Abstract In chronic infection and cancer, a subset of self-renewing, antigen-specific CD8+ T cells, termed progenitors, is crucial for long-term immunity and immunotherapy effectiveness. The mechanisms governing their stemness, however, are not fully understood. Our single-cell RNA + ATAC sequencing analysis revealed that SATB1 (special AT-rich sequence-binding protein) is exclusively expressed in the progenitor CD8+ T cells during chronic infection. Known for its key role in chromatin organization during T-cell development, SATB1’s unique expression in the progenitor CD8+ T cell led us to hypothesize that it plays a pivotal role in regulating the stemness of the antigen-specific CD8+ T cells. Using the lymphocytic choriomeningitis virus (LCMV) Clone 13 mouse model of chronic infection, the SATB1 gene was deleted in CD8+ T cells via CRISPR/ RNP technology. At day 21 post-infection, the SATB1 deletion resulted in a reduction of virus-specific progenitor CD8+ T cells and a decrease of TCF1 expression. The transcriptomic and epigenomic studies further revealed that SATB1 plays a key role in regulating the chromatin accessibility and transcriptional activities of stemness-associated genes, such as Tcf7, Bach2, and Myb. Overall, our results underscore the critical role of SATB1 in maintaining the transcriptional integrity of progenitor CD8+ T cells in response to antigen activation, which sheds light on the genetic pathways that regulate the stemness of antigen-specific CD8+ T cells.

The transcription cofactor TLE3 interacts with RUNX3 and TCF1 to repress the transcription and chromatin accessibility of CD8+ TCM cell signature genes, while simultaneously acting as a coactivator for TBET to facilitate the expression of CD8+ TEM cell signature genes. As such, TLE3 serves as a gatekeeper of CD8+ TCM cell formation.

Current HIV vaccines designed to stimulate CD8+ T cells have failed to induce immunologic control upon infection. The functions of vaccine-induced HIV-specific CD8+ T cells were investigated here in detail. Cytotoxic capacity was significantly lower than in HIV controllers and was not a consequence of low frequency or unaccumulated functional cytotoxic proteins. Low cytotoxic capacity was attributable to impaired degranulation in response to the low antigen levels present on HIV-infected targets. The vaccine-induced T cell receptor (TCR) repertoire was polyclonal and transduction of these TCRs conferred the same reduced functions. These results define a mechanism accounting for poor antiviral activity induced by these vaccines and suggest that an effective CD8+ T cell response may require a vaccination strategy that drives further TCR clonal selection.

CD4 and CD8 T cells are key players in the immune response against both pathogenic infections and cancer. CD4 T cells provide help to CD8 T cells via multiple mechanisms, including licensing dendritic cells (DCs), co-stimulation, and cytokine production. During acute infection and vaccination, CD4 T cell help is important for the development of CD8 T cell memory. However, during chronic viral infection and cancer, CD4 helper T cells are critical for the sustained effector CD8 T cell response, through a variety of mechanisms. In this review, we focus on T cell responses in conditions of chronic Ag stimulation, such as chronic viral infection and cancer. In particular, we address the significant role of CD4 T cell help in promoting effector CD8 T cell responses, emerging techniques that can be utilized to further our understanding of how these interactions may take place in the context of tertiary lymphoid structures, and how this key information can be harnessed for therapeutic utility against cancer.

CD4 T cell help is critical to sustain effector CD8 T cell responses during chronic infection, notably via T follic-ular helper (Tfh)-derived interleukin-21 (IL-21). Conversely, CD4 depletion results in severe CD8 T cell dysfunction and lifelong viremia despite CD4 T cell reemergence following transient depletion. These obser-vations suggest that repopulating CD4 subsets are functionally or numerically insufficient to orchestrate a robust CD8 response. We utilize spatial transcriptomics and single-cell RNA sequencing (scRNA-seq) to investigate CD4 T cell heterogeneity under CD4-replete and-deplete conditions and explore cellular interac-tions during chronic infection. Although IL-21-producing Tfh cells repopulate following transient CD4 deple-tion, they are outnumbered by immunomodulatory CD4 T cells. Moreover, the splenic architecture appears perturbed, with decreases in white pulp regions, coinciding with germinal center losses. These disruptions in splenic architecture are associated with diminished Tfh and progenitor CD8 T cell colocalization, providing a potential mechanism for impaired progenitor-to-effector CD8 T cell differentiation during "un-helped"con-ditions.

