Quiescence of Memory CD8(+) T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4.

Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection yet exist in a functionally quiescent state. The paradigm is that memory T cells remain inactive due to lack of T cell receptor (TCR) stimuli. Here, we report that regulatory T (Treg) cells orchestrate memory T cell quiescence by suppressing effector and proliferation programs through inhibitory receptor, cytotoxic-T-lymphocyte-associated protein-4 (CTLA-4). Loss of Treg cells resulted in activation of genome-wide transcriptional programs characteristic of effector T cells and drove transitioning as well as established memory CD8(+) T cells toward terminally differentiated KLRG-1(hi)IL-7Rα(lo)GzmB(hi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality, and protective efficacy. CTLA-4 functionally replaced Treg cells in trans to rescue memory T cell defects and restore homeostasis. These studies present the CTLA-4-CD28-CD80/CD86 axis as a potential target to accelerate vaccine-induced immunity and improve T cell memory quality in current cancer immunotherapies proposing transient Treg cell ablation.

Cutting Edge: Effect of Disease-Modifying Therapies on SARS-CoV-2 Vaccine-Induced Immune Responses in Multiple Sclerosis Patients.

Multiple sclerosis (MS) is a demyelinating inflammatory disease of the CNS treated by diverse disease-modifying therapies that suppress the immune system. Severe acute respiratory syndrome coronavirus 2 mRNA vaccines have been very effective in immunocompetent individuals, but whether MS patients treated with modifying therapies are afforded the same protection is not known. This study determined that dimethyl fumarate caused a momentary reduction in anti-Spike (S)-specific Abs and CD8 T cell response. MS patients treated with B cell-depleting (anti-CD20) or sphingosine 1-phosphate receptor agonist (fingolimod) therapies lack significant S-specific Ab response. Whereas S-specific CD4 and CD8 T cell responses were largely compromised by fingolimod treatment, T cell responses were robustly generated in anti-CD20-treated MS patients, but with a reduced proportion of CD4+CXCR5+ circulating follicular Th cells. These data provide novel information regarding vaccine immune response in patients with autoimmunity useful to help improve vaccine effectiveness in these populations.

Programming of CD8 T Cell Quantity and Polyfunctionality by Direct IL-1 Signals.

IL-1, generally considered an amplifier of adaptive immune responses, has been proposed for use as adjuvant during immunization with weak immunogens. However, its effects on memory T cell function remain largely undefined. Using the murine model of acute viral infection, in this paper, we show that in addition to augmenting the size of the Ag-specific pool, IL-1 signals act directly on CD8 T cells to promote the quality of effector and memory responses. Ablation of IL-1R1 or MyD88 signaling in T cells led to functional impairment; both the ability to produce multiple cytokines on a per cell basis (polyfunctionality) and the potential for recall proliferation in response to antigenic restimulation were compromised. IL-1 supplementation during priming augmented the expansion of Ag-specific CD8 T cells through the MyD88-IRAK1/4 axis, resulting in a larger memory pool capable of robust secondary expansion in response to rechallange. Together, these findings demonstrate a critical role of the IL-1-MyD88 axis in programming the quantity and quality of memory CD8 T cell responses and support the notion that IL-1 supplementation may be exploited to enhance adoptive T cell therapies against cancers and chronic infections.

MicroRNA-17~92 regulates effector and memory CD8 T-cell fates by modulating proliferation in response to infections.

The precise microRNAs and their target cellular processes involved in generation of durable T-cell immunity remain undefined. Here we show a dynamic regulation of microRNAs as CD8 T cells differentiate from naïve to effector and memory states, with short-lived effectors transiently expressing higher levels of oncogenic miR-17-92 compared with the relatively less proliferating memory-fated effectors. Conditional CD8 T-cell-intrinsic gain or loss of expression of miR-17-92 in mature cells after activation resulted in striking reciprocal effects compared with wild-type counterparts in the same infection milieu-miR-17-92 deletion resulted in lesser proliferation of antigen-specific cells during primary expansion while favoring enhanced IL-7Rα and Bcl-2 expression and multicytokine polyfunctionality; in contrast, constitutive expression of miR-17-92 promoted terminal effector differentiation, with decreased formation of polyfunctional lymphoid memory cells. Increased proliferation upon miR-17-92 overexpression correlated with decreased expression of tumor suppressor PTEN and increased PI3K-AKT-mTOR signaling. Thus, these studies identify miR17-92 as a critical regulator of CD8 T-cell expansion and effector and memory lineages in the physiological context of acute infection, and present miR-17-92 as a potential target for modulating immunologic outcome after vaccination or immunotherapeutic treatments of cancer, chronic infections, or autoimmune disorders.

