cGAS puts gas on anti-tumor effect of checkpoint inhibitor

This week PNAS published new article explaining mechanism of action of checkpoint inhibitors, such as anti-PDL1, in tumor immunity. It shows that cytoplasmic DNA sensor cGAS/STING pathway synergize with anti-PDL1 therapy in mouse model of melanoma.

While this study provides noteworthy observation, it is poorly done. For example, when comparing WT and KO mice (cGAS or STING KO mice), the authors did not mention if they used littermate control in these experiments.

In addition, when the authors used exogenous cGAMP (product of cGAS activity detected by STING), they did not use it on cGAS or STING KO mice as controls to verify relationship between cause and effect.   

    

In summary, the connection between DNA recognition system and checkpoint inhibitors is very interesting. Whether it is a simple generic augmentation of T cell priming or specific stimulation of tumor-specific T cells is to be seen.   

David Usharauli

Foxp3+ Treg-derived IL-35 limits anti-tumor T cell immunity

IL-35 is a cytokine composed of the p35 subunit of IL-12 (encoded by Il12a) and Ebi3 (Ebi3). It was discovered in 2007 at Dario Vignali's lab and since then ~ 200 papers have been published about IL-35, according PubMed database. Most studies indicate that IL-35 has immune inhibitory function.

Now, new study in journal Immunity from the same Dario Vignali's lab provided evidence that Foxp3+ Treg-derived IL-35 inhibits effectiveness of anti-tumor T cell activity, pointing to it as a new immunotherapeutic modality. I am going to highlight the most notable results.

First, the authors showed that tumor (B16 melanoma) growth or its metastasis were inhibited when (a) mice were injected with antibody against IL-35 or when (b) mice lacked IL-35 specifically in Tregs (Foxp3^Cre-YFP. Ebi3^L/L mouse).

Similar effect of IL-35 blockade on tumor growth was seen in genetically-induced tumor model, in KP mouse, that spontaneously develop lung cancer after adenovirus-Cre delivery (KP mouse contains an activating mutation in K-RAS and a loss of function mutation in p53 controlled by Cre mediated recombination).

Mechanistically, the authors showed that IL-35 blockade improved T cell recruitment into tumor tissue.

Finally, the authors found that presence of Treg-derived IL-35 contributes to T cell exhaustion during tumor growth.

In summary, this study indicate the following scenario: certain tumors recruit Tregs. These Tregs secrete IL-35 and inhibit recruitment of effector T cells and/or contributes to their exhaustion. Blocking of IL-35 could provide immunotherapeutic benefit (interestingly, it appears that IL-35 does not synergize with anti-PD1 treatment in tumor models discussed in this paper).

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

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

Bypassing "do not eat me" signal could unleash anti-tumor T cell response

CD47 receptor on healthy cells serves as a "do not eat me" signal when engaged by its ligand, SIRPα, on phagocytic macrophages. As cells ageing they express less of CD47 and become targets for scavenger macrophages. In 2013, a study was published that showed that anti-CD47 antibody therapy could prime anti-tumor CD8 T cell response via cross-priming phagocytic macrophages. 

This time, however, a new paper in Nature Medicine suggested that anti-tumor effect of anti-CD47 antibody therapy is mediated via dendritic cell-enabled cross-priming of CD8 T cells involving intracellular DNA recognition by STING-IFNα pathway. Lets examine the data.

First, the authors showed that the growth of both lymphoma (A20) or solid tumors (MC38) were inhibited in wild-type mice after injection of mouse anti-CD47 monoclonal antibody.

This anti-tumor effect of anti-CD47 antibody was dependent on CD8 T cells.

In addition, using CD11c-DTR BM chimera, the authors showed that anti-tumor effect of anti-CD47 antibody was dependent on CD11⁺ dendritic cells.

Further experiments revealed that IFNα singaling in CD11⁺ dendritic cells played an important role.

Finally, the authors showed that tumor growth in mice deficient for intracellular DNA sensing signaling pathway, called STING (tmem173), was not inhibited by anti-CD47 antibody.

In summary, these results suggest the following scenario: anti-CD47 antibody injection facilitates uptake of tumor DNA and associated antigens by DCs. Within DCs, tumor DNA activates STING-IFNα pathway and allows anti-tumor CD8 T cell priming.

From this study it is not clear whether anti-CD47 antibody has any direct cytotoxic effect on CD47⁺ tumor cells. Also it is not clear how tumor antigen and DNA is delivered to DCs. The authors did not mention anything about any side effects of anti-CD47 therapy either (CD47 is expressed ubiquitously).

There is one figure in this paper that is problematic. While in all other figures anti-CD47 antibody treatment alone has a clear anti-tumor effect in wild-type mice, in Fig. 6a, injection of anti-CD47 antibody alone in wild-type mice shows no anti-tumor effect at all (A20 cells and antibody injection on day 12, 17).

I could not find any explanation in this paper for this major discrepancy. This could question the reliability of anti-CD47 treatment.

David Usharauli