In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity

Abstract

SummaryAccording to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2–16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8+ T cell immunity.

Cortical actin recovery at the immunological synapse leads to termination of lytic granule secretion in cytotoxic T lymphocytes

“…To evaluate cortical actin dynamics in the context of CTL cytolytic activity, we first used live-cell spinning-disk confocal microscopy to follow actin during the entire course of CTL interactions with their targets. CTLs were derived from OT-I T-cell receptor (TCR) transgenic mice that recognize a peptide from ovalbumin (OVA) and were used in conjunction with peptide-pulsed targets expressing the Ca 2+ indicator GCaMP6m, whose intensity directly correlates with intracellular calcium ion concentrations (1,(13)(14)(15). Previous work has shown that calcium increases are associated with very early loss of membrane integrity, as occurs in target cells after CTLs secrete their lytic granules containing perforin, which leads to the formation of pores in the target cell membrane (15)(16)(17)(18)(19).…”

“…CTLs carry out their cytolytic functions through the directed secretion of lytic granules, specialized lysosomes that contain perforin and granzymes that induce death of target cells. Because a single CTL is capable of killing multiple target cells during an immune response (1,2), CTLs are thought to act as "serial killers" (3). As such, CTLs need to regulate secretion tightly, both to kill only appropriate target cells on contact and to facilitate their ability to carry out serial responses.…”

Memory CD8 + T Cells: Innate-Like Sensors and Orchestrators of Protection

“…Lack of IFNγ receptor signaling in these subsets of myeloid cells or lack of memory T cell-derived IFNγ impairs protective recall response in the Lm model of vaccination and challenge infection. Perhaps in support of this concept, and contrasting to the established view [72], a recent study suggested that the rate of effector CD8 + T cell-killing during acute viral infection by intravital time-lapse biphoton microscopy, was only 2–16 target cells per day [73], a finding that suggest that direct killing in acute infections may not be the major mechanism of host protection. Collectively, this body of work highlights novel evidence for the importance of non-cytolytic, memory CD8 + T cell-dependent mechanisms of protection in vaccinated hosts.…”

Pharmacologic HIV-1 Nef blockade promotes CD8 T cell–mediated elimination of latently HIV-1–infected cells in vitro

“…Furthermore, Nef expression within clonally expanded CD4 T cells would promote immune evasion, alluding to how such HIV-1 genomes could persist indefinitely. CD8 CTL are extremely sensitive to MHC-I presentation in vivo, and it has been demonstrated that virus-mediated MHC-I downregulation dramatically diminishes the ability of CD8 T cell clones to interact with and thus eliminate infected cells, even at high antigenic concentrations as demonstrated by Halle et al (54). In the setting of SIV, rhesus macaques infected with a Nef strain incapable of downregulating MHC-I, but not other molecules, reverts to WT early during acute infection in vivo, implicating SIV in imparting strong selective pressure for this feature of immune evasion (55).…”