CD8⁺ T cells retain protective functions despite sustained inhibitory receptor expression during Epstein-Barr virus infection in vivo

Epstein Barr virus (EBV) is one of the most ubiquitous human pathogens in the world, persistently infecting more than 90% of the adult human population. It drives some of the strongest human CD8⁺ T cell responses, which can be observed during symptomatic primary infection known as infectious mononucleosis (IM). Despite high viral loads and prolonged CD8⁺ T cell stimulation during IM, EBV enters latency and is under lifelong immune control in most individuals that experience this disease. We investigated whether changes in T cell function, as frequently characterized by PD-1 up-regulation, occur during IM due to the prolonged exposure to high antigen levels. We readily detected the expansion of PD-1 positive CD8⁺ T cells together with high frequencies of Tim-3, 2B4, and KLRG1 expression during IM and in mice with reconstituted human immune system components (huNSG mice) that had been infected with a high dose of EBV. These PD-1 positive CD8⁺ T cells, however, retained proliferation, cytokine production, and cytotoxic abilities. Multiple subsets of CD8⁺ T cells expanded during EBV infection, including PD-1⁺Tim-3⁺KLRG1⁺ cells that express CXCR5 and TCF-1 germinal center homing and memory markers, and may also contain BATF3. Moreover, blocking the PD-1 axis compromised EBV specific immune control and resulted in virus-associated lymphomagenesis. Finally, PD-1⁺, Tim-3⁺, and KLRG1⁺ CD8⁺ T cell expansion coincided with declining viral loads during low dose EBV infection. These findings suggest that EBV infection primes PD-1 positive CD8⁺ T cell populations that rely on this receptor axis for the efficient immune control of this ubiquitous human tumor virus.