A specific bacteria-infecting virus, bacteriophage, found in gut microflora, augments anti-tumor T cell immunity

Molecular mimicry between microbial and host's antigens could contribute to autoimmunity but also to the protection against tumors through epitope cross-reactivity. A new study in journal Science indicates that those cross-reactive epitopes could come from viruses that infect endogenous microbial species. 

In this study the authors made a surprising observation that only certain Enterococcus hirae microbial strains (E. hirae 13144 or IGR11) augmented anti-cancer effect in experimental cancer model.

 

 

Next, the authors showed that this biological activity was linked to one dominant epitope, TSLARFANI, derived from TMP protein that originated in 39.2-kb prophage only in those specific E. hirae strains. Mice immunized with heat-inactivated E. hirae 13144 strain, or peptide TSLARFANI, or irrelevant E.coli engineered to express TMP, all augmented anti-cancer effect. 

 

 

Mechanistically, the authors showed that epitope, GSLARFRNI, derived from cancer cells used in these experiments, was recognized by the same CD8 T cells which labeled with TSLARFANI epitope tetramers confirming cross-reactivity between these 2 epitopes.

 

 

In summary, this study suggests that microbiota and bacteriophages they carry represent new modality in fight against cancer. In this study overall anti-tumor effect is modest but we need to take into account that this is an effect of just one cross-reactive epitope in one type of MHC inbred mice, and it is likely that many other epitopes will be involved in outbred species such as humans. However, it is still extremely hard to do such analysis in humans in real world scenario due to lack of exact knowledge about human microbiota strains and poor reliability and performances of available bioinformatics approaches. However, once the mechanistic principles underlying anti-cancer effects are uncovered and accepted, then it is much easier to move the field forward.

posted by David Usharauli

Cross-reactivity between microbial-derived antigens and tumor neoantigens correlates with long-term survival

This is a very interesting paper published in Nature few months back. In this study the authors wanted to uncover immune correlates of long-term (>10 yrs) survival from pancreatic ductal adenocarcinoma that normally account for less than 2% of all patients. 

First they found that "patients with both the highest predicted neoantigen number and either the greatest CD3+CD8+, or polyclonal T-cell repertoire, but neither alone, exhibited the longest survival". It suggested that tumor neoantigen-derived epitope quality rather than simply quantity correlated with survival. 

More importantly, bioinformatics analysis of similarities (mimicry or cross-reactivity or poly-reactivity) and neoantigen fitness modelling between tumor neoatigens and microbial-derived antigens significantly stratified short- and long-term survivors independent of confounding factors and adjuvant chemotherapy. 

If these data will  hold true in other cancer settings, it will append how immunotherapy is applied to treat cancer patients. While this finding is potentially hugely important for immunotherapy, the authors tried not to make too big a statement about it (I would imagine it is a result of a typical reviewers conservatism), writing that "This hypothesis does not assume any associations between pre-existing antimicrobial immunity and survival, but rather aims to develop a strategy to identify candidate neoantigens based on defined immunogenic pathogen-derived epitopes" and in other place "Our results do not indicate causal associations of pre-existing microbial and anti-tumour immunity in LTSs [long-term survivors]. Instead, our data suggest that embedding microbial homology in the context of our neoantigen quality model can help to create an effective surrogate for immunogenic neoantigens." 

posted by David Usharauli

Reduced CTLA-4 signaling predisposes to Th2 driven gastric tumorigenesis

Anti-CTLA4 antibodies such as Yervoy, has been used in clinical practice to treat solid tumors. It supposed to work either by augmenting and revitalizing effector T cells function directly or indirectly through temporal silencing of inhibitory Foxp3+ Treg population or both. However, new study from Journal of Experimental Medicine showed that at least in [genetically predisposed] mice reduced CTLA-4 signaling by itself could cause Th2 driven tumorigenic transformation of stomach epithelial tissue.

For this study the authors created transgenic CTLA4 shRNA knockdown (CTLA4KD) mice on the BALB/c × C57BL/6 (B6) mixed genetic background. This they did because it appears that BALB/c but not B6 mice were susceptible developing gastric tumors in this model. CTLA4KD mice showed gastric epithelial transformation by 20w of age. Similarly, month long treatment of newborn BALB/c mice with anti-CTLA4 antibody also led to gastric epithelial transformation.

This tumorigenic transformation was CD4 T cell dependent and effector T cells from CTLA4KD but not from WT mice could mediate it. It indicated that changes in effector T cell composition and functionality were driving de novo inflammatory tumorigenesis.

Interestingly, gastric epithelial transformation were happening even in germ-free CTLA4KD mice lacking microbiota. However, since these mice also harbor increased numbers of inflammatory T cells, in all subsets analyzed such as Th1, Th2, Th17, and independent of microbiota it could indicate that T cells could be responding to antigens from food or environment.

Finally, elimination of canonical T helper cytokines showed that surprisingly neither IFN-γ nor IL-17 but IL-4 deficiency could abolish gastric epithelial transformation under conditions of reduced CTLA-4 activity.

