Generation Of Immunological Memory Research Articles

Infection-induced trained immunity: a twist in paradigm of innate host defense and generation of immunological memory.

In contrast to adaptive immunity, which relies on memory T and B cells for long-term pathogen-specific responses, trained immunity involves the enhancement of innate immune responses through cellular reprogramming. Experimental evidence from animal models and human studies supports the concept of trained immunity and its potential therapeutic applications in the development of personalized medicine. However, there remains a huge gap in understanding the mechanisms, identifying specific microbial triggers responsible for the induction of trained immunity. This underscores the importance of investigating the potential role of trained immunity in redefining host defense and highlights future research directions. This minireview will provide a comprehensive summary of the new paradigm of trained immunity or innate memory pathways. It will shed light on infection-induced pathways through non-specific stimulation within macrophages and natural killer cells, which will be further elaborated in multiple disease perspectives caused by infectious agents such as bacteria, fungi, and viruses. The article further elaborates on the biochemical and cellular basis of trained immunity and its impact on disease status during recurrent exposures. The review concludes with a perspective segment discussing potential therapeutic benefits, limitations, and future challenges in this area of study. The review also sheds light upon potential risks involved in the induction of trained immunity.

Generation of specific immune memory by bacterial exposure in the major malaria vector Anopheles arabiensis (Diptera: Culicidae)

There is a growing body of evidence that invertebrates can generate improved secondary responses after a primary challenge. This immunological memory can be primed by a range of pathogens, including bacteria. The generation of immunological memory has been demonstrated in mosquitoes, with the memory primed by a range of initial stimuli. This study aimed to examine whether insecticide resistance affects the capacity to generate immunological memory. The primary hypothesis was tested by examining the capacity of genetically related laboratory-reared Anopheles arabiensis strains that differ by insecticide resistant phenotype to generate immunological memory. The competing hypothesis tested was that the bacterial virulence was the key determinant in generating immunological memory. Immune memory was generated in F1 females but not males. Immunological memory was demonstrated in both laboratory strains, but the efficacy differed by the insecticide resistant phenotype of the strain. An initial oral challenge provided by a blood meal resulted generated better memory than an oral challenge by sugar. The efficacy of memory generation between the two bacterial strains differed between the two mosquito strains. Regardless of the challenge, the two strains differed in their capacity to generate memory. This study therefore demonstrated that insecticide resistant phenotype affected the capacity of the two strains to generate immunological memory. Although this study needs to be replicated with wild mosquitoes, it does suggest that a potential role for insecticide resistance in the functioning of the immune system and memory generation of An. arabiensis.

Design and Optimization of a Novel Proteomimetic Polymer Nanoplatform for the Co-delivery of Antigens with Adjuvants

Abstract We describe here a novel nanoplatform called the Protein-Like Polymer (PLP) with unique characteristics allowing for sustained delivery of tumor antigens in conjunction with STING agonists. Methods: A library of PLP formulations were synthesized via ROMP and characterized. Cell uptake/functional assays were conducted with payload-specific T Cells. Ability of PLPs to co-deliver adjuvants was tested by electrostatically coupling 2′3′ cGAMP, forming stable nanostructures. Results: Conjugating antigens to the polymer backbone using a cleavable disulfide linkage (reduces in APCs) resulted in increased endosomal localization and T cell proliferation/activation. Incorporating OEG side chains reduced enzymatic degradation while increasing immunogenicity and APC uptake. Increasing the density of side chains further improved vaccine efficacy and resistance to proteolysis. Antigen-PLP conjugates is effective only when paired with cells from their cognate system, demonstrating payload-specificity. Mice bearing established B16F10 tumors treated with gp100-PLPs resulted in increased survival time and reduced tumor burden with corresponding changes in immune cell profiles. Notably, STING-PLP complexes showed significantly smaller tumors at day 14 and allowed for subcutaneous administration of 2′3′ cGAMP. Studies looking at multiplexing pools of neoantigens onto one PLP and generation of immunological memory are ongoing. Conclusion: The modularity of the PLP platform allows for complex nano-architectures including systems capable of delivering challenging compounds, ie small molecule STING agonists, subcutaneously through electrostatic coupling, highlighting its potential to revolutionize cancer vaccinology. Supported by grants from the NIH (5F30CA257519-03)

Impaired tolerance to the autoantigen LL-37 in acute coronary syndrome.

