Tissue retention of antigen-specific resident memory T cells

In recent years research in memory T cell response showed that secondary response to peripheral antigen challenge is driven mostly by locally-residing, tissue resident memory T cells (T_RM). Such studies are important to understand how vaccines affect development of T_RM (previously, main focus was on memory T cells residing in lymphoid tissues, such as spleen and lymph nodes).

Few studies suggested that virus induced T_RM cells could migrate out and reside in tissues not directly affected by virus [i.e. Ag-independent manner]. However, immune response to different viruses can differ fundamentally from each other. For example, new report in Journal of Experimental Medicine (JEM) showed that skin infection with vaccinia virus (VacV, a benign relative of smallpox) instructs development and retention of local T_RM in an antigen-specific manner.

In mice, infection with VacV by skin scarification produces local infection and induction of virus antigen-specific T cell response is local as well.

Importantly, accumulation of CD8 T cells within infected tissue was indeed antigen-specific, and not driven by local virus-induced inflammation per se.

Secondary antigenic challenge confirmed that T_RM response was restricted to local tissue previously infected with virus carrying the same antigen.

In summary, this study showed that in case of VacV infection tissue retention of T_RM cells was antigen-dependent and occurred locally at the site of primary viral challenge.

David Usharauli

Cish is a NK cell checkpoint inhibitor

This week several science news outlets spotlighted new study from Nature Immunology showing anti-tumor effect of Cish deficient NK cells. This study showed that Cish deficient NK cells are hyper-responsive to its canonical cytokine IL-15 and show improved control of experimental tumors.

I analysed this study to determine if the buzz was deserving. In my view this study is in fact two independent [and not connected] studies put together artificially. The finding that Cish deficient NK cells have superior anti-tumor behavior is based on non-physiological experimental model.

First part of this study deals with cytokine sensitivity of Cish deficient NK cells. This is exclusively in vitro experiments. It does show that Cish deficient NK cells are hyper-responsive to NK cell canonical cytokines such as IL-15, or IL-12/IL-18 combo [and other activatory receptors].

Second part of this study, however, is completely dissociated with IL-15 story and jumps directly to tumor protection experiments with WT or Cish deficient NK cells. These experiments showed that Cish deficient mice are resistant to exogenously injected tumor cells and this protection depended on asiolo-GM1+ cells and IFN-γ.

Finally, the authors showed that adoptive transfer of Cish deficient NK cells into NK-deficient hosts (Ncr1^Mcl1Δ/Δ mice) also provided protection against i.v. injected melanoma cell metastasis.

In summary, this study proposed that Cish functions as a checkpoint inhibitor for NK cells.

My view:

(A) It is not clear whether Cish deficient NK cells alone is sufficient for anti-tumor effect [it is independent of CD8 T cells, but CD4 T cells involvement was not tested].

(B) It is not clear what role cytokines such as IL-15 or IL-12/18 play in vivo in Cish deficient mice.  

(C) One of the major differences between NK and T cells is that NK cell effector functions are not regulated in an antigen-specific manner but rather by sensing signaling balance between activatory and inhibitory membrane receptors [functions like a rheostat].

During ontogeny individual NK cell undergoes its own "adjustment" to its environment and can even "tolerate" naturally arising tumor cells. This is why experimental tumor models when tumors are injected exogenously, and appear in the body out of the blue so to speak, do not recapitulate natural interaction with NK cells and easily could produce biased, non-physiological response from first-time encountering NK cells. The more physiological tumor models will be to use spontaneously arising mouse tumor models.

David Usharauli

    

Harnessing donor TCR specificity for cancer immunotherapy

Cancerous tissues harbor protein mutations that can be recognized by immune system as neoantigens. However, when tumor progresses it indicates that either (A) patient lacks T cells with adequate affinity to tumor neoantigens or (B) tumor environment actively suppresses immune response [or both]. 

For example, novel drug class of checkpoint inhibitors targeting CTLA4 and PD1/PD-L1 inhibitory circuits operating in T cells (Keytruda, Opdivo, Yervoy, Tecentriq) work on option B by modulating tumor suppressive micro-environment.

Another approach obviously would be an option A by using engineered T cells expressing tumor specific T cell receptors. Ideally, patient's own T cells can be expanded and re-infused back to attack tumor cells. But, more likely, patient will lack T cells with  adequate affinity to tumor neoantigens due to TCR editing. 

To overcome this limitation, new study published in journal Science suggested to use instead tumor-specific TCRs harvested from healthy donors. Here, the authors led by T cell expert Ton Schumacher, showed that HLA-matched healthy donors contain T cells with sufficient affinity and specificity to recipient's tumor neoantigens (of note, Ton Schumacher is also affiliated with biotech company Kite Pharma).