Vitamin D receptor signals regulate effector and memory CD8 T cell responses to infections in mice.

BACKGROUND: Vitamin D insufficiency is associated with broad-ranging human disease sequelae such as bone disease, cancer, cardiovascular disease, allergy, autoimmune disorders, diabetes, and infectious diseases. Disease risk and severity of a large proportion of the nonskeletal disorders heavily involve the cytotoxic cluster of differentiation (CD) 8 T lymphocyte (CTL) arm of cellular adaptive immunity. Considering the importance of vitamin D in CTL-dependent diseases, there is a critical need for systematic in-depth explorations into the role of vitamin D deficiency in generation and maintenance of CTL immunity during infections and vaccinations. OBJECTIVE: With the use of wild-type (WT) vitamin D-sufficient mice and the vitamin D receptor knockout (Vdr(-/-)) mouse model of in vivo deficiency of vitamin D signaling, we systematically analyzed the impact of vitamin D deficiency on antigen-specific effector and memory CD8 T cell responses to acute viral and bacterial infections. METHODS: WT and Vdr(-/-) mice were infected with lymphocytic choriomeningitis virus, a natural mouse pathogen, and antigen-specific CTL responses were analyzed during priming, expansion, contraction, and memory phases. Magnitude, breadth, cytokine production, and localization of antiviral effector and memory CTLs to lymphoid and nonlymphoid tissues were specifically assessed. RESULTS: The absence of vitamin D signals led to 1) aberrant CD8 T cell effector differentiation (∼2-fold lower granzyme B and reduced B cell lymphoma 2; P ≤ 0.05) and enhanced contraction (∼15% increase; P ≤ 0.05) in antigen-specific CTLs; 2) a significantly restricted (P ≤ 0.05) breadth of the antigen-specific CD8 T cell effector and memory repertoire; and 3) preferential localization of effector (∼2.5-fold increase; P ≤ 0.01) and memory (∼5-fold increase; P ≤ 0.001) CD8 T cells to the lymph nodes compared to nonlymphoid tissues. CONCLUSION: Our data show a previously unrecognized impact of vitamin D deficiency on the quantity, quality, breadth, and location of CD8 T cell immunity to acute viral and bacterial infections.

Diminished responses to mRNA-based SARS-CoV-2 vaccines in individuals with rheumatoid arthritis on immune-modifying therapies.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder that causes debilitating swelling and destruction of the joints. People with RA are treated with drugs that actively suppress one or more parts of their immune system, and these may alter the response to vaccination against SARS-CoV-2. In this study, we analyzed blood samples from a cohort of patients with RA after receiving a 2-dose mRNA COVID-19 vaccine regimen. Our data show that individuals on the cytotoxic T lymphocyte antigen 4-Ig therapy abatacept had reduced levels of SARS-CoV-2-neutralizing antibodies after vaccination. At the cellular level, these patients showed reduced activation and class switching of SARS-CoV-2-specific B cells, as well as reduced numbers and impaired helper cytokine production by SARS-CoV-2-specific CD4+ T cells. Individuals on methotrexate showed similar but less severe defects in vaccine response, whereas individuals on the B cell-depleting therapy rituximab had a near-total loss of antibody production after vaccination. These data define a specific cellular phenotype associated with impaired response to SARS-CoV-2 vaccination in patients with RA on different immune-modifying therapies and help inform efforts to improve vaccination strategies in this vulnerable population.

Autocrine and paracrine IL-2 signals collaborate to regulate distinct phases of CD8 T cell memory.

