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Introduction: Pre-erythrocytic malaria vaccines hold the promise of inducing sterile protection thereby preventing the morbidity and mortality associated with Plasmodium infection. The main surface antigen of P. falciparum sporozoites, i.e., the circumsporozoite protein (CSP), has been extensively explored as a target of such vaccines with significant success in recent years. Systematic adjuvant selection, refinements of the immunization regimen, and physical properties of the antigen may all contribute to the potential of increasing the efficacy of CSPbased vaccines. Protection appears to be dependent in large part on CSP antibodies. However due to a knowledge gap related to the exact correlates of immunity, there is a critical need to improve our ability to down select candidates preclinically before entering clinical trials including with controlled human malaria infections (CHMI). Methods: We developed a novel multiplex competition assay based on wellcharacterized monoclonal antibodies (mAbs) that target crucial epitopes across the CSP molecule. This new tool assesses both, quality and epitope-specific concentrations of vaccine-induced antibodies by measuring their equivalency with a panel of well-characterized, CSP-epitope-specific mAbs. Results: Applying this method to RTS,S-immune sera from a CHMI trial demonstrated a quantitative epitope-specificity profile of antibody responses that can differentiate between protected vs. nonprotected individuals. Aligning vaccine efficacy with quantitation of the epitope fine specificity results of this equivalency assay reveals the importance of epitope specificity. Discussion: The newly developed serological equivalence assay will inform future vaccine design and possibly even adjuvant selection. This methodology can be adapted to other antigens and disease models, when a panel of relevant mAbs exists, and could offer a unique tool for comparing and down-selecting vaccine formulations. [ABSTRACT FROM AUTHOR]

IntroductionPre-erythrocytic malaria vaccines hold the promise of inducing sterile protection thereby preventing the morbidity and mortality associated with Plasmodium infection. The main surface antigen of P. falciparum sporozoites, i.e., the circumsporozoite protein (CSP), has been extensively explored as a target of such vaccines with significant success in recent years. Systematic adjuvant selection, refinements of the immunization regimen, and physical properties of the antigen may all contribute to the potential of increasing the efficacy of CSP-based vaccines. Protection appears to be dependent in large part on CSP antibodies. However due to a knowledge gap related to the exact correlates of immunity, there is a critical need to improve our ability to down select candidates preclinically before entering clinical trials including with controlled human malaria infections (CHMI).MethodsWe developed a novel multiplex competition assay based on well-characterized monoclonal antibodies (mAbs) that target crucial epitopes across the CSP molecule. This new tool assesses both, quality and epitope-specific concentrations of vaccine-induced antibodies by measuring their equivalency with a panel of well-characterized, CSP-epitope-specific mAbs.ResultsApplying this method to RTS,S-immune sera from a CHMI trial demonstrated a quantitative epitope-specificity profile of antibody responses that can differentiate between protected vs. nonprotected individuals. Aligning vaccine efficacy with quantitation of the epitope fine specificity results of this equivalency assay reveals the importance of epitope specificity.DiscussionThe newly developed serological equivalence assay will inform future vaccine design and possibly even adjuvant selection. This methodology can be adapted to other antigens and disease models, when a panel of relevant mAbs exists, and could offer a unique tool for comparing and down-selecting vaccine formulations.

