Category Archives: T regulatory cells

“Combination Cancer Immunotherapy and New Immunomodulatory Targets” published in Nature Reviews Drug Discovery

Part of the Article Series from Nature Reviews Drug Discovery, our paper hit the press today

Combination cancer immunotherapy and new immunomodulatory targets. Nature Reviews Drug Discovery 14, 561–584. 2015.  doi:10.1038/nrd4591

by Kathleen Mahoney, Paul Rennert, Gordon Freeman.

a prepublication version is available here: nrd4591 (1)

ICI15 presentation is now available

Over 100 slides on immune checkpoint combination therapy, novel targets and drug development in immuno-oncology, created for a 3 hour workshop at ICI15 (link).

As always we work from indications to discovery and back again, keeping one eye on the rapid evolution of clinical practice in oncology and the other on novel targets and therapeutics.

on SlideShare now:

The twisted tale of neoantigens and anti-tumor immune responses

Two papers out this week add to a pile of data addressing the role of neoantigens in tumor therapy. While these papers address tumor neoantigen “load” in the context of immune checkpoint therapy the results have implications for TIL therapeutics, TCR therapeutics and onco-vaccine development.

A really dramatic paper from diverse groups at the University of Pennsylvania and their collaborators, just published in Nature (link-1), explores the complex interplay of radiation therapy and anti-CTLA4 antibody therapy (ipilimumab, from BMS) in patients with stage IV metastatic melanoma (relapsed or previously untreated). In this Phase 1/2 clinical trial (NCT01497808) patients with multiple melanoma metastases received various doses of radiation therapy delivered to a single metastasis, termed the “index lesion”. They then received 4 doses of ipilimumab (3 mg/kg, i.v., once every 3 weeks) and non-irradiated lesions were evaluated within 2 months of the last dose.

Although the sample size reported is small (n=22) some interesting lessons emerged from the study. The response rate was low, and the progression free survival (PFS: 3.8 months) and overall survival (OS: 10.7 months) data bear this out. It appears that just shy of 40% of patients were still alive at ~30 months (see Figure 1c in the paper). It is too early to tell if there will be a “long-tail” effect going forward. In the original ipilimumab study a very small percentage of patients lived for a very long time, “pulling” the PFS and OS curves to the right. Regardless, most patients in this study did not respond and the questions posed in this paper are directed to the mechanisms of resistance.

The mouse B16-F10 melanoma model was used to model resistance. Mice with tumors were locally irradiated then treated with an anti-mouse-CTLA4 antibody, to mimic the clinical trial. Only 17% of the treated mice responded. Two predictors of response/non-response were elucidated: 1) the ratio of effector T cells (Teff) to regulatory T cells (Treg) and 2) a gene signature in the tumor cells that is dominated by the expression of PD-L1 and IFNgamma regulated genes. In short, if the melanoma cells are expressing PD-L1 and the tumor infiltrating lymphocyte (TIL) population is dominated by Tregs (which are PD-1+), then the radiation + anti-CTLA4 therapy failed.

To further subset TIL into active Teff versus non-responsive “exhausted” Teff, the authors used an expression profile of PD-1+/Eomes+ to identify exhausted Teff and PD-1+/Eomes+/Ki67+/GzmB+ for active Teff. Importantly, exhausted Teff could be reanimated upon treatment with PD-1 pathway antagonists: anti-PD-1 antibody or anti-PD-L1 antibody. This reanimation led to an improved CD8+ Teff/Treg ratio and led to tumor control in the majority of the mice (up to 80%) when the treatment consisted of irradiation plus anti-CTLA4 plus anti-PD-L1. Of note, radiation plus anti-PD-L1 did not achieve this effect; the triple therapy was required (see Figure 2d).

The striking conclusion is that upregulation of PD-L1 on tumor cells can subvert the effect of anti-CTLA4 antibody therapy, and this therefore qualifies as a mechanism of resistance.

