Failure to tolerate gut microbiota transforms immunodeficiency into immunopathology

Here is a paradox for you that many don't even know that it exists: frequently, genetic immunodeficiency (weak immune response) syndromes are associated with immunopathologies (excessive immune response). So how could it be explained?   

New study published in Journal of Experimental Medicine may have some new answers. This study described mouse model of human immunodeficiency syndrome called, Omenn syndrome (after its discoverer) and showed that immunopathology was driven by gut microbiota.

Omenn syndrome is caused by hypomorphic (low active) RAG mutations. Analysis of intestinal tissue from Rag2^R229Q [Omenn] mice revealed pathological infiltration with inflammatory T cell subsets, T_H17 and T_H1.

Adoptive transfer showed that intestinal immunopathology was mediated by Rag2^R229Q mutant T cells.

Interestingly, Rag2^R229Q mouse harbored comparable numbers of Foxp3⁺ CD4⁺ T cells, implying that simple presence of regulatory T cells [generated in Rag2^R229Q mice] was not enough to control tissue inflammation.

Defect of Tregs derived from Rag2^R229Q mice was confirmed in adoptive transfer experiment with WT Tregs.

Besides T cell-driven immunopathology, Rag2^R229Q mice displayed IgA deficiency (failure to properly coat gut microflora). These data pointed to the possibility of microbial translocation causing excessive inflammatory response.

Indeed, antibiotic treatment of Rag2^R229Q mice could reduce intestinal immunopathology.

The role of antibiotic-sensitive gut flora in driving immunopathology in Rag2^R229Q mice was confirmed in adoptive fecal transfer experiments.

In summary, this study suggests the following scenario: hypomorphic RAG defect in Rag2^R229Q mice leads to "narrowing" of TCR and BCR repertoire. This in turn leads to outgrowth of oligoclonal T and B cells in Rag2^R229Q mice (wherein Rag2^R229Q mice contain T and B cells with limited, restricted, deficient TCR and BCR repertoires). Without proper TCR and BCR repertoire diversity, however, Rag2^R229Q mice fails to develop tolerance (IgA and Tregs) to gut flora or commensal microbial antigens present at mucosal surfaces (such as lung, intestine). Repertoire restriction also leads to failure to mount adequate and proper immune response.

David Usharauli

Deceptive strength applies to T cells as well

This week journal Nature Immunology published interesting study examining the correlation between TCR affinity and T cell competence. Using M. tuberculosis infection model, the authors showed that strength of antigen recognition by TCR reduces T cell competence long-term.

For this study the authors compared control of M. tuberculosis infection by two T cell receptor (TCR) transgenic mouse, named C24 and C7. In C24, CD4+ T cells express a TCR with high avidity [affinity, here] for M. tuberculosis antigen ESAT6, while in C7, CD4+ T cells express a TCR with intermediate avidity for ESAT6. Surprisingly, adoptive transfer of in vitro generated T_H1 cells from high avidity C24 mice provided significantly less protection against lung M. tuberculosis infection compared to Th1 cells from intermediate avidity C7 mice.

Interestingly, C24 Th1 cells showed more significant down-regulation of surface TCR post-transfer in both infected and un-infected hosts [it appears that in vitro activated Th1 continue to proliferate independent of antigen].

In addition, while TCR down-regulation was a general feature of activated CD4+ T cells (including endogenous, non-transgenic ESAT6-specific T cells), such TCR down-regulation was profound and long-term for C24 T cells.

Importantly, unlike C7, C24 Th1 cells transferred to uninfected hosts and harvested at day 13 showed drastic reduction of antigen-specific cytokine production.

Moreover, mice transferred with naive C24 T cells and infected with L. monocytogenes–ESAT6 and reinfected 1 month later showed failure to mount secondary response.

In summary, this study revealed that high avidity (affinity) T cells do not necessarily represent the best T cells to control infection. We could extrapolate this observation to include T cells with high affinity TCR against tumor-specific antigens. Important consideration in designing TCR transduced T cells for adoptive T cell immunotherapy.

David Usharauli

Melanoma patients harbor tumor mutation-specific PD1+ CD8 T cells in the peripheral blood

Steven Rosenberg's research group at National Institutes of Health (NIH) continues to define and refine condition for T cell-based cancer-specific immunotherapy. (for example, introduction of high-throughput personalized screening strategy capable of evaluating T cell reactivity to neo-antigens presented on all of the HLA restriction elements of the individual).