Biomaterials allow for the precision control over the combination and release of cargo needed to engineer cell outcomes. These capabilities are particularly attractive as new candidate therapies to treat autoimmune diseases, conditions where dysfunctional immune cells create pathogenic tissue environments during attack of self-molecules termed self-antigens. Here we extend past studies showing combinations of a small molecule immunomodulator co-delivered with self-antigen induces antigen-specific regulatory T cells. In particular, we sought to elucidate how different ratios of these components loaded in degradable polymer particles shape the antigen presenting cell (APC) -T cell interactions that drive differentiation of T cells toward either inflammatory or regulatory phenotypes. Using rapamycin (rapa) as a modulatory cue and myelin self-peptide (myelin oligodendrocyte glycoprotein- MOG) – self-antigen attacked during multiple sclerosis (MS), we integrate these components into polymer particles over a range of ratios and concentrations without altering the physicochemical properties of the particles. Using primary cell co-cultures, we show that while all ratios of rapa:MOG significantly decreased expression of co-stimulation molecules on dendritic cells (DCs), these levels were insensitive to the specific ratio. During co-culture with primary T cell receptor transgenic T cells, we demonstrate that the ratio of rapa:MOG controls the expansion and differentiation of these cells. In particular, at shorter time points, higher ratios induce regulatory T cells most efficiently, while at longer time points the processes are not sensitive to the specific ratio. We also found corresponding changes in gene expression and inflammatory cytokine secretion during these times. The in vitro results in this study contribute to in vitro regulatory T cell expansion techniques, as well as provide insight into future studies to explore other modulatory effects of rapa such as induction of maintenance or survival cues.

Antigen-experienced memory CD4+ T cells are the major target of HIV infection and support both productive and latent infections, thus playing a key role in HIV dissemination and persistence, respectively. Here, we reviewed studies that have shown direct association between HIV infection and antigen specificity. During untreated infection, some HIV-specific cells host productive infection, while other pathogen-specific cells such as cytomegalovirus (CMV) and Mycobacterium tuberculosis also contribute to viral persistence on antiretroviral therapy (ART). These patterns could be explained by phenotypic features differing between these pathogen-specific cells. Mechanisms involved in these preferential infection and selection processes include HIV entry and restriction, cell exhaustion, survival, self-renewal and immune escape. For instance, MIP-1β expressing cells such as CMV-specific memory cells were shown to resist infection by HIV CCR5 coreceptor downregulation/inhibition. Conversely, HIV-infected CMV-specific cells undergo clonal expansion during ART. We have identified several research areas that need further focus such as the role of other pathogens, viral genome intactness, inducibility and phenotypic features. However, given the sheer diversity of both the CD4+ T cell repertoire and antigenic history of each individual, studying HIV-infected, antigen-experienced cells still imposes numerous challenges.

Antigen-specific T cells play a central role in the adaptive immune response and come in a wide range of phenotypes. T cell receptors (TCRs) mediate the antigen-specificities found in T cells. Importantly, high-throughput TCR sequencing provides a fingerprint which allows tracking of specific T cells and their clonal expansion in response to particular antigens. As a result, many studies have leveraged TCR sequencing in an attempt to elucidate the role of antigen-specific T cells in various contexts. Here, we discuss the published approaches to studying antigen-specific T cells and their specific TCR repertoire. Further, we discuss how these methods have been applied to study the TCR repertoire in various diseases in order to characterize the antigen-specific T cells involved in the immune control of disease. [ABSTRACT FROM AUTHOR]

Antigen-specific T cells play a central role in the adaptive immune response and come in a wide range of phenotypes. T cell receptors (TCRs) mediate the antigen-specificities found in T cells. Importantly, high-throughput TCR sequencing provides a fingerprint which allows tracking of specific T cells and their clonal expansion in response to particular antigens. As a result, many studies have leveraged TCR sequencing in an attempt to elucidate the role of antigen-specific T cells in various contexts. Here, we discuss the published approaches to studying antigen-specific T cells and their specific TCR repertoire. Further, we discuss how these methods have been applied to study the TCR repertoire in various diseases in order to characterize the antigen-specific T cells involved in the immune control of disease.

