Changes in tumor-specific antigenicity overtime in melanoma patients undergoing adoptive T cell therapy

Most tumors, and most likely "all" tumors, express one or more non-synonymous somatic mutations that generate sufficient number of novel HLA/peptides that potentially could be targeted by T cells. However, similar to infections, when under immune pressure tumor can also develop escape variants by modulating its HLA/peptide landscape. 

This is what new study published in journal Nature found in tumor patients. For this study, the authors (who are associated with AIMM Therapeutics) sequenced patients tumor mRNA at different time points (before or after adoptive cell therapy) and found that some of the tumor-specific HLA/peptides had disappeared [overtime] and some new tumor-specific HLA/peptides became enriched instead. 

Of note, within each patient almost all of T cell reactivity to tumors were patient-specific (i.e. private HLA/peptide) and did not share sequence similarities with publicly known shared tumor-associated epitopes. For example, analysis of patient's CD8+ T cell specificity against a panel of >200 MHC-multimers containing known shared tumor-associated epitopes demonstrated that only 1.24% of the CD8+ T cells responded to three different "shared" gp100 epitopes. The rest were patient's specific. 

For this study two melanoma patients had their tumors mRNA sequenced and underwent autologous adoptive tumor-specific T cell therapy. In one patient, initial tumor mRNA sequencing revealed  501 non-synonymous mutated genes. Most of CD8+ T cell reactivity were directed to two HLA/peptides complexes: KIAA0020 p.P451L (KIA P>L) and ribosomal protein RPL28 p.S76F (RPL28 S>F).

Around 1 year later, however, patient's CD8+ T cell reactivity was only observed against the KIAP>L neo-antigen, and not against RPL28S>F and tumor mRNA sequencing confirmed absence of mutant allele encoding the RPL28S>F neo-antigen within tumors harvested at a later time.

Similarly, in an another patient undergoing similar procedures, the authors found that initially melanoma patient's CD8+ T cells were specific for the neo-antigens echinoderm microtubule associated protein like 1 p.R64W (EML1R>W), Septin-2 p.R300C (SEPT2R>C), and CAD protein p.R1854Q (CADR>Q).  

However, later analysis revealed no significant T cell reactivity against EML1R>W, CADR>Q neo-antigen and SEPT2R>C neo-antigens. Instead, it showed T cell response towards the Programmed Cell Death Protein 10 p.P28S (PDCD10P>S) neo-antigen. Indeed, tumor mRNA sequencing confirmed that while mutant allele encoding the SEPT2R>C neo-antigen that was present in the original tumor, it was selectively lost in the tumor samples harvested later, and that RNA for novel PDCD10 neo-antigen had increased > 40-fold in later time points instead.

These data points to two important considerations: first, tumor undergoes changes in their mutational landscape and can evade T cell detection and second, most tumor mutations are patient's specific. This means that adoptive immunotherapy using single T cell specificity would be less efficient overtime and that tumor vaccines using "generic-shared" tumor epitopes would provide no benefits in most patients. 

David Usharauli

      

The adaptation of microglia (brain macrophages) to brain development

Microglia is a brain-specific macrophage lineage that seeds the developing brain tissue in utero (embryonic) and contributes to "patterning and wiring of the brain in early development and contribute to homeostasis throughout life". Since brain is cocooned from the rest of the body by blood-brain barrier (BBB), it is not clear whether microglia, as a member of innate immune system, is responsive to signals generated within immune system.

New study in Science suggests that brain resident microglia undergo time-dependent step-wise upgrades and retains sensitivities to signals derived from immune system and microbiota.

Basically for this study the authors analyzed gene expression profile of microglia (both from single cell and from the pool) harvested from brain at different developmental stage (in utero (embryonic), newborn and adult).      

Interestingly and relevant for medicine, the authors could show that systemic activation of immune system by poly I:C could disrupt normal formation of newborn microglia by switching its developmental program to more adult microglia-like setting. 

Such changes in newborn microglia can potentially underlie neuro-developmental challenges in babies born to mothers experiencing immune activation (viral infections) during pregnancy.   

David Usharauli

Absence of NOD2 signaling protects against chemical[STZ]-induced diabetes

Experimental models developed in "clean" laboratory mice rarely translates to human diseases. For example, NOD mice spontaneously develop Type 1 diabetes, as susceptible humans do, however pancreatic tissue pathology does not match. 

There is another model of T1D in mice called treptozotocin (STZ)-induced diabetes. STZ is toxic to insulin producing β cells in pancreas and drive T1D in a T cell-dependent manner. In new study published in Journal of Experimental Medicine the authors showed that treptozotocin induced T1D depended on gut flora derived muramyl dipeptide → NOD2 signaling.

