Picking up speed: cell cycle regulation during effector CD8+ T cell differentiation.

Clonal expansion and development of immunological memory are two hallmarks of adaptive immune responses. Resolving the intricate pathways that regulate cell cycle activity and lead to the generation of diverse effector and memory T cell subsets is essential for improving our understanding of protective T cell immunity. A deeper knowledge of cell cycle regulation in T cells also has translational implications for adoptive cell therapies and vaccinations against infectious diseases. Here, we summarize recent evidence for an early diversification of effector and memory CD8+ T cell fates and discuss how this process is coupled to discrete changes in division speed. We further review technical advances in lineage tracing and cell cycle analysis and outline how these techniques have shed new light on the population dynamics of CD8+ T cell responses, thereby refining our current understanding of the developmental organization of the memory T cell pool.

Effect of Pregnancy Specific ß1-Glycoprotein on the Replicative Potential of Naïve T Cells and Immune Memory T Cells.

We studied the effects of pregnancy-specific ß1-glycoprotein (PSG) on the replicative potential of naïve T cells (CD45RA+) and immune memory T cells (CD45R0+) in vitro by evaluating the expression of the hTERT gene in combination with the proliferative activity of cells. Human PSG was obtained by the author's patented method of immunopurification using a biospecific sorbent with subsequent removal of immunoglobulin contamination on a HiTrap Protein G HP column. We used monocultures of CD45RA+ and CD45R0+ lymphocytes isolated from peripheral blood mononuclear cells of reproductive-age women. It was found that PSG in physiological concentrations inhibited the expression of the hTERT gene mRNA in naïve T cells and immune memory T cells and simultaneously reduced the number of proliferating T cells estimated by the differential gating method. At the same time, PSG reduced CD71 expression only on naïve T cells without affecting this molecule on immune memory T cells. Thus, PSG decreased the replication potential and suppressed the proliferation of T cells and immune memory T cells, which in the context of pregnancy can contribute to the formation of immune tolerance to the semi-allogeneic embryo.

Molecular and Cellular Mechanisms Modulating Trained Immunity by Various Cell Types in Response to Pathogen Encounter.

The induction of trained immunity represents an emerging concept defined as the ability of innate immune cells to acquire a memory phenotype, which is a typical hallmark of the adaptive response. Key points modulated during the establishment of trained immunity include epigenetic, metabolic and functional changes in different innate-immune and non-immune cells. Regarding to epigenetic changes, it has been described that long non-coding RNAs (LncRNAs) act as molecular scaffolds to allow the assembly of chromatin-remodeling complexes that catalyze epigenetic changes on chromatin. On the other hand, relevant metabolic changes that occur during this process include increased glycolytic rate and the accumulation of metabolites from the tricarboxylic acid (TCA) cycle, which subsequently regulate the activity of histone-modifying enzymes that ultimately drive epigenetic changes. Functional consequences of established trained immunity include enhanced cytokine production, increased antigen presentation and augmented antimicrobial responses. In this article, we will discuss the current knowledge regarding the ability of different cell subsets to acquire a trained immune phenotype and the molecular mechanisms involved in triggering such a response. This knowledge will be helpful for the development of broad-spectrum therapies against infectious diseases based on the modulation of epigenetic and metabolic cues regulating the development of trained immunity.

