Asymmetric inheritance of mTORC1 kinase activity during division dictates CD8(+) T cell differentiation.

The asymmetric partitioning of fate-determining proteins has been shown to contribute to the generation of CD8(+) effector and memory T cell precursors. Here we demonstrate the asymmetric partitioning of mTORC1 activity after the activation of naive CD8(+) T cells. This results in the generation of two daughter T cells, one of which shows increased mTORC1 activity, increased glycolytic activity and increased expression of effector molecules. The other daughter T cell has relatively low mTORC1 activity and increased lipid metabolism, expresses increased amounts of anti-apoptotic molecules and subsequently displays enhanced long-term survival. Mechanistically, we demonstrate a link between T cell antigen receptor (TCR)-induced asymmetric expression of amino acid transporters and RagC-mediated translocation of mTOR to the lysosomes. Overall, our data provide important insight into how mTORC1-mediated metabolic reprogramming affects the fate decisions of T cells.

Store-Operated Ca2+ Entry Controls Clonal Expansion of T Cells through Metabolic Reprogramming.

Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for T cell function and adaptive immunity. SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels that are activated by stromal interaction molecule (STIM) 1 and STIM2. SOCE regulates many Ca2+-dependent signaling molecules, including calcineurin, and inhibition of SOCE or calcineurin impairs antigen-dependent T cell proliferation. We here report that SOCE and calcineurin regulate cell cycle entry of quiescent T cells by controlling glycolysis and oxidative phosphorylation. SOCE directs the metabolic reprogramming of naive T cells by regulating the expression of glucose transporters, glycolytic enzymes, and metabolic regulators through the activation of nuclear factor of activated T cells (NFAT) and the PI3K-AKT kinase-mTOR nutrient-sensing pathway. We propose that SOCE controls a critical "metabolic checkpoint" at which T cells assess adequate nutrient supply to support clonal expansion and adaptive immune responses.

B Cell Subsets Differentially Contribute to the T Cell-Independent Memory Pool.

The roles distinct B cell subsets play in clonal expansion, isotype switching, and memory B cell differentiation in response to T cell-independent type 2 Ags (TI-2 Ags) has been understudied. Using sorted B cells from VHB1-8 knock-in mice, we evaluated B-1b, marginal zone, and follicular B cell responses to the TI-2 Ag, NP-Ficoll. All subsets extensively divided in response to NP-Ficoll. Nonetheless, B-1b cells exhibited significantly increased IgG switching and differentiation into Ab-secreting cells (ASC)-a finding that coincided with increased AgR signaling capacity and Blimp1 expression by B-1b cells. All subsets formed memory cells and expressed markers previously identified for T cell-dependent memory B cells, including CD80, PDL2, and CD73, although B-1b cells generated the greatest number of memory cells with higher frequencies of IgG- and CD80-expressing cells. Despite memory formation, secondary immunization 4 wk after primary immunization did not increase NP-specific IgG. However, boosting occurred in B-1b cell-recipient mice when IgG levels declined. CD80+ memory B-1b cells divided, class switched, and differentiated into ASC in response to Ag in vivo, but this was inhibited in the presence of NP-specific IgG. Furthermore, CD80 blockade significantly increased memory B-1b cell division and differentiation to ASC upon Ag restimulation. Collectively, these findings demonstrate B-1b, marginal zone B, and follicular B subsets significantly contribute to the TI-2 Ag-specific memory B cell pool. In particular, we show B-1b cells generate a functional CD80-regulated memory population that can be stimulated to divide and differentiate into ASC upon Ag re-encounter when Ag-specific IgG levels decline.

ß-Selection-induced proliferation is required for αß T cell differentiation.

Proliferation and differentiation are tightly coordinated to produce an appropriate number of differentiated cells and often exhibit an antagonistic relationship. Developing T cells, which arise in the thymus from a minute number of bone-marrow-derived progenitors, undergo a major expansion upon pre-T cell receptor (TCR) expression. The burst of proliferation coincides with differentiation toward the αß T cell lineage-but the two processes were previously thought to be independent from one another, although both were driven by signaling from pre-TCR and Notch receptors. Here we report that proliferation at this step was not only absolutely required for differentiation but also that its ectopic activation was sufficient to substantially rescue differentiation in the absence of Notch signaling. Consistently, pharmacological inhibition of the cell cycle machinery also blocked differentiation in vivo. Thus the proliferation step is strictly required prior to differentiation of immature thymocytes.

