Food antigens, not microbiota, regulate Foxp3+ regulatory T cells in small intestine

Foxp3⁺ regulatory T cells, shortly Tregs, are an unique subset of CD4 T cells that regulate magnitude and possibly class of immune response. 

In general, Tregs are divided into two subsets referred as thymus-derived Tregs (tTregs) and peripheral tissue-induced Tregs (pTregs). Historically speaking, this division of Tregs is arbitrary [it is assumed that thymus alone could not generate Tregs specific for peripheral antigens, such as tissue-specific antigens or food derived antigens] but till today we have no definite proof whether there is indeed such a thing as a peripheral Tregs.

In this regard new Science paper is of interest. In this "observation" type paper, the authors reported that food antigens, not microbiota, played important role in regulation of small intestinal Tregs, while the reverse was true for large intestinal Tregs.

I would like to point out that in this study the authors have used Neuropilin-1 (Nrp-1) to differentiate between tTregs and pTregs. However, more recent studies questioned the validity of this marker. So, for me, the results in this paper are simply "observations", not necessary "mechanisms".

The main contribution of this study is the use of antigen-free diet [chemically defined food devoid of macro-molecules] to analyse Tregs physiology in mice. It showed that mice on antigen-free diet had reduced number of Tregs in small intestinal tissue (but not in spleen or large intestine).

Next, the authors reported that small intestine of mice fed with antigen-free diet lacked Tregs expressing low level of Neuropilin-1 (that according to the authors represent peripheral Tregs). At least, these results indicate that food derived antigens regulate the fate of Neuropilin-1^low Tregs in small intestine.

Finally, by transferring ovalbumin-specific OT-II CD4 T cells in mice fed with antigen-free diet, the authors showed that lack of host Neuropilin-1^low Tregs in small intestine could lead to excessive proliferation of donor antigen-specific T cells in these mice (that could lead to exaggerated, allergic-type immune response).

In summary, this study showed that solid food derived antigens are important in regulating physiology of small intestinal Tregs. This knowledge could help to understand how food allergies develop.

David Usharauli  

Selective PD-L1/2 expression in lymphoid tissues allows tumor escape during allo-HSCT

The goal of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is to achieve graft-versus-leukemia (GVL) effect to eliminate residual tumor cells in host [left after irradiation]. Since most allo-HSCs are derived from HLA-compatible donors, GVL reaction is mediated by donor T cells reacting against host minor histocompatibility antigens (miHAg; self or tumor-specific Ags). However, even reaction to self-miHAg could still induce graft-versus-host disease (GVHD) because self-miHAgs are broadly expressed in different tissues. Interestingly, GVHD preferentially affects peripheral tissues such as liver or gut. But why?

A new [and very good] paper in journal Immunity may have an answer for this question. In this study, the authors found that selective expression of PD-1 ligands, PD-L1 and PD-L2, in allo-HSCT recipients' lymphoid tissues [but not in peripheral tissues] provide a "cover" against cytotoxic activity exerted by donor CD8 T cells.

To mimic allo-HSCTs, the authors have used female-to-male BM stem cell transplantation model. In this model, self-miHAg is represented by male antigen, HY. To induce GVHD, female BM cells were transplanted alongside with HY-specific transgenic MataHari CD8 T cells. As expected, only male recipients of female HSCs + MataHari CD8 T cells developed GVHD (in liver, gut and skin tissue).

To address the question why only those peripheral tissues were affected by GVHD, the authors examined the hypothesis that HY specific CD8 T cell cytotoxic activity were differentially affected by different tissues. Indeed, co-transfer of labeled male and female targets revealed that recipients of allo-HSCs + MataHari CD8 T cells showed selective reduction of cytotoxic activity against male targets in lymphoid tissues (but not in liver).

This observation was supported by the fact that in contrast to peripheral tissues, MataHari CD8 T cells obtained from lymphoid tissues expressed low level of granzyme B (molecule involved in cytotoxic activity).