Infection with SARS-CoV-2 primes immunological memory in human nasal-associated lymphoid tissue.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, has resulted in considerable morbidity and mortality in humans. Little is known regarding the development of immunological memory following SARS-CoV-2 infection or whether immunological memory can provide long-lasting protection against reinfection. Urgent need for vaccines is a considerable issue for all governments worldwide. METHODS: A total of 39 patients were recruited in this study. Tonsillar mononuclear cells (MNCs) were co-cultured in RPMI medium and stimulated with the full-length SARS-CoV-2 spike protein in the presence and absence of a CpG-DNA adjuvant. An enzyme-linked immunosorbent assay (ELISA) was utilised to measure the specific antibody response to the spike protein in the cell culture supernatants. RESULTS: The SARS-CoV-2 spike protein primed a potent memory B cell-mediated immune response in nasal-associated lymphoid tissue (NALT) from patients previously infected with the virus. Additionally, spike protein combined with the CpG-DNA adjuvant induced a significantly increased level of specific anti-spike protein IgG antibody compared with the spike protein alone (p < 0.0001, n = 24). We also showed a strong positive correlation between the specific anti-spike protein IgG antibody level in a serum samples and that produced by MNCs derived from the same COVID-19-recovered patients following stimulation (r = 0.76, p = 0.0002, n = 24). CONCLUSION: Individuals with serological evidence of previous SARS-CoV-2 exposure showed a significant anti-spike protein-specific memory humoral immune response to the viral spike protein upon stimulation. Additionally, our results demonstrated the functional response of NALT-derived MNCs to the viral spike protein. CpG-DNA adjuvant combined with spike protein induced significantly stronger humoral immune responses than the spike protein alone. These data indicate that the S protein antigen combined with CpG-DNA adjuvant could be used as a future vaccine candidate.

The Bone Marrow as Sanctuary for Plasma Cells and Memory T-Cells: Implications for Adaptive Immunity and Vaccinology.

The bone marrow (BM) is key to protective immunological memory because it harbors a major fraction of the body's plasma cells, memory CD4+ and memory CD8+ T-cells. Despite its paramount significance for the human immune system, many aspects of how the BM enables decade-long immunity against pathogens are still poorly understood. In this review, we discuss the relationship between BM survival niches and long-lasting humoral immunity, how intrinsic and extrinsic factors define memory cell longevity and show that the BM is also capable of adopting many responsibilities of a secondary lymphoid organ. Additionally, with more and more data on the differentiation and maintenance of memory T-cells and plasma cells upon vaccination in humans being reported, we discuss what factors determine the establishment of long-lasting immunological memory in the BM and what we can learn for vaccination technologies and antigen design. Finally, using these insights, we touch on how this holistic understanding of the BM is necessary for the development of modern and efficient vaccines against the pandemic SARS-CoV-2.

Pregnancy Induces an Immunological Memory Characterized by Maternal Immune Alterations Through Specific Genes Methylation.

During pregnancy, the maternal immune system undergoes major adaptive modifications that are necessary for the acceptance and protection of the fetus. It has been postulated that these modifications are temporary and limited to the time of pregnancy. Growing evidence suggests that pregnancy has a long-term impact on maternal health, especially among women with pregnancy complications, such as preeclampsia (PE). In addition, the presence of multiple immunological-associated changes in women that remain long after delivery has been reported. To explain these long-term modifications, we hypothesized that pregnancy induces long-term immunological memory with effects on maternal well-being. To test this hypothesis, we evaluated the immunological phenotype of circulating immune cells in women at least 1 year after a normal pregnancy and after pregnancy complicated by PE. Using multiparameter flow cytometry (FCM) and whole-genome bisulfite sequencing (WGBS), we demonstrate that pregnancy has a long-term effect on the maternal immune cell populations and that this effect differs between normal pregnancy and pregnancy complicated by PE; furthermore, these modifications are due to changes in the maternal methylation status of genes that are associated with T cell and NK cell differentiation and function. We propose the existence of an "immunological memory of pregnancy (IMOP)" as an evolutionary advantage for the success of future pregnancies and the proper adaptation to the microchimeric status established during pregnancy. Our findings demonstrate that the type of immune cell populations modified during pregnancy may have an impact on subsequent pregnancy and future maternal health.

PD-L1 signaling on human memory CD4+ T cells induces a regulatory phenotype.

