregs in a antigen[epitope]-specific manner (OVA cross-reactivity). Second, there is some inconsistency regarding CBir T cell experiment. In the figure legend it says they were transferred into SPF RAG1 KO hosts but in the supplemental data it says "naïve CBir CD4+CD62LhiCD44loFoxp3GFP- T cells (1x106) were transferred intravenously to the C57BL/6 mice maintained in SPF conditions". These difference could make huge difference in data interpretation. Third, CBir T cells specificity to flagellin has now been questioned in new study.

In vitro co-culture of DCs and T cells pretreated with B. bifidum or its cell surface β-glucan/galactan (CSGG) polysaccharides could induce FOXP3+ Treg phenotype in a TGF-β1 dependent manner. Again, Bacteroides fragilis (Bf) could not do it.

Also, TLR2 appears to be involved in the action of B. bifidum cell surface β-glucan/galactan (CSGG) polysaccharides.

In summary, this study tries to suggest that B. bifidum conserved microbial wall component, β-glucan/galactan (CSGG) polysaccharides could convert naive T cells into FOXP3+ Tregs. It is certainly obvious that β-glucan/galactan (CSGG) polysaccharides could increase frequency of FOXP3+ Tregs but whether it is genuine conversion vs. expansion of pre-committed proto-Tregs via direction of existing thymus-derived Tregs were not cleanly verified. And lets remember that B. bifidum monocolonization increased frequency of IFN-γ producing T cells too. How these data fit to tolerance model? I am also puzzled by the fact that the authors primarily or exclusively showing frequency and not the actual numbers of FOXP3+ Tregs without providing any guidance.

Finally, lets step back and think a little bit more about general premise of such papers. The authors concluded that because B. bifidum and its microbial wall component, β-glucan/galactan (CSGG) polysaccharides could increase frequency of FOXP3+ Tregs it indicates it is a tolerogenic effect. But what's biological function or context that would require increase in frequency of Tregs? Lets assume it is to control inflammation. So, without causing inflammation what is the point of increasing the numbers of Tregs? So, does it mean that B. bifidum is actually 'inflammatory' and Treg expansion is a compensatory mechanism to counteract to such inflammation? Second, expansion and maintenance of Tregs would require increase of those enigmatic cells that supply IL-2 to Tregs. So, by definition, expansion of Tregs cannot happen without concurrent expansion of IL-2 producing cells. So what kind of signaling could drive such concurrent expansion of both Tregs and IL-2 producing cells? 

posted by David Usharauli

New auto[cross-reactive]-antigen, GDP-l-fucose synthase, identified in MS patients

Multiple sclerosis (MS) is a debilitating autoimmune disease affecting nervous system. Several auto-antigens have been traditionally suspected for MS pathology, mostly derived from neural tissues such myelin. However, more recently the list of relevant auto-antigens expanded to include such member as RAS guanyl releasing protein (RASGRP1–4) family. Similarly, a new paper in Science Translational Medicine presented evidence that yet another auto-antigen, GDP-l-fucose synthase, is a target antigen in a subset of MS patients.

The authors has used modified version of positional scanning epitope library to identify epitope derived from auto-antigen, GDP-L-fucose synthase, as a target epitope for CD4+ T cell clone, TCC21.1, derived from MS patients with DR15 haplotype. Peptides sequences matching GDP-l-fucose synthase was detected in brain tissue.

MS patients whose T cells displayed high response to peptides from GDP-L-fucose synthase in stimulation assay showed high responses to myelin proteins as well.

Curiously, almost all high responders carried DRB3*02:02 allele. 

Finally, the authors speculated that sequence similarity between human and microbiota-derived evolutionary conserved GDP-L-fucose synthase could be a factor that initiates MS pathology in these patients.

In summary, this study has a lot of nice data that support idea that GDP-L-fucose synthase is a  new auto-antigen that could be relevant in molecular diagnosis of MS pathology.

There are several questions about this study. GDP-L-fucose synthase expression is not restricted to brain tissue. So, it should be relevant to understand if other tissues were affected in those MS patients. Second, cross-reactivity per se is not sufficient to explain how and why autoimmune responses are being initiated. Microbiota expressing GDP-L-fucose synthase most likely reside in patients long before MS, maybe even since birth, and T cells are tolerant to them. So, what has to be changed, in a antigen-specific manner, to make T cells less tolerant to initiate specific autoimmune attack on nervous system and not a total autoimmunity targeting all available auto-antigens? 

posted by David Usharauli