A Cryptosporidium parvum vaccine candidate effect on immunohistochemical profiling of CD4+, CD8+, Caspase-3 and NF-κB in mice.

BACKGROUND: Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4+, CD8+, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads. METHODS: Thirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 105 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 105 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4+, CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively. RESULTS: The IHC results showed that CD4+, CD8+, Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4+ levels and low CD8+ expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4+ level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4+, CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues. CONCLUSIONS: The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.

Shaping immunity for life: Layered development of CD8+ T cells.

Historically, the immune system was believed to develop along a linear axis of maturity from fetal life to adulthood. Now, it is clear that distinct layers of immune cells are generated from unique waves of hematopoietic progenitors during different windows of development. This model, known as the layered immune model, has provided a useful framework for understanding why distinct lineages of B cells and γδ T cells arise in succession and display unique functions in adulthood. However, the layered immune model has not been applied to CD8+ T cells, which are still often viewed as a uniform population of cells belonging to the same lineage, with functional differences between cells arising from environmental factors encountered during infection. Recent studies have challenged this idea, demonstrating that not all CD8+ T cells are created equally and that the functions of individual CD8+ T cells in adults are linked to when they were created in the host. In this review, we discuss the accumulating evidence suggesting there are distinct ontogenetic subpopulations of CD8+ T cells and propose that the layered immune model be extended to the CD8+ T cell compartment.

A conserved Bacteroidetes antigen induces anti-inflammatory intestinal T lymphocytes.

The microbiome contributes to the development and maturation of the immune system. In response to commensal bacteria, intestinal CD4+ T lymphocytes differentiate into functional subtypes with regulatory or effector functions. The development of small intestine intraepithelial lymphocytes that coexpress CD4 and CD8αα homodimers (CD4IELs) depends on the microbiota. However, the identity of the microbial antigens recognized by CD4+ T cells that can differentiate into CD4IELs remains unknown. We identified ß-hexosaminidase, a conserved enzyme across commensals of the Bacteroidetes phylum, as a driver of CD4IEL differentiation. In a mouse model of colitis, ß-hexosaminidase-specific lymphocytes protected against intestinal inflammation. Thus, T cells of a single specificity can recognize a variety of abundant commensals and elicit a regulatory immune response at the intestinal mucosa.

Distinct CD4-CD8- (Double-Negative) Memory T-Cell Subpopulations Are Associated With Indeterminate and Cardiac Clinical Forms of Chagas Disease.

CD4-CD8- (double-negative, DN) T cells are critical orchestrators of the cytokine network associated with the pathogenic inflammatory response in one of the deadliest cardiomyopathies known, Chagas heart disease, which is caused by Trypanosoma cruzi infection. Here, studying the distribution, activation status, and cytokine expression of memory DN T-cell subpopulations in Chagas disease patients without cardiac involvement (indeterminate form-IND) or with Chagas cardiomyopathy (CARD), we report that while IND patients displayed a higher frequency of central memory, CARD had a high frequency of effector memory DN T cells. In addition, central memory DN T cells from IND displayed a balanced cytokine profile, characterized by the concomitant expression of IFN-γ and IL-10, which was not observed in effector memory DN T cells from CARD. Supporting potential clinical relevance, we found that the frequency of central memory DN T cells was associated with indicators of better ventricular function, while the frequency of effector memory DN T cells was not. Importantly, decreasing CD1d-mediated activation of DN T cells led to an increase in IL-10 expression by effector memory DN T cells from CARD, restoring a balanced profile similar to that observed in the protective central memory DN T cells. Targeting the activation of effector memory DN T cells may emerge as a strategy to control inflammation in Chagas cardiomyopathy and potentially in other inflammatory diseases where these cells play a key role.

Identification of Two Subsets of Murine DC1 Dendritic Cells That Differ by Surface Phenotype, Gene Expression, and Function.

