Inflammation modifies pattern of differentiation of commensal-specific T cells

1. #Commnesal S. epidermidis–specific -#MIIINA:H2-M3+ CD8+ T cells ordinarily differentiate into Tc1 or Tc17 subsets. However, when co-exposed to chitin or sand fly (source of #inflammation), Tc17 subset also produce type II cytokines. pic.twitter.com/g9Wy7BncPD

— David Usharauli (@3DiMMUNE) January 10, 2019

3. In any case, such deviation into type II cytokine+ lineage during co-exposure to S. epidermidis and inflammation is specific for CD8+CCR6+ cells (referred to as Tc17). pic.twitter.com/6uNCVHMtEz

— David Usharauli (@3DiMMUNE) January 10, 2019

5. The authors tried to show how skin #Treg deficiency could have the same effect on Tc17 de-differentiation. Again. the authors failed to show that IL-17+ and IL-5/IL-13+ Tc17 cells overlap. They don't. They put in suppl material these #data. Should be in main paper. pic.twitter.com/QupGc7HWLo

— David Usharauli (@3DiMMUNE) January 10, 2019

7. But again, no evidence is provided that IL-17+ T cells co-express IL-5 or IL-13 when exposed to IL-18. Exactly opposite. No overlap. pic.twitter.com/QG8U9EBSSH

— David Usharauli (@3DiMMUNE) January 10, 2019

So, what is my conclusions: this paper does not support the authors' conclusions about Tc17 acquiring Tc2 phenotype. Yes, it is true that RORgt+ or CCR6+ CD8+ T cells could acquire Tc2 phenotype but the same not true IL-17+ Tc17 cells or at least data are not there.

— David Usharauli (@3DiMMUNE) January 10, 2019

Outcome of TCR/pMHC interactions is presently unpredictable

This week journal Cell published a very important paper from KC Garcia's lab at Stanford. The focus of the study was to understand and determine, if possible, all the parameters that makes a given peptide/MHC and its similar looking variants stimulatory versus non-stimulatory to a given TCR molecule.

First, they compared two TCR molecules specific for class II HLA-DR4-HIV_{(Gag164–183)}. While both TCR expressing clones, TCR6 and TCR11, could bind pHLA tetramer, only TCR6 "proliferated, secreted cytokines, and downregulated TCR in response to stimulation with" HLA-DR4-HIV_{(Gag164–183)}. Both TCR clones could respond directly or after transducing them into TCR αβ-deficient cell line to non-specific stimulation such as anti-CD3 (OKT3).

Similar observation were made using TCRs specific for class I HLA-B35-HIV_{(Pol448–456)}. Here too, both TCR clones designated as TCR589 and TCR55 could bind specific pMHC tetramer but only TCR589 showed dose-dependent secretion of IL-2 in response to peptide-pulsed APCs.

Next, the authors determined TCR/pMHC affinity by surface plasmon resonance. For TCR55 (non-stimulatory) and TCR589 (stimulatory) clones it was 17_μM and 4_μM K_D, respectively. Surprisingly, for TCR6 (stimulatory) and TCR11 (non-stimulatory) clones affinity was 10_μM and 1_μM K_D, respectively.

Analysis of TCR55 (non-stimulatory) and TCR589 (stimulatory) TCR/pMHC interaction on lipid bilayers by total internal reflection fluorescence (TIRF) microscopy showed similar mean dwell times of 10.1 ± 2.0 s and 8.7 ± 2.1 s, respectively. Structural analysis of the crystal structures of TCR589 and TCR55 in complex with HLA-B35-HIV_{(Pol448–456)} also showed "no substantial differences in the conformations of the constant regions that would indicate differential allostery correlating with signaling."

Functional analysis of TCR proximal signaling molecules showed that stimulatory TCR589 but not TCR55 could up-regulate CD69, induce calcium release and recruit ZAP70 in response to HLA-B35-HIV_{(Pol448–456)}.

