Almost every cancer patient harbors tumor-specific T cells

We know now that adaptive immune system (T and B cells) can detect single amino acid changes in mutated proteins. However, such efficiency of immune system was (and still) in odds with cancer development since tumors invariably express mutated proteins. So what's the deal?

Until recently in vitro detection of tumor-specific immune response was technically challenging. First, detection of mutated proteins or RNA was not easy task. Second, culture conditions for expansion and identification of viable tumor-infiltrated lymphocytes (TIL) specific for tumor antigens were not easy either. Today we have different situation. We can both detect and identify both tumor antigens and tumor antigen-specific TILs.

For example, new paper in Science showed that 9 out of 10 patients with GI tract cancers harbor tumor-specific T cells. However, out of 4 patients receiving adoptive transfer of in vitro expanded tumor-specific T cells, only 1 patient showed persistent anti-cancer response.

This study is kind of follow up from earlier study published in 2014. Here, for each cancer patient, the authors [led by Steven Rosenberg/NIH] first identified cancer mutations by rapid RNA sequencing and designed dozen of tandem minigene constructs containing mutated RNA sequences. These TMGs were then transfected into patient's autologous DCs and co-cultured with multiple TIL cultures harvested from metastatic tumors. This technique revealed presence of tumor-specific T cells (example below, TMG7, 14).

These tumor-specific TCRs were cancer antigen specific [showed no reactivity towards wild-type epitopes].

In 1 patient, the authors found TIL culture reactive to cancer driver gene KRAS (KRAS^G12D). The authors could identify HLA allele presenting mutated peptide (the authors even filed a patent for TCR directed to mutant KRAS. But the TCR itself is a product of nature and not patent eligible and TCR transduction into other T cells is by now considered "prior art". So I am not sure if they have a valid patent claim here. Another point is that it is safe to assume the authors already have earlier patents for transduction techniques and other TCRs. If so, then why they would need a new patent for just another TCR?).

however, the most relevant results are not shown in the paper but just discussed. The authors mentioned that 4 patients were treated with adoptively transferred T cells. However, only 1 patient receiving tumor-specific CD4 T cells showed persistent response and ongoing tumor regression, while other 3 patients who received tumor-specific CD8 T cells either do not show any response or showed only transient response. This suggest that unlike CD4 T cells, tumor-specific CD8 T cells may be not very efficient against solid tumors [lack of persistence in transferred hosts].

In summary, this article supports a notion that cancers do not go unnoticed by immune system. It appears that almost every cancer patient harbor tumor specific T cells that can be harvested, expanded and re-introduced back to patients to target tumors [alone or in combination of checkpoint inhibitors]. However, understanding biological difference between effectiveness of tumor-specific CD8 and CD4 T cells surely requires further research.  

David Usharauli

Adaptive immune cells within ectopic lymphoid-like structures are nurturing hepatocellular carcinoma cells

Lymph nodes represent secondary lymphoid tissues. They are designed to facilitate detection and initiation of adaptive immune response to foreign antigens. There are, however, tertiary lymphoid-like structures, also called ectopic [out-of-place] lymphoid-like structures, that are usually associated with chronic inflammation or tumors. 

Generally, scientists assumed that ectopic lymphoid-like structures provided additional help to control tumors or chronic inflammation. However, paradoxically, new study in journal Nature Immunology provided evidence that ectopic lymphoid-like structures associated with liver tumor, hepatocellular carcinoma, were actively supporting progenitor cancer cell growth and tumor expansion.

First the authors found that presence of ectopic lymphoid-like structures [and its signature] were negatively correlated with survival in hepatocellular carcinoma (HCC) patients.

Next, the authors have used genetically modified mice expressing liver-specific constitutively active NF-κB [to drive inflammation]. Starting at 7 months of age, these mice, IKKβ(EE)^Hep, developed ectopic lympoid-like structures (ELS) in the liver.

Interestingly, IKKβ(EE)^Hep mice on RAG KO background, that lack adaptive T and B cells and ectopic lymphoid-like structures (ELS), showed resistance for HCC development when exposed to carcinogen diethylnitrosamine (DEN). 

Similar effect on HCC tumor burden were seen in diethylnitrosamine treated IKKβ(EE)^Hep mice exposed to anti-Thy-1.2 antibody injection [that depletes T cells and some NKT cells].