What about the role of irradiation? In both the patients and the mouse model irradiation was local, not systemic. Further, this local irradiation was required to achieve complete responses in the mouse model. What is going on here? Irradiation was linked to a modest increase in TIL infiltration of melanoma tumors in the mouse model, but sequencing of the T cell receptors (TCR) revealed that there was an increase in the diversity of TCRs, meaning that more antigens were being recognized and responded to by TIL after irradiation. In this context then, anti-CTLA4 reduced the Treg population, anti-PD-L1 allowed CD8+ TIL expansion, and irradiation set the antigenic landscape for response.

Returning to the patients armed with this information from the mouse study, the authors find that low PD-L1 expression on the melanoma cells correlates with productive response to irradiation plus ipilimumab therapy, while PD-L1 high expressing tumors were associated with persistent T cell exhaustion. In addition, monitoring the state of the CD8+ T cell population (PD-1+/Eomes+ versus PD-1+/Eomes+/Ki67+/GzmB+) suggested that these phenotypes might be useful as peripheral blood biomarkers. The patient numbers are very small for this analysis however, which awaits further validation.

The conclusion: irradiation combined with ipilimumab plus anti-PD-L1 antibody therapy should be a productive therapeutic combination in PD-L1+ stage IV melanoma. Similar strategies may be beneficial in other solid tumor types. This is interesting news for companies developing anti-PD-L1 antibodies, including BMS-936559 (also from BMS), MPDL3280A (Roche/Genentech), MEDI4736 (AZN) and MSB0010718C (Merck Serono).

A second paper (link) bring our focus back to PD-1, in the context of non-small cell lung cancer (NSCLC). Using the anti-PD-1 antibody pembrolizumab (from Merck) a group from the Memorial Sloan-Kettering Cancer Center sought to determine correlates of response of NSCLC patients to anti-PD-1 therapy. Their findings again hone in on neoantigen load, as the best predictors of response were the non-synonymous mutational burden of tumors, including neoantigen burden and mutations in DNA repair pathways. What all this means is that mutations that change the amino acid sequence (thus, are non-synonymous) can produce neoantigens that can be recognized by CD8+ T cells; mutations in the DNA repair pathways increase the rate that such mutations go uncorrected by a cell.

The authors sequenced the exomes (expressed exons – these encode proteins) from tumors versus normal tissue, as a measure of non-synonymous mutational burden that could produce neoantigens. Patients were subsetted based on response: those with durable clinical benefit (DCB) and those with no durable benefit (NDB). High mutational burden was correlated with clinical efficacy: DCB patients averaged 302 such mutations, while NDB patients averaged 148; ORR, PFS and OS also tracked with mutational burden. In a validation cohorts the number of non-synonymous mutations was 244 (DCB) versus 125 (NDB).

Examination of the pattern of exome mutations across both cohorts was studied in an attempt to discern a pattern of response to pembrolizumab treatment. The mutational landscape was first refined using an algorithm that predicts neoepitopes that can be expressed in the context of each patients specific class I HLA repertoire – these are the molecules that bring antigens to cell surfaces and present them to T cells for recognition (I’m simplifying this process but that is the gist of it). The algorithm identified more potential neoepitopes in the DCB patient tumors than in the NDB cohort, more impressively, a dominant T cell epitope was identified in an individual patient using a high-throughput HLA multimer screen. At the start of therapy this T cell clone represented 0.005% of peripheral blood T cells, after therapy the population had risen 8-fold, to 0.04% of peripheral blood T cells. Note that most of this clone of T cell would be found in the tumor, not in circulation, so that 8-fold increase is impressive. The T cells were defined as activated CD8+ Teff cells by expression markers: CD45RA-/CCR7-/LAG3-. As in the first paper we discussed, it is useful that these markers of systemic response to immunotherapy treatment are being developed.

There is an interesting biology at work here. It is often noted that high mutational burden is associated with better outcome, for example to chemotherapy in ovarian cancer, and irrespective of therapy across different tumor types (link-2). This suggests that tumor neoepitopes are stimulating an ongoing immune response that is stifled by active immunosuppression, yet is still beneficial. Once unleashed by immune checkpoint blockade, the immune system can rapidly expand it’s efforts.