The authors found that NOD2^-/- mice, but not NOD1^-/- or Rip2^-/- mice, were resistant to STZ effect.

Interestingly, similar protective effect against T1D were seen in WT mice treated with broad-spectrum antibiotics and STZ. However, addition of NOD2 ligand muramyl dipeptide reversed protection in [antibiotic + STZ] treated mice.

This study showed that NOD2 ligand, muramyl dipeptide derived from gut microbiota, contributes to T1D in STZ-treated mice.

David Usharauli

Tissue-specific immunopathology initiated by microbiota

Frequently, autoimmune disorders come in combinations. For example, a subset of human patients with type 1 diabetes (T1D) develop autoimmune polyglandular syndrome (APS) that depending on type (APS types 1-4) involves other tissues such as thyroid, adrenal, submandibular, and lacrimal glands. 

New study published in Journal of Immunology and conducted in autoimmune-prone mouse strain, NOD, showed that presence of microbiota modifies immunopathology in a tissue-specific manner.

For instance, female NOD mice devoid of microbiota (GF NOD mice) showed reduced pathology in salivary glands (sialitis) but as expected no change or worsening of pathology in pancreas (insulitis).  

Similarly, MyD88 deficiency completely abolished sialitis in both WT and GF female mice but it couldn't rescue insulitis phenotype on GF background (as reported prevously).

In summary, this short study suggests that microbiota → MyD88 pathways directly influence development of sialitis, while for insulitis (and diabetes development) microbiota/MyD88 pathways diverge.

David Usharauli

Route of vaccination determines level of protection against coronaviruses (SARS, MERS)

The coronaviruses (CoV) such as Middle East respiratory syndrome (MERS)-CoV and severe acute respiratory syndrome (SARS)-CoV "have crossed species to cause severe human respiratory disease."

New study in Immunity tested vaccine design based on Venezuelan equine encephalitis replicons (VRP) encoding a SARS-CoV CD4+ T cell epitope derived from nucleocapsid (N) protein of SARS-CoV (N353). VRPs are non-replicating viral vectors that preferentially "infect human and mouse dendritic cells and serve as self-adjuvants". The N protein is "conserved among different coronaviruses" and unlike highly variable S protein, it could induce cross-protection at T cell level.

Interestingly, when tested on laboratory mice, protection from lethal disease [upon challenge] was nearly complete after i.n. (intra-nasal) but not s.c. (subcutaneous) administration of VRP-SARS-N vaccine, demonstrating the importance of the route of vaccination. 

I reviewed this paper to emphasize that "route of vaccination" that induces antigen-specific T cells residing at the port of [pathogen] entry is crucial in vaccine effectiveness.   

David Usharauli

Pre-clinical animal model of CD47-blocking immunotherapy in small-cell lung cancer

This week Journal of Clinical Investigation (JCI) published a new study examining CD47-blocking immunotherapy in small-cell lung cancer. This is a joint study by people affiliated with Forty Seven Inc. and Alexo Therapeutics Inc., two biotech companies which focus on clinical application of anti-CD47 targeting immunotherapy.

Just to remind the readers, that simply put, CD47 is an inhibitory molecule expressed on multiple cell types, including tumors, that act as a "do not eat me" signaling. It is believed that by removing this negative signaling, macrophages could phagocytose tumor cells efficiently and thus restrict tumor growth.

In this study, the authors analyzed effectiveness of blocking of CD47/SIRPα pathway in NSG mouse small-cell lung cancer (SCLC) model. First, the authors showed that human small cell lung cancer cell lines or patient-derived SCLC cells express CD47 and when co-incubated with macrophage and anti-CD47 antibody, such tumor cells are efficiently phagocytosed (though there is no correlation between level of CD47 expression and phagocytosing activity).

Next, the authors showed that treatment with anti-human CD47 antibody inhibited growth of human SCLC cells implanted in immunodeficient NSG mice.

Anti-human CD47 antibody was active in PDX model as well (patient-derived xenograft tumor model in NSG mice).

Similar trend, though less potent tumor growth inhibition was observed with CD47KO SCLC cell line derived via Cas9 editing [suggesting that anti-CD47 antibody provides additional signaling beyond CD47 blocking]. 

To answer some of the criticism of earlier studies, the authors conducted CD47KO mouse tumor cell transplantation experiments in immunocompetent mice and observed similar outcome.  