Common flow cytometry-based methods used for functional assessment of antigen-specific T cells rely on de novo expression of intracellular cytokines or cell surface activation induced markers. They come with some limitations such as complex experimental setting, loss of cell viability and often high unspecific background which impairs assay sensitivity. We have previously shown that staining of activated ß2-integrins either with multimers of their ligand ICAM-1 or with a monoclonal antibody can serve as a functional marker detectable on T cells after minutes (CD8+) or few hours (CD4+) of activation. Here, we present a simple method for detection of activated ß2-integrins in combination with established cell surface activation induced markers. We observed that activated ß2-integrins were still detectable after 14 hours of stimulation, allowing their detection together with CD137 and CD154. Combinatorial gating of cells expressing activated ß2-integrins and CD137 or CD154 reduced background in unstimulated samples, increasing the signal-to-noise ratio and allowing improved assessment of low-frequency T cell responses. Extracellular staining of these markers highly correlated with production of intracellular cytokines IL-2, TNF or IFNg in CD4+ and CD8+ T cells. As an exemplary application, SARS-CoV-2 spike-specific T cell responses were assessed in individuals after COVID-19 vaccination. This method should be useful for epitope discovery projects and for the simultaneous monitoring of lowfrequency antigen-specific CD4+ and CD8+ T cell responses in various physiological situations. [ABSTRACT FROM AUTHOR]

Common flow cytometry-based methods used for functional assessment of antigen-specific T cells rely on de novo expression of intracellular cytokines or cell surface activation induced markers. They come with some limitations such as complex experimental setting, loss of cell viability and often high unspecific background which impairs assay sensitivity. We have previously shown that staining of activated ß2-integrins either with multimers of their ligand ICAM-1 or with a monoclonal antibody can serve as a functional marker detectable on T cells after minutes (CD8+) or few hours (CD4+) of activation. Here, we present a simple method for detection of activated ß2-integrins in combination with established cell surface activation induced markers. We observed that activated ß2-integrins were still detectable after 14 hours of stimulation, allowing their detection together with CD137 and CD154. Combinatorial gating of cells expressing activated ß2-integrins and CD137 or CD154 reduced background in unstimulated samples, increasing the signal-to-noise ratio and allowing improved assessment of low-frequency T cell responses. Extracellular staining of these markers highly correlated with production of intracellular cytokines IL-2, TNF or IFNγ in CD4+ and CD8+ T cells. As an exemplary application, SARS-CoV-2 spike-specific T cell responses were assessed in individuals after COVID-19 vaccination. This method should be useful for epitope discovery projects and for the simultaneous monitoring of low-frequency antigen-specific CD4+ and CD8+ T cell responses in various physiological situations.

Abstract Backgound Although Myeloid-derived suppressor cells (MDSCs) have a prominent ability to suppress the immune responses of T lymphocytes and propel tumor immune escape, a lack of profound systemic immunesuppression in tumor-bearing mice and tumor patients. The underlying mechanism of these remains unclear. Methods For this purpose, renal cancer-derived exosomes (RDEs) were first labeled with PKH67 and been observed the internalization by MDSCs. Flow cytometry analysis showed the proportion and activity change of MDSCs in spleen and bone marrow induced by RDEs. Further, western blot experiments were used to verify triggered mechanism of MDSCs by RDEs. Finally, proliferation and cytotoxicity of cytotoxic T lymphocytes (CTLs) co-cultured with MDSCs in vitro and a series of experiments in vivo were performed to demonstrate the specific inhibitory effect of RDEs-induced MDSCs. Results This study suggested that RDEs crucially contributed to presenting antigenic information, activating and driving specific immunosuppressive effect to MDSCs. HSP70, which is highly expressed in RDEs, initiate this process in a toll like receptor 2 (TLR2)-dependent manner. Importantly, RDEs-induced MDSCs could exert an antigen-specific immunosuppression effect on CTL and specific promote renal tumors-growth and immune escape in consequence. Conclusion The immunosuppression mediated by MDSCs which is induced by RDEs is antigen-specific. HSP70, which is highly expressed in RDEs, plays a pivotal role in this process. Targeted abrogating the function of MDSCs, or eliminating the expression of HSP70 in exosomes, or blocking the crosstalk between them provides a new direction and theoretical support for future immunotherapy. Video abstract Graphical abstract

The induction of specific immunological tolerance represents an important therapeutic goal for multiple sclerosis and other autoimmune diseases. Sound knowledge of the target antigens, the underlying pathomechanisms of the disease and the presumed mechanisms of action of the respective tolerance-inducing approach are essential for successful translation. Furthermore, suitable tools and assays to evaluate the induction of immune tolerance are key aspects for the development of such treatments. However, investigation of the mechanisms of action underlying tolerance induction poses several challenges. The optimization of sensitive, robust methods which allow the assessment of low frequency autoreactive T cells and the long-term reduction or change of their responses, the detection of regulatory cell populations and their immune mediators, as well as the validation of specific biomarkers indicating reduction of inflammation and damage, are needed to develop tolerance-inducing approaches successfully to patients. This short review focuses on how to demonstrate mechanistic proof-of-concept in antigen-specific tolerance-inducing therapies in MS.