LL-37 is the only member of the cathelicidin family of antimicrobial peptides in humans and is an autoantigen in several autoimmune diseases and in acute coronary syndrome (ACS). In this report, we profiled the specific T cell response to the autoimmune self-antigen LL-37 and investigated the factors modulating the response in peripheral blood mononuclear cells (PBMCs) of healthy subjects and ACS patients. The activation induced marker (AIM) assay demonstrated differential T cell profiles characterized by the persistence of CD134 and CD137, markers that impair tolerance and promote immune effector and memory response, in ACS compared to Controls. Specifically, CD8+CD69+CD137+ T cells were significantly increased by LL-37 stimulation in ACS PBMCs. T effector cell response to LL-37 were either HLA dependent or independent as determined by blocking with monoclonal antibody to either Class-I HLA or Class-II HLA. Blocking of immune checkpoints PD-1 and CTLA-4 demonstrated the control of self-reactive T cell response to LL-37 was modulated predominantly by CTLA-4. Platelets from healthy controls down-modulated CD8+CD69+CD137+ T cell response to LL-37 in autologous PBMCs. CD8+CD69+CD137+ T cell AIM profile negatively correlated with platelet count in ACS patients. Our report demonstrates that the immune response to the autoantigen LL-37 in ACS patients is characterized specifically by CD8+CD69+CD137+ T cell AIM profile with persistent T cell activation and the generation of immunologic memory. The results provide potentially novel insight into mechanistic pathways of antigen-specific immune signaling in ACS.

Abstract 1796: PD-L1 photoimmunotherapy kills immunosuppressive myeloid cells to activate local and systemic antitumor immunity in syngeneic mouse models

Abstract Introduction: Photoimmunotherapy is an investigational anticancer treatment platform that combines the cell surface binding of an antibody conjugated to a light activatable dye (IRDye® 700DX, IR700) with non-thermal red light illumination for selective cell killing. PD-L1, the target of anti-PD-L1 inhibitors, is a suppressive checkpoint marker expressed on tumor cells and immunosuppressive myeloid cells in the tumor microenvironment. Here we examined the anticancer activity and immune activation elicited by anti-PD-L1-IR700 photoimmunotherapy (PD-L1 photoimmunotherapy). Methods: CT26 or LL/2 tumors were used to assess antitumor immune response following PD-L1 photoimmunotherapy. CT26PD-L1-/- tumors were generated by CRISPR/Cas9. Tumor volume was determined by caliper measurements in illuminated and non-illuminated tumors. Intratumoral immune responses were assessed by flow cytometry. Complete responder (CR) mice were challenged with either CT26 or 4T1 tumors to evaluate immune memory. Results: Mice bearing CT26 or LL/2 tumors treated with PD-L1 photoimmunotherapy exhibited a notable reduction of tumor growth compared to mice that received control treatments (saline, anti-PD-L1-IR700 conjugate alone without illumination, or multi-dosing with anti-PD-L1 antibody). Pre-treatment depletion of CD8+ T cells in the mice abrogated the antitumor activity of PD-L1 photoimmunotherapy, demonstrating a key role of CD8+ T-cell effector activity in the responses. PD-L1 photoimmunotherapy induced CRs in 7/15 mice, and all CR mice rejected CT26 tumor growth after re-challenge, indicating the generation of immunological memory. Furthermore, 6/8 mice rejected inoculation of 4T1 tumors, suggesting an enhanced ability to prime new T cells with tumor neoantigens. Intratumoral immune cell analysis showed a reduction of myeloid cells 2 hours following illumination, an increase of CD103+ dendritic cells 2 days following illumination, and an increase of non-exhausted PD-1-CD8+ T effector cells 8 days after illumination. In a bilateral tumor model, PD-L1 photoimmunotherapy resulted in tumor reduction in the non-illuminated tumor. Mice bearing CT26PD-L1-/- tumor cells similarly exhibited tumor reduction after PD-L1 photoimmunotherapy, suggesting that antitumor activity resulted from the elimination of PD-L1+ non-tumor cells. Conclusions: PD-L1photoimmunotherapy induces anticancer responses by killing PD-L1+ myeloid cells and possibly PD-L1+ cancer cells within the tumor, resulting in augmentation of local and systemic antitumor immunity. These results indicate that PD-L1 photoimmunotherapy can elicit anticancer immune responses in target and distal tumors, including syngeneic mouse models refractory to checkpoint inhibition. Citation Format: Amy H. Thorne, Michelle H. Hsu, Daniel Mendoza, Jason Lapetoda, Christopher M. Parry, Jerry J. Fong, Miguel Garcia-Guzman. PD-L1 photoimmunotherapy kills immunosuppressive myeloid cells to activate local and systemic antitumor immunity in syngeneic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1796.