Programmed cell death protein 1 (PD-1) is expressed on T cells upon T cell receptor (TCR) stimulation. PD-1 ligand 1 (PD-L1) is expressed in most tumor environments, and its binding to PD-1 on T cells drives them to apoptosis or into a regulatory phenotype. The fact that PD-L1 itself is also expressed on T cells upon activation has been largely neglected. Here, we demonstrate that PD-L1 ligation on human CD25-depleted CD4+ T cells, combined with CD3/TCR stimulation, induces their conversion into highly suppressive T cells. Furthermore, this effect was most prominent in memory (CD45RA-CD45RO+) T cells. PD-L1 engagement on T cells resulted in reduced ERK phosphorylation and decreased AKT/mTOR/S6 signaling. Importantly, T cells from rheumatoid arthritis patients exhibited high basal levels of phosphorylated ERK and following PD-L1 cross-linking both ERK signaling and the AKT/mTOR/S6 pathway failed to be down modulated, making them refractory to the acquisition of a regulatory phenotype. Altogether, our results suggest that PD-L1 signaling on memory T cells could play an important role in resolving inflammatory responses; maintaining a tolerogenic environment and its failure could contribute to ongoing autoimmunity.

Interferon regulatory factor 4 controls effector functions of CD8+ memory T cells.

The transcription factor IRF4 is required for CD8+ T cell activation, proliferation, and differentiation to effector cells and thus is essential for robust CD8+ T cell responses. The function of IRF4 in memory CD8+ T cells yet needs to be explored. To investigate the role of IRF4 for maintaining differentiation state and survival of CD8+ memory T cells, we used a mouse model with tamoxifen-inducible Irf4 knockout to preclude effects due to inefficient memory cell differentiation in absence of IRF4. We infected mice with ovalbumin-recombinant listeria and induced Irf4 knockout after clearance of the pathogen. Loss of IRF4 resulted in phenotypical changes of CD8+ memory T cells but did not cause a reduction of the total memory T cell population. However, upon reencounter of the pathogen, CD8+ memory T cells showed impaired expansion and acquisition of effector functions. When compared to CD8+ effector memory T cells, CD8+ tissue-resident memory T cells (TRM cells) expressed higher IRF4 levels. Mice with constitutive Irf4 knockout had diminished CD8+ TRM-cell populations, and tamoxifen-induced Irf4 deletion caused a reduction of this cell population. In conclusion, our results demonstrate that IRF4 is required for effective reactivation but not for general survival of CD8+ memory T cells. Formation and maintenance of CD8+ TRM cells, in contrast, appear to depend on IRF4.

Human Lung-Resident Macrophages Colocalize with and Provide Costimulation to PD1hi Tissue-Resident Memory T Cells.

Rationale: Tissue-resident memory T cells (TRM) play a critical role in the defense against inhaled pathogens. The isolation and study of human lung tissue-resident memory T cells and lung-resident macrophages (MLR) are limited by experimental constraints. Objectives: To characterize the spatial and functional relationship between MLR and human lung tissue-resident memory T cells using ex vivo lung perfusion (EVLP). Methods: TRM and MLR were isolated using EVLP and intraperfusate-labeled CD45 antibody. Cells isolated after 6 hours of EVLP were analyzed using spectral flow cytometry. Spatial relationships between CD3+ and CD68+ cells were explored with multiplexed immunohistochemistry. Functional relationships were determined by using coculture and T-cell-receptor complex signal transduction. Measurements and Main Results: Lungs from 8 research-consenting organ donors underwent EVLP for 6 hours. We show that human lung TRM and MLR colocalize within the human lung, preferentially around the airways. Furthermore, we found that human lung CD8+ TRM are composed of two functionally distinct populations on the basis of PD1 (programed cell death receptor 1) and ZNF683 (HOBIT) protein expression. We show that MLR provide costimulatory signaling to PD1hi CD4+ and CD8+ lung TRM,, augmenting the effector cytokine production and degranulation of TRM. Conclusions: EVLP provides an innovative technique to study resident immune populations in humans. Human MLR colocalize with and provide costimulation signaling to TRM, augmenting their effector function.

Long-term survival of patients with stage III colon cancer treated with VRP-CEA(6D), an alphavirus vector that increases the CD8+ effector memory T cell to Treg ratio.