Differential interleukin-2 (IL-2) signaling and production are associated with disparate effector and memory fates. Whether the IL-2 signals perceived by CD8 T cells come from autocrine or paracrine sources, the timing of IL-2 signaling and their differential impact on CD8 T cell responses remain unclear. Using distinct models of germline and conditional IL-2 ablation in post-thymic CD8 T cells, this study shows that paracrine IL-2 is sufficient to drive optimal primary expansion, effector and memory differentiation, and metabolic function. In contrast, autocrine IL-2 is uniquely required during primary expansion to program robust secondary expansion potential in memory-fated cells. This study further shows that IL-2 production by antigen-specific CD8 T cells is largely independent of CD4 licensing of dendritic cells (DCs) in inflammatory infections with robust DC activation. These findings bear implications for immunizations and adoptive T cell immunotherapies, where effector and memory functions may be commandeered through IL-2 programming.

Metabolic regulation by PD-1 signaling promotes long-lived quiescent CD8 T cell memory in mice.

Inhibitory signaling in dysfunctional CD8 T cells through the programmed cell death 1 (PD-1) axis is well established in chronic viral infections and cancers. PD-1 is also transiently induced to high concentrations during priming of acute infections and immunizations, yet its impact on the development of long-lived antigen-independent T cell memory remains unclear. In addition to its expected role in restraining clonal effector expansion, here, we show that PD-1 expression on antigen-specific CD8 T cells is required for the development of a durable CD8 T cell memory pool after antigen clearance. Loss of T cell­specific PD-1 signaling led to increased contraction and a defect in antigen-independent renewal of memory CD8 T cells in response to homeostatic cytokine signals, thus resulting in attrition of the memory pool over time. Whereas exhausted CD8 T cells regain function after PD-1 checkpoint blockade during chronic viral infection, the preexisting pool of resting functional bystander memory CD8 T cells established in response to a previously administered immunogen decreased. Metabolically, PD-1 signals were necessary for regulating the critical balance of mTOR-dependent anabolic glycolysis and fatty acid oxidation programs to meet the bioenergetic needs of quiescent CD8 T cell memory. These results define PD-1 as a key metabolic regulator of protective T cell immunity. Furthermore, these results have potential clinical implications for preexisting CD8 T cell memory during PD-1 checkpoint blockade therapy.

Development of a novel Francisella tularensis Live Vaccine Strain expressing ovalbumin provides insight into antigen-specific CD8+ T cell responses.

Progress towards a safe and effective vaccine for the prevention of tularemia has been hindered by a lack of knowledge regarding the correlates of protective adaptive immunity and a lack of tools to generate this knowledge. CD8+ T cells are essential for protective immunity against virulent strains of Francisella tularensis, but to-date, it has not been possible to study these cells in an antigen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) in F. tularensis, which allows for the study of CD8+ T cell responses to the bacterium. We demonstrate that in response to intranasal infection with the F. tularensis Live Vaccine Strain, adoptively transferred OVA-specific CD8+ T cells expand after the first week and produce IFN-γ but not IL-17. Effector and central memory subsets develop with disparate kinetics in the lungs, draining lymph node and spleen. Notably, OVA-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more antigen-specific CD8+ T cells in the lung-draining lymph node compared to scarification vaccination, but that an intranasal booster overcomes this difference. Together, our data show that this novel tool can be used to study multiple aspects of the CD8+ T cell response to F. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

Dicer Regulates the Balance of Short-Lived Effector and Long-Lived Memory CD8 T Cell Lineages.

MicroRNAs constitute a major post-transcriptional mechanism for controlling protein expression, and are emerging as key regulators during T cell development and function. Recent reports of augmented CD8 T cell activation and effector differentiation, and aberrant migratory properties upon ablation of Dicer/miRNAs in naïve cells have established a regulatory role of miRNAs during priming. Whether miRNAs continue to exert similar functions or are dispensable during later stages of CD8 T cell expansion and memory differentiation remains unclear. Here, we report a critical role of Dicer/miRNAs in regulating the balance of long-lived memory and short-lived terminal effector fates during the post-priming stages when CD8 T cells undergo clonal expansion to generate a large cytotoxic T lymphocyte (CTL) pool and subsequently differentiate into a quiescent memory state. Conditional ablation of Dicer/miRNAs in early effector CD8 T cells following optimal activation and expression of granzyme B, using unique dicerfl/fl gzmb-cre mice, led to a strikingly diminished peak effector size relative to wild-type antigen-specific cells in the same infectious milieu. Diminished expansion of Dicer-ablated CD8 T cells was associated with lack of sustained antigen-driven proliferation and reduced accumulation of short-lived effector cells. Additionally, Dicer-ablated CD8 T cells exhibited more pronounced contraction after pathogen clearance and comprised a significantly smaller proportion of the memory pool, despite significantly higher proportions of CD127Hi memory precursors at the effector peak. Combined with previous reports of dynamic changes in miRNA expression as CD8 T cells differentiate from naïve to effector and memory states, these findings support distinct stage-specific roles of miRNA-dependent gene regulation during CD8 T cell differentiation.