This study focused on HLA-A*02:01-restricted neoantigens from stage IV melanoma patients. Neoantigens were identified with whole-exome and RNA sequencing and selected for further analysis based on high predicted binding affinity to HLA-A*02:01. Autologous monocyte-derived dendritic cells transfected with mRNA encoding the candidate epitopes and cultured with healthy donor T cells. All 4 healthy donor  T cells specifically detected mutated tumor neoantigens with greater sensitivity.

Tumor neoantigen-specific T cell response was confirmed in epitope pulse experiment using WT or mut epitopes.

Furthermore, when donor T cell derived TCRs were re-introduced by gene transfer, resulting T cells were specific to patient's mut neoantigen and did not recognize, for example, 3rd party tumor cells.

In summary, this short but definitive study points to a growing and undeniable evidence in support for T cell based cancer immunotherapy. By incorporating donor derived TCR specificity this strategy vastly expands the reach of T cells immunotherapy. The challenge remains how to streamline this process (exome sequencing, MHC:peptide binding prediction, TCR identification, TCR transduction and re-infusion) to make it affordable for every cancer patient.  

David Usharauli

  

Why anti-viral immune serum doesn't work in every infected patient?

Many viruses, such as Ebola or avian-origin Flu viruses, are extremely virulent and can cause death of infected individuals within days. However, some individuals are naturally resistant to such virulent infections and generate protective, neutralizing antibodies in response to them. Many organizations across world responsible for public health safety [and also military institutions] try to stockpile such anti-viral sera obtained from individuals who survive natural infection. It is believed that injection of anti-viral serum into infected individuals could help them to survive.

However, new study in journal Nature suggests that anti-viral serum effectiveness depends on viral tropism. It turns out that protection against viruses that infect so called immune privileged tissues, such as brain tissue, required presence of both anti-viral neutralizing antibodies and anti-viral CD4 T cells. Specifically, the authors, led by Akiko Iwasaki from Yale School of Medicine, showed that anti-viral CD4 T cell's role is to open the "gate" to privileged tissues for anti-viral antibodies that usually can not cross such barrier on their own.

For this study the authors used herpes simplex virus type 2 (HSV-2) virus challenge model that infects mouse immune privileged tissues such as the innervating neurons in the dorsal root ganglia (DRG). Initially the authors reported and re-confirmed that if mice were vaccinated with attenuated strain of HSV-2 before WT virus challenge, then these vaccinated mice were protected against WT virus challenge and this protection depended on antibody.

Strangely, however, immune serum could not protect naive, non-vaccinated mice against WT virus challenge. Moreover, immune serum protected vaccinated mice even if these mice did not make antibodies on their own. This suggested that something else  besides antibodies [generated during vaccination] needed alongside with anti-viral antibodies for virus protection. 

Indeed, vaccinated mice depleted of anti-viral memory CD4 T cells just prior to WT virus challenge were not protected.

Next, the authors found that presence of anti-viral CD4 T cells (but not of irrelevant CD4 T cells) were necessary for anti-viral antibodies to enter and accumulate within infected neuronal tissue.

Finally, the authors showed that neuronal tissue recruitment of anti-viral CD4 T cells depended on α4β1 (VLA4 integrin) interactions and subsequent CD4 T cell-derived IFN-γ secretion mediated local vascular permeability to enable antibody access to neuronal tissue.

In summary, this study explains why simple application of immune serum is not always sufficient for protection against neurotropic infections. Neutralizing immune serum on its own is not able to penetrate barrier, immune privileged tissues in infected recipients if they lack anti-viral CD4 T cells. This could explain why anti-Ebola serum was not effective in all Ebola-infected patients. This study also provides mechanistic explanation for current paradigm for rabies virus protection that requires application of both vaccine [to induce T cells] alongside anti-rabies immune serum.

David Usharauli

Foxp3+ Tregs go against tumor in the "dark"

This week PNAS published follow-up study from Harvey Cantor's lab examining role of Helios (the Sun in Greek mythology) in Foxp3+ T_REG stability. In this new study they showed that antibody [anti-GITR]-mediated down-regulation of Helios in Foxp3+ T_REG causes them to acquire effector function and participate in anti-tumor response.

I will highlight relevant findings. The first finding was that mice with Helios-deficient Foxp3+ T_REG showed additional resistance to tumor (however, please note that tumor growth pattern and mice survival do not correlate in Helios^fl/fl.FoxP3-Cre mice).