Although the sialyltransferases ST3GAL1 and ST3GAL2 are known to transfer sialic acid to the galactose residue of type III disaccharides (Gal81,3GalNAc) in vitro, sialylation of O-linked glycosylated proteins in living cells has been largely attributed to ST3GAL1. To examine the role of ST3GAL2 in O-sialylation, we examined its expression during differentiation of human-induced pluripotent stem cells (iPSCs) into hematopoietic progenitor cells (HPCs) and megakaryocytes (MKs). ST3GAL1 and ST3GAL2 each became highly expressed during the differentiation of iPSCs to HPCs but decreased markedly in their expression upon differentiation into MKs, suggesting coordination of expression during megakaryopoiesis. To further delineate their role in these processes, we generated ST3GAL1-, ST3GAL2-, and doubly deficient human iPSC lines. Binding of the peanut agglutinin lectin, which reports the presence of unsialylated Gal81,3GalNAc glycan chains, was strongly increased in HPCs and MKs derived from double-knockout iPSCs and remained moderately increased in cells lacking either one of these sialyltransferases, demonstrating that both can serve as functional cellular O-glycan sialyltransferases. Interestingly, the HPC markers CD34 and CD43, as well as MK membrane glycoprotein (GP) GPIba, were identified as major GP substrates for ST3GAL1 and ST3GAL2. In contrast, O-sialylation of GPIIb relied predominantly on the expression of ST3GAL2. Finally, although disruption of ST3GAL1 and ST3GAL2 had little impact on MK production, their absence resulted in dramatically impaired MK proplatelet formation. Taken together, these data establish heretofore unknown physiological roles for ST3GAL1 and ST3GAL2 in O-linked glycan sialylation in hemato- and megakaryocytopoiesis.

BACKGROUNDHIV-infected patients with poor virologic control and multidrug-resistant virus have limited therapeutic options. The current study was undertaken to evaluate the safety, immunologic effects, and antiviral activity of peripheral lymphocytes transferred from an elite controller, whose immune system is able to control viral replication without antiretroviral medications, to an HLA-B*2705-matched progressor.METHODSApproximately 22 billion cells were collected from an elite controller by lymphapheresis and infused within 6 hours into a recipient with a preinfusion CD4+ T cell count of 10 cells/μL (1%) and HIV plasma viral load of 114,993 copies/mL.RESULTSDonor cells were cleared from the recipient's peripheral blood by day 8. A transient decrease in viral load to 58,421 (day 3) was followed by a rebound to 702,972 (day 6) before returning to baseline values by day 8. The decreased viral load was temporally associated with peak levels of donor T cells, including CD8+ T cells that had high levels of expression of Ki67, perforin, and granzyme B. Notably, recipient CD8+ T cells also showed increased expression of these markers, especially in HIV-specific tetramer-positive cells.CONCLUSIONThese results suggest that the adoptive transfer of lymphocytes from an HIV-infected elite controller to an HIV-infected patient with progressive disease may be able to perturb the immune system of the recipient in both positive and negative ways.TRIAL REGISTRATIONClinicalTrials.gov NCT00559416.FUNDINGIntramural Research Programs of the US NIH Clinical Center and the National Institute of Allergy and Infectious Diseases (NIAID); the National Cancer Institute.

The speed at which a molecular motor operates is critically important for the survival of a virus or an organism but very little is known about the underlying mechanisms. Tailed bacteriophage T4 employs one of the fastest and most powerful packaging motors, a pentamer of gp17 that translocates DNA at a rate of up to ∼2000-bp/s. We hypothesize, guided by structural and genetic analyses, that a unique hydrophobic environment in the catalytic space of gp17-adenosine triphosphatase (ATPase) determines the rate at which the 'lytic water' molecule is activated and OH- nucleophile is generated, in turn determining the speed of the motor. We tested this hypothesis by identifying two hydrophobic amino acids, M195 and F259, in the catalytic space of gp17-ATPase that are in a position to modulate motor speed. Combinatorial mutagenesis demonstrated that hydrophobic substitutions were tolerated but polar or charged substitutions resulted in null or cold-sensitive/small-plaque phenotypes. Quantitative biochemical and single-molecule analyses showed that the mutant motors exhibited 1.8- to 2.5-fold lower rate of ATP hydrolysis, 2.5- to 4.5-fold lower DNA packaging velocity, and required an activator protein, gp16 for rapid firing of ATPases. These studies uncover a speed control mechanism that might allow selection of motors with optimal performance for organisms' survival.