Interferon stimulation creates chromatin marks and establishes transcriptional memory.

Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNß stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of ∼2,000 IFNß-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.

The Effects of Sperm and Seminal Fluid of Immunized Male Mice on In Vitro Fertilization and Surrogate Mother-Embryo Interaction.

The latest vaccination campaign has actualized the potential impact of antigenic stimuli on reproductive functions. To address this, we mimicked vaccination's effects by administering keyhole limpet hemocyanin (KLH ) to CD1 male mice and used their sperm for in vitro fertilization (IVF). Two-cell embryos after IVF with spermatozoa from control (C) or KLH-treated (Im) male mice were transferred to surrogate mothers mated with vasectomized control (C) or KLH-treated (Im) male mice, resulting in four experimental groups: C-C, Im-C, C-Im, and Im-Im. The pre-implantation losses were significantly lower in the Im-C group than in the C-Im group. At the same time, the resorption rates reduced markedly in the C-Im compared to the Im-C group. Embryo and placenta weights were significantly higher in the Im-Im group. Although the GM-CSF levels were lower in the amniotic fluid of the gestating surrogate mothers in the Im-Im group, they were strongly correlated with embryo mass. The number-size trade-off was only significant in the Im-Im group. This suggests a positive, cooperative effect of spermatozoa and seminal fluid from immune-primed males on embryo growth and the optimal distribution of surrogate mother maternal resources despite the negative impact of males' antigenic challenge on the IVF success rate.

New putative vaccine candidates against Acinetobacter baumannii using the reverse vaccinology method.

Infections caused by multi-drug resistance Acinetobacter baumannii are increasing worldwide. Discovery of the vaccine against this bacterium as a cost-effective and preventive strategy seems necessary. This study has introduced 11 new putative vaccine candidates against A. baumannii using the reverse vaccinology method. We considered 33 genomes of A. baumannii strains and selected the outer membrane and secreted proteins as putative vaccine candidates using Vaxign web tool. Finally, 11 proteins were confirmed as promising vaccine candidates. These targets belonged to proteins involved in cell division (NlpD), fimbria or pili assembly (FimA, PapC, and PapC associated with usher system), iron acquisition (FhuA, BfnH, FatA-like protein, and IutA), DcaP-like protein and two novel hypothetical proteins (HP-1 and HP-2). The analysis of linear and conformational B-cell epitopes showed that the outer membrane proteins including DcaP-like protein and HP-2 had high conserved surface-exposed epitopes that they can consider as excellent putative vaccine targets in the upcoming immunological assays.

2B4 Mediates Inhibition of CD8+ T Cell Responses via Attenuation of Glycolysis and Cell Division.

We recently showed that 2B4 expression on memory T cells in human renal transplant recipients was associated with reduced rates of rejection. To investigate whether 2B4 functionally underlies graft acceptance during transplantation, we established an experimental model in which 2B4 was retrogenically expressed on donor-reactive murine CD8+ T cells (2B4rg), which were then transferred into naive recipients prior to skin transplantation. We found that constitutive 2B4 expression resulted in significantly reduced accumulation of donor-reactive CD8+ T cells following transplantation and significantly prolonged graft survival following transplantation. This marked reduction in alloreactivity was due to reduced proliferation of CD8+ Thy1.1+ 2B4rg cells as compared with control cells, underpinned by extracellular flux analyses demonstrating that 2B4-deficient (2B4KO) CD8+ cells activated in vitro exhibited increased glycolytic capacity and upregulation of gene expression profiles consistent with enhanced glycolytic machinery as compared with wild type controls. Furthermore, 2B4KO CD8+ T cells primed in vivo exhibited significantly enhanced ex vivo uptake of a fluorescent glucose analogue. Finally, the proliferative advantage associated with 2B4 deficiency was only observed in the setting of glucose sufficiency; in glucose-poor conditions, 2B4KO CD8+ T cells lost their proliferative advantage. Together, these data indicate that 2B4 signals function to alter T cell glucose metabolism, thereby limiting the proliferation and accumulation of CD8+ T cells. Targeting 2B4 may therefore represent a novel therapeutic strategy to attenuate unwanted CD8+ T cell responses.