Similar tumor growth retardation was observed in adoptive co-transfer experiment with WT T cells and Helios-KO Foxp3+ T_REG.

Mechanistically, Helios^fl/fl.FoxP3-cre T_REG up-regulated effector cytokines specifically at tumor site.

Finally, treatment of mice with antibody, DTA-1, directed to GITR (glucocorticoid induced TNF receptor), caused Helios down-regulation in FoxP3+ T_REG and improved tumor protection.

In summary, the authors proposed that therapeutic targeting of Helios expression in tumor-associated FoxP3+ T_REG could convert them from tolerogenic cells into anti-cancer fighting cells (however, please note the authors did not formally test whether anti-cancer effect of anti-GITR antibody was indeed mediated via its effect on Helios in FoxP3+ T_REG).

David Usharauli

PSGL-1, a ligand for the selectin family of receptors, controls T cell immunopathology

The Selectin receptors, L, E and P, control T cell migration. New study in Immunity showed that Selplg-KO T cells (lacking selectin receptor ligand PSGL-1) display enhanced effector differentiation and greater control of chronic viral infection and tumor, though it comes at the expense of tissue pathology.  

In this paper the authors referred to PSGL-1 as checkpoint inhibitor. However, unlike CTLA4 and PD1 deficient mice, Selplg ^-/- naive mice don't show any abnormality. Interestingly, the authors found that Selplg ^-/- mice efficiently controlled chronic LCMV infection (C13).

However, this enhanced protection against chronic LCMV infection led to severe tissue pathology.

Additionally, and most likely reason why this paper ended up in Immunity, the authors showed that Selplg ^-/- mice could better control tumor (Yumm1.5 melanoma cells).

At present it is not clear whether PSGL-1 signaling works as an independent "checkpoint inhibitor" in vivo or modulates functionality of other canonical checkpoint inhibitors such as PD-1 .

David Usharauli

Experimental malaria vaccine shows 55% efficacy in small trial

This week Nature Medicine published results from small trial involving controlled human malaria infection (CHMI) and experimental malaria vaccine developed by Sanaria (Sanaria Inc., Rockville, Maryland, USA). This vaccine based on attenuated Plasmodium falciparum sporozoite (abbreviated as PfSPZ vaccine) showed 55% efficacy at 1 year post-immunization.

Currently, only malaria vaccine available on market, RTS,S, has efficacy of ~ 22%. In this new study, scientists tested different dosages and routes of immunization for new PfSPZ vaccine as follows:

The results of the controlled human malaria infection are shown below. Here, healthy malaria-naive volunteers were first vaccinated and then received malaria infection from actual mosquitoes bites that carry malaria clone 3D7. The best outcomes were achieved with groups 4 and 5 (i.v. administration, 4 doses of 2.7 × 10⁵ PfSPZ).

Next step was to understand immunological correlates of protection. However, this task was quite challenging, as it turned out. All vaccination protocols, with the exception of i.m. immunization, induced anti-malaria Ab or T cell responses. However, when vaccine trial participants were divided based on blood parasitemia following malaria challenge, it was observed that participants without parasitemia developed higher levels of anti-malaria serum Abs.

At the cellular level, however, the only marker that correlated with vaccine efficacy was frequency of unstimulated Vγ9+Vδ2+ γδ T cells which comprises ~75% of γδ T cells in blood. Actually, the frequency of Vγ9+Vδ2+ γδ T cells in pre-vaccinated individuals was the only marker that correlated with vaccine efficacy in challenge model.

In summary, this study showed that there is still room to improve malaria vaccine. Vaccine efficacy of 55% in 1-year followup is a significant progress when considering that In 2015 there were an estimated 214 million clinical cases of malaria in the world. Another outcome of this study is that fact that it is quite hard to find or define immune correlates of protection and this is especially true for parasitic infection such as malaria or dengue.

David Usharauli

Maternal HBV infection induces HBVeAg/PD-L1 dependent tolerance in offspring

Hepatitis B virus (HBV) can be vertically transmitted from mother to babies. Unlike exposure at adulthood, immunological consequences of such exposure to HBV in newborns is "tolerance" to chronic HBV infection.

Ordinarily mice are not naturally infected with HBV. New paper in Immunity reported development of mouse model to study immunology of vertical transmission of HBV.

This model is a combination of two independent processes: 1st, use of HBV transgenic mice and 2nd, hydrodynamic injection of plasmid that contained 1.3-mer HBV genomic DNA. Initially, the authors showed mice born to HBV Tg females (referred here as TGD mouse), but not controls, developed "chronic HBV infection" when exposed to HBV plasmid. 