Anticancer immunity induced by a synthetic tumor-targeted CD137 agonist

BackgroundIn contrast to immune checkpoint inhibitors, the use of antibodies as agonists of immune costimulatory receptors as cancer therapeutics has largely failed. We sought to address this problem using a...

To Remember or to Forget: The Role of Good and Bad Memories in Adoptive T Cell Therapy for Tumors.

The generation of immunological memory is a hallmark of adaptive immunity by which the immune system “remembers” a previous encounter with an antigen expressed by pathogens, tumors, or normal tissues; and, upon secondary encounters, mounts faster and more effective recall responses. The establishment of T cell memory is influenced by both cell-intrinsic and cell-extrinsic factors, including genetic, epigenetic and environmental triggers. Our current knowledge of the mechanisms involved in memory T cell differentiation has instructed new opportunities to engineer T cells with enhanced anti-tumor activity. The development of adoptive T cell therapy has emerged as a powerful approach to cure a subset of patients with advanced cancers. Efficacy of this approach often requires long-term persistence of transferred T cell products, which can vary according to their origin and manufacturing conditions. Host preconditioning and post-transfer supporting strategies have shown to promote their engraftment and survival by limiting the competition with a hostile tumor microenvironment and between pre-existing immune cell subsets. Although in the general view pre-existing memory can confer a selective advantage to adoptive T cell therapy, here we propose that also “bad memories”—in the form of antigen-experienced T cell subsets—co-evolve with consequences on newly transferred lymphocytes. In this review, we will first provide an overview of selected features of memory T cell subsets and, then, discuss their putative implications for adoptive T cell therapy.

An Integrated Multi-omic Single-Cell Atlas of Human B Cell Identity

SummaryB cells are capable of a wide range of effector functions including antibody secretion, antigen presentation, cytokine production, and generation of immunological memory. A consistent strategy for classifying human B cells by using surface molecules is essential to harness this functional diversity for clinical translation. We developed a highly multiplexed screen to quantify the co-expression of 351 surface molecules on millions of human B cells. We identified differentially expressed molecules and aligned their variance with isotype usage, VDJ sequence, metabolic profile, biosynthesis activity, and signaling response. Based on these analyses, we propose a classification scheme to segregate B cells from four lymphoid tissues into twelve unique subsets, including a CD45RB+CD27− early memory population, a class-switched CD39+ tonsil-resident population, and a CD19hiCD11c+ memory population that potently responds to immune activation. This classification framework and underlying datasets provide a resource for further investigations of human B cell identity and function.

T cell help to B cells: Cognate and atypical interactions in peripheral and intestinal lymphoid tissues.

