Unlocking CAR-T’s Full Potential: Why Targeting AHR Matters

Hey there! Let’s chat about something pretty exciting happening in the world of cancer treatment. You’ve probably heard of CAR-T therapy, right? It’s one of those cutting-edge approaches that uses a patient’s own immune cells, specifically T cells, engineers them in the lab to become super cancer-fighters, and then puts them back in to track down and destroy tumor cells. It’s shown some truly remarkable results, especially for certain blood cancers like leukemia and lymphoma. It feels like we’ve finally got these amazing immune superheroes on our side!

But, like with any superhero story, there are challenges. While CAR-T has been a game-changer for many, it doesn’t work for everyone, and tackling solid tumors is still a big hurdle. Why? Well, the tumor environment itself can be a pretty hostile place for our engineered T cells. It’s like sending our superheroes into a toxic swamp – they can get tired, lose their power, and become dysfunctional. This “dysfunction” means they don’t proliferate well, their killing abilities weaken, and they start displaying markers that basically tell them to stand down.

Meeting AHR: A Potential Culprit?

So, scientists are constantly looking for ways to make these T cells tougher, more resilient, and better equipped to handle that tough tumor environment. One interesting character that’s popped up in this story is something called the Aryl Hydrocarbon Receptor, or AHR for short.

Think of AHR as a kind of sensor inside our cells. It was first identified because it reacts to external chemicals (like pollutants), but it turns out it’s also deeply involved in regulating our immune system. And here’s the kicker: studies, particularly in mice, have suggested that AHR might actually be *bad* news for antitumor T cell function. It seemed to be putting the brakes on their ability to fight cancer.

This got us thinking. If AHR is hindering T cells in mice, could it be doing the same in humans? And if so, could we somehow target AHR to make human T cells, especially our powerful CAR-T cells, more effective?

Our Mission: Characterizing AHR in Human T Cells

While the mouse studies were insightful, the role of AHR in human T cells wasn’t totally clear – it’s actually been a bit controversial! So, our goal was to get a much better look at what AHR is doing in human T cells and understand how it might be contributing to that T cell dysfunction we see in the tumor microenvironment.

To do this, we decided to use a pretty cool tool called CRISPR-Cas9. You might have heard of it – it’s like a super precise molecular editing tool that allows us to cut out or modify specific genes. We used it to effectively “knock out” the AHR gene in human T cells. This way, we could see what happens when AHR isn’t there to do its thing.

We then put these engineered T cells through their paces in the lab, using a model that mimics the kind of chronic stimulation they’d face when constantly exposed to tumor cells. We wanted to see if removing AHR would help them stay strong and functional under pressure.

What We Discovered: AHR is Active and Involved

First off, we looked at AHR itself in normal human T cells. We found that when T cells get activated (which is what happens when they encounter something they need to fight, like a tumor cell), AHR expression goes up. And importantly, in our model of chronic stimulation, AHR stayed highly expressed over time. This suggests AHR is actively involved when T cells are repeatedly challenged, like they are in a persistent tumor environment.

We also confirmed that AHR in human T cells can be activated by substances like kynurenine, which is a molecule often found in higher levels in tumors and is known to suppress the immune system. When activated by kynurenine, AHR moves from the cytoplasm into the nucleus, where it can start influencing gene expression.

AHR Knockout: Boosting T Cell Power

Now for the exciting part! When we knocked out AHR in human T cells using CRISPR-Cas9, we saw some really promising changes.

• Enhanced Profiles: The engineered T cells started looking more like “effector” and “memory” cells. Think of effector cells as the immediate fighters and memory cells as the ones that stick around long-term, ready for action. This is great because you want your cancer-fighting T cells to be both potent and persistent.
• Gene Expression Shifts: Looking at their genes, we saw an upregulation of genes associated with strong antitumor responses and better persistence. Genes like TBX21 and EOMES (linked to cytotoxic T cells) and TCF7 and BACH2 (linked to less differentiated, longer-lasting T cells) were expressed at higher levels when AHR was gone.
• Increased IL-2 Production: We also saw an increase in the production of IL-2, a cytokine that helps T cells grow and survive. This makes sense, as AHR had previously been linked to repressing IL-2 production.