We recently reviewed the importance of neoantigens in anti-tumor therapy (link-3) although the focus then was on cellular therapeutics rather than on immune checkpoint modifiers such as anti-CTLA4 and anti-PD-1 or PD-L1 antibodies. We can mow add that our ability to track neoantigens and the immune response to neoantigens is opening new avenues for investigating immuno-oncology therapeutics and their efficacy.

Last Week’s Immune Checkpoint Papers In Nature Are Complicated!

Last week we were treated to a barrage of good news regarding PD-1/PD-L1 therapeutics and the ability to select responders. The centerpiece was a trio of papers in Nature.

Powles et al. presented data on the use of MPDL3280A, an anti-PD-L1 IgG1 antibody that has been engineered to lack all ADCC function (link 1). The antibody blocks the interaction of PD-L1 with PD-1 and with CD80, two receptors found primarily on lymphocytes. The paper focused on the application of ’3280′ therapy in chemotherapy-resistant metastatic urothelial bladder cancer (UBC). Nearly all patients (93%) had failed platinum-based chemotherapy; 72% had failed 2 or more lines of prior therapy. 75% had visceral metastases, most had poor renal function and the majority (59%) had a performance score of 1 (very poor). In a word, these patients were incurable. Preliminary Phase 1 data demonstrating efficacy in UBC was presented at ASCO and led to breakthrough designation for ’3280 for the treatment of UBC in June 2014.

The original Phase 1 trial had enrolled UBC patients whose resection or biopsy tissue demonstrated the presence of tumor-infiltrating lymphocytes (TIL) with dark staining (score 2 or 3) for PD-L1. The expansion cohort allowed for the enrollment of patients whose tissue specimens contained TIL which were PD-L1 dim (score = 1) or negative. 205 patient tissues were analyzed (see table 1 in the paper). 67 patients were enrolled and evaluable with PD-L1 staining results as follows:

Screen Shot 2014-12-04 at 11.14.57 AM

A total of 17 patients responded and 16/17 responses were ongoing (i.e. durable) at the time of data cutoff. The longest duration of response was a remarkable 30 weeks in the cohort with the brightest PD-L1 TIL staining, although the range was broad (from 1 week to 30). Median duration of treatment was 9 weeks, so this is really an early snapshot. Regardless, the ability to invoke an anti-tumor response in a cohort of patients that are this ill, and deemed incurable, is remarkable.

With reference to the staining pattern of PD-L1 and the relevance of PD-L1 expression to successful response, the authors came to the following conclusions:

1) therapy triggered expansion of the circulation CD8+ T cell population, and transient elevation of IL-18 and IFNgamma was observed; these systemic changes reflect the proposed mechanism of action of ’3280 but did not correlate with response.

2) expression of PD-L1 on TIL, but not on tumor cells, was predictive of response to therapy. On note, this was true whether the available tissue sample was new acquired or archival (up to 10 years old). This suggests that there is an ongoing and futile immune response in these PD-L1+/TIL+ tumors. The lack of association with tumor PD-L1+ status is discussed more extensively in the companion paper (see below).

3) the efficacy of PD-L1-directed therapy in UBC and also NSCLC and melanoma, all tumors with very high mutational burdens, suggests that antigen diversity or antigen “burden” may be important for successful induction of an anti-tumor immune response in ’3280-treated patients.

The UBC cohort was part of a much larger clinical trial that included diverse solid tumors. A companion paper by Herbst et al. investigates the utility of PD-L1 TIL expression in other cohorts (link 2). The focus of this work is on the biomarker application, particularly with respect to PD-L1+ TIL staining, as defined in the prior paper. Patients (n=277) with advanced incurable cancers were enrolled in a ’3280 dose ranging study, given drug iv every 3 weeks. Across tumor types high PD-L1 expression on TIL, but not tumor cells, was associated with response and increased PFS. Note here that the PFS gain, while encouraging, does not suggest that we will see a high percentage of truly durable (“long tail”) responses in this particular patient population, even in those patients with PD-L1 bright (score of 3) staining:

Screen Shot 2014-12-04 at 9.27.50 AM

There were some interesting additional analyses. In NSCLC patients who had been smokers, 43% responded to therapy, while only 10% of non-smokers responded. Such data have been reported before, and are often taken to mean that the higher mutational burden seen in smokers with NSCLC biases their tumor toward immune recognition (this echoes the mutational diversity/mutational burden argument made in the Powles UBC paper). Sticking with NSCLC, 83% of patients with a PDL1+ TIL staining score of 3 (lots of cells and therefore dense/dark staining) responded versus 38% of patients with a PDL1+ TIL staining score of 2 (diffuse staining, fewer cells). Response was positively correlated with CTLA4+ staining on TIL, and negatively correlated with fractalkine expression. In melanoma (but not NSCLC or RCC) response was associated with elevated IFNgamma and IDO1 and CXCL9 that are induced by IFN gamma. Strikingly, positive anti-tumor responses were not associated with a measureable change in FoxP3 expression, suggesting the T regulatory T cells were not playing a role in the setting of ’3280 therapy.

What about the non-responders, as these make up the majority of the patients across indications? Progressing tumors were characterized into three classes:

1) few or no TIL present – “immune ignorance”

2) TIL present but little or no PD-L1 expression – “non-functional immune response”

3) TIL present and PD-L1+ but located on the edge of the tumor – “excluded infiltrate”

Missing here I think is an analysis of tumors with PD-L1+ TIL with high staining scores (2 or 3) that progressed, i.e. did not respond to therapy. It seem to me unlikely that these all fell into category “3″ above, so this analysis may be coming in a follow-up paper.

The authors make a very interesting point about this data, which is that they seem to refute the consensus model of “immune resistance” in which it is postulated that CD8+ T cells infiltrating tumors secrete IFNgamma and other cytokines that induce PD-L1 expression on the tumor cells themselves, and these tumor cells in turn produce factors that create an immunosuppressive environment that includes potently immunosuppressive, PD-L1 bright T regulatory cells. The “immune resistance” model further postulates that the expression of PD-L1 on tumor cells and T regulatory cells is responsible for shutting down CD8+ T cells by binding to PD-1.

There are several key messages in this paper – first, responses in these incurable patients are measureable and remarkable, if they respond (most do not). Second, CD8+/PD-L1+ TIL are highlighted as a potential prognostic indicator of the potential for response the ’3280 therapy. Finally, it is clear that other signals will have to be disabled or enhanced in order to induce a productive and durable immune response in more patients and/or move PD-1/PD-L1-directed therapies to front line.

Now, the final paper in this triad turns things upside down. Tumeh et al. analyzed tumor tissue samples from 46 metastatic melanoma patients treated with pembrolizumab, an anti-PD-1 antibody  (link 3). The analytic methods used are elegant and overlap but also extend the analyses used in the prior 2 papers: quantitative immunohistochemistry, quantitative multiplex immunofluorescence, and TCR deep sequencing (NGS).

This paper is strictly about melanoma. The ORR in this small study was 48% (22/46). The authors focused on expression of PD-L1 on tumor cells and of PD-1 on CD8+ T cells. Doing so they come to strikingly different conclusions than the papers discussed above. Responders in this study had PD-1+ CD8 T cells massed on the tumor margin, adjacent to PD-1+ tumor cells. Response was associated with infiltration of the tumor by those CD8+ T cells, which also increased in density (proliferated). Therefore the paper specifically supports the “immune resistance” model in which tumor-expressed PD-L1 suppresses PD-1+ CD8 T cells. CD8 T cell proliferation was associated with expression of granzyme B within the tumor and phosphorylated STAT1 at the tumor margin where CD8+ T cells were infiltrating (phospho-STAT1 in induced by IFNgamma receptor signaling). Finally, response was associated with T cell (TCR) clonality, i.e. the fewer tumor antigens, and thus the lower the antigen burden that is invoking a response, the better. This is a different take than we got from the prior papers.

So, perhaps melanoma is distinctly different.

Aside from that, these papers provide critical take-home messages and perhaps even more critical questions to be addressed:

1) CD8 T cells are good. That’s pretty clear, whatever they are expressing. We can argue more about their geography, but if they are not present, you will not respond.