The induction of specific immunological tolerance represents an important therapeutic goal for multiple sclerosis and other autoimmune diseases. Sound knowledge of the target antigens, the underlying pathomechanisms of the disease and the presumed mechanisms of action of the respective tolerance-inducing approach are essential for successful translation. Furthermore, suitable tools and assays to evaluate the induction of immune tolerance are key aspects for the development of such treatments. However, investigation of the mechanisms of action underlying tolerance induction poses several challenges. The optimization of sensitive, robust methods which allow the assessment of low frequency autoreactive T cells and the long-term reduction or change of their responses, the detection of regulatory cell populations and their immune mediators, as well as the validation of specific biomarkers indicating reduction of inflammation and damage, are needed to develop tolerance-inducing approaches successfully to patients. This short review focuses on how to demonstrate mechanistic proof-of-concept in antigen-specific tolerance-inducing therapies in MS. [ABSTRACT FROM AUTHOR]

Ischemic stroke causes brain tissue damage and may release central nervous system (CNS)-specific peptides to the periphery. Neural antigen presentation in the lymphoid tissue could prime immune cells and result in adaptive immune response. However, autoimmune responses against neural antigens are not commonly uncovered after stroke. We studied the brain tissue of nine fatal stroke cases and the blood of a cohort of 13 patients and 11 controls. Flow cytometry carried out in three of the brain samples showed CD8 and CD4 T cells in the cerebrospinal fluid (CSF) of the ventricles in the patient deceased 1 day poststroke, T cells with an activated phenotype in the CSF of the patient that died at day 6, and T cells in the ischemic brain tissue in the patient deceased 140 days after stroke onset. Immunohistochemistry showed higher T cell numbers in the core of the lesion of the patient deceased 18 days post-stroke than in the patients deceased from 1 to 5 days post-stroke. In blood samples, we studied whether lymphocytes were primed in the periphery against neural antigens at sequential times (on admission, day 5, and day 90) after stroke. T lymphocytes of stroke patients produced IFN-γ and TNF-α and responded to MBP peptides by increasing their production of TNF-α and IL-10 at admission, but not at later time points. In contrast, IL-4 producing T cells showed progressive increases. Higher percentages of TNF-α producing T lymphocytes at admission were independently associated with poorer outcomes at 90 days. However, we did not detect T cell responses to neural-antigen stimulation 90 days post-stroke. Altogether the results suggest acute T cell priming in the periphery in acute stroke, T cell trafficking from the CSF to the ischemic brain tissue, and the existence of active mechanisms preventing autoreactivity.

Backgound: Although Myeloid-derived suppressor cells (MDSCs) have a prominent ability to suppress the immune responses of T lymphocytes and propel tumor immune escape, a lack of profound systemic immunesuppression in tumor-bearing mice and tumor patients. The underlying mechanism of these remains unclear. Methods: For this purpose, renal cancer-derived exosomes (RDEs) were first labeled with PKH67 and been observed the internalization by MDSCs. Flow cytometry analysis showed the proportion and activity change of MDSCs in spleen and bone marrow induced by RDEs. Further, western blot experiments were used to verify triggered mechanism of MDSCs by RDEs. Finally, proliferation and cytotoxicity of cytotoxic T lymphocytes (CTLs) co-cultured with MDSCs in vitro and a series of experiments in vivo were performed to demonstrate the specific inhibitory effect of RDEs-induced MDSCs. Results: This study suggested that RDEs crucially contributed to presenting antigenic information, activating and driving specific immunosuppressive effect to MDSCs. HSP70, which is highly expressed in RDEs, initiate this process in a toll like receptor 2 (TLR2)-dependent manner. Importantly, RDEs-induced MDSCs could exert an antigen-specific immunosuppression effect on CTL and specific promote renal tumors-growth and immune escape in consequence. Conclusion: The immunosuppression mediated by MDSCs which is induced by RDEs is antigen-specific. HSP70, which is highly expressed in RDEs, plays a pivotal role in this process. Targeted abrogating the function of MDSCs, or eliminating the expression of HSP70 in exosomes, or blocking the crosstalk between them provides a new direction and theoretical support for future immunotherapy. Video abstract [ABSTRACT FROM AUTHOR]