Next, they generated peptide library similar to HIV_{(Pol448–456)} with potencies "ranging from full agonism to partial agonism to no detectable signaling". Importantly, "the peptides that elicited these disparate signaling outcomes were remarkably similar in sequence."

One common feature of agonist peptides such as pep20 and SQL that could signal TCR55 was that such interaction excluded CD45 with varying degrees of efficiency compared to non-stimulatory peptide ligands in "a cell-free system using giant unilamellar vesicles (GUVs) to eliminate effects of signaling in live cells".

In addition, another common feature of stimulatory peptide ligands were so called catch bond formation between TCR and stimulatory pMHC "under conditions of force using a Biomembrane Force Probe (BFP) bond lifetime assay". In contrast, non-stimulatory ligands formed slip bond formations.

In summary, this study indicates that "catch bond formation and CD45 exclusion as principal emergent properties differentiating stimulatory from non-stimulatory TCR-pMHC interactions" but "the same pMHC can be an agonist or non-agonist for different TCRs, although it is difficult to assign causality to either the TCR or the peptide-MHC. The formation of a catch or slip bond is a collective property of the entire TCR-pMHC interface."

My opinion: one drawback of this study is that not many labs can verify its conclusions. I have not even heard of some of the techniques they used here. I could only trust that the authors tried to interpret their data as unbiased as it is rationally possible. Another weakness of the study is a failure to identify parameters that control CD45 exclusion or catch bond formation when TCR interacts with pMHC. Basically, with the exception of few very high affinity ligands (< 2_μM), we cannot predict in silico whether a given peptide will be stimulatory or non-stimulatory without actually doing an experiment on it.   

posted by David Usharauli

First-born advantage of early produced CD8 T cells

During immune response to infection or vaccine antigen-specific T cells differentiate into various categories of effector/memory population. This is a stochastic process that follows some not yet fully understood "rules". New study in Cell suggests that one of those rules is a "date of birth" of individual CD8 T cells that participate in immune response. 

For this study the authors used genetically modified mice where T cells could be permanently color-labeled after injection of tamoxifen (CD4 promoter-driven tamoxifen-inducible CRE mice, CD4^cre-ERT2). When compared to each other, CD8 T cells produced early on during neonatal stage (day 1-7) harbored high proportion of cells with innate-like phenotype (CD44⁺/CD122⁺) than those CD8 T cells produced after day 28.

Similar phenotype was maintained even in mice where both day 1 and day 28 CD8 T cells were produced in the same mouse (following neonatal RFP⁺ thymus transplantation into YFP⁺ adult mice and analyzed at indicated time period post transplantation).

Notably, early-born CD8 T cells were characterized by heightened sensitivity to innate cytokines such IL-18 and IL-12 and rapid initial response to cognate antigen (between day 1-7 post-infection).

The authors opined that various categories of effector/memory T cells observed in other studies should be re-interpreted in light of "layered" CD8 T cells wherein CD8 T cells of different "date of birth" are producing different effector/memory T cell population.

It is certainly interesting observation. But there are several missing opportunities in this study. First, due to their innate-like phenotype, the authors should have looked at the role of microbiota in imprinting these features of early-born CD8 T cells. Second, it is not clear and the authors did not examined if biologically, lack of early-born vs. adult-born CD8 T cells, would have modified the host's response to infection. Third, the authors did not analyze whether early-born versus adult-born CD8 T cells differed in their TCR profile (even using transgenic CD8 T cells on WT background is not proper control). Fourth, different effector/memory categories have been produced by injection of a single CD8 T cells so individual CD8 T cells can indeed produce diverse phenotype of effector/memory T cells. 

posted by David Usharauli

T1D target epitope from zinc transporter 8 (ZnT8) cross-reacts with commensal bacteria

Type 1 diabetes (T1D) is considered autoimmune disease. Of course, in humans, we don't have a direct evidence that islet-specific auto-reactive T cells and auto-antibodies found in peripheral blood from T1D patients are indeed responsible for tissue damage. Such evidence would require T/B cell and Ab depletion experiment that is not feasible. Autoimmune nature of T1D is basically extrapolated from mouse studies or in vitro antigen binding assays.   