These findings strongly suggest that AHR might be actively suppressing these desirable traits in human T cells. Removing it seems to unleash their potential!

Putting the Brakes On: AHR and Inhibitory Receptors

Remember those “brakes” on T cells we talked about – the inhibitory receptors? We looked at those too. Our analysis showed that knocking out AHR led to a *decrease* in the expression of certain inhibitory receptors, notably CD39 and TIGIT.

CD39 is particularly interesting because it’s part of a pathway that generates adenosine, a molecule that can suppress immune responses in the tumor microenvironment. TIGIT is another inhibitory receptor that’s often found alongside PD-1 on exhausted T cells, and blocking it is a strategy being explored to revitalize these cells.

While we didn’t see a significant change in PD-1 or TIM-3 expression in our model, the reduction in CD39 and TIGIT is a big deal. It means that without AHR, the T cells are less likely to have these “stand down” signals telling them to back off from the fight.

The Ultimate Test: AHR Knockout in CAR-T Cells

Okay, so AHR knockout looks good in regular T cells. But what about our specialized CAR-T superheroes? We applied the same CRISPR-Cas9 technique to CAR-T cells designed to target a specific cancer marker (CD123).

First, we checked if knocking out AHR messed with their basic ability to kill cancer cells in a short-term test. Good news – it didn’t! The AHR-knockout CAR-T cells were just as effective at killing tumor cells as the regular ones in the short term. We also confirmed that knocking out AHR in CAR-T cells prevented the increase in CD39 expression, just like we saw in regular T cells.

The Real Game Changer: Persistence!

Now for the most crucial part: how do these AHR-knockout CAR-T cells perform under *chronic* stress, like facing tumor cells repeatedly over a longer period? We set up an experiment where we co-cultured the CAR-T cells with tumor cells at an unfavorable ratio (many tumor cells for each CAR-T cell) to really challenge them.

The results were incredibly encouraging! The AHR-knockout CAR-T cells persisted for a significantly longer time compared to the regular CAR-T cells. After several rounds of being challenged by tumor cells, a much higher percentage of the AHR-knockout CAR-T cells were still around and fighting compared to the control group. This is HUGE because persistence is key for CAR-T therapy to work effectively against solid tumors or prevent relapse.

We also saw that the persisting AHR-knockout CAR-T cells showed weaker expression of CD39 and TIGIT, reinforcing the idea that they were in a less dysfunctional state than the control CAR-T cells that managed to survive.

Interestingly, we also saw that adding kynurenine (the AHR activator) negatively impacted the regular CAR-T cells’ antitumor activity, but this negative effect was *not* observed when AHR was knocked out. This further supports the idea that AHR is mediating some of kynurenine’s immunosuppressive effects on CAR-T cells.

Why This All Matters

So, what’s the takeaway from all this? Our research strongly suggests that AHR acts as a brake on human T cells, limiting their ability to stay functional and persistent, especially when they’re constantly challenged by tumor cells in a tough environment. By knocking out AHR using genetic engineering tools like CRISPR-Cas9, we can potentially remove this brake.

This could be a game-changer for improving CAR-T therapy, particularly for solid tumors where T cell dysfunction and lack of persistence are major problems. Genetically reprogramming T cells by targeting AHR could lead to therapies that are more effective, last longer, and work for a wider range of cancers.

It’s still early days, of course, and more research is needed to fully understand all the nuances of AHR’s role and how best to target it safely and effectively in a clinical setting. But these findings are incredibly exciting and point towards AHR being a really valuable target for making our T cell-based cancer therapies even better. It feels like we’ve found a key piece of the puzzle to help our immune superheroes win the fight!

Source: Springer