Misty R. Jenkins

The strength of T cell receptor signal controls the polarization of cytotoxic machinery to the immunological synapse.

Summary Killing by cytotoxic T lymphocytes (CTLs) is mediated by the secretion of lytic granules. The centrosome plays a key role in granule delivery, polarizing to the central supramolecular activation complex (cSMAC) within the immunological synapse upon T cell receptor (TCR) activation. Although stronger TCR signals lead to increased target cell death than do weaker signals, it is not known how the strength of TCR signal controls polarization of the centrosome and lytic granules. By using TCR transgenic OT-I CTLs, we showed that both high- and low-avidity interactions led to centrosome polarization to the cSMAC. However, only high-avidity interactions, which induced a higher threshold of intracellular signaling, gave rise to granule recruitment to the polarized centrosome at the synapse. By controlling centrosome and granule polarization independently, the centrosome is able to respond rapidly to weak signals so that CTLs are poised and ready for the trigger for granule delivery.

Addition of a Prominent Epitope Affects Influenza A Virus-Specific CD8+ T Cell Immunodominance Hierarchies When Antigen Is Limiting

A reverse genetics strategy was used to insert the OVA peptide (amino acid sequence SIINFEKL; OVA257–264) into the neuraminidase stalk of both the A/PR8 (H1N1) and A/HK×31 (H3N2) influenza A viruses. Initial characterization determined that KbOVA257 is presented on targets infected with PR8-OVA and HK-OVA without significantly altering Db nucleoprotein (NP)366 presentation. There were similar levels of KbOVA257- and DbNP366-specific CTL expansion following both primary and secondary intranasal challenge. Interestingly, while variable, the presence of the immunodominant KbOVA257-specific response resulted in diminished Db acidic polymerase224- and Kb basic polymerase subunit 1703-, but not DbNP366-specific responses and didn’t alter endogenous influenza A virus-specific immunodominance hierarchies. However, challenging PR8-OVA-primed mice with HK-OVA via the i.p. route, and thereby limiting Ag dose, led to a reduction in the magnitude of all the influenza A virus-specific responses measured. A similar reduction in CTL response to native epitopes was also seen following primary respiratory HK-OVA infection of mice that received substantial numbers of KbOVA257-specific TCR transgenic T cells. Thus, during the course of infection, the generation of individual virus-specific CTL responses is independently regulated. However, in cases in which Ag is limiting, or high precursor frequency, the presence of immunodominant CTL responses can impact on the magnitude of other specific populations. Therefore, depending on both the size of the T cell precursor pool and the mode of Ag presentation, the addition of a major epitope can diminish the size of endogenous, influenza-specific CD8+ T cell responses, although never to the point that these are totally compromised.

Perforin forms transient pores on the target cell plasma membrane to facilitate rapid access of granzymes during killer cell attack

Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of proapoptotic granzymes. However, the mechanism of granzyme entry into cells remains unresolved. Using biochemical approaches combined with time-lapse microscopy of human primary cytotoxic lymphocytes engaging their respective targets, we defined the time course of perforin pore formation in the context of the physiological immune synapse. We show that, on recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilization in as little as 30 seconds. Within the synaptic cleft, target cell permeabilization by perforin resulted in the rapid diffusion of extracellular milieu-derived granzymes. Repair of these pores was initiated within 20 seconds and was completed within 80 seconds, thus limiting granzyme diffusion. Remarkably, even such a short time frame was sufficient for the delivery of lethal amounts of granzymes into the target cell. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2 minutes of perforin permeabilization. This study defines the final sequence of events controlling cytotoxic lymphocyte immune defense, in which perforin pores assemble on the target cell plasma membrane, ensuring efficient delivery of lethal granzymes.