Several islet antigens are known to represent targets in T1D, such as preproinsulin (PPI), glutamic acid decarboxylase (GAD), insulinoma-associated protein-2 (IA-2), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) and more recently described zinc transporter 8 (its peptide ZnT8_186–194).

New study in Science Immunology found that ZnT8_186–194-specific CD8 T cells are largely similarly present in both T1D patients and healthy controls and that ZnT8_186–194-specific CD8 T cells could recognize (cross-react) peptide derived from gut commensal microbe Bacteroides stercoris.

Staining with ZnT8_186–194-specific HLA class I multimers (MMr) and other functional antigen-specific assays found that T1D and healthy controls harbored largely similar number and functional ZnT8_186–194-specific CD8 T cells.

Interestingly, ZnT8_186–194-specific CD8 T cells could be double stained with HLA class I multimer + peptide derived from B. stercoris, a commensal bacterial species found in gut flora. It is not the first time such cross-reactivity has been observed between islet-specific CD8 T cells and commensal bacteria. Previously, at least two bacterial species have been identified to cross-react with IGRP-specific CD8 T cells.

In summary, this study indicates that deletion of auto-reactive CD8 T cells in the thymus is not sufficient to prevent autoimmunity and that regulatory mechanisms operating in the periphery is necessary to prevent initiation of auto-reactive attack by circulating ZnT8_186–194-specific CD8 T cells (probably Tregs the authors had in mind). Cross-reactive peptide derived from B. stercoris could play role in priming of ZnT8_186–194-specific CD8 T cells in absence of regulatory circuit. But how and why such antigen-specific tolerance breakdown happens in one and not in another is not clear at this stage.

posted by David Usharauli

Recognition of microbiota-derived N-formyl methionine peptides by non-classical MHC class I, H2-M3-restricted CD8 T cells

A new study in journal Cell revealed that a specialized subset of CD8 T cells recognize conserved microbial derived N-formyl methionine peptides in context of non-classical MHC class I molecule H2-M3.

Using skin commensal Staphylococcus epidermidis (S. epidermidis) challenge model, the authors showed that recognition of this microbe by CD8+ T cells specifically relied on H2-M3 rather than other MHC molecules.

H2-M3 has been known to bind peptides that contain an N-formyl methionine (fMet), which is required to initiate protein translation in bacteria and mitochondria. Indeed, fMet peptide:H2-M3 tetramer (f-MIIINA:H2-M3) could stain a population of CD8 T cells from skin or lymph nodes. Interestingly, half of tetramer positive CD8 T cells were CD44^hi even in germ-free mice indicating cross-reactivity with other antigens or "ready-made" origin similar to thymus-derived Tregs.

To show biological significance of these CD8 T cell population recognizing commensals, the authors used H2-M3 KO mice and wound healing experiment. The authors claim that in the absence of H2-M3 wound healing was delayed, though data do not strongly support such assertion.

In summary, the authors described yet another T cell population recognizing products from bacteria. Because such recognition does not produce inflammation the authors suggest it could be involved in wound repair. But wound repair model provided very minimal support for such hypothesis. Basically, they missed the central point to make this paper relevant. So how it ended up in Cell? Ask editors. I could come up with couple of suggestions. When the paper's authors list includes Giorgio Trinchieri, John O’Shea and senior author, Yasmine Belkaid, all heads of big labs at NIH, it is quite difficult to say no.

posted by David Usharauli

Molecular mimicry to gut microbiota antigen protects against colitis but induces diabetes

Current issue of journal Cell has one very interesting but at the same time confusing research paper. In it, the authors proposed that

(a) diabetes susceptible mice strain, NOD, harbor CD8 T cells specific for microbiota antigen that cross-react with β cell antigen, IGRP, and

(b) such molecular mimicry prevents colitis but at the same time could induce diabetes.