Finally, the authors showed that in IKKβ(EE)^Hep mice early application of antibody directed to lymphotoxin-β receptor (LTβR-Ig) signaling could prevent HCC "addiction" to stimulating immune cytokines (LTβ and LTα or LIGHT).

In summary, this study indicates that chronic liver inflammation (in response to genotoxic reagents or viral infection) promotes tertiary lymphoid tissue development that in turn could harbor and nurture early cancer progenitors by providing immune cytokines such as LTβ and LTα or LIGHT.

This observation is in contrast to the role of ectopic lymphoid-like structures play in several other tumors (colon, mammary, breast, skin). These data would require rethinking of how immunotherapy could be applied for HCC treatment.

David Usharauli

Epigenetic modification silence cancer responsiveness to IFN-γ and immunotherapy

Epigenetics is a study of modulation of gene function through non-heritable mechanisms such as histone modification and DNA methylation. These mechanisms are so potent that observed phenotypes could be mistaken for genomic mutations. In clinics, epigenetics plays especially important role during therapeutic treatment where drug effectiveness varies widely among pool of patients, for example, during immunotherapy in cancer patients.      

For example, a new paper recently in Nature showed that both (a) EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) and (b) DNA methyltransferase 1-mediated DNA methylation independently silence cancer cells' responsiveness to IFN-γ/T cells and their activity inversely correlated with patients outcome.

The authors have used EZH2 inhibitor GSK126 or DNMT1 inhibitor 5-AZA-dC. Using humanized mouse model of ovarian cancer, the authors showed that combination of GSK126 and 5-AZA-dC synergized with tumor-specific T cells in tumor protection (inhibitors alone or in absence of T cells had no/minimal effect).

Interestingly, combination of GSK126 and 5-AZA-dC specifically enhanced tumor expression of Th1 chemokines CXCL9 and CXCL10 in response to IFN-γ.

Further experiments showed that both inhibitors could independently enhanced cancer CXCL10 expression in response to IFN-γ (here, primary ovarian cancer cells were pre-treated with shEZH2 or shDNMT and exposed to 5-AZA-dC and GSK126 respectively).

As an additional control, the authors showed that GSK126 had no effect on DNMT1 and 5-AZA-dC had no effect on H3K27me3. 

Finally, the authors found negative correlation between expression level of EZH2 and DNMT1 and cancer patients survival.

In summary, this study expands our understanding of cancer tissue sensitivity to immunotherapy (including to anti-PD1 therapy). It appears that in some patients EZH2 and DNMT1 selectively silence TH1 chemokine locus and inhibit cancer cell responsiveness to IFN-γ derived from tumor infiltrated T cells thus blocking positive loop for attracting and recruiting additional tumor-specific T cells. Accordingly, application of selective inhibitors of EZH2 and DNMT1 could improve cancer patients survival.

Of note: it is not clear why would cancer cells silence only Th1 chemokine locus. While it is true that Th1 play important role in cancer protection, several other cancer models have shown protective role for Th17 and even Th2 cells. Since we don't know whether cancer protection in this paper is dependent of IFN-γ, we have no way to make any conclusion in this matter.      

David Usharauli

   

Specialized monocyte subset protects lung vasculature from tumor metastases

Immune system's cellular lineage diversity is expanding and quite rapidly. Now we have several types of T cells, several types of NK cells, whole new family of innate lymphoid cells (ILCs), cytokine-secreting B cells, specialized subset of neutrophils using sticky DNA nets to trap the microbes. 

Here comes a paper in Science showing a new subset of orphan nuclear receptor Nur77-positive monocytes specializing in protection of lung vasculature from tumor metastases.

First, using reporter mice that tracks Nur77⁺monocytes (encoded by Nr4a1, a Nr4a1-GFP green mice), the authors showed that after intravenous injection of red fluorescent Lewis Lung Carcinoma cells (LLC-RFP), Nur77⁺monocytes are rapidly accumulating in the lung vasculature, then slowing down their speed in close proximity to tumor cells.

To confirm that Nur77⁺monocytes play a role in tumor defense, the authors first repeated the same experiment with total Nr4a1-KO mice, which according to the authors, selectively lack Nur77⁺monocytes. Nra1-KO mice appeared to harbor more lung metastasis (but not in liver), even if tumor cells [B16 melanoma] were injected subQ.