Early CD8 T-cell memory precursors and terminal effectors exhibit equipotent in vivo degranulation.

Early after priming, effector CD8 T cells are distinguished into memory precursor and short-lived effector cell subsets (MPECs and SLECs). Here, we delineated a distinct in vivo heterogeneity in killer cell lectin-like receptor G1 (KLRG-1) expression, which was strongly associated with diverse MPEC and SLEC fates. These in vivo MPECs and SLECs expressed equivalent levels of cytotoxic molecules and effector cytokines. Using a unique in vivo degranulation assay, we found that the MPECs and SLECs similarly encountered infected target cells and elaborated equivalent levels of cytotoxicity in vivo. These data provide direct in vivo evidence that memory-fated cells pass through a robust effector phase. Additionally, the preferential localization of the MPECs in the lymph nodes, where a lesser degree of cytotoxicity was elaborated, suggests that the MPECs may be protected from excessive stimulation and terminal differentiation by virtue of their differential tissue localization. These data provide novel mechanistic insights into the linear decreasing potential model of memory differentiation.

Synthesis and characterization of homo- and heterodinuclear M(II)-M'(III) (M(II) = Mn or Fe, M'(III) = Fe or Co) mixed-valence supramolecular pseudo-dimers. The effect of hydrogen bonding on spin state selection of M(II).

Reaction of H(3)L(1), the Schiff base condensate of tris(2-aminoethyl)amine with three equivalents of 5-methyl-1H-pyrazole-3-carboxaldehyde, with manganese(II)perchlorate or iron(II)tetrafluoroborate results in the isolation of [MH(3)L(1)]X(2) (M = Mn and X = ClO(4) and M = Fe and X = BF(4)). These complexes are high spin d(5) and d(6), respectively, as inferred from the long M-N bond distances obtained by single crystal X-ray diffraction for both and variable temperature magnetic susceptibility and Mössbauer spectroscopy for the iron complex. Aerobic treatment of a solution of [CoH(3)L(1)](2+) with three equivalents of potassium hydroxide produced [CoL(1)]. Homonuclear pseudo-dimers were prepared by the aerobic reaction of [FeH(3)L(1)](BF(4))(2) with 1.5 equivalents of potassium hydroxide to give {[FeH(1.5)L(1)](BF(4))}(2) or by the metathesis reaction of [FeH(2)L(1)][FeHL(1)](ClO(4))(2) with sodium hexafluorophosphate to give [FeH(3)L(1)][FeL(1)](PF(6))(2). The complexes were characterized by EA, IR, ESI-MS, variable temperature single crystal x-ray diffraction and Mössbauer spectroscopy. The iron(III) atom is low spin while the iron(II) atom is spin crossover. Heteronuclear pseudo-dimers were prepared by the 1:1 reaction of [FeH(3)L(1)](BF(4))(2) or [MnH(3)L(1)](ClO(4))(2) with [CoL(1)]. [MH(3)L(1)][CoL(1)](X)(2) (M = Fe and X = BF(4) or M = Mn and X = ClO(4)), were characterized by IR, EA, variable temperature single crystal X-ray diffraction and Mössbauer spectroscopy in the iron case. The data support a spin crossover and high spin assignment for the iron(II) and manganese(II), respectively. DFT calculations demonstrate that the spin state of the iron(II) atom in {[FeH(3)L(1)][FeL(1)]}(2+) changes from high spin to low spin as the iron(II)-iron(III) distance decreases. This is supported by experimental results and is a result of hydrogen bonding interactions which cause a significant compression of the M(II)-N(pyrazole) bond distances.