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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.

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 cell–specific 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.

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.

Differential IL-2 signaling is implicated in memory CD8 T cell fates. Autocrine IL-2 production is a hallmark of polyfunctional, long-lived lymphoid memory cells. Whether the IL-2 signals perceived by CD8 T cells come from autocrine or paracrine sources, the timing of these signals, and their differential impact on the qualitative outcomes of CD8 T cell responses are not clearly understood. We used two distinct models of germline and conditional autocrine IL-2 ablation in post-thymic CD8 T cells to specifically dissect the requirement and timing of autocrine IL-2. Our studies show that autocrine IL-2 is uniquely required during primary CTL responses to program the key memory property of robust recall responses. Ablation of autocrine IL-2 in fully differentiated wild type memory cells immediately prior to rechallenge did not impact their recall expansion potential. Furthermore, we found that paracrine IL-2 could effectively supplant autocrine IL-2 for primary expansion, effector differentiation and memory maintenance, but not recall expansion. Likewise, our data highlight the redundancy of autocrine and paracrine IL-2 in optimally driving equivalent metabolic programs associated with effector and memory states. While CD4 licensing of DCs is needed for optimal programming of autocrine IL-2 in CD8 T cells, we found that the requirement for CD4 help is less critical for autocrine IL-2 production in the context of inflammatory infections. These findings have implications in adoptive T cell immunotherapies where expansion of engineered cells is largely driven by paracrine IL-2, which can potentially impact their long-term sentinel function.

The general paradigm is that the tumor microenvironment is non-conducive to sustaining productive cytotoxic T cell responses due to suppressive and counter-immunity T cell extrinsic factors. It has been shown that the tumor microenvironment can harbor large numbers of bystander memory cells. However, it is not very clear if given the right stimulation these bystander memory cells can be commandeered to utilize the available resources of the tumor microenvironment, get optimally activated and elaborate productive immune responses. This has wide-ranging implications in hijacking the tumor-resident bystander memory cells for efficacious tumor control. In this study we show that the tumor microenvironment is capable of harboring ultra-functional bystander memory cells, which display complete panoply of memory markers. These tumor–resident bystander memory cells are not only capable of elaborating robust ex vivo and transplantable memory T cell functions, but they can also efficiently utilize the resources within the tumor microenvironment to elaborate in situ recall effector functions. We further show that these memory T cells utilize CXCR3 as a key chemokine receptor for trafficking to the tumor sites. Our data identify bystander tumor-resident memory cells as Spartan warriors to mediate enhanced tumor control during adoptive T cell immunotherapy.

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.

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 ofFrancisella tularensis, but to-date, it has not been possible to study these cells in a pathogen-specific manner. Here, we report the development of a tool for expression of the model antigen ovalbumin (OVA) inF. tularensis, which allows for the study of CD8+T cell responses to the bacterium. We demonstrate that in response to intranasal infection with theF. tularensisLive Vaccine Strain, pathogen-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,F. tularensis-specific cells are poorly retained in the lungs after clearance of infection. We also show that intranasal vaccination leads to more pathogen-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 toF. tularensis. Use of this tool will enhance our understanding of immunity to this deadly pathogen.

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.

IL-2 is a key driver of T cell responses critical for the conversion of resting antigen specific T cells into glycolytic powerhouses that undergo massive expansion and elicit cytolytic function to clear infections. Recent studies have used ex vivo antigen challenge combined with somatic IL-2 deletion to show that both paracrine and autocrine IL-2 signals are indispensable for the formation of highly functional memory CD8 T cells. Despite this critical role of autocrine IL-2 for memory T cell fate, the in vivo history and fate of autocrine IL-2 producing CD8 T cells remains unknown. We generated a mouse model in which autocrine IL-2 production leads to transcription of TdTomato, leading to an indelibly marked T cell. Following acute infection, a proportion of antigen specific CD8 T cells made autocrine IL-2, however, more than half of the T cell response had no history of IL-2 production. Furthermore, contrary to their ex vivo functionality, memory precursor and terminal effector T cells had similar history of in vivo IL-2 production. Despite autocrine IL-2 production being a benchmark of highly functional memory T cells, we observed no increase in the ex vivo polyfunctionality of T cells that produced in vivo IL-2 during infection. Finally, while strong TCR engagement is critical for the recruitment of robust T cell responses, we saw that in vivo autocrine IL-2 production is more effectively elicited by lower affinity TCR signals. Thus, we see that while proportion of T cells rely on autocrine IL-2 during infection, the ability of a CD8 T cell to recruit autocrine IL-2 function heavily depends on the avidity of the TCR to the peptide-MHC complex. Together these data provide the first insights into the in vivo function of IL-2 in antigen specific CD8 T cells.