To understand why it is the case, the authors examined expression of inhibitory molecules on CD8 T cells. This revealed that while PD-1 on CD8 T cells were similarly expressed irrespective of tissue origin, its ligands, PD-L1 and PD-L2 were selectively up-regulated in lymphoid tissues.

The role of PD-L1 and PD-L2 in inhibition of CD8 T cell cytotoxicity in lymphoid tissues were confirmed in  experiment with anti-PD1 antibody.

Finally, using anti-PD-1 antibody injection, the authors showed that B cell leukemia cells that were hiding in lymphoid tissues of allo-HSCs male recipients could be now eliminated by MataHari CD8 T cells.

In summary, this study showed that (a) during GVHD donor CD8 T cell activity is differentially regulated by different tissues based on availability of inhibitory PD-1 signaling and that (b) this is exploited by leukemia cells to hide in lymphoid tissues but it could be overcome by anti-PD1 antibody injection.

David Usharauli

Gut microbiota, not IL-17, is required for spontaneous arthritis in K/BxN mice

IL-17 has been implicated in autoimmune inflammatory diseases such as arthritis. It was proposed that gut microbiota, [SFB]-induced Th17 cells were mediating autoimmune arthritis. Experiments with K/BxN mouse, a human model of spontaneous autoimmune [rheumatoid] arthritis, provided the initial support for this hypothesis. 

However, a new paper in Journal of Immunology, suggests that IL-17 may have nothing to do with arthritis in K/BxN mice.

For this study, the authors have generated K/BxN mice deficient for IL-17A. Surprisingly, IL-17 deficient K/BxN mice showed no difference in arthritis development compared to WT K/BxN mice.

Even though IL-17 deficient K/BxN mice had no Th17 cells, levels of anti-GPI IgG auto-antibodies were not affected by absence of IL-17. 

Next, the authors showed that antibiotic treatment could inhibit arthritis development in K/BxN mice independent of IL-17 expression.  

Interestingly, the authors found that K/BxN mice with CD4-specific deletion of Bcl6 [and lacking T follicular helper T cells, Tfh] were resistant for arthritis development, implying role of Tfh and auto-antibodies in arthritis pathology [independent of IL-17].

In summary, this study suggests that IL-17 may not be involved in development of autoimmune [rheumatoid] arthritis in all cases. Rather, it is gut microbiota and Tfh/Ab that play dominant "pathogenic" role in this autoimmune disease.

David Usharauli

Anti-PD1 antibody treatment shows beneficial effect in human Alzheimer disease (AD) mouse model

This week journal Nature Medicine published a short paper from Michal Schwartz lab at Weizmann Institute of Science (Israel) showing beneficial effect of anti-PD1 treatment in human Alzheimer disease (AD) mouse model.

Michal Schwartz lab is famous in immunology circles for producing data showing the role of immune system in CNS function. For example, earlier their lab showed that mice deficient for adaptive immune system display decline in cognitive functions.

In this new paper the authors showed that two consecutive injections of anti-PD1 antibody (checkpoint inhibitor used in cancer immunotherapy) reduces CNS tissue pathology in mice with human AD phenotype [in two different models, (a) five familial AD mutations (5XFAD) and (b) APP/PS1 mouse models]. This beneficial effect correlated with the accumulation of peripheral macrophages into CNS and were dependent on IFN-γ.

It is not clear why systemic immune activation improves AD tissue pathology. Simple explanation is that peripheral "activated" macrophages that migrate to CNS are better equipped to digest and clean up AD-associated amyloid depositions. However, it is not clear whether CNS with AD pathology sends out any specific signals to recruit those peripheral macrophages or it is just nonspecific migration into CNS and other tissues [not examined in this paper].