Heterogeneity of Effector Phenotype for Acute Phase and Memory Influenza A Virus-Specific CTL

Ag-specific, CD8+ CTLs clear influenza A viruses from the lung via granzyme (Gzm) and perforin-dependent mechanisms. Ex vivo analysis of perforin-Gzm mRNA profiles demonstrated substantial heterogeneity in patterns of effector mRNA transcription of CD8+ DbNP366- or DbPA224-specific CTL. The only difference between the two epitope-specific sets was apparent very early after infection with similar molecular profiles seen in peak primary and secondary responses and in long-term memory. Surprisingly, memory T cells also expressed a diverse pattern of effector mRNA profile with an emphasis on GzmB and, surprisingly, GzmK. This analysis thus defines how naive, effector, and memory T cells differ in cytotoxic potential and provides novel insight into the molecular signatures of effector molecules observed at various stages after infection.

Centrosome docking at the immunological synapse is controlled by Lck signaling

The T cell receptor–activated tyrosine kinase Lck controls docking of the centrosome at the plasma membrane within the immunological synapse but not polarization of the centrosome around the nucleus.

Failed CTL/NK cell killing and cytokine hypersecretion are directly linked through prolonged synapse time

Jenkins et al. discover that failure of perforin and granzyme cytotoxicity by human and mouse CTLs/NK cells prolongs the immunological synapse, leading to repetitive calcium signaling and hypersecretion of inflammatory mediators that subsequently activate macrophages. Disengagement from target cells is dependent on apoptotic caspase signaling. The findings may provide mechanistic understanding for immunopathology in familial hemophagocytic lymphohistiocytosis.

The synapse and cytolytic machinery of cytotoxic T cells

Cytotoxic T lymphocytes (CTLs) rapidly kill target cells via the release of lytic granules into the immunological synapse, a process directed by the docking of the centrosome at the plasma membrane. New evidence highlights how signal strength and avidity influence the recruitment of cytolytic machinery to the synapse, and the role of each synaptic compartment. Release of cytolytic effector proteins, including perforin and FasL, is controlled at multiple levels and is also influenced by the avidity of the interaction. New imaging technologies and the use of photoactivatable peptides have allowed the dissection of signalling molecules involved in each step of the cytolytic process. This review highlights the important role of avidity in controlling how a T cell kills its target.

Location rather than CD62L phenotype is critical in the early establishment of influenza-specific CD8+ T cell memory

The rapid recall of influenza virus-specific CD8+ T cell effector function is protective, although our understanding of T cell memory remains incomplete. Recent debate has focused particularly on the CD62L lymph node homing receptor. The present analysis shows that although functional memory can be established from both CD62Lhi and CD62Llo CD8+ T cell subsets soon after initial encounter between naïve precursors and antigen, the optimal precursors are CD8+CD44hiCD25lo immune lymphocytes isolated from draining lymph nodes on day 3.5 after influenza virus infection. Analysis of primed T cells at different times after challenge indicates that the capacity to transfer memory is diminished at the peak of the primary cytotoxic T lymphocyte response, challenging speculations that the transition to memory first requires full differentiation to effector status. It seems that location rather than CD62Lhi/lo phenotype may be the more profitable focus for further dissection of the early establishment of T cell memory.

Cell cycle-related acquisition of cytotoxic mediators defines the progressive differentiation to effector status for virus-specific CD8+ T cells.

Although analysis of virus-specific CTL function at the peak of infection suggests that granzyme (grz) and perforin (pfp) gene expression is not coregulated, early differentiation events leading to acquisition of function are poorly understood. Using a combination of CFSE dilutions and single-cell RT-PCR, effector gene expression was determined early after CTL activation. There were low levels of pfp and grz expression at division 3, with increased expression by divisions 6–8. The increase in effector mRNA expression with division correlated with increasing ex vivo cytotoxicity. Of the mRNA transcripts detected at division 3, there was an increased frequency of grzB and grzK (compared with grzA or pfp), and this pattern was also observed at later divisions. The prevalence of OT-I CTL expressing grz/pfp mRNA was equivalent for the divided CD62Lhigh and CD62Llow sets, but the concentrations of grzB protein, levels of CTL activity, and the absolute amounts of grzB transcript were substantially greater for the CD62Llow population. Thus, while effector gene expression can be acquired early, maturation of cytotoxic capacity requires extended differentiation.

Rapid and Unidirectional Perforin Pore Delivery at the Cytotoxic Immune Synapse

The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector–target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65–100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8+ T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.