First, the authors showed that MHC I alelle expressed in NOD mice (H2K^d) could bind IGRP_206-214 homologue derived from integrase family expressed by some gut Bacteroides species (BacIYL_36–44). 

At high dosage, such binding was functional in stimulating high affinity IGRP_206-214-specific T cells (17.4+ CD8 T cells).

Human T cells from PBMCs could apparently respond to it as well (though it is strange that it generated better stimulation index than Tetanus toxoid).

Then, the authors did the following experiment. They exposed IGRP^-/- 17.4+ TCR transgenic mice to chemical irritant (DSS) and observed that high affinity IGRP_206-214-specific T cells, 17.4+ CD8 T cells, but not low affinity ones (17.6+), could protect against colitis (I assume that they used IGRP^-/-mice to avoid diabetes development).

It appears that colitis protection depended on perforin expression by 17.4+ T cells. The authors speculated that 17.4+ CD8 T cells prevented colitis by eliminating dendritic cells laden with microbiota-derived antigen (BacIYL_36–44).

As a confirmation, the authors showed that germ-free TCR Tg NOD mice colonized with Bacteroides species expressing BacIYL_36–44 were protected against colitis.

Colitis protection was observed even in classical, adoptive naive CD4+ T cell transfer colitis model.

Interestingly, however, transfer of T cells from pre-diabetic NOD mice into germ-free NOD.scid mice colonized with Bacteroides species expressing BacIYL_36–44 did not accelerate diabetes development (here I assume DSS is required to accelerate T cells priming against IGRP by creating dysbiosis).

In summary, this study suggests the following scenario: diabetes-inducing CD8+ T cells cross-react with gut microbiota-derived antigen. When such microbiota-derived antigens become visible to T cells (during dysbiosis?) CD8+ T cells migrate to gut and eliminate dendritic cells laden with cross-reactive antigens. By eliminating DCs, other T cells are not able to induce inflammation in the gut, thus no colitis. However, the same beneficial CD8+ T cells later migrate to β cells, recognize similar looking antigen, IGRP, and mediate its destruction and diabetes.

Does such circuit makes any evolutionary sense? 

Update: Interestingly, other research group previously detected different set of gut microbiota antigens cross-reactive to IGRP_206-214. They used TCR NY8.3 transgenic NOD mice (that recognize the same IGRP epitope) and found that these CD8 T cells cross-reacted with IGRP_206–214 homologous peptide, W15944, derived from L. goodfellowii, a member of the phylum Fusobacteria (gram-negative anaerobe), a human and NOD mouse oral commensal. 

posted by David Usharauli   

Foxp3+ Tregs control CD8 T cells, but not CD4 T cells, by IL-2 deprivation

This week Nature Immunology published another interesting study from Rudensky's lab done in collaboration with Jason Fontenot (who apparently moved from Biogen to Juno Therapeutics). So, if you are a fan of Tregs, below is my short scientific overview of that paper.

This study tries to answer what role IL-2 signaling plays in already established Foxp3+ Tregs. For such study mice with germline deficiency in IL-2 signaling cascade would have been impractical since IL-2's effect on thymocytes and etc. Instead, the authors went to already well established path of using mice lacking "molecule of interest" specifically in Tregs. In this case several Foxp3-cre mice were used, such as: l2rb^fl/flFoxp3^Cre, Il2ra^fl/flFoxp3^Cre, Stat5a^fl/flStat5b^fl/flFoxp3^Cre, Rosa26^Stat5bCAIl2rb^fl/flFoxp3^Cre and Rosa26^Stat5bCAIl2ra^fl/flFoxp3^Cre, Rosa26^Stat5bCAFoxp3^Cre–ERT2. All these gene-modified mice allows specific targeting of Foxp3+ Tregs.

Unsurprisingly, mice lacking IL-2Rβ or IL-2Rα or STAT5 signaling specifically in Tregs developed autoimmunity.