In summary, this study suggests that inherited or clinically-induced reduction of CTLA-4 signaling in predisposed individuals could paradoxically lead to inflammatory tumorigenesis driven by type II immunity.

posted by David Usharauli

Effort to identify tumor-specific antigens: The University Industrial Complex study results

New study in journal Cell is prime example why utilization of sophisticated high-throughput methods and computer technologies does not guarantee generation of clinically useful results. I imagine the only reason this study was even accepted in Cell was the fact that list of authors included many well-known scientists with links to both academia and silicon valley (Stanford University School of Medicine, Chan Zuckerberg Biohub, Parker Institute for Cancer Immunotherapy).   

Idea of this study was to develop techniques to quickly identify tumor-specific antigens (most likely mutated antigens) that could be used in immunotherapy (though there is no evidence that any cancer vaccines based on mutant protein sequences actually work in humans using available practices). 

For this task, the authors took advantage of yeast-display library expressing random peptide covalently linked to the HLA-A*02:01 molecule, an allele which is present in up to 50% of a number of populations. The authors estimated that "approximately 400 million unique peptides ranging from 8 to 11 amino acids are represented in the combined [yeast-display] libraries."

To validate this approach, they used three recombinant 'blinded' positive control TCRs derived from a melanoma patient (their antigen specificity had been identified independently by exome sequencing, tetramer staining and binding prediction algorithms). However, antigen-specificity of only 1 TCR (NKI 2) could be validated using their yeast-display library. As the authors said "targets of NKI 1 and NKI 3 could not be unambiguously identified through this blinded validation."

Of note, in these validation experiments with NKI 2 (specific for ALDPHSGHFV, a peptide neoantigen derived from CDK4 and other DMF5 TCR specific for EAAGIGILTV derived from the MART-1 melanoma antigen, successful validation [specific enrichment + TCR staining] occurred when HA tagged 10-mer epitope library were used. 

The authors anyway went ahead with this "less than perfect" approach to try to identify tumor antigen specificity of T cells derived from 2 patients with colorectal adenocarcinoma and homozygous for the HLA-A*02 allele. The authors focused on 20 TCR most enriched in tumor tissues (based on frequency of occurrence of the same TCR genes). 

Out of these 20, only 4 TCRs could enrich peptide from the library (only with c-Myc tagged 9-mer epitope library) and only 3 TCR could stain yeast samples.  

Next, the authors try to identify epitopes from potential landscape of sequences for each TCR. Several algorithms were deployed (at least 3 or more such as a modified variant of the previous statistical method using a position weight matrix and a method utilizing a two-layer convolutional neural network). They found 1 peptide sequence EYGVSYEW, which closely matches the peptide motif for TCR 1A, however, neither this exome peptide or the anchor-modified exome peptide (EMGVSYEM), nor the human peptide predictions stimulated the cell line modified to express the TCR 1A. TCR 4B was stimulated with several peptides and as the authors write "true in vivo specificity cannot be unambiguously identified without additional tumor information". Regarding TCR 2A and 3B, only 1 peptide stimulated cell line expressing these TCRs. This peptide was MMDFFNAQM, which is derived from U2AF2, a protein involved in an RNA splicing complex. However, in both patients, no mutations were found in U2AF2.

In summary, the authors wrote "although we cannot definitively determine an immune response targeting the peptide derived from U2AF2, the evidence from the yeast-display screen, prediction algorithm, and in vitro stimulation identify this peptide as the likely target". However, when reading this study it is clear that none of the components worked: yeast-display screen performed suboptimally, prediction algorithms provide little clue and in vitro stimulation made it even more confusing. So, what have we learned from all of these? I would say maybe don't do what they did.

posted by David Usharauli    

Hidden arm against tumors: microbiota-enabled checkpoint immunotherapy

This week Science published two studies showing how diverse microbiota directly contributes to efficacy of PD-1 checkpoint immunotherapy in several tumors.

First, we need to mention that senior authors from both papers disclosed associations with for-profit pharma/biotech companies (as cofounders, stockholders, paid consultants or advisory board members). Such associations could, in general, be seen as problematic if one promotes therapy lacking particularities.     

Second, data presented do not advance our understanding how microbiota contributes to the effectiveness of checkpoint immunotherapy. The sole conclusion from both papers is that the more diverse cancer patient's microbiota the more benefit it provides during PD-1 immunotherapy. However, when it comes to narrow down beneficial correlation to particular species we find that one paper reported enrichment of Akkermansia muciniphila while other paper reported enrichment of Faecalibacterium and Clostridiales in Responders (as opposed to Non-Responders).

We still don't know much about the role of microbiota in cancer immunotherapy. I think real advance will come when we define how antigens derived from specific microbiota contribute to anti-cancer immunotherapy either by amplifying existing cross-reactive effector T cells or Foxp3+ Tregs. 

posted by 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