BACKGROUND: There remains a significant need to eliminate the risk of recurrence of resected cancers. Cancer vaccines are well tolerated and activate tumor-specific immune effectors and lead to long-term survival in some patients. We hypothesized that vaccination with alphaviral replicon particles encoding tumor associated antigens would generate clinically significant antitumor immunity to enable prolonged overall survival (OS) in patients with both metastatic and resected cancer. METHODS: OS was monitored for patients with stage IV cancer treated in a phase I study of virus-like replicon particle (VRP)-carcinoembryonic antigen (CEA), an alphaviral replicon particle encoding a modified CEA. An expansion cohort of patients (n=12) with resected stage III colorectal cancer who had completed their standard postoperative adjuvant chemotherapy was administered VRP-CEA every 3 weeks for a total of 4 immunizations. OS and relapse-free survival (RFS) were determined, as well as preimmunization and postimmunization cellular and humoral immunity. RESULTS: Among the patients with stage IV cancer, median follow-up was 10.9 years and 5-year survival was 17%, (95% CI 6% to 33%). Among the patients with stage III cancer, the 5-year RFS was 75%, (95%CI 40% to 91%); no deaths were observed. At a median follow-up of 5.8 years (range: 3.9-7.0 years) all patients were still alive. All patients demonstrated CEA-specific humoral immunity. Patients with stage III cancer had an increase in CD8 +TEM (in 10/12) and decrease in FOXP3 +Tregs (in 10/12) following vaccination. Further, CEA-specific, IFNγ-producing CD8+granzyme B+TCM cells were increased. CONCLUSIONS: VRP-CEA induces antigen-specific effector T cells while decreasing Tregs, suggesting favorable immune modulation. Long-term survivors were identified in both cohorts, suggesting the OS may be prolonged.

Area under Immunosurveillance: Dedicated Roles of Memory CD8 T-Cell Subsets.

Immunological memory, defined as the ability to respond in an enhanced manner upon secondary encounter with the same pathogen, can provide substantial protection against infectious disease. The improved protection is mediated in part by different populations of memory CD8 T cells that are retained after primary infection. Memory cells persist in the absence of pathogen-derived antigens and enable secondary CD8 T-cell responses with accelerated kinetics and of larger magnitude after reencounter with the same pathogen. At least three subsets of memory T cells have been defined that are referred to as central memory CD8 T cells (Tcm), effector memory CD8 T cells (Tem), and tissue-resident memory CD8 T cells (Trm). Tcm and Tem are circulating memory T cells that mediate bodywide immune surveillance in search of invading pathogens. In contrast, Trm permanently reside in peripheral barrier tissues, where they form a stationary defensive line of sentinels that alert the immune system upon pathogen reencounter. The characterization of these different subsets has been instrumental in our understanding of the strategies that memory T cells employ to counter invading pathogens. It is clear that memory T cells not only have a numerical advantage over naive T cells resulting in improved protection in secondary responses, but also acquire distinct sets of competencies that assist in pathogen clearance. Nevertheless, inherent challenges are associated with the allocation of memory T cells to a limited number of subsets. The classification of memory T cells into Tcm, Tem, and Trm may not take into account the full extent of the heterogeneity that is observed in the memory population. Therefore, in this review, we will revisit the current classification of memory subsets, elaborate on functional and migratory properties attributed to Tcm, Tem, and Trm, and discuss how potential heterogeneity within these populations arises and persists.

Divergence of Tissue-Memory T Cells: Distribution and Function-Based Classification.

Tissue-resident memory T cells (Trm) comprise the majority of memory cells in nonlymphoid tissues and play a predominant role in immunity at barrier surfaces. A better understanding of Trm cell maintenance and function is essential for the development of vaccines that confer frontline protection. However, it is currently challenging to precisely distinguish Trm cells from other T cells, and this has led to confusion in the literature. Here we highlight gaps in our understanding of tissue memory and discuss recent advances in the classification of Trm cell subsets based on their distribution and functional characteristics.