Chimeric antigen receptor (CAR) T cells therapies have made substantial progress in treating blood cancers in humans. However, CAR T therapies currently have several limitations, including the lack of a persistent anti-tumor memory population to limit relapse and poor efficacy against solid tumors. Many studies on CAR T therapies have used immunocompromised xenograft mouse models, limiting the translational impact of the results. Furthermore, mouse models have largely focused on cancers in the blood while neglecting solid tumors. To improve the relevance of preclinical studies of CAR T cell therapy and to understand the mechanisms underlying some of the current CAR T therapy limitations, we have created a novel immunocompetent mouse model. This model consists of three components: murine CD8 T cells expressing the human CD19 CAR, syngeneic murine tumor cell lines expressing a truncated human CD19 (CD19t) serving as the tumor antigen, and an immunocompetent mouse tolerized to human CD19t and the CD19 CAR. Our results demonstrate that the CD19 CAR T cells can efficiently target the CD19t-expressing tumor lines. Furthermore, tolerized recipient mice do not reject implanted syngeneic CD19t-expressing tumors or adoptively transferred CD19 CAR-expressing T cells. This model now provides a platform to study the in vivo biology of CAR T cells with respect to localization, exhaustion, and functional reinvigoration; to investigate best approaches to treating multiple solid tumors with distinct microenvironments; and to test the preclinical efficacy of novel CAR T strategies.

The high affinity IL-2 receptor (IL-2Ra, CD25) is a key activation marker, which is transiently and heterogeneously expressed during the priming . While IL-2 signals are deemed critical for expansion of antigen-specific CD8 T cells, the role of IL-2 in programing the fate of exhausted CD8 T cells in vivo is poorly understood. Exhausted CD8 T cells are broadly divided into 2 subsets: one with brightest levels of inhibitory receptors such as PD-1, and lower mitochondrial spare respiratory capacity (SRC) – these cells are terminally exhausted and are largely unresponsive to PD-1 checkpoint blockade immunotherapy. The other subset has intermediate levels of inhibitory receptors and retains higher mitochondrial SRC – these are the clinically relevant cells that are highly responsive to PD-1 blockade. Based on the ability of IL-2 signals to drive glycolytic burnout in T cells, we hypothesized that the stochastic heterogeneity in in vivo assimilation of IL-2 signals during priming drives distinct CD8 T cell fates late during exhaustion. We sorted in vivo primed CD8 T cells based on their heterogeneity in CD25 expression, and adoptively transferred them into infection-matched chronically infected recipients. We observed that the exhausted CD8 T cells generated from CD25Hi donor cells phenotypically and functionally resembled terminally exhausted cells. Furthermore, knocking down the expression of CD25 or Special AT-rich sequence binding protein 1 (SATB1), which is known to regulate CD25 expression, resulted in better survival and function of exhausted CD8 T cells. Our data underscores the critical role of IL-2 signals in the exhaustion of CD8 T cells and has clinical significance in current combination checkpoint blockade immunotherapies.

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.

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.

PD-1 is highly expressed on CD8 T cells during activation, both in acute and chronic viral infections. Inhibitory signaling in cytotoxic T cells through the PD-1 axis is well characterized during chronic infections and has led to the development of potent blockade therapies that restore function to otherwise exhausted CD8 T cells. However, the functional significance of transiently increased PD-1 expression on CD8 T cells early after activation and its rapid down-regulation following clearance of antigen during acute infections, remains to be characterized. The enigma is – expression of PD-1 (an immunological brake) during activation temporally coincides with rapid proliferation and production of copious amounts of signature effector cytokines (IFN-γ and TNF-α). We generated PD-1−/− antigen-specific CD8 T cells to study its cell-intrinsic role during acute infection with LCMV. Unexpectedly, PD-1−/− cells did not exhibit increased proliferation early during infection, or enhanced accumulation at the peak of expansion. PD-1−/− CD8 T cells were also unaltered in their polyfunctionality and granzyme B expression. Despite robust effector properties, PD-1−/− CD8 T cells underwent precipitous contraction, leading to near ablation of the memory pool. Mechanistically, in vivo analysis of PD-1−/− memory cells showed a severe defect in antigen-independent homeostatic proliferation due to decreased sensitivity to IL-2, −7, and −15 signals, without evident change in the expression of common gamma-chain cytokine receptors. These studies demonstrate a previously unrecognized role of PD-1 signaling in programming of memory CD8 T cell maintenance and could make it a novel target for manipulating vaccine-induced memory T cell longevity.