Similarly, increased tumor metastases (but not primary tumors) were seen with mouse model of spontaneous mammary tumor, MMTV-PyMT, receiving Nr4a1-KO bone marrow transfer, supporting the role of Nur77⁺ hematopoietic cells in prevention of tumor metastases.

To more specifically address the role of Nur77⁺monocytes in tumor protection, the authors have used two different myeloid-specific Nr4a1 conditional knockout models, CSF1R-Cre⁺ Nr4a1^fl/fl and LysM-Cre⁺ Nr4a1^fl/fl. In both models, there was an increase in tumor metastases to the lung. Interestingly, T cell-specific Nur77 deletion showed no effect on tumor metastases.

Finally, the authors found that receptor CX3CR1 could play an important role in tumor recognition by Nur77⁺monocytes.

In summary, this study suggest that there is a specialized monocytes lineage defined by Nr4a1 expression that specifically patrol and protects the lung vasculature against tumor metastases.

David Usharauli

Depletion of "hybrid" B cells could explain benefits of anti-CD20 immunotherapy in MS patients

Multiple Sclerosis (MS) is believed to be an autoimmune disease. During MS, the patient's own immune cells attack peripheral nerves' myelin sheath disrupting proper signal transmission. In mouse model of MS, called experimental autoimmune encephalomyelitis (EAE), several myelin proteins have been identified as such targets (such as MBP and MOG). 

While self-specific T cells are widely thought to play a pathogenic role in MS progression, clinical trials revealed surprising benefits of B cell depletion in MS. Mechanism is unknown.    

Now new paper is Science Translational Medicine provided initial data suggesting that depletion of pro-inflammatory GM-CSF secreting B cell subset could explain the clinical benefits of anti-CD20 antibody therapy in MS.

First, the authors showed that activated peripheral B cells from healthy donors express pro-inflammatory cytokine GM-CSF. Interestingly, this B cell population does not overlap with B cell population expressing anti-inflammatory cytokine IL-10.

Next, the authors showed that activated B cells from MS patients tend to express more GM-CSF compared to B cells from healthy donors.

In vitro co-culture of B cells and macrophages revealed that B cell-derived GM-CSF contributed to Th1-type macrophage polarization.

More complex co-culture experiments showed that macrophages derived from MS patients soon after B cell depletion produced less of pro-inflammatory cytokine IL-12p40.

And more importantly, this tendency was maintained even after 1 year of B cell depletion, even though new B cell came back by this time.

This points to a re-configuration of cytokine network in anti-CD20 antibody treated MS patients such as reversal of B cell-derived GM-CSF/IL-10 ratio.

In summary, this study provides additional clues why anti-CD20 antibody depletion might benefit MS patients. Antibody-independent function of B cells is a novel concept [and not widely known or accepted]. One of the reasons for this resistance is lack or inadequate knowledge of B cell receptor specificity of such specialized B cell subset. We need to go one step further and connect the dots between BCR specificity or other innate receptors and B cell's "hybrid" function. Only by developing this biological "framework" can we fully understand the role B cells play in health or diseases.

If you are interesting in science of MS, I will recommend a book by Susan Quinn's "Human Trials: Scientists, Investors, And Patients In The Quest For A Cure" (2001).

David Usharauli

Hair follicles attract normal and lymphoma T cells via common gamma chain (γc) cytokines IL-15 and IL-7

Skin is a body's second largest surface that is continuously exposed to exogenous antigens. So, no wonder that it attracts and harbors specialized immune cells. One of these immune cells are skin resident memory T cells (T_RM cells) who play an important role in local skin epidermal defense as a rapid deployment tactical units. Besides this protective function however, T_RM cells can contribute to pathological conditions such a drug allergy or lymphomas.

New paper just published in Nature Medicine showed that accumulation of normal or oncogene-transformed T_RM cells in skin epidermal layer is directed by hair follicle derived IL-15 and IL-7.

First, the authors confirmed that T_RM cells are present in both skin dermal and epidermal layers (but CD8 T cells were present only in epidermal layer).