2) IFNgamma is good. We see this especially in the melanoma setting as detailed in two of the papers.

Neither of these conclusions is novel nor surprising.

3) Biomarker development beyond CD8+ T cell staining remains complex.

4) Regardless of their biomarker status most patients still do not respond and we do not know why. As we consider combination therapy, will other markers be used to further sort patients into rational combination buckets, or will this simply too complex to be useful?

5) Finally, what about those T regulatory cells we’ve been obsessed with for the last decade? These are hardly mentioned in the context of PD-1/PD-L1 therapeutics in the three studies.

next time:

>>> back to those tumor antigens? New papers, preclinical and clinical, shed some light… and

>>> those T regulatory cells may be important in some settings, but were betting on the tumor microenvironment to yield interesting new targets for therapy

stay tuned

High Fiber, Butyrate, Tregs & Immune Responses – Evaluating Recent Papers in Nature, Nature Medicine and Immunity

Last week I reviewed four recent papers on the impact of gut microbial commensals and pathogens on immune function, focusing on regulatory T cell (Treg) generation and on the role of effector Th17 T cells (Th17s) on disease. See the post here: http://www.sugarconebiotech.com/?p=6.

The other day another paper on the role of dietary fatty acids in the regulation of immune responses appeared (see http://www.nature.com/nm/journal/vaop/ncurrent/full/nm.3444.html). Honestly, the results presented in this new paper are sufficiently distinct from the prior two papers in Nature that a reappraisal makes sense, as there are clearly a whole host of unresolved questions in this body of work. Also, we will touch briefly on an Immunity paper, also just out.

The driver for all of these studies is the extensive observations on the ability of particular fatty acids to modulate the immune response. There are similar observations on the role of fatty acids in regulating metabolism – that work will not be discussed here.

In particular, the investigators are trying to understand if, and how, short-chain fatty acids (SCFAs) produced by gut bacteria, such as butyrate, propionate and acetate, can modulate the immune system. The stakes are high, as there is early clinical work aimed at manipulating the microbiome in order to treat diseases, notably gut diseases such as inflammatory bowel disease (IBD) and severe diarrhea. Also there are the massive supplement and wellness industries already selling such SCFAs, without much understanding of the science.

Suffice to say we are entering the high fiber diet metaverse, cautiously.

Here is a quick recap of the earlier papers, taken one at a time and in a little more detail. I’ll highlight some similarities and differences. The first two studies were published in Nature on 19 December 2013. The key findings are summarized below. Note that this and the other studies discussed here are done in pure strain inbred mice.

  • commensal microbes support Treg development
  • specifically, large bowel (colonic) production of the SCFA butyrate induces Tregs
  • butyrate functions by inhibiting HDAC IIa
  • HDAC inhibition allows more extensive acetylation of FoxP3 and other genes
  • expression of FoxP3 and other genes drives Treg differentiation
  • butyrate blocks the development of IDB in a T-cell dependent mouse colitis model

The first study was led by Hiroshi Ohno from the RIKEN Center in Kanagawa, Japan. The colitis experiment was shown in that paper. Now onto some of the other details – there are 4 figures in the text and 22 supplemental figures so we can’t cover everything.

The Tregs under discussion in this paper are peripherally generated (i.e. not thymic in origin) and are identified in flow cytometry (FACS) experiments as FoxP3+/neuropilin-/Helios-. About half the high-fiber diet (HFD) induced Tregs were activated and therefore CD103+. Critically, Ohno and colleagues show that this Treg population in restricted to the local (colonic lamina propria) environment. There are plenty of Tregs in the lymphoid organs – mesenteric lymph nodes and spleen – but the percentage of FoxP3+/neuropilin-/Helios- cellsdoes not change in these organs in response to the HFD. The investigators then demonstrate that the microbes responsible for fermenting the HFD into beneficial SCFAs are bacteria of the class Clostridiales and that the potentially beneficial SCFAs include the very potent butyrate and the less potent propionate. Acetate had no effect.