This time, their group showed that peripheral blood of melanoma patients harbors small but distinct PD1+ CD8 T cell population enriched in specificities for cancer associated neo-antigens.

If one compares CD8 T cells from PBMC v Tumor sites (TIL), blood derived T cells contains few PD1⁺/PD1^high CD8 T cells.

However, when the authors has expanded in vitro those sorted PD1⁺ CD8 T cells and co-cultured them with autologous dendritic cells expressing tumor neo-antigens (as tandem minigenes, TMG), they could identify circulating neoantigen-reactive CD8 T cells in three of the four melanoma patients evaluated.

Then the authors re-constructed blood PD1⁺/PD1^high CD8 T cell TCR specificity by (a) pairing the sequences encoding the two most-dominant TCR-α and TCR-β sequences, (b) cloning them into retroviral vectors and (c) transducing autologous PBMC. This TCR construct could [for example] detect neo-antigens derived from mutations in the genes MAGE family member A6 (MAGEA6).

Importantly, both PD1⁺/PD1^high CD8 T cells enriched from peripheral blood or T lymphocytes transduced with retroviruses expressing neo-antigen-specific TCRs could detect autologous tumor cell lines.

Finally, the authors found that blood and tumor site derived PD1⁺ CD8 T cell showed high degree of overlap in their TCR specificity [to tumor neo-antigens], suggesting that analysis of peripheral PD1⁺ CD8 T cells from cancer patients could reveal TCR specificities of tumor infiltrated lymphocytes.

In summary, this study is another evidence that cancer immunotherapy holds great promise in providing cancer antigen-tailored treatments. Identification of cancer neo-antigen specific T cells (TCRs) as shown in this study, would accelerate development of tumor-specific TCR constructs and could contribute in overcoming precursor limitation inherent to endogenous T cell clones.

David Usharauli

pMHC tetramer-coated nanoparticles treat autoimmune conditions in mice

This week Nature published "figure-dense" paper showing that nanoparticles coated with auto-antigen+MHC complexes (pMHC) could treat spontaneous or experimentally-induced autoimmune diseases in mice. In fact, this study contains so many experimental results it should have been published in two part, in my view. I will try to condense its basic findings in few paragraphs and provide its interpretation below.

Non-obese diabetic (NOD) mice spontaneously develop [autoimmune] diabetes. Part of T cells that infiltrate and damage β cells in NOD mice express T-cell receptor (TCR) resembling diabetogenic BDC2.5-specific TCR that can be recognized by pMHC–2.5mi/I^Ag7 tetramers. When the authors injected nanoparticles (dextran-coated or pegylated iron oxide NPs) coated with 2.5mi/I^Ag7 tetramers (pMHC–NP) it induced expansion of cognate CD4+ T cells in blood and spleens of NOD mice. These cells had a memory-like (CD44^hiCD62L^low) FOXP3− T_R1-like phenotype.

These "activated/experienced" CD4+ T_R1 cells from donor mice treated with pMHC–2.5mi/I^Ag7-NPs suppressed diabetes development in T-cell-reconstituted NOD-scid hosts upon adoptive transfer. This effect of T_R1 cells was augmented by treating hosts with pMHC–NPs.

Moreover, 90–100% of the already diabetic mice that received nanoparticles coated with β cell antigens, 2.5mi/I^Ag7, IGRP_4–22/I^Ag7 or IGRP_128–145/I^Ag7, reverted to stable normoglycaemia.

Of note, treatment withdrawal resulted in loss of the normoglycaemic state in 25–60% of mice, in association with the loss of the tetramer+CD4+ T-cell pools. In other words, these data suggest that almost half of treated diabetic mice maintained normoglycaemic state after 5 weeks of treatment.

Beyond diabetes, the authors showed that nanoparticles coated with myelin oligodendrocyte glycoprotein, pMOG_38–49/IA^b–NP, dampended progression of experimental autoimmune encephalomyelitis (EAE, a model of multiple sclerosis), when given on day 14 after immunization and even restored motor function in paralytic mice when given on day 21.

Similar therapeutic effects were seen in 3rd autoimmune disease model, collagen-induced arthritis (CIA). HLA-DR4-IE-transgenic mice receiving nanoparticles displaying mouse collagen (mCII)_259–273/DR4-IE showed reduced joint inflammation. In all these models, pMHC–NPs effect was antigen-specific.