Next, the authors generated RAG-deficient BM chimera where the host's non-hematopoietic tissue [such as keratinocytes] also lacked IL-15 expression. Transfer of wild-type T cells into these IL-15/RAG double-deficient host showed that non-hematopoietic cell-derived IL-15 was important for CD8 T cell [but not CD4 T cell] recruitment to the epidermis [though it is not clear why would the authors suggest that non-hematopoietic tissue-specific IL-15 deficiency equals to hair follicle-specific IL-15 deficiency].

Further experiments with mice with keratinocyte-specific conditional IL-7 deletion revealed that cytokine IL-7 also played important role in recruitment of both CD4 and CD8 T cells to the skin [again, the authors have used this model as an example of hair follicle-specific IL-7 deletion. Not sure how accurate is this notion].

Accordingly, the authors showed that skin contact hypersensitivity response to hapten DNFB that requires T cell activation were reduced in mice with non-hematopoietic tissue-specific IL-15 and IL-7 deficiency.

Finally, using mouse T cell lymphoma model, that mimics human cutaneous T cell lymphoma (CTCL), the authors showed that keratinocytes-derived IL-7 was important to direct accumulation of T lymphoma cells to the epidermal layer.

In summary, this study provided mechanistic explanation for some of the clinical features of human T cell lymphomas. It appears that keratinocytes [hair follicle]-derived IL-15 and IL-7 actively attract both normal and cancerous T cells to the epidermis. Since both IL-15 and IL-7 are common gamma chain (γc) cytokines, inhibition of their signaling with JAK3 inhibitors [such as Pfizer's JAK1/3 inhibitor Tofacitinib] may provide some relieve in pathological situations such as drug [hapten]-induced allergy and T cell lymphomas (CTLC, mycosis fungoides, Sezary's syndrome).

David Usharauli

Food allergies caused by mast cells with a serial number 9

Allergies are complex immune responses. Whether allergic reaction have any "protective" function or represent a purely pathological reaction is still debated. Classical form of allergy is IgE mediated and require IL-4 and Th2 cells. However, the list of effector molecules and cells responsible for various forms of allergic reactions are continuously expanding.

For example, in a recent paper in Immunity the authors showed that certain mouse models of food allergy were driven by mucosal mast cells secreting high level of IL-9.

This is quite messy paper with lots of figures. In fact, it was under review for one year [and it shows by its lack of harmonized relationship between figures]. 

Initially, using mouse model of food sensitization [intra-gastric antigen gavage], the authors showed that allergic reactions (e.g. diarrhea) to antigen in susceptible mouse strains correlated with the number of GI mast cells, not serum IgE. Moreover, such correlation was highly significant for IL-9 producing lamina propria Lin^-/- population in susceptible mouse strains [since not every mouse strain develop allergic response in this setting].

Further investigation with IL-4eGFP reporter mice revealed that this IL-9 producing Lin^-/- population belonged to mast cell lineage, rather than innate lymphoid cell type 2 (that express IL-25 cytokine receptor IL-17RB).

Next, the authors showed that induction of IL-9⁺ mast cells (MMC9) required IL-4, STAT6 and T cells.

Finally, using BM chimera experiments the authors showed that while active signaling through IL-9 was dispensable for MMC9 induction, it was required to produce allergic phenotype.

 

In summary, this study suggests that GI tissue associated mast cell secreting high levels of IL-9 play a role in allergic response to sensitized antigens. The development of this MMC9 requires signaling via IL-4, STAT6 and T cells, implying typical Th2-driven immune response. It is possible [but not formally tested in this study] that therapeutic targeting of IL-9 pathway may benefit patients suffering from GI tract allergies to food.

David Usharauli

Treg cells need a little Helios to function

Regulatory T cells (T_REG cells) are required to maintain tolerance to self and maybe even to foreign antigens, such as commensal bacteria-derived antigens. But how exactly T_REG cells keep other immune cells in check is not clear. 

Simply speaking, paradox with T_REG cells is that if T_REG cells are too potent or constantly "active" then they should prevent any immune response to any antigens. If T_REG cells are not constantly "active", then what signal(s) activate them? and if active T_REG cells are "inhibited" by infection and they regain their inhibitory functions once infection is cleared, how is such system coordinated? Basically, none of the available models can satisfactorily explain full spectrum of T_REG cells biology. Hence the reason why translation of basic knowledge about T_REG cells for clinical application is so slow.