Now this is where is starts to get a little complicated. Having demonstrated that the gut SCFAs induce Treg differentiation only in the colonic lamina propria, the authors next show that T cells isolated from the mouse spleen can be differentiated into Tregs using stimulation conditions that include TGFbeta and butyrate plus stimulatory antibodies to CD3 and CD28. This might indicate that there is some barrier that prevents butyrate reaching sites other than the gut wall, and that this accounts for the local aspect of the Treg response to butyrate. However, while most of these SCFAs are passively and actively taken up by intestinal cells, SCFAs can also be detected in circulation. The absorption of SCFAs can be saturating, i.e. above some concentration SCFA uptake into the circulation is maxed out. These observations suggest that there is a requirement for locally high concentrations of SCFAs in order to induce Treg differentiation – this would explain why these induced Tregs were only found in colonic tissue and not in spleen or LN. I can’t find the relevant concentration data nor is there any dose response data – this is disconcerting. They also report that butyrate can drive naive T cells to a Treg phenotype irrespective of pro-inflammatory Th1, Th2 or Th17 inducing conditions. This is a very dramatic result but suffers from the same lack of critical experimental detail.

The observations made using butyrate stimulation of T cell are followed up in vivo using SCFA supplemented diets. As noted earlier the MOA here is the antagonism of the deacetylase HDAC IIa, thereby allowing increased acetylation and activation of the FoxP3 gene. Finally, using an activated CD4+ T cell transfer colitis model (a model in which Tregs are specifically excluded from the transferred cells) the investigators show protection from disease when the mice are fed diets containing butyrate.

OK, we still have no idea how this is mediated, but the observation is in line with other papers that have examined to ability of butyrate to control colitis (its a large body of work). So, we are not criticizing this whole story, but just suggesting that more experimental detail would be useful, especially in a world where one can buy butyrate capsules or arrange for butyrate enema treatment. A more general critique is offered at the end of this post.

The second Nature paper is by Alexander Rudensky and colleagues from Memorial Sloan-Kettering in NY and reaches broadly similar conclusions as the Ohno paper. Their naive T cell culture conditions are a little different, substituting dendritic cells and Il-2 for the anti-CD28 antibody signal, and they do show dose response data. The latter results indicate a sharp rise in Treg induction above 30uM butyrate. To manipulate the system in vivo these investigators used antibiotics to clear the SCFA producing bacteria. Thus the model is rigged to show an increase above an artificially low background. That’s OK, but let us just be clear about it.

Using sodium butyrate in the drinking water, the investigators induced Treg differentiation in the peripheral LN and spleen. The serum concentration achieved with the drinking water regimen was 500pg/ul which is ~ 4.5uM, a physiological concentration in wildtype, untreated mice. In other words, they brought the SCFA level back to normal, and that induced T regs. In order to induce Tregs in the colonic lamina propria they used either butyrate-enriched food, or a butyrate enema.

The conclusion of all that work is that systemic exposure (drinking water) can induce Tregs in the periphery (spleen, LN) but that local exposure (food, enema) is required to induce Tregs in the gut. Note that this latter conclusion echoes the Ohno paper. Turning to propionate and acetate, they next show that propionate in the drinking water can induce peripheral Tregs in the spleen and LN, and that both propionate and acetate can induce local Tregs in the colon. However, these latter cells are possibly thymic-derived, not extrathymic Tregs, as shown by independence from the CNSI gene (required for extrathymic Treg differentiation). The other option is that these cells are preferentially recruited from the circulation. Note that the acetate result is at odds with this prior paper.

So this has now gotten very complicated, with three abundant starch-derived fatty acids being endowed with both unique and overlapping abilities to induce different types of Tregs in different geographies. Just to make this even more complicated, the Rudensky team next shows that this biology is not just T cell specific, but that butyrate can also endow dendritic cells (DCs) with the ability to preferentially induce Tregs. Lets not go into detail except to say that this effect on DCs did not depend on GPR109a, the niacin and butyrate specific G-protein-coupled receptor (GPCR). More on GPCRs later. The rest of the story – HDAC inhibition and FoxP3 induction – is familiar from the Ohno paper (and many others, the HDAC mechanism is pretty well known).