Indeed, the effects of pMHC–NP therapy were not associated with impaired systemic immunity because pMHC–NP-treated mice showed unimpaired anti-viral immunity and mounted antibodies against an exogenous antigen as efficiently as control mice.

Interestingly, pMHC–NPs could not expand tetramer+ T cells in non-diabetic control mice which harbor mostly naive T cells. Further experiments confirmed that therapeutic effect of pMHC–NP required presence of "experienced" T cells. For example, whereas pMHC–NP therapy afforded 100% diabetes protection to T-cell-reconstituted NOD-scid hosts bearing memory BDC2.5 T cells, such therapy was inconsequential in hosts receiving naive BDC2.5 T cells.

The role of "experienced", but not naive T cells, in therapeutic effect of pMHC–NP was also supported by observation that whereas diabetic NOD G6pc2−/− mice (which lack IGRP antigen and corresponding memory/experienced T cells, but most likely harbor IGRP_4–22 specific [aka, nonself-specific] naive T cells) responded to 2.5mi/I^Ag7–NPs like wild-type NOD mice, they did not respond to IGRP_4–22/I^Ag7–NPs.

At molecular level, the authors showed blockade of IL-10, TGF-β and IL-21R (but not IFNγ) abrogated the anti-diabetogenic properties of 2.5mi/I^Ag7–NPs or IGRP_4–22/I^Ag7–NPs in diabetic NOD mice.

However, to make things more complicated, the authors also showed that the development of the T_R1 precursors and/ or T_R1-like cells that expand in response to pMHC–NP therapy required IFNγ in addition to IL-10 in pre-diabetic NOD mice.

Finally, the authors found that at the cellular level, pMHC–NP treated NOD mice harbored increased numbers of so called regulatory B cells, B_REG cells (but only in pancreatic lymph nodes, PLNs). These B_REG cells were producing IL-10 in response to lipopolysaccharide (LPS). In vivo, these B cells suppressed diabetes development in T-cell-reconstituted NOD-scid hosts as compared to PLN B cells from control mice (and worked together with T_R1 cells). The role of B_REG cells was supported by observation that treatment of newly diabetic NOD mice with a B-cell depleting anti-CD20 mAb abrogated the anti-diabetogenic activity of 2.5mi/I^Ag7–NPs (see above).

In summary, this study suggests that when injected into mice these pMHC–nanoparticles expand antigen-specific and antigen-experienced T cells with regulatory properties. These T_R1 work with B_REG cells to block the chain of events that perpetuates autoimmune cycle. 

David Usharauli

In vitro comparison of human CAR-T cells expressing either CD28 or 4-1BB costimulatory domains

Today journal Immunity published new [short] study from Carl June's lab describing some functional and metabolic differences between CAR-T cells expressing CD28 or 4-1BB costimulatory domains.

It is not a particularly cutting-edge study. But since its focus is human CAR-T cells such limited experiments are still expected. Though I will anticipate that soon publications of in vitro studies on human primary cells [in top journals] will require incorporation of specific gene targeting experiments using CRISPR-Cas9 technology. Such experiments are now technically feasible and will be more informative.

In short, for this study the authors compared proliferation/expansion and metabolic/energy source of two CAR constructs, one with CD28 and another with 4-1BB (aka CD137) signaling domains, as shown below. FMC63 is anti-CD19 scFv and SS1 is anti-mesothelin scFv.

Human primary T cells were electroporated with these two CAR constructs. Efficacy of CAR expression was 90%.

Then, these transduced CAR-T cells were stimulated with magnetic beads coated with a recombinant anti-CD19 or mesothelin-Fc (such stimulation allowed selective activation of CAR-T cells via CAR construct). It revealed that CAR construct with 4-1BB domain imparted superior long-term expansion functionality on CAR-T cells.

Metabolic analysis revealed that unlike CD28 construct, 4-1BB CAR-T cells showed enrichment for genes responsible for lipid oxidation, implying 4-1BB signaling activates metabolic pathway resembling that of naive and memory T cells (involving mitochondrial oxidation of free fatty acids).

Indeed, transmission electron microscopy data confirmed that 4-1BB CAR-T cells were enriched for mitochondria. 