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

Anti-tumor effect of adoptive CD4 T cells positively correlates with high precursor frequency

It is now well accepted that similar to cytotoxic CD8 T cells, antigen-specific effector CD4 T cells can show a direct anti-tumor effect both in  mice and humans. This concept is still relatively new (actually first paper about it was published only in 2003). Incorporation of CD4 T cells and MHC II + peptides in tumor immunotherapy strategy increases chances of finding tumor-specific antigens (epitopes) relevant for personalized cancer medicine.

In this regard, new paper from journal Immunity is of interest. There, the authors showed that unlike CD8 T cells, intra-clonal competition of adoptive transfer CD4 T cells [at high precursor frequency] improved rather than diminished its anti-tumor effect and productive effector differentiation.  

In this study, the authors transferred different number (10³, 10⁴, 10⁵, 10⁶) of melanoma antigen, TRP-1 specific CD4 T cells into mice implanted with melanoma. Despite significant expansion at low precursor frequency, only high frequency transferred TRP-1 specific CD4 T cells managed to control and eradicate established tumors.

Moreover, the authors showed that in this settings, only TRP-1 specific CD4 T cells from high frequency adoptive transfer hosts underwent productive effector differentiation.

Even addition of anti-PD1 antibody to [low frequency] TRP-1 specific CD4 T cell adoptive transfer hosts failed to rescue their differentiation (increase in IL-21 is a marker of CD4 T cell exhaustion).

In summary, this study suggests that for CD4 T cells intra-clonal competition does not prevent efficient effector differentiation and establishment of productive anti-tumor immunity. It appears that CD4 T cells undergoing significant expansion at low precursor frequency develop "early and checkpoint inhibition-refractory exhaustion" preventing them to participate in effective anti-tumor response. This knowledge should be taken into account when considering adoptive T cell tumor therapy and checkpoint inhibition (according this study, anti-PD1 therapy could potentially accelerate tumor-specific CD4 T cell exhaustion at low precursor frequency).

David Usharauli

How to make vaccine immunity to persist longer?

Most vaccines, if not all, work through antibody mediated neutralization of infection. Within few days of vaccination initial waves of low-affinity antibodies are produced by antigen-specific plasmablasts via extra-germinal center (GC) reactions. However, those plasmablast are short-lived and disappear within next 7-14 days. 

Long-term vaccine-induced immunity is maintained by long-lived plasma cells (LLPCs) and memory B cells (MBCs), both cell types generated within germinal-center (GC) reactions. The difference between these cell types is that only LLPCs can secrete high affinity antibodies and this happens spontaneously, in a steady-state. Level of neutralizing antibodies secreted by LLPCs determine degree of immunity in vaccinated individuals. 

But what is the function of MBCs and how are they related to LLPCs? Its not clear. MBCs can express high affinity IgM or IgG but they do not secrete antibodies. However, they can rapidly respond to secondary infection by differentiating into antibody forming cells (maybe even into LLPCs), if LLPC-mediated Ab protection is breached. Since antibody specificity produced by LLPCs are "fixed" during primary infection, they lack flexibility and are in fact useless if secondary infection is slightly different (mutated) from primary infection. 

Precise mechanisms how MBCs or LLPCs are formed during primary infection is lacking. Are they related (derived from the same B cell clones?) or independently generated (based on affinity or time of entering GC?).  

In this regard, it was interesting to read new paper in journal Immunity from Mark Shlomchik's lab. There, the authors showed that majority of MBCs were formed before day 14 during primary response, while majority of LLPCs required much longer time to form (between day 21 - 40).

In this paper, the authors have used quite old technique, BrdU pulse chase, to label proliferating naive B cells and follow their fate up to 2 months. BrdU is incorporated into DNA of actively proliferating cells and can be later detected by anti-BrdU antibody. BrdU+ cells are those cells which at some point previously underwent a brief burst of proliferation and then entered a quiet state as expected for MBCs or LLPCs. By "pulsing" mice with BrdU at different times post antigen injection and then "chasing" BrdU+ cells, one could determine what percentage of MBCs or LLPCs are formed at what time.