Finally, I mentioned at the top the Nature Medicine paper that triggered this reappraisal (http://www.nature.com/nm/journal/vaop/ncurrent/full/nm.3444.html). Using a mouse model of house dust mite (HDM) antigen allergic asthma, Benjamin Marsland and colleagues from the University of Lausanne, Switzerland, demonstrate that the susceptibility to and severity of HDM-induced asthma was worsened on a low fiber diet and improved on a high fiber diet. They traced the change in asthmatic response to a change in the gut microbiota supported by the different diets, bringing us again to the SCFAs produced. Higher concentrations of the usual suspects (butyrate, propionate, acetate) were produced when the microbiome was dominated by the Phylum Bacteroidetes, to which the class Clostridiales belongs. Note that the differences in the composition of the microbiome on the low and high fat diets were not significant. This is an issue we will revisit.

The asthma paper is strikingly different from the two Treg papers. In this paper the focus in on propionate, not butyrate, and on systemic effects, not local effects. The premise is that the dietary changes impact the bone marrow, not the local lung tissue. Indeed SCFAs could not be detected in the lung. Propionate treatment reduced Th2 immune responses to HDM in a manner that was dependent on GPR41, a SCFA receptor. This receptor is expressed at high levels in the colon where it mediates a variety of responses to SCFAs, however, in this paper the impact of propionate was traced to the CD11bhisubset of DC in the lung-training LN. How this impacts the allergic-asthmatic response is hypothesized to be (and I’m quoting the paper here) “after inflammation, the lung DC compartment is replenished with inflammatory monocyte-derived DCs that have been exposed to SCFAs in the bone marrow and circu­lation, leading to a maturation profile that is ineffective at driving Th2 cell responses.”

Therefore the authors conclude that they have elucidated a “gut-lung axis for the formation of the airway microbiota” and therefore I suppose, lung immune responses.

Really?

What we have here are three reductionist tales, necessary to help us understand the rules of the system but perhaps not sufficient in themselves to draw sweeping biological and pathological conclusions. It’s very clear from the disparate results obtained that we are still working out the rules. Also, one cautionary note, a recent study in human subjects showed that plasma butyrate concentration remained very close to 2uM under a variety of high fiber meal and fasting conditions, suggesting that this SCFA may not be as variable in concentration as is seen in mice (http://jn.nutrition.org/content/140/11/1932.full).

A somewhat more straightforward study just published last week in Immunity (http://download.cell.com/immunity/pdf/PIIS1074761313005645.pdf?intermediate=true). Vadivel Ganapathy and colleagues from the Georgia Regents University in Augusta show that GPR109 signaling is required to maintain IL-10 dependent Treg activity in the colon, and they trace this function to DC and macrophage responses. Butyrate (or niacin, the nominate ligand for Niacr1 aka GPR109a) treatment of DCs and macrophages induced a phenotype that supported Treg differentiation. Note that this result contradicts the Rudensky paper, in which GPR109a was ruled out as the causative receptor, at least on DCs.

GPR109a gene-deficient mice were then shown to be more susceptible to colitis and inflammation induced colon carcinogenesis, and this effect was shown to be dependent on both the hematopoietic compartment and colonic tissue cells. This final study is satisfying, as now we are seeing pharmacological manipulation of a defined receptor, albeit with a molecule (niacin) that has a pretty checkered history as a therapeutic.

Where does this leave us? I think the take home message is that these systems are very complex, and by trying to simplify them we have the benefit of gaining some insight but the risk of over-interpretation. The human microbiome is incredibly variable, over time and between individuals. The fact that we are seeing different results from manipulation of highly inbred strains of mice on very carefully defined diets should give us pause, especially when some studies can’t statistically distinguish between components of the microbiota they are describing. However, at the very least these studies support the belief that high fiber diets that producing lots of butyrate and propionate should be beneficial, and we have identified some targetable GPCRs, which should drive further research. Finally, we are perhaps a step or two closer to understanding how to manipulate Treg cell populations in human disease. This last goal, the ability to regulate immune responses via regulatory T cell modulation, has proved to be an elusive one so far.