TBL1XR1 Mutations Drive Extranodal Lymphoma by Inducing a Pro-tumorigenic Memory Fate.

The most aggressive B cell lymphomas frequently manifest extranodal distribution and carry somatic mutations in the poorly characterized gene TBL1XR1. Here, we show that TBL1XR1 mutations skew the humoral immune response toward generating abnormal immature memory B cells (MB), while impairing plasma cell differentiation. At the molecular level, TBL1XR1 mutants co-opt SMRT/HDAC3 repressor complexes toward binding the MB cell transcription factor (TF) BACH2 at the expense of the germinal center (GC) TF BCL6, leading to pre-memory transcriptional reprogramming and cell-fate bias. Upon antigen recall, TBL1XR1 mutant MB cells fail to differentiate into plasma cells and instead preferentially reenter new GC reactions, providing evidence for a cyclic reentry lymphomagenesis mechanism. Ultimately, TBL1XR1 alterations lead to a striking extranodal immunoblastic lymphoma phenotype that mimics the human disease. Both human and murine lymphomas feature expanded MB-like cell populations, consistent with a MB-cell origin and delineating an unforeseen pathway for malignant transformation of the immune system.

Still naïve or primed: Anticoccidial vaccines call for memory.

Despite decades of investigation to clarify protective mechanisms of anticoccidial responses, one crucial field is neglected, that is, protective memory responses in primed birds. Protective memory immunity is critical for host resistance to reinfection and is the basis of modern vaccinology, especially in developing successful subunit vaccines. There are important differences between the immune responses induced by infections and antigens delivered either as killed, recombinant proteins or as live, replicating vector vaccines or as DNA vaccines. Animals immunized with these vaccines may fail to develop protective memory immunity, and is still naïve to Eimeria infection. This may explain why limited success is achieved in developing next-generation anticoccidial vaccines. In this review, we try to decipher the protective memory responses against Eimeria infection, assess immune responses elicited by various anticoccidial vaccine candidates, and propose possible approaches to develop rational vaccines that can induce a protective memory response to chicken coccidiosis.

Discordant rearrangement of primary and anamnestic CD8+ T cell responses to influenza A viral epitopes upon exposure to bacterial superantigens: Implications for prophylactic vaccination, heterosubtypic immunity and superinfections.

Infection with (SAg)-producing bacteria may precede or follow infection with or vaccination against influenza A viruses (IAVs). However, how SAgs alter the breadth of IAV-specific CD8+ T cell (TCD8) responses is unknown. Moreover, whether recall responses mediating heterosubtypic immunity to IAVs are manipulated by SAgs remains unexplored. We employed wild-type (WT) and mutant bacterial SAgs, SAg-sufficient/deficient Staphylococcus aureus strains, and WT, mouse-adapted and reassortant IAV strains in multiple in vivo settings to address the above questions. Contrary to the popular view that SAgs delete or anergize T cells, systemic administration of staphylococcal enterotoxin B (SEB) or Mycoplasma arthritidis mitogen before intraperitoneal IAV immunization enlarged the clonal size of 'select' IAV-specific TCD8 and reshuffled the hierarchical pattern of primary TCD8 responses. This was mechanistically linked to the TCR Vß makeup of the impacted clones rather than their immunodominance status. Importantly, SAg-expanded TCD8 retained their IFN-γ production and cognate cytolytic capacities. The enhancing effect of SEB on immunodominant TCD8 was also evident in primary responses to vaccination with heat-inactivated and live attenuated IAV strains administered intramuscularly and intranasally, respectively. Interestingly, in prime-boost immunization settings, the outcome of SEB administration depended strictly upon the time point at which this SAg was introduced. Accordingly, SEB injection before priming raised CD127highKLRG1low memory precursor frequencies and augmented the anamnestic responses of SEB-binding TCD8. By comparison, introducing SEB before boosting diminished recall responses to IAV-derived epitopes drastically and indiscriminately. This was accompanied by lower Ki67 and higher Fas, LAG-3 and PD-1 levels consistent with a pro-apoptotic and/or exhausted phenotype. Therefore, SAgs can have contrasting impacts on anti-IAV immunity depending on the naïve/memory status and the TCR composition of exposed TCD8. Finally, local administration of SEB or infection with SEB-producing S. aureus enhanced pulmonary TCD8 responses to IAV. Our findings have clear implications for superinfections and prophylactic vaccination.