In summary, this study provided supporting evidence that 4-1BB CAR construct endows T cells with superior expansion/persistence quality. Initial studies on this topic suggested that CD28 CAR construct displayed superior "effector" activity against tumor, while 4-1BB CAR construct showed longer survival [superior memory function]. Subject is not settled, but in discussion section, the authors do note that mixing both CAR constructs could provide better outcome compared to individual construct.

David Usharauli

     

Hybrid peptides represent novel target for auto-reactive T cells in autoimmune diabetes

This week Science published intriguing study showing that in type I diabetic individuals hybrid peptides derived from two unrelated proteins represent novel auto-antigens.  

Initially, the authors showed that chemically cross-linked or synthetic peptide that is made one half of proinsulin C-peptide and another half from chromogranin A (ChgA) peptide (both β cell proteins), could activate diabetogenic mouse T cell clones.

Next, the authors showed that T cells specific for such hybrid peptides can be detected in un-manipulated diabetes susceptible NOD female mice (it would have been more informative if the authors have included data from non-susceptible WT mice as a control).

Finally, the authors showed in T cells from T1D individuals could also recognize hybrid peptide made of human proinsulin C-peptide and neuropeptide Y.

In summary, this study suggests that in T1D susceptible individuals, diabetogenic auto-antigens could be generated by fusion of peptide derived from two unrelated proteins (a side reaction of the proteolytic hydrolysis of peptide in secretory granules). Whether generation of such immunogenic hybrid peptides happens only T1D susceptible individuals remains to be determined.  

David Usharauli

Flexing muscles with IL-33 responsive Tregs

Just read another paper about Foxp3+ Tregs published in journal Immunity. Not clear what to make out of it. It came from Christophe Benoist and Diane Mathis lab, both wellknown immunologists, but it feels if it was done by non-immunologist. It does have Amy J. Wagers as a co-authors. She is known for her studies of stem cells.

So, the basic premise of this study is that aging decreases muscle regeneration [after injury] due to failure to recruit IL-33 responsive Tregs.

Now, how does one test this hypothesis? I will highlight what is good in this paper and what is missing. Initially, the authors showed that in old mice muscle injury (cryo-injury or by toxin) does not attracts as much Tregs as in young mice. Strangely, Tregs recruitment within injured muscle tissue was examined using anti-Foxp3 antibody rather more widely accepted model of Foxp3 reporter mice [which they indicated in methods section they had].

Next, the authors found that cytokine IL-33 was up-regulated in injured muscle tissue (young mouse).

Correspondingly, Tregs recruited and enriched within injured muscle tissue expressed IL-33 receptor called ST2.

Next, the authors speculated that there was maybe some connection between IL-33 responsive Tregs (ST+ Tregs) and muscle regeneration. Indeed, when they have repeated the same experiment with mice deficient for IL-33 receptor specifically on Tregs, muscle regeneration were impaired [after injury]. Though the authors did not show data whether there was no difference between muscle regeneration between young and old mice when both were deficient for IL-33 receptor specifically on Tregs. Without such data it is not possible to conclude whether difference in IL-33 responsiveness in Tregs accounts for difference between young and old mice with respect to muscle regeneration.  

  

Analysis of IL-33 producing cell within muscle tissue showed that fibro/adipogenic progenitor (FAP) cells were the main producer of muscle IL-33 and they produced less of IL-33 after muscle injury in old mice.

Finally, injection of IL-33 into old mice muscle improved its regenerative potential after injury (however, the authors did not repeat the same experiment with Treg-specific IL-33 receptor deficient mice to formally confirm that beneficial effect of exogenous IL-33 was indeed mediated via Tregs).

In summary, this study suggests that IL-33 improves muscle regeneration, in general. Whether reduced number of IL-33 responsive Tregs in old mice represents only variable between young or old mice with respect of muscle regeneration is not clear from this paper. Basically, In my opinion, the results do not support the paper's conclusions with respect to Tregs.

David Usharauli

Foxp3+ Treg-derived IL-35 limits anti-tumor T cell immunity

IL-35 is a cytokine composed of the p35 subunit of IL-12 (encoded by Il12a) and Ebi3 (Ebi3). It was discovered in 2007 at Dario Vignali's lab and since then ~ 200 papers have been published about IL-35, according PubMed database. Most studies indicate that IL-35 has immune inhibitory function.