Surprisingly, however, while constitutive expression of STAT5 in Tregs lacking IL-2 signaling receptors (IL-2Rβ or IL-2Rα) could rescue mice from "CD4 effector phenotype" and early death, these mice still developed immunopathology later due to massive expansion of CD8 effector/memory cells, suggesting that sensing of IL-2 by Tregs, so called "IL-2 sink", was necessary to specifically control CD8 T cells, but not CD4 T cells.

In summary, this study suggests that Tregs are using distinct mechanisms to control CD4 and CD8 T cells. It kind of makes sense because MHC class-II restricted CD4+ Tregs cannot interact the same way with MHC class-I restricted CD8+ T cells as they could with conventional MHC class-II restricted conventional CD4+ T cells.

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

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

Tissue-resident regulatory T cells are balancing between immunity and immunopathology

Yesterday journal Nature published very interesting study related to Foxp3+ regulatory T cells. This research revealed that "over-active" Foxo1 within Tregs specifically reduces number of peripheral tissue-resident active Tregs that control CD8 T cell-mediated tumor immunity and immunopathology.

Initially, using parabiotic mice pairs the authors showed that Tregs too undergo mixing and achieve equilibrium within 4 weeks. This indicates that Tregs are not maintained locally and require replenishment. 

Next, using Foxp3-cre driven Foxo1 "active" mutant mice the authors showed that when both alleles of normal Foxo1 genes were replaced by "active" Foxo1, it led to dramatic reduction of tissue-resident Tregs (lymphoid tissue resident Tregs were less affected).

Surprisingly, when these Foxo1 homozygous mutant mice were followed for longer time (6-8 weeks), they displayed spontaneous CD8 T cell-mediated wasting disease and peripheral tissue pathologies in liver and intestine (though these tissue pathologies were different from those observed in Foxp3 mutant mice). Hemizygous Foxo1 mutant mice with only one mutant Foxo1 allele were healthy.

Interestingly, reduction of peripheral Tregs in hemizygous Foxo1 mutant mice were sufficient to confer CD8 T cell-mediated anti-tumor effect without immunopathology. 

In summary, this study revealed three important results: 

1. Lymphoid resident Tregs are not sufficient to control autoimmunity/immunopathology. Tissue-resident Tregs play unique and essential role in this process.

2. Level of active Foxo1 molecule control frequency of tissue-resident Tregs

3. Manipulation [reduction] of level of active Foxo1 within Tregs could improve anti-tumor immunity.

David Usharauli

CD4 T cells provide universal "help" to CD8 T cells via pathogen-tailored DCs

Ordinarily [but not always] naive CD8 T cells require "help" from CD4 T cells to undergo full differentiation and to develop into memory. Such CD4 T cell help is provided via so called "licensed" antigen-presenting cells, DCs. In a simple scenario, when pathogen invades tissue, local DCs will pick up its antigens and present them to both CD4 and CD8 T cells. In turn, activated antigen-specific CD4 T cell "licenses" the same DCs to up-regulate or secrete necessary molecules to complete priming of naive CD8 T cells (I am going to use terms "help and "license" interchangeably).

This simple model is complicated by fact those "licensing" molecules for CD8 T cells differ depending on pathogens. The most well described "helps" include IL-12, IL-15 or type I IFNs. So, how CD4 T cells are able to deliver so many different licensing signals?  

Apparently, CD4 T cells don't. According to new paper published in Cell Reports, CD4 T cells simply amplifies pre-existing pathogen-tailored signals within DCs. Lets see if data are convincing (note, this paper was under review process for > 2 years)

Initially, the authors confirmed that CD8 T cell priming/expansion during viral infection, HSV-1, required presence of CD4 T cells, MHC II, CD40L or CD40.

Next, the authors showed that CD8 T cells priming/expansion during HSV-1 infection required signaling via either IFNαR or IL-15.