Proliferation is a defining feature of clonal cytotoxic T lymphocyte (CTL) responses upon activation. However, how proliferative events during primary expansion relate to divergent CD8 T cell fate outcomes remains poorly defined. Emerging evidence from our laboratory and others in the field suggest that death-fated effectors undergo more pronounced proliferation compared to memory-fated cells during antigen-driven T cell expansion. Adoptive transfer of activated CD8 T cell-subsets, purified based on their extent of proliferation, showed that in vivo differentiation of CD8 T cells and memory fate are linked to proliferation. We found that the less-divided effector CTL subset preferentially contributed to the central memory lineage. Interestingly, these less-divided memory precursor cells were also programmed to produce more IL-2 compared to their more-divided terminal effector counterparts. How do memory-fated cells that make their own IL-2 (a pro-proliferative cytokine) proliferate less and how does this make them better fit for the long-lived memory lineage? To answer this question, we generated CD8 T cells that could be conditionally ablated for il2. We ablated IL-2 specifically in antigen-specific CD8 T cells, during distinct phases of effector and memory differentiation. Our data showed that while paracrine IL-2 is critical for driving the early in vivo proliferative burst, the programmed life-long autochthonous IL-2 in memory-fated cells was necessary for antigen-independent maintenance, and robust secondary expansion of memory cells. Thus, autocrine and paracrine IL-2 signals collaborate during distinct stages of T cell differentiation to direct diverse CTL lineages through effects on proliferation.

MicroRNAs are small RNAs (∼22 nucleotides) that regulate gene expression via post-transcriptional mechanisms, and are emerging as major players in immune cell development and function. Using dual strategy of conditional deletion or constitutive overexpression of miR-17∼92, we have recently shown that miR-17∼92 has a critical CD8 T cell-intrinsic role in driving terminal effector differentiation by promoting proliferation (Khan, et al., 2013, Blood). We hypothesized that miR-17∼92 increases the sensitivity of CD8 T cells to TCR stimulation, thus driving proliferation and terminal differentiation. Indeed, when we overexpressed miR-17∼92 in effector CD8 T cells, we observed heightened levels of proximal phosphorylation events downstream of TCR. While PTEN and PD-1 (negative regulators of T cell stimulation) were downregulated upon overexpression of miR-17∼92, dramatic potentiation of TCR signal transduction occurred as evidenced by 50-fold upregulation of ribosomal S6 Kinase activity compared to WT. Converse results were obtained when miR-17∼92 was deleted, and about 10-folds higher levels of peptide-MHC were needed to induce TCR signals to levels similar to WT. Consistent with these observations, under conditions of limiting TCR stimulation, in vivo expansion of virus-specific miR-17∼92-deleted CD8 T cells was also severely compromised. Our data, altogether present a novel role of miR-17∼92 in regulating TCR sensitivity during CD8 T cell activation and effector differentiation.

Epidemiological studies implicate a potential role of vitamin D in modulating immune responses to infections and vaccines. In this study we analyzed the contribution of vitamin D signals to the development and maintenance of antigen-specific memory CD8 T cell responses during a viral infection. Using the vitamin D receptor (VDR)-deficient murine model, we found that lack of vitamin D signals resulted in a dysregulated CD8 T cell expansion program, which was largely attributable to cell-extrinsic effects. Investigation of events proximal to TCR activation demonstrated a delayed yet sustained priming of antigen-specific CD8 T cells in the absence of vitamin D signals. In spite of a larger ensuing effector CD8 T cell pool generated in the absence of vitamin D signals, the final memory pool was smaller. Interestingly, within both effector and memory CD8 T cell pools, we observed a bias towards CD8 T cells specific for immunodominant determinants, resulting in a restricted repertoire of antigen-specific CD8 T cells in the absence of vitamin D signals. Collectively, these studies delineate a critical role of vitamin D in regulating the quantity and quality of adaptive T cell immunity. Importantly, our studies have direct impact on the efficacy of vaccines, owing to disparate nutritional states of vitamin D that are currently seen cross-sectionally around the world.