Enduring immunity against harmful pathogens depends on the generation of immunological memory. Serum immunoglobulins are constantly secreted by long-lived antibody-producing cells, which provide extended protection from recurrent exposures. These cells originate mainly from germinal center structures, wherein B cells introduce mutations to their immunoglobulin genes followed by affinity-based selection. Generation of high-affinity antibodies relies on physical contacts between T and B cells, a process that facilitates the delivery of fate decision signals. T-B cellular engagements are mediated through interactions between the T cell receptor and its cognate peptide presented on B cell major histocompatibility class II molecules. Here, we describe the cellular and molecular aspects of these cognate T-B interactions, and highlight exceptional cases, especially those arising at intestinal lymphoid organs, at which T cells provide help to B cells in an atypical manner, independent of T cell specificity.

Goodbye SIRS? Innate, trained and adaptive immunity and pathogenesis of organ dysfunction.

The novel concepts within Sepsis‑3 criteria include afocus on dysregulated host responses, removal of the systemic inflammation response syndrome (SIRS) criteria from sepsis diagnosis, the use of Sepsis-related (Sequential) Organ Failure Assessment (SOFA) scores to define organ dysfunction, and the explicit recognition of the septic shock as asubset of sepsis. Protection against infection requires asurveillance system, an effector response against "perceived" pathogens, amethod for regaining immune homeostasis following an immune response, and generation of immunological memory. In comparison to normally regulated responses to infection, the innate immune system shows profoundly abnormal neutrophil and macrophage function. Similarly, the adaptive immune system is typically depleted numerically of lymphocytes and functionally with Tand Bcell exhaustion. Although there are numerous proposed mechanisms by which these dysregulated immune responses may be associated with organ failure, it is unclear what the unifying organ failure mechanisms in sepsis are. Furthermore, in sepsis survivors, the epigenetic changes on immune cells and widespread changes to lymphocyte populations may increase the risk of adverse events such as rehospitalisation and mortality. Finally, our current gaps in understanding of the immune response trajectory and the associated modifiable mechanisms in sepsis leave us along way from successful immunomodulation for these patients. This article is freely available.

Delivery of 5'-triphosphate RNA with endosomolytic nanoparticles potently activates RIG-I to improve cancer immunotherapy.

RNA agonists of the retinoic acid gene I (RIG-I) pathway have recently emerged as a promising class of cancer immunotherapeutics, but their efficacy is hindered by drug delivery barriers, including nuclease degradation, poor intracellular uptake, and minimal access to the cytosol where RIG-I is localized. Here, we explore the application of pH-responsive, endosomolytic polymer nanoparticles (NPs) to enhance the cytosolic delivery and immunostimulatory activity of synthetic 5' triphosphate, short, double-stranded RNA (3pRNA), a ligand for RIG-I. Delivery of 3pRNA with pH-responsive NPs with an active endosomal escape mechanism, but not control carriers lacking endosomolytic activity, significantly increased the activity of 3pRNA in dendritic cells, macrophages, and cancer cell lines. In a CT26 colon cancer model, activation of RIG-I via NP delivery of 3pRNA induced immunogenic cell death, triggered expression of type I interferon and pro-inflammatory cytokines, and increased CD8+ T cell infiltration into the tumor microenvironment. Consequently, intratumoral (IT) delivery of NPs loaded with 3pRNA inhibited CT26 tumor growth and enhanced the therapeutic efficacy of anti-PD-1 immune checkpoint blockade, resulting in a 30% complete response rate and generation of immunological memory that protected against tumor rechallenge. Collectively, these studies demonstrate that pH-responsive NPs can be harnessed to strongly enhance the immunostimulatory activity and therapeutic efficacy of 3pRNA and establish endosomal escape as a critical parameter in the design of carriers for immunotherapeutic targeting of the RIG-I pathway.

Abstract 4586: Intratumoral Treg cell depletion by local administration of IL-2-Diphteria toxin fusion protein E7777 induces a therapeutic and memory anti-tumor immune response in preclinical models