Experiments with BM chimeras, IL-15^KO:CD11c^DTR and IFNαR^KO:CD11c^DTR, revealed that DCs-specific expression of IFNαR and IL-15 were required for CD8 T cell priming during HSV-1 infection.

However, production of IL-15 by DCs in response to IFNα also required presence of CD4 T cells.

In fact, ex vivo stimulation of CD8α⁺ DCs with IFNα and αCD40-mimetic (as a surrogate for CD4 T cell help) showed that CD4 T cell "help" amplified IL-15 induced by innate [viral-induced] IFNα (since αCD40-mimetic alone had no effect). However, it is not clear whether αCD40-mimetic could fully recapitulate CD4 T cell function. So, this requires additional tests.

Dominant role of innate signaling in determining the nature of CD4 T cell "help" was revealed in experiments in which mice were challenged with cell-associated OVA in combination with LPS or Poly(I:C). In presence of LPS, "help" was IL-12 dependent, while in presence of Poly(I:C), "help" was IL-15 dependent.

In summary, the conclusion of this study, according to the authors, is that CD4 T cells simply amplify pathogen-tailored innate signals already generated within DCs, rather than proving unique maturation signals. My interpretation of these results is not very different from earlier models. I don't think that anyone claimed that CD4 T cell "help" and innate signals were completely interchangeable. For me, "licensing" and in this case "amplification" are very same concepts. For me, more important question is how those CD4 T cells that deliver "help" are getting activated in first place (basically, who primes the "primers").

David Usharauli

Neither apoptosis nor necrosis but necroptosis allows cross-priming of CD8 T cells

Some pathogens, especially viruses, infect only epithelial cells but not dendritic cells (DCs). This creates dilemma for immune system since activation of naive CD8 T cells require dendritic cells. So how can DCs present cytoplasmic [viral] antigens to CD8 T cells without having one?

This process is called cross-presentation. Since discovery of programmed cell death called apoptosis or silent cell death, it was proposed that apoptosis-derived antigens were cross-presented for tolerance while necrosis-derived antigens [from virus infected cells] were cross-presented for priming. However, data were not consistent and frequently contradictory results were produced. 

Now new paper in journal Science provided support for the alternative model for cross-presentation that could reconcile and explain earlier observations. It found that it is 3rd pathway, called programmed necrosis or necroptosis, rather than apoptosis or necrosis, that provides cargo proteins for cross-presentation by DCs.

To study effect of apoptosis or necroptosis, the authors transduced NIH-3T3 cell line with fusion constructs containing caspase 8 (involved in apoptosis), or RIPK3 (involved in ripoptosome-mediated necroptosis) or RIPK3ΔC (inducing cell death without ripoptosome). When exposed to dimerization reagent, these transduced cells underwent cell death according to the predicted pathway.

Next, the authors showed that dimerization of RIPK3 (referred here as acR3) or RIPK3ΔC (referred here as ac3ΔC) constructs, but not caspase 8, could induce release of damage-associate molecular patterns (DAMPs) such as HMGB1 or ATP.

More importantly, however, when these transduced cell lines [also expressing OVA antigen] were injected into mice to induce CD8 T cell response, the authors found that only wild-type RIPK3 construct-induced cell death promoted CD8 T cell expansion and effector differentiation.

In addition, the authors found that neither secondary necrosis nor mechanical [freeze-thaw] necrosis could prime CD8 T cells.

Next, the authors observed that cells undergoing RIPK3-ripoptsome mediated necroptosis selectively release IL-6 and show rapid RIPK1-mediated IκB degradation.

Finally, using CRISPR/cas9 modified CT26 tumor cell line, the authors showed that RIPK3-mediated ripoptosome assembly involving RIPK1-NF-κB pathway was crucial for immunogenic necroptosis in tumor challenge model.

In summary, this study further refined our understanding of immunogenic cell death and further defined molecular components essential to achieve it. Of note, the role of necroptosis in immunogenic cell death may finally harmonize prior data regarding necrosis or apoptosis in cross-presentation and improve our therapeutic tool box.

David Usharauli