Systems-guided forward genetic screen reveals a critical role of the replication stress response protein ETAA1 in T cell clonal expansion.

T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8+ and CD4+ T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adult mice homozygous for two different Etaa1 mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity.

Cigarette smoke extract acts directly on CD4 T cells to enhance Th1 polarization and reduce memory potential.

Although cigarette smoke is known to alter immune responses, whether and how CD4 T cells are affected is not well-described. We aimed to characterize how exposure to cigarette smoke extract impacts CD4 T cell effector generation in vitro under Th1-polarizing conditions. Our results demonstrate that cigarette smoke directly acts on CD4 T cells to impair effector expansion by decreasing division and increasing apoptosis. Furthermore, cigarette smoke enhances Th1-associated cytokine production and increases expression of the transcription factor T-bet, the master regulator of Th1 differentiation. Finally, we show that exposure to cigarette smoke extract during priming impairs the ability of effectors to form memory cells. Our findings thus demonstrate that cigarette smoke simultaneously enhances effector functions but promotes terminal differentiation of CD4 T cell effectors. This study may be relevant to understanding how smoking can both aggravate autoimmune symptoms and reduce vaccine efficacy.

Activation and propagation of tumor-infiltrating lymphocytes from malignant pleural effusion and ascites with engineered cells for costimulatory enhancement.

Adoptive cell therapy (ACT) of autologous tumor-infiltrating lymphocytes (TILs) has shown an effect on mediating tumor regression in some patients with highly advanced, refractory metastatic malignancy. Here, the in vitro generation of TILs isolated from malignant pleural effusion and ascites was compared with which using engineered cells for costimulatory enhancement (ECCE) and 3 common γ-chain cytokines, interleukin (IL)-2, IL-7, and IL-15, alone or in combination. We showed the robust clinical-scale production of TILs with a less differentiated 'young' phenotype by expansion in the presence of ECCE combined with IL-2/7/15. Furthermore, a major fraction of the TILs generated in this fashion was shown to produce much more IFN-γ and TNF-α, and displayed cytolytic activity against target cells expressing the relevant antigens. To our knowledge, this is the first time that the combination of ECCE and IL-2/7/15 has been applied for the generation of TILs isolated from malignant pleural effusion and ascites.

The Hayflick Limit May Determine the Effective Clonal Diversity of Naive T Cells.

Having a large number of sufficiently abundant T cell clones is important for adequate protection against diseases. However, as shown in this paper and elsewhere, between young adulthood and >70 y of age the effective clonal diversity of naive CD4/CD8 T cells found in human blood declines by a factor of >10. (Effective clonal diversity accounts for both the number and the abundance of T cell clones.) The causes of this observation are incompletely understood. A previous study proposed that it might result from the emergence of certain rare, replication-enhancing mutations in T cells. In this paper, we propose an even simpler explanation: that it results from the loss of T cells that have attained replicative senescence (i.e., the Hayflick limit). Stochastic numerical simulations of naive T cell population dynamics, based on experimental parameters, show that the rate of homeostatic T cell proliferation increases after the age of ∼60 y because naive T cells collectively approach replicative senescence. This leads to a sharp decline of effective clonal diversity after ∼70 y, in agreement with empirical data. A mathematical analysis predicts that, without an increase in the naive T cell proliferation rate, this decline will occur >50 yr later than empirically observed. These results are consistent with a model in which exhaustion of the proliferative capacity of naive T cells causes a sharp decline of their effective clonal diversity and imply that therapeutic potentiation of thymopoiesis might either prevent or reverse this outcome.

Existence and stability of periodic solutions of an impulsive differential equation and application to CD8 T-cell differentiation.