  

Analysis of HBV-specific CD8 T cells from HBV-DNA injected TGD mice liver showed up-regulation of checkpoint inhibitor PD-1 on T cells.

Treatment of TGD mice with anti-PDL1 antibody enabled them to control HBV-DNA infection.

Moreover, the authors found that clodronate-liposome depletion of macrophages from TGD mice also enabled them to control HBV-DNA infection.

Finally, the authors found that HBVeAg played important role in viral persistence and tolerance by observing that (a) mice born to HBV-mut Tg females (HBV lacking eAg) do not develop persistent HVB-DNA infection, and (b) TGD mice exposed to HBV-DNA lacking eAg also were able to eliminate virus.

In summary, this study suggests that HBVeAg controls newborn's tolerance to HBV via inhibitory PD-L1 signaling.

Few additional thoughts: first, the authors reported that after HBV-DNA injection, around 15% of liver macrophages, called Kupffer cells, expressed viral antigen. However, they also found that >75% of Kupffer cells expressed PD-L1 upon HBV-DNA injection. Second, since HBV virus from HBV Tg pregnant females cannot directly infect newborns, it is not clear how HBVeAg is able to modulate macrophages and tolerize newborn's CD8 T cells (the authors also acknowledged this inconsistency).

David Usharauli

CARD9 modulates gut inflammation via microbiota-specific tryptophan metabolism

In my previous blogpost I discussed new Science paper that showed relationship between autophagy pathway (ATG16L1) and microbiota-secreted product (PSA-OMV) in driving Crohn's disease (CD) risk. 

As it happens frequently in scientific publication, yesterday another prestigious journal Nature Medicine published another study from another research group that showed relationship between Card9 (C-type lectin sensor, also involved in NOD2 signaling) and microbiota-specific tryptophan metabolism in driving inflammatory bowel disease (Crohn’s disease and ulcerative colitis) risk.  

It is known that Card9^−/− mice are more susceptible to colitis. Here, the authors noticed that Card9^−/− mice are slow to recover after dextran sulfate sodium (DSS)-induced colitis (a self-limiting colitis model) and expressed fewer IL-22⁺ cells, a cytokine with well-known beneficial effects on intestinal homeostasis.

Examination of composition of the fecal bacterial microbiota using 16S rDNA sequencing revealed differences between WT and  Card9^−/− mice gut flora.

Interestingly, germ-free mice transplanted with gut flora from Card9^−/− mice were more susceptible to DSS-induced colitis. This indicated that unlike WT microbiota, Card9^−/− mice microbiota failed to provide "healing" signaling to host's gut epithelium.

One mechanism for this "healing" could be the modulation of aryl hydrocarbon receptor (AhR) activation. Tryptophan can be metabolized either by the gut bacteria into indole derivatives (i.e IAA) or by host cells into kynurenine (Kyn) via indoleamine 2,3-dioxygenase 1 (IDO1). Indole derivatives are AHR ligands and promote IL-22. Indeed, WT microbiota, but not Card9^−/− microbiota, promoted AhR ligand production (Of note, exogenous IL-22 could normalize AhR ligand production and colitis susceptibility in Card9−/− mice).

In vitro assay with AhR reporter system confirmed that Card9^−/− microbiota were defective in activation of AhR signaling.  

Furthermore, supplementation with Lactobacilli strains that are able to produce AhR ligands could restore "healing" effect of Card9^−/− microbiota on gut epithelium.

Finally, the authors showed that fecal samples from IBD patients were indeed deficient in promoting AhR signaling via indole products (genotyping confirmed that fecal samples from IBD patients with Card9 risk allele was associated with reduced AHR activation in their in vitro reporter assay). Of note, the authors said that no such association was observed among other major IBD risk alleles, such as NOD2, ATG16L1 and LRRK2.

In summary, this new study suggests that alterations in IBD risk genes modifies microbiota composition that in turn could tip the balance in favor of "non-healing" microbiota resulting in reduced level of "healing"AhR ligands.

David Usharauli

Gut flora antigen packaged within outer membrane vesicles mediates natural tolerance to microbiota

Crohn’s disease (CD) and ulcerative colitis (UC) are gut inflammatory conditions of unknown etiology. Current hypothesis suggests that these conditions develop due to failure of body's immune system to tolerate antigen processed or generated within gut. 

New paper in Science has pointed to one of the mechanisms for such failure. This study showed that CD's risk gene, ATG16L1, is involved in response to microbiota and promotes gut tolerance via IL-10 producing Foxp3+ Tregs cells.