Abstract T regulatory (Treg) cells play an important role in maintaining immunological tolerance to self-antigens, thus limiting immune responses to tumor antigens. Therefore, depleting or suppressing Tregs is one strategy by which anti-tumor immunity can be restored. The immunotoxin ONTAK® is an IL-2-diphteria toxin fusion protein that has been shown to diminish Tregs in patients and animal models of cancer in peripheral blood using a systemic intravenous (i.v.) administration route. E7777 is a new version of ONTAK®. In this study we tested the hypothesis that locally-diminished Tregs by intratumoral (i.t.) administration of E7777 generate effective anti-tumor immune response at both local and systemic levels. First, we showed superior anti-tumor activity and safety of E7777 i.t. over E7777 i.v., where i.t. administration resulted in complete tumor regressions in both moderately immunogenic CT26 and non-immunogenic B16F10 tumors with minimal animal body weight loss. In contrast, only tumor growth delay was observed for E7777 i.v. with dose-limiting animal body weight reduction in the same models. Immune phenotyping showed a 4 fold reduction of intratumoral Tregs in treated CT-26 tumors without significant change of Tregs in the spleens of treated animals, confirming a local Treg-depleting effect of E7777 i.t. In contrast, intratumoral CD8+ T cells were not reduced. Second, E7777 i.t. enhanced overall anti-tumor immune response, manifested by significantly increased numbers of CD45+ hematopoietic cells, Granzyme B+ CD8+ cytotoxic T cells, and ratios of cytotoxic T cells/Tregs in the treated tumors. Importantly, E7777 i.t. also resulted in distant effects in the spleen characterized by increased ratio of T lymphocytes to myeloid cells and increased frequencies of both effector memory CD8+ T cells (CD8+CD62L-CD44+) and central memory CD8+ T cells (CD8+CD62L+CD44+) indicative of systemic immune activation. Consistent with the generation of immunological memory, 60% of the tumor-free animals treated with E7777 i.t. rejected completely and 40% displayed delayed tumor growth of B16F10 cell challenge while all naïve control animals grew tumors. Taken together, our results demonstrate that intratumoral Treg depletion by local administration of E7777 leads to an effective local and memory anti-tumor response in preclinical models and support further evaluation of local E7777 delivery as a cancer immunotherapy. Citation Format: Diana I. Albu, Christy Ingersoll, Kuan-Chun Huang, Mary Woodall-Jappe, Xingfeng Bao. Intratumoral Treg cell depletion by local administration of IL-2-Diphteria toxin fusion protein E7777 induces a therapeutic and memory anti-tumor immune response in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4586. doi:10.1158/1538-7445.AM2017-4586

Abstract 5673: Large established B16 tumors in mice are eradicated by ZVex® (dendritic cell-targeting lentiviral vector) and G100 (TLR4 agonist) combination immunotherapy through increasing tumor-infiltrating effector T cells and inducing antigen spreading

