There is continued interest in developing novel vaccine strategies that induce establish optimal CD8 + cytotoxic T lymphocyte (CTL) memory for pathogens like the influenza A viruses (IAVs), where the recall of IAV-specific T cell immunity is able to protect against serologically distinct IAV infection. While it is well established that CD4 + T cell help is required for optimal CTL responses and the establishment of memory, when and how CD4 + T cell help contributes to determining the ideal memory phenotype remains unclear. We assessed the quality of IAV-specific CD8 + T cell memory established in the presence or absence of a concurrent CD4 + T cell response. We demonstrate that CD4 + T cell help appears to be required at the initial priming phase of infection for the maintenance of IAV-specific CTL memory, with "unhelped" memory CTL exhibiting intrinsic dysfunction. High-throughput RNA-sequencing established that distinct transcriptional signatures characterize the helped vs. unhelped IAV-specific memory CTL phenotype, with the unhelped set showing a more "exhausted T cell" transcriptional profile. Moreover, we identify that unhelped memory CTLs exhibit defects in a variety of energetic pathways, leading to diminished spare respiratory capacity and diminished capacity to engage glycolysis upon reactivation. Hence, CD4 + T help at the time of initial priming promotes molecular pathways that limit exhaustion by channeling metabolic processes essential for the rapid recall of memory CD8 + T cells.I t is well accepted that the activation of CD4 + T helper cells is key for ensuring the maturation of protective humoral and cellular immunity following pathogen challenge. Even so, when it comes to generating effective cytotoxic T lymphocyte (CTL) responses in naïve individuals, the need or otherwise, for CD4 + T cell involvement is highly dependent on the nature of the immune challenge. For example, CD4 + T cell-independent primary CTL effectors can be readily induced in the context of robust acute viral (1-3) or bacterial infections that induce a strong inflammatory response (4). In contrast, the acute response to immunogens that induce low levels of inflammation looks to be more CD4 + T cell help-dependent (2, 5).Beyond the primary CTL response, the precise role CD4 + T help in the establishment of optimal CD8 + T cell memory after immunization or infection remains less clear. Initial work suggested that regardless of whether the primary CTL response was CD4 + T cell-dependent or -independent, CD4 + T help during the initial priming phase was necessary for the generation of memory T cells capable of responding to secondary challenge (2, 3). The proposed mechanism is that these helpers induced, at least in a subset of activated CTLs, molecular profiles that ensure optimal CD8 + T cell memory (6). Such programming likely reflects augmented signaling from cytokines, such as IL-2 (7), and the delivery of costimulatory signals that promote dendritic cell (DC) activation (5) to ensure that, at least for some CTL precursors (...

Single molecule (SM) super-resolution microscopies bypass the diffraction limit of conventional optical techniques and provide excellent spatial resolutions in the tens of nanometers without overly complex microscope hardware. SM imaging using optical astigmatism is an efficient strategy for visualizing subcellular features in 3D with a z-range of up to ∼1 µm per acquisition. This approach however, places high demands on fluorophore brightness and photoswitching resilience meaning that imaging entire cell volumes in 3D using SM super-resolution remains challenging. Here we employ SM astigmatism together with multiplane acquisition to visualize the whole nuclear lamina of COS-7 and T cells in 3D. Nuclear lamina provides structural support to the nuclear envelope and participates in vital nuclear functions including internuclear transport, chromatin organization and gene regulation. Its position at the periphery of the nucleus provides a visible reference of the nuclear boundary and can be used to quantify the spatial distribution of intranuclear components such as histone modifications and transcription factors. We found Alexa Fluor 647, a popular photoswitchable fluorophore, remained viable for over an hour of continuous high laser power exposure, and provided sufficient brightness detectable up to 8 µm deep into a cell, allowing us to capture the entire nuclear lamina in 3D. Our approach provides sufficient super-resolution detail of nuclear lamina morphology to enable quantification of overall nuclear dimensions and local membrane features.

The differentiation of naive CD8+ cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naive, effector and memory virus-specific CD8+ T cells. We observed that the architecture of the naive CD8+ T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naive chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naive CD8+ T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naive chromatin state.