Immunological memory and tolerance at the maternal-fetal interface: Implications for reproductive management of mares.

The development of placentation that coincided with the evolution of mammals presented new challenges to the transmission of life from one generation to the next, particularly with regard to the possibility of maternal immunological recognition and destruction of the developing conceptus. The balance between immunity and tolerance dominates the immunological relationship between mother and fetus during mammalian pregnancy, and the focal point of this relationship lies at the interface between the trophoblast cells that comprise the outermost layer of the placenta and the maternal endometrial tissues. Immune memory and tolerance are two of the cardinal characteristics of the immune system. Immune memory is essential in preventing or lessening the effect of infections to the mother or conceptus, but may also be a threat to the semi-allogeneic tissues of the fetus and placenta. The mother must develop functional immune tolerance to her fetus, but at the same time retain her ability to combat infections while pregnant. To address this imperative, mammals have developed overlapping and independent mechanisms for evading maternal anti-fetal immune responses that could result in pregnancy loss. Studies of the unusual component of equine invasive trophoblast in the epitheliochorial placenta have illuminated aspects of immune memory and tolerance that have relevance to fertility in the horse and other mammalian species.

Epigenetic Memory: Lessons From iPS Cells Derived From Human ß Cells.

Incomplete reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) may be responsible for the heterogeneity in differentiation capacity observed among iPSC lines. It remains unclear whether it results from stochastic reprogramming events, or reflects consistent genetic or cell-of-origin differences. Some evidence suggests that epigenetic memory predisposes iPSCs to enhanced differentiation into the parental cell type. We investigated iPSCs reprogrammed from human pancreatic islet ß cells (BiPSCs), as a step in development of a robust differentiation protocol for generation of ß-like cells. BiPSCs derived from multiple human donors manifested enhanced and reproducible spontaneous and induced differentiation towards insulin-producing cells, compared with iPSCs derived from isogenic non-ß-cell types and fibroblast-derived iPSCs (FiPSCs). Genome-wide analyses of open chromatin in BiPSCs and FiPSCs identified thousands of differential open chromatin sites (DOCs) between the two iPSC types. DOCs more open in BiPSCs (Bi-DOCs) were significantly enriched for known regulators of endodermal development, including bivalent and weak enhancers, and FOXA2 binding sites. Bi-DOCs were associated with genes related to pancreas development and ß-cell function. These studies provide evidence for reproducible epigenetic memory in BiPSCs. Bi-DOCs may provide clues to genes and pathways involved in the differentiation process, which could be manipulated to increase the efficiency and reproducibility of differentiation of pluripotent stem cells from non-ß-cell sources.

Restricted Clonality and Limited Germinal Center Reentry Characterize Memory B Cell Reactivation by Boosting.

Repeated exposure to pathogens or their antigens triggers anamnestic antibody responses that are higher in magnitude and affinity than the primary response. These involve reengagement of memory B cell (MBC) clones, the diversity and specificity of which determine the breadth and effectiveness of the ensuing antibody response. Using prime-boost models in mice, we find that secondary responses are characterized by a clonality bottleneck that restricts the engagement of the large diversity of MBC clones generated by priming. Rediversification of mutated MBCs is infrequent within secondary germinal centers (GCs), which instead consist predominantly of B cells without prior GC experience or detectable clonal expansion. Few MBC clones, generally derived from higher-affinity germline precursors, account for the majority of secondary antibody responses, while most primary-derived clonal diversity is not reengaged detectably by boosting. Understanding how to counter this bottleneck may improve our ability to elicit antibodies to non-immunodominant epitopes by vaccination.