Unequal partitioning of the molecular content at cell division has been shown to be a source of heterogeneity in a cell population. We propose to model this phenomenon with the help of a scalar, nonlinear impulsive differential equation (IDE). To study the effect of molecular partitioning at cell division on the effector/memory cell-fate decision in a CD8 T-cell lineage, we study an IDE describing the concentration of the protein Tbet in a CD8 T-cell, where impulses are associated to cell division. We discuss how the degree of asymmetry of molecular partitioning can affect the process of cell differentiation and the phenotypical heterogeneity of a cell population. We show that a moderate degree of asymmetry is necessary and sufficient to observe irreversible differentiation. We consider, in a second part, a general autonomous IDE with fixed times of impulse and a specific form of impulse function. We establish properties of the solutions of that equation, most of them obtained under the hypothesis that impulses occur periodically. In particular, we show how to investigate the existence of periodic solutions and their stability by studying the flow of an autonomous differential equation. Then we apply those properties to prove the results presented in the first part.

Distinct modes of antigen presentation promote the formation, differentiation, and activity of foxp3+ regulatory T cells in vivo.

How the formation and activity of CD4(+)Foxp3(+) regulatory T cells (Tregs) are shaped by TCR recognition of the diverse array of peptide:MHC complexes that can be generated from self-antigens and/or foreign Ags in vivo remains poorly understood. We show that a self-peptide with low (but not high) stimulatory potency promotes thymic Treg formation and can induce conventional CD4(+) T cells in the periphery to become Tregs that express different levels of the transcription factor Helios according to anatomical location. When Tregs generated in response to this self-peptide subsequently encountered the same peptide derived instead from influenza virus in the lung-draining lymph nodes of infected mice, they proliferated, acquired a T-bet(+)CXCR3(+) phenotype, and suppressed the antiviral effector T cell response in the lungs. However, these self-antigen-selected Tregs were unable to suppress the antiviral immune response based on recognition of the peptide as a self-antigen rather than a viral Ag. Notably, when expressed in a more immunostimulatory form, the self-peptide inhibited the formation of T-bet(+)CXCR3(+) Tregs in response to viral Ag, and Ag-expressing B cells from these mice induced Treg division without upregulation of CXCR3. These studies show that a weakly immunostimulatory self-peptide can induce thymic and peripheral Foxp3(+) Treg formation but is unable to activate self-antigen-selected Tregs to modulate an antiviral immune response. Moreover, a strongly immunostimulatory self-peptide expressed by B cells induced Tregs to proliferate without acquiring an effector phenotype that allows trafficking from the draining lymph node to the lungs and, thereby, prevented the Tregs from suppressing the antiviral immune response.

Regulation of asymmetric division and CD8+ T lymphocyte fate specification by protein kinase Cζ and protein kinase Cλ/ι.

During an immune response against a microbial pathogen, activated naive T lymphocytes give rise to effector cells that provide acute host defense and memory cells that provide long-lived immunity. It has been shown that T lymphocytes can undergo asymmetric division, enabling the daughter cells to inherit unequal amounts of fate-determining proteins and thereby acquire distinct fates from their inception. In this study, we show that the absence of the atypical protein kinase C (PKC) isoforms, PKCζ and PKCλ/ι, disrupts asymmetric CD8(+) T lymphocyte division. These alterations were associated with aberrant acquisition of a pre-effector transcriptional program, detected by single-cell gene expression analyses, in lymphocytes that had undergone their first division in vivo and enhanced differentiation toward effector fates at the expense of memory fates. Together, these results demonstrate a role for atypical PKC in regulating asymmetric division and the specification of divergent CD8(+) T lymphocyte fates early during an immune response.

Spaceflight impairs antigen-specific tolerance induction in vivo and increases inflammatory cytokines.

The health risks of a dysregulated immune response during spaceflight are important to understand as plans emerge for humans to embark on long-term space travel to Mars. In this first-of-its-kind study, we used adoptive transfer of T-cell receptor transgenic OT-II CD4 T cells to track an in vivo antigen-specific immune response that was induced during the course of spaceflight. Experimental mice destined for spaceflight and mice that remained on the ground received transferred OT-II cells and cognate peptide stimulation with ovalbumin (OVA) 323-339 plus the inflammatory adjuvant, monophosphoryl lipid A. Control mice in both flight and ground cohorts received monophosphoryl lipid A alone without additional OVA stimulation. Numbers of OT-II cells in flight mice treated with OVA were significantly increased by 2-fold compared with ground mice treated with OVA, suggesting that tolerance induction was impaired by spaceflight. Production of proinflammatory cytokines were significantly increased in flight compared with ground mice, including a 5-fold increase in IFN-γ and a 10-fold increase in IL-17. This study is the first to show that immune tolerance may be impaired in spaceflight, leading to excessive inflammatory responses.