So, doing this type of analysis, the authors showed that antigen-specific MBCs and LLPCs have different kinetics of formation. Namely, most MBCs are formed before day 14, and most LLPCs are formed after day 14.

This dichotomy between MBCs and LLPCs formation was supported by finding that disruption of GC reaction by anti-CD40L on days 12-14, reduced number of antigen-specific LLPCs but has no or little effect on antigen-specific MBCs formation.

Interestingly, V gene sequencing for mutational analysis revealed that some MBCs are generated at the same time as LLPCs and are similarly enriched with mutations.  

In summary, using simple method of cell labeling, the authors showed that in mice majority of GC-derived MBCs are formed earlier than GC-derived LLPCs.

How this results could applied to vaccination? Clearly, this study suggests that vaccines that allow maintenance of "prolonged" GC reaction would produce more of LLPCs and high levels of serum antibodies.

However, one drawback of this study is that the authors did not analyze actual antigen-specific antibody level in the serum and correlate it with the main findings. For example, would early MBCs compensate for the loss of LLPCs in anti-CD40L injected mice? Without such data, we cannot be sure how "time-span" of vaccine-induced GC reaction would affect level of neutralization antibodies in the serum 

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

Monocyte hyperactivity at birth correlates with susceptibility to food allergy in infants

Every time I read a new article from Science Translational Medicine (STM) I have a distinct feeling that something is not right with its editorial board. For some reason, immunology papers published at STM usually start strong and clear and but end weak and confused, as if two different teams worked on them.

For example, this week STM published a very interesting study about immune correlates of food allergy in infants. Journal Science itself had a news/views story about it on its website.

Now, I did analysis of this study for my immunology blog. So I wanted to share my reading experience of this paper.

Basically, for this study the authors had first collected cord blood cells from large cohort of newborn babies. Then, they have conducted surface phenotype and functional assays on those cord blood cells. In parallel, they have followed up with those babies up to 1 year to see who would show any susceptibility to allergens in skin prick test (SPT).       

In Figure 1, the authors showed that compared to non-allergic infants, monocytes from newborns who later developed allergies showed hyperactivity (IL-6↑, TNF-α↑, IL-1β↑) when stimulated in vitro with 1 µg/ml LPS (this dose of LPS is quite high. However they did not show dose titration results to see if this difference is apparent at high LPS dose only).

In the same Figure 1, the authors also showed that cord blood of infants who later developed food allergies had high ratio of monocytes to CD4 T cells (CD14⁺monocyte / CD4⁺ T cell ratio). Its all.

Afterwards, in Fig 2 and 3, the authors went on to show how exogenous cytokines influenced "generic, pooled" cord blood-derived CD4⁺ T cell differentiation. However, they did not compare CD4⁺ T cells derived from allergic-prone and non-allergic infants. So, basically these Fig 2 and 3 are completely uninformative.

In summary, this study analysed cord blood samples from 697 newborn babies and found that presence of large number of hyperactive monocytes in newborn babies correlated with food allergy development by age 1.

So what could we conclude from study that analysed cord blood samples from so large newborn cohort and found only 1 non-specific correlate to food allergy? And what presence of hyperactive monocytes really means? I would expected that STM would demand more vigorous quality.

David Usharauli

Type I IFNs produced during viral infection promotes bystander regulatory T cells

Foxp3⁺ regulatory T cells are one of my favorite topics. Foxp3⁺ T cells are the most powerful regulatory subset within immune system. It is unbelievable but true that as of today only sure thing we know about Foxp3⁺ T cells is the fact that their absence or ablation leads to severe and total autoimmune organ disorders and death. No other T or B cells or innate subsets produce such a dramatic effect on the body.

At the same time, we are still have no clear answer whether Foxp3⁺ regulatory T cells operate in an antigen-specific manner or whether they non-specifically suppress "overly" excessive immune response (both innate or adaptive driven).

