HIV infects immune cells and establishes long-lasting infections that can progress to acquired immunodeficiency syndrome (AIDS) if left untreated. With the advancement of antiviral therapy (ART), HIV infection has become a manageable life-long disease. However, even with antiviral therapy, lingering viral transcription is linked to heightened risk of developing various other maladies, including cardiovascular diseases and cancers. “For 30 years, T follicular helper cells have been thought to be a major site of this HIV reservoir,” which allows the infection to persist during ART, explains Dr. Karsten Eichholz, a staff scientist in Dr. Larry Corey’s lab in the Vaccine and Infectious Disease Division at Fred Hutch. Despite this knowledge, there are no therapies developed to target T follicular helper cells and directly go after what is thought to be a major source of HIV persistence. “We’re interested in depleting those cells,” states Dr. Eichholz. In a recent study led by Dr. Eichholz that was published in The Journal of Clinical Investigation, Eichholz and colleagues developed a creative strategy to deplete T follicular helper cells in vivo, using chimeric antigen receptor (CAR) T cells.
“One of the best markers for T follicular helper cells is PD-1 (programmed cell death protein 1),” which is highly expressed on their cell surface,” states Eichholz. The authors of this study hypothesized that using engineered CAR T cells to recognize and attack PD-1+ cells might be effective at depleting this HIV reservoir. Previously, the Corey lab tested antibody-based anti-SIV (simian immunodeficiency virus, the non-human primate version of HIV) envelope CAR T cells which were ineffective in vivo despite being potent in vitro killers. While this approach using CAR T cells to target the HIV envelope would be a more targeted way to kill only HIV-infected cells, it was not successful likely due to the relatively low abundance of HIV-infected cells during antiviral therapy, in addition to anti-CAR antibody and anti-SIV envelope immune responses which can inhibit CAR function. The Corey lab sought to overcome these obstacles by targeting PD-1 as a cellular marker for HIV persistence and replication. Eichholz admits that this approach involves some collateral damage, namely the loss of PD-1-expressing memory T cells.
After generating anti-PD-1 CAR T cells, thoroughly testing their ability to kill off PD-1+ cells in vitro using a cell culture system and demonstrating that their engineered CAR T cells do not express PD-1, which would promote the killing of other CAR T cells, the researchers turned to test this approach in vivo. For this, they used rhesus macaques infected with SIV that were receiving ART as their model, in addition to uninfected controls. Excitingly, the anti-PD-1 CAR T cells expanded in vivo, having two phases of expansion which is not typical of other CAR T cells, and persisted for over 100 days without signs of exhaustion. This approach was also successful at depleting PD-1-expressing cells in blood and tissues, including lymph nodes and the germinal centers— immune-privileged sites where T follicular helper cells reside and are an important zone for HIV persistence. “We actually generated two types of anti-PD-1 CAR T cells, ones with or without expressing CXCR5. We thought expression of CXCR5 would help target the CAR T cells to germinal centers,” and thus more effectively deplete target cells, Dr. Eichholz says. It turned out that it didn’t make a difference and CAR T cells lacking CXCR5 were just as effective at targeting PD-1+ cells in germinal centers.
While it was encouraging to see that the CAR T cells were effective at depleting T follicular helper cells, the real test was withdrawing the antiviral therapy and measuring whether HIV infection would rebound. Unfortunately, ART withdrawal led to an increase in the viral load detected in the blood plasma. Furthermore, this viral rebound was magnitudes higher than expected, likely due to the depletion of memory T cells. Although this was not the result the researchers hoped for, Eichholz emphasizes that this result is very telling about the biology of the source of residual HIV, stating “there are clearly other reservoirs the virus is coming from which opens more doors” to study these unknown viral sources.
This research was the first to demonstrate that anti-PD-1 CAR T cells are able to expand in vivo without signs of exhaustion and effectively kill PD-1+ T follicular helper cells in germinal centers, which serve as a major reservoir for persistent HIV infection. Currently, Dr. Eichholz is continuing to work with this anti-PD-1 CAR T cell system to make it more specific and controllable using drug-inducible on/off switches, or as Eichholz put it, it is sort of like making “remote control CARs.”
Unrelated to HIV infection, T follicular helper cells can also develop into a number of rare PD1-+ cancers, namely different subtypes of T cell lymphomas, for which curative therapies are not available. The present study suggests that the development of a safe anti-PD1 CAR T cell therapy may be a potentially curative approach to target these cancers and Dr. Eichholz is actively pursuing this direction currently as well.
This work was supported by Gilead Sciences HIV Cure Grant Program and the National Institutes of Health.
Fred Hutch/UW/Seattle Children’s Cancer Consortium members Drs. Larry Corey and Hans-Peter Kiem contributed to this work.
Eichholz K, Fukazawa Y, Peterson CW, Haeseleer F, Medina M, Hoffmeister S, Duell DM, Varco-Merth BD, Dross S, Park H, Labriola CS, Axthelm MK, Murnane RD, Smedley JV, Jin L, Gong J, Rust BJ, Fuller DH, Kiem HP, Picker LJ, Okoye AA, Corey L. Anti-PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4+ T cells in germinal centers. J Clin Invest. 2024.