Ischemic stroke causes brain tissue damage and may release central nervous system (CNS)-specific peptides to the periphery. Neural antigen presentation in the lymphoid tissue could prime immune cells and result in adaptive immune response. However, autoimmune responses against neural antigens are not commonly uncovered after stroke. We studied the brain tissue of nine fatal stroke cases and the blood of a cohort of 13 patients and 11 controls. Flow cytometry carried out in three of the brain samples showed CD8 and CD4 T cells in the cerebrospinal fluid (CSF) of the ventricles in the patient deceased 1 day poststroke, T cells with an activated phenotype in the CSF of the patient that died at day 6, and T cells in the ischemic brain tissue in the patient deceased 140 days after stroke onset. Immunohistochemistry showed higher T cell numbers in the core of the lesion of the patient deceased 18 days post-stroke than in the patients deceased from 1 to 5 days post-stroke. In blood samples, we studied whether lymphocytes were primed in the periphery against neural antigens at sequential times (on admission, day 5, and day 90) after stroke. T lymphocytes of stroke patients produced IFN-γ and TNF-α and responded to MBP peptides by increasing their production of TNF-α and IL-10 at admission, but not at later time points. In contrast, IL-4 producing T cells showed progressive increases. Higher percentages of TNF-α producing T lymphocytes at admission were independently associated with poorer outcomes at 90 days. However, we did not detect T cell responses to neural-antigen stimulation 90 days post-stroke. Altogether the results suggest acute T cell priming in the periphery in acute stroke, T cell trafficking from the CSF to the ischemic brain tissue, and the existence of active mechanisms preventing autoreactivity. [ABSTRACT FROM AUTHOR]

Cellular therapies with polyclonal regulatory T-cells (Tregs) in transplantation and autoimmune diseases have been carried out in both animal models and clinical trials. However, The use of large numbers of polyclonal Tregs with unknown antigen specificities has led to unwanted effects, such as systemic immunosuppression, which can be avoided via utilization of antigen-specific Tregs. Antigen-specific Tregs are also more potent in suppression than polyclonal ones. Although antigen-specific Tregs can be induced in vitro, these iTregs are usually contaminated with effector T cells during in vitro expansion. Fortunately, Tregs can be efficiently engineered with a predetermined antigen-specificity via transfection of viral vectors encoding specific T cell receptors (TCRs) or chimeric antigen receptors (CARs). Compared to Tregs engineered with TCRs (TCR-Tregs), CAR-modified Tregs (CAR-Tregs) engineered in a non-MHC restricted manner have the advantage of widespread applications, especially in transplantation and autoimmunity. CAR-Tregs also are less dependent on IL-2 than are TCR-Tregs. CAR-Tregs are promising given that they maintain stable phenotypes and functions, preferentially migrate to target sites, and exert more potent and specific immunosuppression than do polyclonal Tregs. However, there are some major hurdles that must be overcome before CAR-Tregs can be used in clinic. It is known that treatments with anti-tumor CAR-T cells cause side effects due to cytokine “storm” and neuronal cytotoxicity. It is unclear whether CAR-Tregs would also induce these adverse reactions. Moreover, antibodies specific for self- or allo-antigens must be characterized to construct antigen-specific CAR-Tregs. Selection of antigens targeted by CARs and development of specific antibodies are difficult in some disease models. Finally, CAR-Treg exhaustion may limit their efficacy in immunosuppression. Recently, innovative CAR-Treg therapies in animal models of transplantation and autoimmune diseases have been reported. In this mini-review, we have summarized recent progress of CAR-Tregs and discussed their potential applications for induction of immunological tolerance.