Now, new study in journal Immunity from the same Dario Vignali's lab provided evidence that Foxp3+ Treg-derived IL-35 inhibits effectiveness of anti-tumor T cell activity, pointing to it as a new immunotherapeutic modality. I am going to highlight the most notable results.

First, the authors showed that tumor (B16 melanoma) growth or its metastasis were inhibited when (a) mice were injected with antibody against IL-35 or when (b) mice lacked IL-35 specifically in Tregs (Foxp3^Cre-YFP. Ebi3^L/L mouse).

Similar effect of IL-35 blockade on tumor growth was seen in genetically-induced tumor model, in KP mouse, that spontaneously develop lung cancer after adenovirus-Cre delivery (KP mouse contains an activating mutation in K-RAS and a loss of function mutation in p53 controlled by Cre mediated recombination).

Mechanistically, the authors showed that IL-35 blockade improved T cell recruitment into tumor tissue.

Finally, the authors found that presence of Treg-derived IL-35 contributes to T cell exhaustion during tumor growth.

In summary, this study indicate the following scenario: certain tumors recruit Tregs. These Tregs secrete IL-35 and inhibit recruitment of effector T cells and/or contributes to their exhaustion. Blocking of IL-35 could provide immunotherapeutic benefit (interestingly, it appears that IL-35 does not synergize with anti-PD1 treatment in tumor models discussed in this paper).

David Usharauli

Tumor-tailored "immunogenic" chemotherapy sensitizes tumor to immune attack

Today journal Immunity published an excellent preclinical research study about tumor immunity. In this paper the authors conducted methodical analyses of the role of tumor-tailored chemotherapy on its immunogenicity and synergy with checkpoint immunotherapy.

It is now universally accepted that T cell-oriented immunotherapy (with anti-PD1, anti-CTLA4) represents important medical progress in treatment of [solid] cancers. Still, not every cancer type is responsive to such immunotherapy. However, this is about to change when personalized, molecular evidence-based tumor therapy becomes widely affordable [technology already exists]. This paper is an example how this would be accomplished.

For this study the authors have used KP lung adenocarcinoma mouse model that express "endogenous mutant Kras and deleted Trp53 alleles in lung epithelial cells upon administration of adenovirus expressing Cre recombinase". This tumor was non-responsive to T cell infiltration or checkpoint inhibition or combination of paclitaxel (Ptax) and carboplatin (Carbo), Ptax-Carbo (chemotherapeutics).

Next, the authors hypothesized that this non-responsiveness could be due to lack of "immunogenicity" of KP cells. To examine this, they conducted in vitro "immunogenic cell death" test with several KP cell lines using FDA approved chemotherapy drugs. High mobility group box 1 (HMGB1) release was used as a surrogate marker for chemotherapy drug-induced tumor cell immunogenicity. They showed that combination of mafosfamide (Maf), which is the active metabolite of cyclophosphamide (Cyc), and oxaliplatin (Oxa) stimulated HMGB1 release by all KP tumor cell lines both in vitro and in vivo.

As expected, in vivo application of Oxa-Cyc combination reduced tumor burden in KP mice.

Effect of Oxa-Cyc was mediated via T cells since its effect was abolished in T cell-deficient RAG KO mice or in KP mice depleted of CD8 T cells.

Interestingly, Oxa-Cyc effect was also depended on TLR4 expression on CD11b⁺/CD11c⁺ myeloid cells.

More importantly, the authors showed that Oxa-Cyc treatment synergized with checkpoint inhibitors, anti-PD-1 + anti-CTLA-4, to control lung tumor burden in KP mice.

Finally, the authors expanded this observation to include two other tumor types, MCA205 fibrosarcoma and CT26 colon carcinoma, and showed selective tumor-tailored chemotherapy synergized with checkpoint inhibitors to control tumor burden in these models as well.

In summary, the results in this study indicate that checkpoint inhibitors alone may not provide any benefits to patients where tumor lack T cell infiltration due to absence of tumor "immunogenic death". However, application of selective, tumor-tailored chemotherapeutics (to induce tumor immunogenic cell death and attract T cells) would synergize with checkpoint inhibitors (working then on tumor-infiltrating T cells) in non-responsive tumor patients and improve clinical outcome of the tumor therapy. In practical terms this would mean development of in vitro tests to examine patient's tumor cell "immunogenicity" response to chemotherapeutics on a personalized basis.

David Usharauli