Initially the authors showed that DCs deficient for ATG16L1 failed to induce IL-10 producing Foxp3+ regulatory T cells in response to WT PSA-OMV (Polysaccharide A derived from gut commensal Bacteroides fragilis and packaged within outer membrane vesicles). Conversely, WT DCs failed to induce IL-10 producing regulatory T cells in response to OMVs harvested from isogenic B. fragilis mutant lacking PSA (ΔPSA-OMV).

Similarly, NOD2-deficient DCs pulsed with WT PSA-OMV failed to support IL-10 production from Foxp3+ Tregs during in vitro co-cultures (NOD2 has been shown to physically interact with ATG16L1).

In vivo experiments confirmed that unlike WT mice, mice deficient for DC-specific ATG16L1 or NOD2 were not protected by WT PSA-OMV from chemical-induced colitis (though it is not clear whether chemically-induced mouse colitis models, i.e. 2,4-dinitrobenzenesulfonic acid (DNBS)-induced colitis or dextran sulfate sodium (DSS)-induced colitis, are representative of inflammations in Crohn's disease)

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Finally, the authors showed that monocyte-derived dendritic cells (MoDC) from Crohn's patients harboring ATG16L1 T300A risk variant did not respond to WT PSA-OMV and failed to induce IL-10 production from Foxp3+ Tregs.

In summary, this study showed that failure to sense gut microbiota components by NOD2-ATG16L1 axis could predispose individuals to gut inflammation.

David Usharauli

Maternal IgG fully compensates IgA to influence newborn's immune system maturation

The mammalian babies are born with immature immune system. It is not clear why it is the case, though in my view, most likely explanation would be that it has something to do with initial encounter with and adaptation to endogenous commensal gut [and other tissue] microbiota. 

Since babies are born more or less sterile [devoid of microorganisms], their immune system has to learn to set a "baseline" for what is a "normal" microbial community [i.e. microbiota]. Babies immune system needs to tolerate this local microbial community. Once such "baseline" and tolerance threshold are set, only then baby's immune system can recognize to respond to "foreign" microbe and reject it. 

One way for a mammalian babies to set normal tolerance threshold and "baseline" is via instructions from their mom's [experienced] immune system. Maternally derived IgA and IgG accumulate in newborns [from milk or during gestation] and transfer initial instructions for proper immune maturation in babies.

In this regard new study in Cell is interesting. By utilizing several of genetically-modified mouse strains, the authors were able to show that maternal milk-derived IgG3 and IgG2b (subclasses of IgG), could fully compensate IgA in "educating" newborn's immune system.

First, the authors showed that normal WT mouse serum detects gut microbiota. As expected, microbiota-bound IgA was detected even in absence of added serum. As expected, serum from µMT^{- /-} mice that lack most of B cells did not stain gut flora.

Presence of microbiota-specific IgG in the serum was unexpected [unlike IgA or IgM]. Further analysis revealed that these IgG was made mostly of IgG3 and IgG2b subclasses.

And, these IgG3 and IgG2b were indeed microbiota-specific because no staining was observed with serum from germ-free mice that lack microbiota.

Presence of microbiota-specific IgG3 and IgG2b were independent of adaptive T cells and dependent on TLR signaling.

Microbiota staining dynamic revealed that serum IgG in pups before age of 4 weeks were maternally derived [between weeks 4-8, pups were starting to make their own microbiota-specific IgG antibodies].

Next big question that the authors answered was to show what was the immunological consequences of failure to transfer maternal antibodies to offspring. First, they showed that specific absence of milk-derived antibodies (pups born to µMT^{- /-} parents or WT pups cross-fostered by µMT^{- /-} moms) increased both microbiota translocation and effector CD4 T cells differentiation in pups.

Second, pups born to IgA-KO parents showed no such changes indicating something else compensated IgA in "educating" newborn's immune system.

Indeed, if mom's milk did not contain both IgA and IgG (in pups born to IgA/FcRn double KO parents), then pup's immune system showed over-activation. This indicates that microbiota-specific IgG could fully compensate microbiota-specific IgA deficiency with regard of immune system education [however, "cleaner" model would be to use IgG-KO mouse here, if available].

Finally, at cellular level, absence of milk derived IgG was associated with over-expansion of antibody-inducing follicular T helper cells (Tfh cells) and germinal-center B cells in 3-4 weeks old pups.

In summary, this fundamental study showed that in mice milk-derived antibodies, both IgG and IgA subtypes, play important role in setting normal threshold of immune maturation [activation] in newborns, thus contributing to proper education of developing immune system.

David Usharauli