Tissue-resident memory T (T_RM) cells serve as vanguards of antimicrobial host defense in nonlymphoid tissues, particularly at barrier epithelia and in organs with nonrenewable cell types (e.g., brain). In this study, we asked whether an augmented ability to sense Ag complemented their role as early alarms of pathogen invasion. Using mouse polyomavirus, we show that brain-resident mouse polyomavirus–specific CD8 T cells, unlike memory cells in the spleen, progressively increase binding to MHC class I tetramers and CD8 coreceptor expression. Using the two-dimensional micropipette adhesion-frequency assay, we show that T_RM cells in brain, as well as in kidney, express TCRs with up to 20-fold higher affinity than do splenic memory T cells, whereas effector cells express TCRs of similar high affinity in all organs. Together, these data demonstrate that T_RM cells retain high TCR affinity, which endows them with the high Ag sensitivity needed for front-line defense against infectious agents.

Infection of the central nervous system (CNS) by murine polyomavirus (MuPyV), a persistent natural mouse pathogen, establishes brain‐resident memory CD8 T cells (bT_RM) that uniformly and chronically express programmed cell death protein 1 (PD‐1) irrespective of the expression of α_E integrin CD103, a T_RM cell marker. In contrast, memory antiviral CD8 T cells in the spleen are PD‐1⁻, despite viral loads being similar in both the brain and spleen during persistent infection. Repetitive antigen engagement is central to sustained PD‐1 expression by T cells in chronic viral infections; however, recent evidence indicates that expression of inhibitory receptors, including PD‐1, is part of the T_RM differentiation program. Here we asked whether PD‐1 expression by CD8 bT_RM cells during persistent MuPyV encephalitis is antigen dependent. By transferring MuPyV‐specific CD8 bT_RM cells into the brains of naive mice and mice infected with cognate epitope‐sufficient and ‐deficient MuPyVs, we demonstrate that antigen and inflammation are dispensable for PD‐1 maintenance. In vitro and direct ex vivo analyses indicate that CD103⁻ MuPyV‐specific CD8 bT_RM retain functional competence. We further show that the Pdcd‐1 promoter of anti‐MuPyV bT_RM cells is epigenetically fixed in a demethylated state in the brain. In contrast, the PD‐1 promoter of splenic antiviral memory CD8 T cells undergoes remethylation after being demethylated during acute infection. These data show that PD‐1 expression is an intrinsic property of brain T_RM cells in a persistent CNS viral infection.

High-affinity CD4 T cells produce IL-21 to help CD8 T cells differentiate into tissue-resident memory cells in the brain.

Programmed cell death-1 (PD-1) receptor signaling dampens the functionality of T cells faced with repetitive antigenic stimulation from chronic infections or tumors. Using intracerebral (i.c.) infection of mice with mouse polyomavirus (MuPyV), we have shown that CD8 T cells establish a PD-1hi, tissue-resident memory population in the brains (bTRM) of mice with a low-level persistent infection. In MuPyV encephalitis, PD-L1 was expessed on infiltrating myeloid cells, microglia and astrocytes, but not on oligodendrocytes. Engagement of PD-1 on anti-MuPyV CD8 T cells limits their effector activity. NanoString gene expression analysis showed that neuroinflammation is higher in PD-L1-/- than wild type mice at day 8 post-infection, the peak of the MuPyV-specific CD8 response. During the persistent phase of infection, however, the absence of PD-1 signaling was found to be associated with a lower inflammatory response than in wild type mice. Genetic disruption and intracerebroventricular blockade of PD-1 signaling resulted in an increase in number of MuPyV-specific CD8 bTRM and the fraction expressing CD103, the αE integrin commonly used to define tissue-resident T cells. However, PD-L1-/- mice persistently infected with MuPyV showed impaired virus control upon i.c. re-infection with MuPyV. Collectively, these data reveal a temporal duality in PD-1-mediated regulation of MuPyV-associated neuroinflammation. PD-1 signaling limited the severity of neuroinflammation during acute infection but sustained a level of inflammation during persistent infection for maintaining control of virus re-infection.