There are several hypothetical questions regarding Foxp3⁺ T cells that would require experimental proof before we can make any significant breakthrough. 

For example, 

"if Foxp3⁺ T cells are so potent, how immune response is initiated in the first place?" 

"If initial inflammatory stimuli temporary inactivates Foxp3⁺ T cells to allow initiation of immune response, how long such inactivation lasts?" 

"if Foxp3⁺ T cells regulate excessive immune response (excessive inflammation), how they can sense what is excessive?"

In this regard new paper in Nature Immunology from Steven Ziegler's lab is interesting to read. Here, the authors showed that pre-exposure of naive antigen-inexperienced CD4 T cell to type I IFNs initiates their differentiation towards regulatory pathway rather than effector. Such scenario possibly prevents excessive bystander activation of naive T cells and reduces overall tissue damage.

In vitro studies showed that CD4 T cells initially pre-exposed in vivo to polyI:C (as a source of type I IFN) and then co-cultured with antigen-pulsed DCs and TGF-β tended to preferentially develop into Foxp3⁺ T cells.

Such preference for Foxp3⁺ T cell development were abolished with CD4 T cells from IFNαR1^KO mice.

RIP-mOva × Rag2KO hosts receiving naive OVA-specific T cells pre-exposed to polyI:C do not develop diabetes (c), while RIP-mOva × Rag2KO hosts receiving control naive OVA-specific T cells together with polyI:C rapidly developed diabetes. Importantly, secondary exposure to polyI:C of RIP-mOva × Rag2KO hosts transferred with naive OVA-specific T cells pre-exposed to polyI:C still did not break tolerance (d).

These experiments indicated that simultaneous exposure of naive T cells to antigen and type I IFNs drove effector differentiation, but if they were first pre-exposed to type I IFNs and then to antigens it drove their Foxp3⁺ T cell differentiation. Indeed, this observations were confirmed in subsequent experiments that revealed that exposure to type I IFNs 2-3 days before antigenic exposure provided the most optimal condition for Foxp3⁺ T cell development.

In summary, this study suggests the following scenario: type I IFNs released during viral infection conditioned bystander, nonspecific naive T cells for Foxp3⁺ T cell development. This mechanism potentially prevents excessive activation and effector differentiation of naive T cells encountering antigens later during immune response (including tissue-derived self-antigens).

David Usharauli

Tumor suppressor PTEN promotes anti-viral immunity

In recent years new and powerful immunothereutic strategies such as checkpoint inhibitors (anti-CTLA4/anti-PD1) and CAR-T cells entered into clinics. Moreover, just few months ago, another immunotherapeutic strategy, called oncolytic viral therapy, received FDA approval. This latter approach is very interesting. Basic claim is that this "so called" oncolytic viruses selectively infect and target tumor cells. Of course, this is a nonsense. No virus, including modified oncolytic viruses, could selectively infect tumor cells (and sparing healthy cells). However, analysis of the following paper would explain how oncolytic viruses would "prefer" cancer cells.

This new paper, published in Nature Immunology showed that tumor suppressor phosphatase PTEN is involved in type I IFN production in response to viral infection.   

To understand significance of this finding in oncolytic immunotherapy, we need to remember that PTEN is a classic tumor suppressor (like p53), which is frequently mutated in cancer cells. Here, the authors initially found that PTEN ^-/- cells were deficient in type I IFN (but not IL-6) production upon viral infection.

PTEN ^-/- cells could be rescued to produce type I IFN by transfection with WT PTEN, but not phosphatase[activity]-deficient PTEN (though this PTEN anti-viral phosphatase activity was independent of its phosphatase activity for PI(3)K-Akt pathway).

In vivo experiments with tamoxifen-inducible Cre recombinase PTEN^LoxP/LoxP mice confirmed essential role of PTEN in anti-viral immunity.

