Hidden arm against tumors: microbiota-enabled checkpoint immunotherapy

This week Science published two studies showing how diverse microbiota directly contributes to efficacy of PD-1 checkpoint immunotherapy in several tumors.

First, we need to mention that senior authors from both papers disclosed associations with for-profit pharma/biotech companies (as cofounders, stockholders, paid consultants or advisory board members). Such associations could, in general, be seen as problematic if one promotes therapy lacking particularities.     

Second, data presented do not advance our understanding how microbiota contributes to the effectiveness of checkpoint immunotherapy. The sole conclusion from both papers is that the more diverse cancer patient's microbiota the more benefit it provides during PD-1 immunotherapy. However, when it comes to narrow down beneficial correlation to particular species we find that one paper reported enrichment of Akkermansia muciniphila while other paper reported enrichment of Faecalibacterium and Clostridiales in Responders (as opposed to Non-Responders).

We still don't know much about the role of microbiota in cancer immunotherapy. I think real advance will come when we define how antigens derived from specific microbiota contribute to anti-cancer immunotherapy either by amplifying existing cross-reactive effector T cells or Foxp3+ Tregs. 

posted by David Usharauli

PD-1 expression on tumor-infiltrating T cells does not correlate with antigen-specific response

Patients selection [stratification] and then monitoring for immunotherapy effectiveness is part of precision medicine. In humans, especially, when determination of cancer neo-antigens is not always feasible, clinical diagnostic tests are focused on surrogate markers to tell whether patient has a tumor antigen-specific immune response. More recently, PD-1 has become one of such surrogate markers. However it is not clear whether expression of PD-1 could truly correlate with T cell antigen-specific response.

New paper in Journal Immunology provided data to suggest that at least in mice tumor models PD-1 expression does not correlate with T cell antigen-specific response within tumor tissue.

For example, they showed that OT-I CD8 T cells would express PD-1 irrespective whether tumor expresses or not specific OVA antigen. In contrast, Nur77 (part of TCR signaling) expression correlated with tumor neo-antigen expression. 

This study indicate that diagnostic tests measuring PD-1 expression on tumor-infiltrating T cells may over-estimate tumor antigen-specific immune response and lead to unpredictable outcomes during antibody immunotherapy.

David Usharauli

PD-1 signaling assists regulatory T cells when Foxp3 is down

Foxp3⁺ regulatory T cells (Tregs) maintain peripheral tolerance to self. Several molecules expressed by Tregs, such as CTLA-4, play crucial role in maintaining this state of tolerance to self. PD-1 is another such molecule, though its role in Tregs function is less clear.

New paper in PNAS shed some light in PD-1 role in Tregs biology. It found that PD-1 keeps Tregs in shape when Foxp3 level is unstable.

This study arose from unexpected observation in new gene-modified mice where GFP was inserted in Foxp3 locus (mice carrying the IRES-GFPcre reporter KI at the 3′ untranslated region of the FoxP3 gene, FoxP3-GFPcreKI). When these mice were crossed with PD-1KO mice, it was found that male offspring of such cross showed early death, reminiscence of Foxp3KO mice. This was surprising since PD-1KO mice ordinarily do not show such phenotype.

Further experimentation found that GFP insertion affected Foxp3 stability thus resulted in reduced Foxp3 levels in FoxP3-GFPcreKI mice.

Indeed, FoxP3-GFPcreKI/PD-1KO male mice could be rescued with transfer of WT Tregs irrespective of their PD-1 expression indicating that PD-1 function were dispensable Tregs with normal level of Foxp3.

Finally, the authors showed that absence of PD-1 could further destabilize "Foxp3-low" Tregs function (conversion into ex-Foxp3 Tregs) resulting in lethal autoimmunity.

In summary, this study revealed that PD-1 could contribute to Tregs function in situations that affects Foxp3 stability.

David Usharauli

Availability of free cytoplasmic cholesterol augments TCR signaling in CD8 T cells

Few days ago Nature published new research study from mainland China wherein the authors showed that availability of free cholesterol in CD8 T cells augments TCR signaling and contributes to better control of experimental tumor.

This study focus on Acat1 that encodes cholesterol esterification enzymes that convert free cholesterol to cholesteryl esters for storage. The scientists noticed that chemical inhibition of Acat1 in CD8 T cells could augment their cytolytic effector differentiation (granzyme B ↑).

Similar results were obtained with CD8 T cells from Acat1 conditional knockout mice (Acat1^CKO).

Boost of CD8 T cell cytolytic functions with Acat1 inactivation yielded better tumor protection in adoptive transfer experiments as well.

Mechanistically, it appears that availability of excess free cholesterol in Acat1^CKO CD8 T cells improved TCR downstream signaling.

Finally, the authors showed that avasimibe, an Acat inhibitor with a good safety profile in humans, could delay tumor progression in mice and even show synergy with checkpoint inhibitor, anti-PD1 antibody.

In summary, this study suggests that commonly used cholesterol modulating drugs could have important role in CD8 T cells effector function and could influence tumor immunotherapy results.

Here I would like to note that one earlier study showed that reduction of cellular free cholesterol triggered universal STING-cGAS mediated anti-viral type I IFN response. This suggests that on one hand free cholesterol could augment CD8 T cell cytolytic function, but on the other hand it could diminish cell-autonomous anti-viral response.  

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

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