Cellular therapies with polyclonal regulatory T-cells (Tregs) in transplantation and autoimmune diseases have been carried out in both animal models and clinical trials. However, The use of large numbers of polyclonal Tregs with unknown antigen specificities has led to unwanted effects, such as systemic immunosuppression, which can be avoided via utilization of antigen-specific Tregs. Antigen-specific Tregs are also more potent in suppression than polyclonal ones. Although antigen-specific Tregs can be induced in vitro , these iTregs are usually contaminated with effector T cells during in vitro expansion. Fortunately, Tregs can be efficiently engineered with a predetermined antigen-specificity via transfection of viral vectors encoding specific T cell receptors (TCRs) or chimeric antigen receptors (CARs). Compared to Tregs engineered with TCRs (TCR-Tregs), CAR-modified Tregs (CAR-Tregs) engineered in a non-MHC restricted manner have the advantage of widespread applications, especially in transplantation and autoimmunity. CAR-Tregs also are less dependent on IL-2 than are TCR-Tregs. CAR-Tregs are promising given that they maintain stable phenotypes and functions, preferentially migrate to target sites, and exert more potent and specific immunosuppression than do polyclonal Tregs. However, there are some major hurdles that must be overcome before CAR-Tregs can be used in clinic. It is known that treatments with anti-tumor CAR-T cells cause side effects due to cytokine "storm" and neuronal cytotoxicity. It is unclear whether CAR-Tregs would also induce these adverse reactions. Moreover, antibodies specific for self- or allo-antigens must be characterized to construct antigen-specific CAR-Tregs. Selection of antigens targeted by CARs and development of specific antibodies are difficult in some disease models. Finally, CAR-Treg exhaustion may limit their efficacy in immunosuppression. Recently, innovative CAR-Treg therapies in animal models of transplantation and autoimmune diseases have been reported. In this mini-review, we have summarized recent progress of CAR-Tregs and discussed their potential applications for induction of immunological tolerance. [ABSTRACT FROM AUTHOR]

Abstract: HLA/peptide tetramers are frequently used for ex vivo monitoring of disease- or vaccine-induced T cell immune responses and for T cell epitope identification. However, when low-levels HLA/peptide tetramer-positive T cell populations are encountered, it is difficult to ascertain whether this represents a true T cell receptor (TCR)-mediated interaction or background signal. To address this issue, we have developed a method for both HLA class I and class II tetramer assays to confirm tetramer-binding to the TCR/CD3 complex. Preincubation of T cells with anti-CD3 mAb SPV-T3b and subsequent crosslinking interferes with the binding of HLA/peptide tetramers to the TCR/CD3 complex and thereby indicates to what extent HLA/peptide tetramer binds through interaction with TCR/CD3 complex. SPV-T3b pretreatment results in a 2- to 10-fold decrease in tetramer-binding intensity to antigen-specific CD8+ or CD4+ T cells, whereas background reactivity of HLA/peptide tetramers containing HIV-derived peptide in HIV-negative donors remained unchanged. SPV-T3b pretreatment forms a valuable tool to verify tetramer-based detection of antigen-specific T cells during the monitoring of immune responses in clinical studies. [Copyright &y& Elsevier]

The concept of T-cell mediated suppression evolved more than 30 years ago. At that time it spawned many claims that have not stood the test of time. The rediscovery of suppression phenomena and regulatory T cells over the past 15 years created schizophrenic responses amongst immunologists. Some claimed that the new proponents of suppression were, once again, bringing immunology into disrepute, whilst others have embraced the field with great enthusiasm and novel approaches to clarification. Without faithful repetition of the “old” experiments, it is difficult to establish what was right and what was wrong. Nevertheless, immunologists must now accept that a good number of the old claims were overstated, and reflected poor scientific discipline. [ABSTRACT FROM AUTHOR]