Antigen-specific naive CD8+ T cells produce a single pulse of IFN-γ in vivo within hours of infection, but without antiviral effect.

In vitro studies have shown that naive CD8(+) T cells are unable to express most of their effector proteins until after at least one round of cell division has taken place. We have reassessed this issue in vivo and find that naive CD8(+) T cells mount Ag-specific responses within hours of infection, before proliferation has commenced. Newly activated naive Ag-specific CD8(+) T cells produce a rapid pulse of IFN-γ in vivo and begin to accumulate granzyme B and perforin. Later, in vivo cytolytic activity is detectable, coincident with the initiation of cell division. Despite the rapid development of these functional attributes, no antiviral effect was observed early during infection, even when the cells are present in numbers similar to those of virus-specific memory cells. The evolutionary reason for the pulse of IFN-γ synthesis by naive T cells is uncertain, but the lack of antiviral impact suggests that it may be regulatory.

Regulatory T-cell therapy: is it ready for the clinic?

The identification of suppressor T cells as important regulators of basic processes that are designed to maintain tolerance has opened an important area of potential clinical investigation in autoimmunity, graft-versus-host disease and transplantation. However, the field has been limited by an inability to define the antigenic specificities of these cells and by the small numbers of circulating regulatory T cells. Recently, new methods for expanding polyclonal and antigen-specific regulatory T cells have emerged. This article summarizes efforts to exploit regulatory T-cell therapy for the treatment of immunological diseases and poses the question of when and where regulatory T cells will first impact on clinical diseases.

NK cell responses to cytomegalovirus infection lead to stable imprints in the human KIR repertoire and involve activating KIRs.

Human natural killer (NK) cells are functionally regulated by killer cell immunoglobulin-like receptors (KIRs) and their interactions with HLA class I molecules. As KIR expression in a given NK cell is genetically hard-wired, we hypothesized that KIR repertoire perturbations reflect expansions of unique NK-cell subsets and may be used to trace adaptation of the NK-cell compartment to virus infections. By determining the human "KIR-ome" at a single-cell level in more than 200 donors, we were able to analyze the magnitude of NK cell adaptation to virus infections in healthy individuals. Strikingly, infection with human cytomegalovirus (CMV), but not with other common herpesviruses, induced expansion and differentiation of KIR-expressing NK cells, visible as stable imprints in the repertoire. Education by inhibitory KIRs promoted the clonal-like expansion of NK cells, causing a bias for self-specific inhibitory KIRs. Furthermore, our data revealed a unique contribution of activating KIRs (KIR2DS4, KIR2DS2, or KIR3DS1), in addition to NKG2C, in the expansion of human NK cells. These results provide new insight into the diversity of KIR repertoire and its adaptation to virus infection, suggesting a role for both activating and inhibitory KIRs in immunity to CMV infection.

Phase 1 trial of IL-15 trans presentation blockade using humanized Mikß1 mAb in patients with T-cell large granular lymphocytic leukemia.

In the present study, Hu-Mikß1, a humanized mAb directed at the shared IL-2/IL-15Rß subunit (CD122) was evaluated in patients with T-cell large granular lymphocytic (T-LGL) leukemia. Hu-Mikß1 blocked the trans presentation of IL-15 to T cells expressing IL-2/IL-15Rß and the common γ-chain (CD132), but did not block IL-15 action in cells that expressed the heterotrimeric IL-15 receptor in cis. There was no significant toxicity associated with Hu-Mikß1 administration in patients with T-LGL leukemia, but no major clinical responses were observed. One patient who had previously received murine Mikß1 developed a measurable Ab response to the infused Ab. Nevertheless, the safety profile of this first in-human study of the humanized mAb to IL-2/IL-15Rß (CD122) supports its evaluation in disorders such as refractory celiac disease, in which IL-15 and its receptor have been proposed to play a critical role in the pathogenesis and maintenance of disease activity.