In summary, this study showed that anti-CD47 therapy could be effective against CD47+ small cell lung cancer which originates from neuroendocrine cells of the lung (however keep in mind that in many models, CD47 blockade does not completely eliminate tumors). 

David Usharauli

Mutation in DNAse1L3 triggers systemic lupus erythematosus (SLE)-like condition in mice

Autoimmune disease, as word implies, is an immune response directed to self. Traditionally we used to think that autoimmune diseases arise as a result of failure of adaptive immune system (T and B cells) to distinguish between self and nonself antigens. However, as with many inherited immunodeficiencies, frequently we see that inherited innate genetic mutations play pivotal part in autoimmune phenotypes as well.  

For example, few days ago journal Cell published study showing that mice deficient for DNAse1L3, an enzyme capable of degrading extracellular chromatin associated DNA, develop systemic lupus erythematosus (SLE)-like condition.

DNAse1L3, a homologue to DNAse1, contains a short, positively charged C-terminal peptide that allows it to uniquely digest DNA chromatin in microparticles released from apoptotic cells.

Mice deficient for DNAse1L3 develop anti-dsDNA Ab response and Ab deposition in the kidney glomeruli [clinical feature of SLE in humans].

Unlike other genetic DNA/RNA housekeeping mutations (Trex1^-/-, DNase-II^-/-, RNase H2B^-/- ) phenotype of DNAse1L3-KO mice was independent of STING activity (but was dependent on MyD88, though was not clear how or why).

In summary, This study revealed that DCs/Mac derived secreted DNAse1L3 is intimately involved in digestion of apoptotic microparticle associated DNA and in prevention of anti-DNA antibody formation (of note, one of the authors is a co-founder and consultant of Resolve Therapeutics, which develops soluble nucleases for therapeutic purposes).

David Usharauli

  

Do innate NK cells truly show memory-like response to antigens? Monobenzone [hapten] study

This week Immunity published study claiming NK cell response to melanocyte-targeting hapten, monobenzone, display adaptive-like behavior with memory component.

The concept itself is quite new and not fully developed  [first report of this kind was published in 2006]. I personally do not see that data presented so far support proposal that NK cells have canonical "adaptive memory". For example, one of the hallmarks of adaptive memory response is that secondary response is stronger and more durable compared to primary response. However, in this new paper both primary and secondary hapten-specific response by NK cells show equal magnitude.

To confirm memory response by NK cells, in my view, the authors should show that (a) NK cell response is antigen-[hapten] specific, (b) it is long-lived and (c) secondary response is stronger. 

There is no doubt that in this paper NK cells show antigen-specific response (condition A) and such response could persist for relatively long term (ranging from few weeks to few months, condition B). However, condition C has not been met. In crucial experiment, the authors transferred NK cells recovered after primary response into naive host and then challenged the host 1 week later. However, for some reason, the authors also applied hapten 1 day after NK cells transfer [and before secondary challenge]. This basically prevented testing of recall response.

Only experiment that the authors performed to show long-lived NK cell memory response was the experiment wherein hapten-primed mice were left to sit around for 4 months and then challenged for the second time. However, in this scenario it is hard to tell whether it is true memory response or hapten simply persisted in mice and kept NK cells in active "effector" phase. In addition, in this experiment, both primary and secondary responses were of equal magnitude.

In summary, so far I have not seen definite results in support of "canonical" NK cell memory.

David Usharauli

Runx3-dependent intraepithelial lymphocytes (CD4IELs) control gut tolerance to dietary antigens

Few days ago journal Science published another immunological study. This hard to read research paper showed that special subset of Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs) found within intestinal epithelium play distinct and dominant role in preventing gut inflammation to dietary antigens.

It appears that induction or maintenance of Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs) depends on antigens derived from either from gut flora [microbiome] or food.

The main finding of this paper was observation that antigen-specific T cell monoclonal mouse strain deficient for Foxp3 expression [but not Foxp3 wt] depleted of CD4IELs using anti-CD8α antibodies during antigen feeding showed severe intestinal inflammation and diarrhea. It suggests Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs) play distinct and separate [that from Foxp3wt T cells] role in protecting intestinal tissue against antigenic inflammation.

Why is this paper relevant? For one, it indicates that tolerance to food antigens in physiological situations, for example during feeding, could be mediated by Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs), and not by Foxp3+ CD4+ T cells, as scientists ordinarily assume. However, for now, we know relatively little about tissue-resident innate-like or adaptive-like cells, such as these Foxp3–CD8αα+CD4+ intraepithelial lymphocytes (CD4IELs).