Tissue-resident memory CD8 T (T_RM) cells defend against microbial reinfections at mucosal barriers; determinants driving durable T_RM cell responses in non-mucosal tissues, which often harbor opportunistic persistent pathogens, are unknown. JC polyomavirus (JCPyV) is a ubiquitous constituent of the human virome. With altered immunological status, JCPyV can cause the oft-fatal brain demyelinating disease progressive multifocal leukoencephalopathy (PML). JCPyV is a human-only pathogen. Using the mouse polyomavirus (MuPyV) encephalitis model, we demonstrate that CD4 T cells regulate development of functional antiviral brain-resident CD8 T cells (bT_RM) and renders their maintenance refractory to systemic CD8 T cell depletion. Acquired CD4 T cell deficiency, modeled by delaying systemic CD4 T cell depletion until MuPyV-specific CD8 T cells have infiltrated the brain, impacted the stability of CD8 bT_RM, impaired their effector response to reinfection, and rendered their maintenance dependent on circulating CD8 T cells. This dependence of CD8 bT_RM differentiation on CD4 T cells was found to extend to encephalitis caused by vesicular stomatitis virus. Together, these findings reveal an intimate association between CD4 T cells and homeostasis of functional bT_RM to CNS viral infection. Tissue resident memory cells (T_RM) persist in nonlymphoid organs serving as frontline defense against microbial reinfection. The requirements for generating pathogen-specific T_RM to acutely resolved infections is well documented; however, little is known about the development of T_RM to persistent infections. In this study, we investigated the importance of CD4 T cell availability to CD8 T_RM development during persistent viral encephalitis. Using mouse polyomavirus (MuPyV) brain infection and systemic CD4 T cell insufficiency, we found that loss of CD4 T cells abrogated brain T_RM (bT_RM) development, disrupted the metabolic homeostasis of CD8 T cells, and reduced CD8 T cell responses upon viral reinfection. Additionally, CD8 T cells in CD4 T cell-deficient mice required resupply from circulating CD8 T cells, directly contrasting the independence from circulation in canonical T_RM. Upon delayed CD4 T cell depletion and brain infection with an acutely resolving viral infection, CD8 T cells also had aberrant bT_RM development and required resupply from the vasculature. Our findings demonstrate that CD4 T cells are essential for establishing long-lasting, virus-specific CD8 bT_RM for antiviral CNS immunosurveillance. Our data also raise important clinical implications for developing therapies to augment CD4 T cell help to bolster protective CD8 bT_RM responses.

Establishing functional tissue-resident memory (T_RM) cells at sites of infection is a newfound objective of T cell vaccine design. To directly assess the impact of antigen stimulation strength on memory CD8 T cell formation and function during a persistent viral infection, we created a library of mouse polyomavirus (MuPyV) variants with substitutions in a subdominant CD8 T cell epitope that exhibit a broad range of efficiency in stimulating TCR transgenic CD8 T cells. By altering a subdominant epitope in a nonstructural viral protein and monitoring memory differentiation of donor monoclonal CD8 T cells in immunocompetent mice, we circumvented potentially confounding changes in viral infection levels, virus-associated inflammation, size of the immunodominant virus-specific CD8 T cell response, and shifts in TCR affinity that may accompany temporal recruitment of endogenous polyclonal cells. Using this strategy, we found that antigen stimulation strength was inversely associated with the function of memory CD8 T cells during a persistent viral infection. We further show that CD8 T_RM cells recruited to the brain following systemic infection with viruses expressing epitopes with suboptimal stimulation strength respond more efficiently to challenge CNS infection with virus expressing cognate antigen. These data demonstrate that the strength of antigenic stimulation during recruitment of CD8 T cells influences the functional integrity of T_RM cells in a persistent viral infection. Tissue-resident memory (T_RM) cells are a subset of memory T cells that primarily reside in non-lymphoid tissues and serve as sentinels and effectors against secondary infections. T_RM cells have been extensively characterized in mucosal barriers, but much less is known about this population in non-barrier sites such as the brain. In this study, we designed a novel strategy to evaluate the impact of T cell stimulation strength on the generation and functionality of memory CD8 T cells in both lymphoid and nonlymphoid tissues. Using a mouse polyomavirus (MuPyV) library expressing variants of a subdominant epitope recognized by TCR transgenic CD8 T cells, we found that systemic infection producing weaker responses during T cell priming was sufficient for recruitment of effector cells to the brain. Furthermore, lower stimulation conferred greater functionality to memory T cells in the spleen and to brain T_RM cells. Our findings demonstrate that the strength of antigenic stimulation experienced by a naïve T cell early in infection is a determinant of memory functional integrity during viral persistence in a non-barrier organ.