Abstract INTRODUCTION: Effective immunotherapy requires the presence of effector T cells penetrating the tumor. ZVex is a hybrid lentiviral vector platform that targets dendritic cells in vivo to express genes of tumor-associated antigen (TAA)-of-interest and activate TAA-specific CD8 T cells. G100 is the intratumoral (IT) injection of formulated glucopyranosyl lipid A (a synthetic TLR4 agonist) and has been shown to induce T cell homing chemokines, CXCL9 and CXCL10. We report here that G100 promoted an inflamed tumor microenvironment (TME) and improved infiltration of ZVex-induced TAA-specific CD8 T cells to the TME, thereby eradicating large established B16 tumors. This was previously achieved only with a complex vaccine/anti-tumor antibody/checkpoint inhibitor/IL-2 regimen (Moynihan, Nature, 2016). RESULTS: B16-OVA tumor-bearing mice were randomized into 4 treatment cohorts: 1) untreated; 2) ZVex expressing ovalbumin (ZVex/OVA), subcutaneously (SC); 3) G100 (IT); 4) ZVex/OVA (SC) and G100 (IT) combination. While mice in Cohorts 2 and 3 exhibited delayed tumor growth, nearly all mice (16/18) from Cohort 4 had completely regressed tumors and survived tumor-free until end of study (109 days). CD8 T cell depletion abrogated this anti-tumor response. Cohort 4 mice with regressed tumors were then randomized and re-challenged with either a) B16-OVA or b) parental B16 (lacking expression of the ZVex-targeted antigen, OVA): a) B16-OVA re-challenge was rejected by 100% of recipient mice - consistent with induction of T cell memory; and b) B16 re-challenge was rejected by 30%-50% of recipient mice - demonstrating functional antigen spreading. T cell receptor deep sequencing showed that ZVex/OVA alone increased tumor-infiltrating T cell clones specific for OVA, validated by pMHC-multimer staining. ZVex/OVA and G100 combination expanded additional T cell clones, further evident of antigen spreading. Lastly, the TME of Cohort 4 mice showed the most profound pro-inflammatory changes, as assessed by RNA transcriptional profiling. CONCLUSIONS: These data collectively demonstrate that anti-tumor efficacy observed in Cohort 4 mice was mediated largely by ZVex/OVA-induced effector T cells and that TME modulation with G100 drastically shifted the TME to a more inflamed milieu, promoting T cell proliferation, antigen spreading, and generation of immunological memory. To our knowledge, this is the first time that large established B16 tumors have been completely eradicated using a combination of systemic and in situ immunizations, a potentially effective strategy to convert non-inflamed tumors to inflamed tumors. Both ZVex and G100 are in multiple clinical trials, and their combination is currently being investigated in a phase I trial in soft tissue sarcoma patients with injectable tumors. Citation Format: Tina C. Albershardt, Andrea J. Parsons, Jardin Leleux, Peter Berglund, Jan ter Meulen. Large established B16 tumors in mice are eradicated by ZVex® (dendritic cell-targeting lentiviral vector) and G100 (TLR4 agonist) combination immunotherapy through increasing tumor-infiltrating effector T cells and inducing antigen spreading [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5673. doi:10.1158/1538-7445.AM2017-5673

Frontiers in Live Bone Imaging Researches. In vivo imaging of immune tissues

In vivo imaging analysis of the immune tissues, especially secondary lymphoid tissues such as lymph nodes, has greatly increased our understanding of how immune responses are promoted and regulated by immune cell trafficking. Recently, in vivo tracking of follicular helper T (Tfh) cells, a vital T cell subset for B cell responses to produce antibodies, by imaging analysis and light-induced cell labeling not only revealed their migration dynamics, but also provided new insights into how Tfh cells may be involved in the generation of immunological memory.

Editorial: Activation, functions, and generation of immunological memory in γδ T lymphocytes: lessons from nonhuman primates

Discussion of how HMBPP-deficient Listeria mutant in macaques elicits lower pulmonary trafficking of Vγ9Vδ2 T cells, as compared to the HMBPP-producing strain.

Follicular helper T‐cell memory: establishing new frontiers during antibody response

The generation of immunological memory during an immune response is a hallmark of the adaptive immune system. Follicular helper T (Tfh) cells are a CD4(+) T-cell subset specialised to regulate antibody response. Emerging evidence suggests that during antibody response, Tfh memory is generated along with the generation of B-cell memory. There are multiple layers for the differentiation and function of memory Tfh cells. Both early committed precursor Tfh cells and effector Tfh cells exiting germinal centres can contribute to the memory Tfh pool. Functionally, memory Tfh cells not only enhance a secondary response upon antigen rechallenge but also circulate to non-draining lymph tissues to differentiate into effector Tfh cells in the face of systemic antigen/pathogen spreading, thus also promoting a primary response. Circulating memory Tfh cells are a valuable marker to monitor the Tfh programme in human autoimmune diseases, infections and vaccinations. Future studies are required to understand the molecular mechanisms determining the commitment and plasticity of Tfh memory and hence the physiological functions of Tfh memory.