So, every time we see new paper about T_REG cells we hope that we can acquire "some" missing information. In this regards, lets examine new paper in Science from Harvey Cantor's lab. There, the authors found that presence of transcription factor Helios is necessary to stabilize T_REG cells phenotype.

This is a simple, observation-type paper. Initially, the authors showed that starting from 5 months of age, mice deficient for Helios develop auto-antibodies to self antigens.

Next, using adoptive transfer experiments in combination with bone marrow chimera, the authors showed that Helios-deficient T_REG cells lacked suppressive function.

Finally, the authors showed that Helios-deficient T_REG cells displayed unstable phenotype and could be induced to express effector cytokines.

In summary, this study showed that Helios played an important role in stability of T_REG cells phenotype, especially during the ageing process. Of note, other studies with Helios-deficient mice did not observe the same phenotype. The authors claim that it has to do with the age difference of the experimental mice used in those earlier experiments. But genetic difference between mice colony itself or their gut flora could not be dismissed without proper experimentation.

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

          

Costimulation design for next-gen CAR T cells. A case study

Engineered (C)himeric (A)ntigen (R)eceptor (CAR) T cells show unprecedented level of protection against fluid tumors such as myelomas and lymphomas in clinical trials. The vast majority of these trials are based on CAR T cells with α-CD19 specificity. CD19 is a B cell specific marker and it is involved in B cell receptor signaling.

It is not immediately obvious why α-CD19 CAR T cells show such superior activity compared to CAR T cells with other specificity. One study suggested that α-CD19 CAR construct itself is uniquely effective. Another possibility is that since α-CD19 CAR T cells targets are a fluid populations such as B cells and B cell-derived tumors, they are highly sensitive for T cell mediated cytotoxicity.

In this regard, it is of interest to review a new paper published in Cancer Cell (part of Cell publication). Here, the authors have assessed efficacy of next-gen α-CD19 CAR constructs that incorporated several co-stimulatory molecules.

Initially, the authors compared 1st and 2nd generation α-CD19 CAR T cells designated here as 19z (α-CD19 CAR + CD3zeta), 1928z (α-CD19 CAR + CD28 + CD3zeta) and 19BBz (α-CD19 CAR + 4-1BB + CD3zeta). Here, 1928z CAR construct showed superior activity against myeloma, as measured in overall survival assay.

However, when the authors have analyzed CAR T cells expansion and tumor cell reduction in bone marrow at day 21, no difference was found between 1928z and 19BBz. Thus mice survival data and bone marrow data did not match. This complicates interpretation of this study.

Next, the authors showed that next-gen α-CD19 CAR construct incorporating two set of co-stimulatory molecules showed enhanced anti-tumor activity compared to 2nd generation α-CD19 CAR T cells (but not all combinations were effective and some were even detrimental).

Yet again, when the authors have analyzed CAR T cells expansion and tumor cell reduction in bone marrow at day 21, minimal difference was found between "active" CAR constructs. Thus, mice survival data and bone marrow data did not match. This complicates interpretation of this study.

In summary, we can conclude that incorporation of several co-stimulatory molecules in next-gen CAR constructs may enhance their anti-tumor activity, but we need better models to study relationship between CAR T cells anti-tumor activity and their cellular response.

David Usharauli

Mismatch between CD47 and SIRPα was found to explain age-old immunogenicity mystery

Sheep erythrocytes (SRBCs) have been used in immunological research for a very long time. It was well documented that SRBCs were highly immunogenic if injected in mice, so researchers were using it to serve as a "carrier" to enhance immune response to other, less immunogenic antigens, epitopes or haptens. Its mechanism of action was not clear.  

Now, new study in journal Immunity has showed that (a) mismatch between sheep CD47 and mouse SIRPα determined SRBCs immunogenicity in vivo, and (b) that absence of CD47 from mouse erythrocytes could convert mouse erythrocytes into "immunogenic".  

First, the authors showed that plate coated mouse SIRPα would only bind wild-type mouse erythrocytes (red blood cells). This confirmed that "do not eat me" inhibitory circuit mediated by CD47-SIRPα interaction are species-specific (though interestingly SIRPα from non-obese diabetic-prone NOD mice strain does bind strongly to human CD47).