David Usharauli

What could be learned from vaccines that did not work?

It is >30 years since HIV discovery and we still don't have vaccine that can protect against it. Billions of dollars spent. And still no one could explain why HIV vaccines developed thus far are failing one after another (RV144 HIV-vaccine trial is one of those).

Here is what I think. Modern vaccinology is based on concept introduced 50 years ago. It is based on assumption that adaptive immune system (canonical B and T cells) is the main driver of protection. 20 years ago, after discovery of TLRs, there was a conceptual shift towards dendritic cell (DCs)-oriented vaccines. Nonetheless, the main focus of vaccine development still remains adaptive immune system (B cells, T cells and DCs). I think it is time to rethink our approach to vaccination. Adaptive immunity is one part of equation. There are growing lists of innate/innate-like cells that could play crucial role in next-generation of vaccines.

For example, following the RV144 HIV-vaccine trial, there was lots of head scratching. Some thought we need to use stronger adjuvants [compared to Alum]. However new study conducted on monkeys showed that "the higher vaccine immunogenicity of MF59 [adjuvant] does not directly translate to higher vaccine efficacy against SIV [monkey HIV] acquisition."

Moreover, this study found that higher percentage of SIV Env-reactive innate NKp44+IL-17+ cells  following alum-vaccine "were associated with a reduced risk of acquisition [of SIV in monkeys] in the multivariate analysis" (in other immune parameters MF59 was better than alum).

In summary, we need to acknowledge that the more learn about immune system the more we understand how little we know about it. 

David Usharauli

Tumor protein encoding [negatively charged] RNA-nanoparticles induced robust anti-tumor response

This week journal Nature published study that showed that negatively charged [cancer antigen encoding] RNA-nanoparticles are selectively take up by antigen-presenting cells in lymphoid tissues and induced robust type I IFN driven anti-tumor immune response to all tumors tested.

In earlier studies for in vivo delivery of RNA/DNA nucleic acids into antigen-presenting cells scientists have used positively charged [cationic] nanoparticles. However, such particles were mostly trapped within lungs rather than lymphoid tissues where most antigen-presenting cells, such as dendrtic cells, reside. Surprisingly, here the authors showed that negatively charged RNA-nanoparticles selectively accumulated in spleen within antigen-presenting cells after in vivo injection.

Strangely, accumulation of RNA-nanoparticles were reduced when mice were depleted of conventional DCs (cDCs) after diphtheria toxin injection (CD11-DRT BM chimera mice), even though these mice retain both plasmacytoid DCs (pDCs) and macrophages which can themselves capture RNA-nanoparticles.

RNA-nanoparticle injection was associated with rapid IFN-α secretion in a manner that depended on TLR7 expression.

In addition, RNA-nanoparticles induced robust priming of antigen-specific adaptive immune response.

More importantly, injection of tumor protein encoding RNA-nanoparticles delivered incredibly robust prophylactic and therapeutic anti-tumor effect (article has some preliminary  results from phase I study showing T cell priming in cancer patient after RNA-nanoparticle delivery).

In summary, the authors of this study believe that they found universal antigen delivery RNA-nanoparticle construct that induces robust adaptive immune response.

There is one thing that is puzzling about this study which the authors did not tried explain: in mice depletion of cDCs eliminated RNA-nanoparticle uptake and T cell priming. However, pDCs are not depleted in CD11c-DTR chimera mice and the data showed that pDCs are themselves could capture RNA-nanoparticles and are the main source of type I IFNs. So, the question then is why cDCs depletion has such disproportional effect?

David Usharauli

Combination of checkpoint inhibitor and IL-21-primed melanoma-specific T cells produced durable response, "cure", in melanoma patient

This week Journal of Experimental Medicine published a case study of a single melanoma patient who achieved durable response (>5 years), "cure", after combination therapy (IL-21 primed MART1-reactive "polyclonal" CD8+ T cells and Yervoy). 

Of note, earlier attempts to stop melanoma progression in this patient with IL-2/IL-7/IL-15 primed MART1-reactive "monoclonal" CD8+ T cells and/or Yervoy were not successful. As seen before, successful anti-melanoma immunotherapy produced autoimmune skin/hair disorder, vitiligo (loss of melanocytes). 

As the cellular level, IL-21 priming resulted in better survival of infused polyclonal T cells and epitope spreading targeting other melanoma antigens such as NY-EOS1, gp100, tyrosinase and MAGE-A3.

In summary, this study clearly shows the vast [not yet fully tapped] potential of cancer immunotherapy.

David Usharauli