English

Introduction Inflammation is triggered when innate immune cells detect microbial infection or tissue damage. Surveillance mecha­ nisms involve pattern recognition receptors on the cell surface and in the cytoplasm. Most pattern recognition receptors respond to pathogen­ associated molecular patterns or host­derived damage­associated mole­ cular patterns by triggering activation of various transcription factors. Induction of cytokines promotes the activation and recruitment of leukocytes, which are critical for elim­ inating pathogens and host debris. In order to avoid immunopathology, the system is very tightly regulated by numerous molecules that limit the magnitude and duration of the inflammatory response. In this review we present current knowledge on pathogen recognition through diff­ erent pattern recognition receptors and the complex signalling pathways responsible for activation of inflam­ matory and antimicrobial responses. Conclusion There are still many unresolved questions, such as the exact nature of the molecular events leading to diff­ erent immune receptor activation and also identity of some unknown ligands for the receptors. Unravelling of these will offer insight into what critical components might be target­ ed for better therapeutic benefits in inflammatory disorders. Introduction The role of inflammatory response is to combat infection and tissue injury. The innate immune system constitutes the first line of host defence during infection and therefore plays a very crucial role in the early recognition and subsequent trigger­ ing of a pro-inflammatory response to invading pathogens1. The adaptive immune system, on the other hand, is responsible for elimination of pathogens during the late phase of infection and in the generation of immunological memory. The innate immune response is mediated pri­ marily by phagocytic cells and antigen­presenting cells (APCs), such as macrophages, and dendritic cells (DCs), and has been regarded as relatively non-specific2, whereas the adaptive immune response is charac­ terized by antigen-specific receptors on lymphocytes generated by clonal gene rearrangements. Innate immune cells of tissues such as macrophages, fibroblasts and dendritic cells, as well as circulating leukocytes, recognize pathogen inva­ sion or cellular damage with pattern recognition receptors (PRRs). These receptors detect pathogen­associated molecular patterns (PAMPs), such as pathogen­derived nucleic acids and cell wall components, fungal β-glucan, bacterial flagellin, and lipopolysaccharide (LPS) from Gram­ negative bacteria. PRRs also recog­ nize damage­associated molecular patterns (DAMPs), released from injured cells during apoptosis or necrosis. DAMPs include uric acid, ATP, and the DNA­binding nuclear protein HMGB1 and amyloid β fibrils. Activated PRRs then initiate signal­ ling cascades to trigger the release of factors that promote recruitment of leukocytes to the infected region. In this review, we look into the inflammatory signalling response emanating from the recognition of microbial infection and cellular injury by PRRs. Among PRRs, membrane­bound toll­like receptors (TLRs) play a cen­ tral role in the initiation of immune response against pathogen invasion. However, other PRRs are also involved—including membrane­bound C­type lectin receptors (CLRs), cyto­ solic proteins such as NOD­like recep­ tors (NLRs) and RIG­I­like receptors (RLRs). Among these receptor types, TLRs and RLRs are primarily important for the production of type I interfer­ ons (IFNs), whereas NLRs are known to regulate interleukin-1β (IL-1β) production through activation of caspase­1. Discussion Toll-like receptors TLRs were the first PRRs to be iden­ tified. They are also the most well characterized and recognize a wide range of PAMPs3–5. TLRs are trans­ membrane proteins which comprise an ectodomain, which contains leucine­rich repeats that mediate the recognition of PAMPs, a transmem­ brane region, and cytosolic Toll­IL­1 receptor (TIR) domains that activate downstream signalling pathways. TLRs are expressed either on the cell surface or on intracellular vesicles. To date, 10 and 12 functional TLRs have been identified in human and mice, respectively. Each TLR detects distinct PAMPs from bacteria, viruses, fungi and parasites. Upon recognition of respective PAMPs, TLRs recruit a specific set of adaptor molecules that have TIR doma ins, such as MyD88 and TRIF, and initiate downstream signalling

Macropinocytosis is responsible for the uptake of pathogenic and non-pathogenic mycobacteria by B lymphocytes (Raji cells)