Next the authors showed that similar to SRBCs but unlike to wild-type mouse RBCs, erythrocytes from CD47^-/- mice could activate mouse spleen dendritic cells upon adoptive transfer. (A) Interestingly, however, transfer of white blood cells from CD47^-/- mice did not activate spleen DCs; (B) Also, note that the authors failed to observe activation of spleen DCs cultured with SRBCs or CD47-deficient mouse erythrocytes in vitro.

More importantly, additional experiments showed that RBCs from CD47^-/- mice could serve as a "carrier" to enhance CD4 T cell immune response to nominal antigen, such as ovalbumin.

In summary, this study provided long overdue explanation as for immunogenicity of SRBCs. It appears that in absence of CD47-SIRPα inhibitory engagement between SRBCs and mouse spleen DCs, signals [driven by integrins and Src-family tyrosine kinases] dominates that lead to DC activation.

David Usharauli

MHC II cargo detour during live viral infection

Ordinarily, MHC class II molecules present epitopes derived from endocytosed protein cargo, while MHC class I molecules present epitopes derived from cytosolic protein degradation. These observations, however, were based on classic studies done in 1980s and 1990s, when for the purpose of simplification, research scientists were using "nominal", non-replicative proteins. But what about protein presentation during live, replicating viral infection? Will it follow the same rule [I-in/II-out]? 

New study in Nature Medicine tried to answer this question. Here, the authors showed that live viral infection elicited broader CD4 T cell epitope response and that to some extent, this broader CD4 T cell response to live viral infection was driven via non-classical class II presentation.    

This is surprisingly quite simple study [done in a manner characteristic to research from early 2000s], and I am not entirely sure whether it really has answers for its claims. Lets examine it.

First, the authors showed that unlike priming with inactivated influenza virus (BLP-PR8), i.n. infection with live influenza virus (live PR8) elicited broader and stronger CD4 T cell ex vivo IFN-γ response.

Direct, in vitro exposure of BmDCs to live or inactivated influenza virus confirmed that live infection produced broader response (notable for NA-25 and NP-47 epitopes).

More importantly, i.m. or s.c. priming with live PR8 or inactivated PR8 produced similar results: live PR8 infection produced stronger and broader immune [CD4 or Ab] response. Note, i.m. priming does not support live influenza virus replication, so it could be assumed that antigen load should be very similar here between live and inactivated PR8.

To rule out the role of inflammatory milieu [produced during live infection] affected the strength of immune response to inactivated PR8 infection, mice were challenged with inactivated PR8 virus in combination with non-cross-reactive live B/Lee virus. However even the presence of live B/Lee virus did not modify immune response to inactivated PR8 virus.

Next, the authors conducted several in vitro experiments to further evaluate antigen presentation pathways. In first set of experiments, MHC-II negative fibroblast were infected and then exposed to BmDCs and T cell hybridomas [in presence of anti-influenza Ab that supposedly prevents direct infection of BmDCs]. Here, out of six T cell hybridomas tested, only one hybridoma, specific for NP-47, showed response in this classical MHC II presentation assay.  

In second set of experiments, MHC II-positive cells were harvested ex vivo from live PR8 infected donors, sorted into surface viral antigen HA⁺ [indicative of live infection] or HA^- populations and cultured with primed CD4 T cells or CD4 T cell hybridomas. Here too, classical exogenous MHC II presentation pathway [HA^- population] produced minimal response.

Afterwards, significance of rest of the data are not clear: (a) the authors showed that cells from both H2-DMa^-/- and CD74^-/- hosts that lack the classical MHC II presentation pathway molecules could still present CD4 T cell epitopes after live viral infection; (b) however, TAP^-/- hosts that lack the classical MHC I presentation pathway molecule were capable of producing CD4 T cell epitopes as well; (c) proteosome inhibitor epoxomicin could reduce presentation of some of the epitopes from live PR8 viral infection.

However, none of these above mentioned experiments, strictly speaking, provide a definite answer that epitope presentation during live viral infection goes through endogenous cytosolic pathway. First, experiments are missing with the positive control epitope(s) from classical, exogenous, H2-DMa and CD74-dependent pathways. Second, live viral infection in vivo could infect or taken up by different cell or DC population compared to inactivated virus [even BmDC culture used here does not represent a homogeneous cell system at all]. This could introduce variability in antigen presentation pathway not directly addressed by this study.

David Usharauli