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Turning Tregs From Enemies to Allies

May 29, 2019

T regulatory cells (Tregs) are known for their ability to suppress effector T cell functions in the tumor microenvironment. In an effort to better understand and possibly disrupt Treg-mediated immunosuppression, Di Pilato and Kim et al. investigated the CARMA1-BCL10-MALT1 (CBM) signalosome complex within Tregs. Interestingly, disruption of the CBM complex in all or some Tregs not only reduced their suppressive functions, but endowed them with antitumor effector functions sufficient to initiate tumor control. The results were recently published in Nature.

Di Pilato and Kim et al. began their investigation of the CBM complex by targeting CARMA1, which activates several key pathways during the thymic development of Tregs and plays a role in the assembly of the CBM complex following TCR-dependent stimulation. To better understand the role of CARMA1 and the CBM complex in mature Tregs, the researchers developed murine models in which they could knock out one or both alleles of CARMA1 in all or half of Tregs.

Complete knockout of both alleles of CARMA1 in all Tregs led to the development of multiorgan inflammatory disease that was fatal in mice within four weeks, indicating that CARMA1 plays a key role in maintaining Treg homeostasis and preventing autoimmunity. Further investigation revealed that CARMA1 knockout increased the absolute numbers of immune cells in the lymph nodes, and that while the proportion of Tregs remained unchanged, the proportion of effector Tregs (eTregs) was significantly reduced. Interestingly, nearly all of the Tregs retained Foxp3 expression, though they began to produce IFNγ, and to a lesser extent, TNF, IL-4, and IL-17. Only the small portion of eTregs that remained expressed TH1 lineage markers, including T-bet, RORγt, and some GATA-3. Neutralizing IFNγ in CARMA1-deficient mice restored their lifespans to the length of the typical lifespan of Treg-deficient mice, indicating that it was the altered cytokine expression that significantly impacted lifespan. Global gene expression analysis later confirmed that Treg secretion of IFNγ under inflammatory conditions further accelerated existing inflammation.

While total knockout of CARMA1 induced lethal autoimmunity, knockout of CARMA-1 in only half of Tregs (taking advantage of random X-chromosome inactivation in heterozygous female mice), was well tolerated. Like in the inflammatory setting, the portion of Tregs remained unchanged while the portion of eTregs was selectively decreased. In this non-inflammatory setting, however, Tregs did not secrete effector cytokines. Overall, CARMA1-deficient Tregs were found to be less competitive for niche space, less suppressive, and less persistent compared to their CARMA1-sufficient counterparts. Protein expression of proapoptotic BIM and anti-apoptotic BCL2 were both high in this setting, suggesting that CARMA1 deficiency impaired differentiation from central Tregs to eTregs (which demonstrate lower expression of both proteins), and evidence from global gene expression analysis validated this observation.

To determine the mechanism through which CARMA1 deletion and CBM dysfunction impair differentiation and induce IFNγ secretion in Tregs, Di Pilato and Kim et al. tested whether disabled NF-κB signaling, which plays a known role in thymic development and function of Tregs, might be significant. Restoring NF-κB signaling in CARMA1-deficient cells limited the secretion of TNF, but did not limit the secretion of IFNγ, restore differentiation of eTregs, nor increase the lifespan of mice, indicating that the CBM complex maintains Treg homeostasis through mechanisms beyond just NF-κB activation. Early studies suggest a possible role for CARMA1 in the regulation of AKT activity.

Having observed the powerful pro-inflammatory potential of CARMA1-deficient Tregs, the researchers implanted D4M.3A melanoma into fully and partially CARMA1-deficient mice. In these settings, both eTregs and total Tregs were reduced within tumors, proportional to the extent of CARMA1 reduction, and tumor growth was slowed. Because a loss of function in only half of Tregs was not expected to break tumor tolerance, the researchers hypothesized that Tregs might be exerting active antitumor immunity. The researchers observed production of IFNγ and TNF by destabilized Tregs exclusively in the tumor microenvironment, and IFNγ was found to be essential and sufficient to reduce tumor growth.

To further study the selective destabilization of CARMA1-deficient Tregs, the researchers engineered mice in which they could delete CARMA1 in established tumors. In this setting tumor growth decreased and cytokine production could be detected shortly after CARMA1 was switched off. Intratumoral Treg destabilization was accompanied by increased MHC-II expression on macrophages and increased MHC-I expression on tumor cells, sensitizing them to CTL-mediated lysis. However, the increased IFNγ also triggered upregulation of PD-L1, suggesting adaptive tumor resistance. The addition of PD-1 blockade to CARMA-1 deletion enhanced tumor control.

While CARMA1 would seem to be a promising target for immunotherapy, there are currently no pharmacological inhibitors to serve this purpose. As a possible alternative, Di Pilato and Kim et al. showed that pharmacological inhibitors for MALT1, another protein component of the CBM complex, had effects similar to deleting CARMA1 in Tregs. MALT1 inhibition decreased tumor growth, induced IFNγ and TNF production by Tregs in the TME, and induced tumor expression of MHC-I and PD-L1 in D4M.3A melanoma-bearing female mice. In D4M.3A melanoma-bearing male mice, which are fully resistant to anti-PD-1 monotherapy, the addition of MALT1 inhibition to PD-1 blockade sensitized tumors to treatment and slowed tumor growth. When mice bearing an ovalbumin-expressing D4M.3A melanoma, which initially responds to anti-PD-1 therapy but frequently relapses, were treated with combination MALT1 inhibition and PD-1 blockade, strong tumor control was observed and most mice did not relapse.

Targeting the immunosuppressive function of Tregs has been a key goal for improving immunotherapy, but turning Tregs into active antitumor agents goes the extra mile. Inhibition of the CBM complex through inhibition of MALT1 or CARMA1 is an interesting and potentially useful new tool in the immunotherapy toolbox.

by Lauren Hitchings

Meet the Researcher

This week, we interviewed first co-author Mauro Di Pilato.

First co-author Mauro Di Pilato and lead author Thorsten R. Mempel.

What prompted you to tackle this research question?
Cancer is one of the leading causes of death globally, and while immunotherapy is one of the most promising treatments, few people actually benefit from it. When I joined the laboratory of Dr. Mempel, the goal of my research was to increase the number of patients that will respond to immunotherapy.

What was the most surprising finding of this study for you?
Many previous studies have focused on removing regulatory T cells from tumors. I was surprised to find that reprogramming these cells was enough to dramatically increase the efficacy of immunotherapy.

What was the coolest thing you’ve learned (about) recently outside of the lab?
I learned that the Boston area during the summer offers very nice weather to do outdoor activities like hiking and biking. I found that many of my colleagues participate in these as well. We often bike along the esplanade and hike in the nearby mountains.

References:

Di Pilato M., Kim E.Y., Cadilha B.L., Prüßmann J.N., Nasrallah M.N., Seruggia D., Usmani S.M., Misale S., Zappulli V., Carrizosa E., Mani V., Ligorio M., Warner R.D., Medoff B.D., Marangoni F., Villani A.C., Mempel T.R. Targeting the CBM complex causes T(reg) cells to prime tumours for immune checkpoint therapy. Nature. 2019 May 15.

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