These results could explain why tumors maybe selectively "susceptible" for oncolytic virus immunotherapy. If tumor cell lacks PTEN it becomes sensitive to viral infection since it will fail to produce basic defense anti-viral molecules such as type I IFNs. In a sense, a tumorigenic transformation of healthy cells (as a result of PTEN mutation) comes at cost of reduced anti-viral defense that could be exploited by therapy. However, oncolytic viruses are not regular WT viruses either. They are modified for reduced virulence (WT viruses would not get FDA approval, for sure). So, in the end, positive outcome with oncolytic virus therapy is a sum of (1) reduced anti-viral defense of cancer cells, (2) reduced virulence of oncolytic virus, (3) direct viral cytotoxicity of infected cancer cells, and (4) priming of oncolytic virus-specific T cells (and then cancer epitope spreading).

David Usharauli

DC vaccine synergizes with αCTLA4/αOX40 against solid tumors

The Objective of any tumor immunotherapy is to achieve tumor-specific T cell response while avoiding any bystander T cell activation. The reason why checkpoint inhibitors, such as αPD-1 and αCTLA4 antibodies work only in a fraction of patients have to do with the fact that only those "responders" have enough pre-existing tumor-specific T cells "responsive" to αPD-1 and αCTLA4 inhibitions. 

Other patients are refractory because their immune system is "blind" or "ignorant" of tumor antigens, i.e. first stage of T cell priming has failed due to lack of antigenic load in DCs (for example, if tumor express few mutated antigens). This situation, however, is potentially reversible with DC vaccines, wherein tumor-specific antigens/epitopes are loaded into DCs. Such "loaded" DCs would then prime T cells which in turn become responsive to checkpoint inhibitors.

So, solid tumor immunotherapy would require multi pronged approach to improve its efficacy. In this regard, I found this new paper in PNAS worth reviewing. Here, the authors showed that combination of "loaded" DC vaccine with αCTLA4/αOX40 treatment were able to overcome dormant state of T cells and allowed efficient control of solid tumors.

Initially, the authors showed that dual αCTLA4/αOX40 antibody treatment allowed substantial expansion of antigen-specific CD8 T cells (in a CD4 T cell-dependent manner).

However, these expanded CD8 T cells were not able to control solid tumor in mice challenged with HER2-expressing TUBO mammary cancinoma cells. However, when αCTLA4/αOX40 antibody treatment were combined with DC vaccine (DEC-205/HER2/PolyI:C), half of tumor challenged mice achieved long-term tumor-free status. 

Now, this is just an example of what potentially could be accomplished with the right concept. In general, OX40 is a complex molecule that has additional role in TH2 immunity, so it is not clear whether this particular combination would be useful in humans. PolyI:C, TLR3 agonist, is not approved for human use either, so it is also not very relevant right now.

In summary, combination of DCs vaccine with checkpoint inhibitors could add missing piece necessary to arm T cells against solid tumors.

David Usharauli

Systems vaccinology is anything but useful

Systems vaccinology is a field of immunology that tries to apply "big data" analysis to predict vaccine efficacy based on responder/non-responder signature. These signatures involve readouts such as mRNA or cytokine signature. Notwithstanding its purpose and scientific justification, systems vaccinology generated very little useful information thus far.

This week Nature Immunology has published another such paper. However, as previously, when it comes to predictions of vaccine efficacy, this new paper also concluded that "in sum, neither molecular nor cellular data offered any consensus prevaccination predictor of nonresponsiveness akin to those proposed in studies of nonadjuvanted vaccines".

I will only discuss those few data that were interesting. In this paper the authors has analysed PMBC and serum response from individuals vaccinated with adjuvanted 2009 H1N1 vaccine, Pandemrix (Of note, this is the same vaccine that caused spike in autoimmune sleeping disorder called narcolepsy in vaccinated individuals in EU). 

20% of vaccinated individuals were classified as non-responders (<4-fold increase in HAI titres). 