BackgroundThe classical roles of B cells include the production of antibodies and cytokines and the generation of immunological memory, these being key factors in the adaptive immune response. However, their role in innate immunity is currently being recognised. Traditionally, B cells have been considered non-phagocytic cells; therefore, the uptake of bacteria by B cells is not extensively documented. In this study, we analysed some of the features of non-specific bacterial uptake by B lymphocytes from the Raji cell line. In our model, B cells were infected with Mycobacterium tuberculosis (MTB), Mycobacterium smegmatis (MSM), and Salmonella typhimurium (ST).ResultsOur observations revealed that the Raji B cells were readily infected by the three bacteria that were studied. All of the infections induced changes in the cellular membrane during bacterial internalisation. M. smegmatis and S. typhimurium were able to induce important membrane changes that were characterised by abundant filopodia and lamellipodia formation. These membrane changes were driven by actin cytoskeletal rearrangements. The intracellular growth of these bacteria was also controlled by B cells. M. tuberculosis infection also induced actin rearrangement-driven membrane changes; however, the B cells were not able to control this infection. The phorbol 12-myristate 13-acetate (PMA) treatment of B cells induced filopodia and lamellipodia formation, the production of spacious vacuoles (macropinosomes), and the fluid-phase uptake that is characteristic of macropinocytosis. S. typhimurium infection induced the highest fluid-phase uptake, although both mycobacteria also induced fluid uptake. A macropinocytosis inhibitor such as amiloride was used and abolished the bacterial uptake and the fluid-phase uptake that is triggered during the bacterial infection.ConclusionsRaji B cells can internalise S. typhimurium and mycobacteria through an active process, such as macropinocytosis, although the resolution of the infection depends on factors that are inherent in the virulence of each pathogen.

Heterogeneity within T Cell Memory: Implications for Transplant Tolerance

Adaptive immunity in both mouse and man results in the generation of immunological memory. Memory T cells are both friend and foe to transplant recipients, as they are intimately involved and in many cases absolutely required for the maintenance of protective immunity in the face immunosuppression, yet from the evidence presented herein they clearly constitute a formidable barrier for the successful implementation of tolerance induction strategies in transplantation. This review describes the experimental evidence demonstrating the increased resistance of memory T cells to many distinct tolerance induction strategies, and outlines recent advances in our knowledge of the ways in which alloreactive memory T cells arise in previously untransplanted individuals. Understanding the impact of alloreactive memory T cell specificity, frequency, and quality might allow for better donor selection in order to minimize the donor-reactive memory T cell barrier in an individual transplant recipient, thus allowing stratification of relative risk of alloreactive memory T cell mediated rejection, and conversely increase the likelihood of successful establishment of tolerance. However, further research into the molecular and cellular pathways involved in alloreactive memory T cell-mediated rejection is required in order to design new strategies to overcome the memory T cell barrier, without critically impairing protective immunity.

Ocular Surface APCs Are Necessary for Autoreactive T Cell-Mediated Experimental Autoimmune Lacrimal Keratoconjunctivitis

As specialized sentinels between the innate and adaptive immune response, APCs are essential for activation of Ag-specific lymphocytes, pathogen clearance, and generation of immunological memory. The process is tightly regulated; however, excessive or atypical stimuli may ignite activation of APCs in a way that allows self-Ag presentation to autoreactive T cells in the context of the necessary costimulatory signals, ultimately resulting in autoimmunity. Studies in both animal models and patients suggest that dry eye is a chronic CD4(+) T cell-mediated ocular surface autoimmune-based inflammatory disease. Using a desiccating stress-induced mouse model of dry eye, we establish the fundamental role of APCs for both the generation and maintenance of ocular-specific autoreactive CD4(+) T cells. Subconjunctival administration of liposome-encapsulated clodronate efficiently diminished resident ocular surface APCs, inhibited the generation of autoreactive CD4(+) T cells, and blocked their ability to cause disease. APC-dependent CD4(+) T cell activation required intact draining cervical lymph nodes, as cervical lymphadenectomy also inhibited CD4(+) T cell-mediated dry eye disease. In addition, local depletion of peripheral conjunctival APCs blocked the ability of dry eye-specific CD4(+) T cells to accumulate within the ocular surface tissues, suggesting that fully primed and targeted dry eye-specific CD4(+) T cells require secondary activation by resident ocular surface APCs for maintenance and effector function. These data demonstrate that APCs are necessary for the initiation and development of experimental dry eye and support the standing hypothesis that dry eye is a self-Ag-driven autoimmune disease.