However, no "signature" was found that correlated with vaccination outcome. Interestingly, the majority of non-responders actually had high levels of preexisting HAI titres, suggesting cross-reactivity with previous flu infection/immunization.

Since data generated by these analyses failed to provide any signature that could have predicted vaccine efficacy, the authors tried to salvage the data by analyzing signature for vaccine "side effects" also known as adverse events. This analysis revealed a correlation between number of "transitional" B cells (and high level of pre-vaccination titres of auto-antibodies) and severity of adverse events. 

What can we conclude from this "big data" study: nothing much (very close what the authors themselves said). No cytokine(s) or gene(s) expression pattern showed any correlation with vaccine efficacy. There was some unusual correlation between high level of pre-vaccination auto-antibodies and vaccine adverse events. However, this observation would require confirmation in other studies with other vaccines since this particular vaccine, Pandemrix, showed unusual tendency to induce narcolepsy, an autoimmune disease. This particular effect could have skewed their analysis.  

Overall, the results from systems vaccinology studies suggest need to develop alternative readouts, beyond PBMCs and serum cytokines, that would have more predictive powers. 

David Usharauli  

Thymic epitope expression determines pattern of CD4 T cell peripheral tolerance

A few months ago I posted my analysis of one of the important papers from journal of Immunity discussing mechanisms of CD4 T cell peripheral tolerance. In that paper, the authors led by J.J. Moon, showed that number and functionality of CRE recombinase-specific endogenous CD4 T cells were determined by antigen expression in the thymus. 

Today, journal Nature Immunology published very similar study from Marc Jenkins' lab (JJ Moon's former supervisor) where the authors showed that number and functionality of eGFP epitope-specific endogenous CD4 T cells were also determined by level of specific epitope expression in the thymus. These studies have important implications predicting the efficacy of vaccines and mechanisms of autoimmune diseases, so lets review it.

As some of you might know Marc Jenkins' lab pioneered technique for analysis of endogenous antigen[epitope]-specific T cells in WT mice. Here, the authors have analysed expansion of endogenous eGFP epitope:MHC II tetramer⁺ CD4 T cells in dozen of transgenic mice expressing eGFP protein under the guidance of different ubiquitous or tissue-specific promoters. 

This analysis revealed 3 patterns of T cell tolerance: ignorance, thymic Treg development and thymic deletion. Mechanistically, level of antigen expression in the thymus determined which out of these 3 tolerance patterns were operational.   

Tolerance by ignorance: Ins1^eGFP mice express eGFP exclusively in pancreas. Analysis showed that both Ins1^eGFP  and WT mice harbor similar number of eGFP-specific naive CD4 T cells, implying ignorance.

Tolerance by Treg induction: Ins2^eGFP mice express eGFP in pancreas and in the thymus in a AIRE-dependent manner.

Presence of eGFP in the thymus in Ins2^eGFP mice slightly reduced tet⁺ CD4 T cell numbers and correspondingly increased frequency of tet⁺ Foxp3⁺ CD4 T cells. 

Immunization with CFA-eGFP peptide showed that tolerance in Ins2^eGFP mice depended on AIRE-driven eGFP epitope expression in the thymus.

Tolerance by thymic deletion: UBC^eGFP mice displayed a profound thymic deletion of eGFP-specific tet⁺ CD4 T cells. 

The authors clearly showed that eGFP expression in the thymus inversely correlated with the number of tet⁺ CD4 T cells. 

Similar pattern of T cell "tolerance" were found for truly nonself- and self-epitope-specific T cells.

In summary, this study confirmed that level of antigen expression in the thymus (and not in periphery) determined overall T cell tolerance status. By analyzing the number of antigen-specific human T cells [in comparison to reference "self"-specific T cell numbers] the scientist could predict efficacy of vaccines, tumor vaccine for example, or predict the potential for development of autoimmune diseases.

David Usharauli