Classical dendritic cells (cDCs) in mice have been divided into 2 major subsets based on the expression of nuclear transcription factors: a CD8+Irf8+Batf3 dependent (DC1) subset, and a CD8-Irf4+ (DC2) subset. We found that the CD8+DC1 subset can be further divided into CD8+DC1a and CD8+DC1b subsets by differences in surface receptors, gene expression, and function. Whereas all 3 DC subsets can act alone to induce potent Th1 cytokine responses to class I and II MHC restricted peptides derived from ovalbumin (OVA) by OT-I and OT-II transgenic T cells, only the DC1b subset could effectively present glycolipid antigens to natural killer T (NKT) cells. Vaccination with OVA protein pulsed DC1b and DC2 cells were more effective in reducing the growth of the B16-OVA melanoma as compared to pulsed DC1a cells in wild type mice. In conclusion, the Batf3-/- dependent DC1 cells can be further divided into two subsets with different immune functional profiles in vitro and in vivo.

Reducing Hinge Flexibility of CAR-T Cells Prolongs Survival In Vivo With Low Cytokines Release.

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in B cell malignancies. However, high tumor burden limits clinical efficacy and increases the risk of cytokine release syndrome and neurotoxicity, which is associated with over-activation of the CAR-T cells. The hinge domain plays an important role in the function of CAR-T cells. We hypothesized that deletion of glycine, an amino acid with good flexibility, may reduce the flexibility of the hinge region, thereby mitigating CAR-T cell over-activation. This study involved generating a novel CAR by deletion of two consecutive glycine residues in the CD8 hinge domain of second-generation (2nd) CAR, thereafter named 2nd-GG CAR. The 2nd-GG CAR-T cells showed similar efficacy of CAR expression but lower hinge flexibility, and its protein affinity to CD19 protein was lower than that of 2nd CAR-T cells. Compared to the 2nd CAR-T cells, 2nd-GG CAR-T cells reduced proinflammatory cytokine secretion without diminishing the specific cytotoxicity toward tumor cells in vitro. Furthermore, 2nd-GG CAR-T cells prolonged overall survival in an immunodeficient mouse model bearing NALM-6 when tumor burden was high. This study demonstrated that a lower-flexibility of CD8α hinge improved survival under high tumor burden and reduced proinflammatory cytokines in preclinical studies. While there is potential for improved safety and efficacy, yet this needs validation with clinical trials.

Infection-induced type I interferons critically modulate the homeostasis and function of CD8+ naïve T cells.

Naïve T (Tn) cells require two homeostatic signals for long-term survival: tonic T cell receptor:self-peptide-MHC contact and IL-7 stimulation. However, how microbial exposure impacts Tn homeostasis is still unclear. Here we show that infections can lead to the expansion of a subpopulation of long-lived, Ly6C+ CD8+ Tn cells with accelerated effector function. Mechanistically, mono-infection with West Nile virus transiently, and polymicrobial exposure persistently, enhances Ly6C expression selectively on CD5hiCD8+ cells, which in the case of polyinfection translates into a numerical CD8+ Tn cell increase in the lymph nodes. This conversion and expansion of Ly6C+ Tn cells depends on IFN-I, which upregulates MHC class I expression and enhances tonic TCR signaling in differentiating Tn cells. Moreover, for Ly6C+CD8+ Tn cells, IFN-I-mediated signals optimize their homing to secondary sites, extend their lifespan, and enhance their effector differentiation and antibacterial function, particularly for low-affinity clones. Our results thus uncover significant regulation of Tn homeostasis and function via infection-driven IFN-I, with potential implications for immunotherapy.

Ribosylation of the CD8αß heterodimer permits binding of the nonclassical major histocompatibility molecule, H2-Q10.

The CD8αß heterodimer plays a crucial role in the stabilization between major histocompatibility complex class I molecules (MHC-I) and the T cell receptor (TCR). The interaction between CD8 and MHC-I can be regulated by posttranslational modifications, which are proposed to play an important role in the development of CD8 T cells. One modification that has been proposed to control CD8 coreceptor function is ribosylation. Utilizing NAD+, the ecto-enzyme adenosine diphosphate (ADP) ribosyl transferase 2.2 (ART2.2) catalyzes the addition of ADP-ribosyl groups onto arginine residues of CD8α or ß chains and alters the interaction between the MHC and TCR complexes. To date, only interactions between modified CD8 and classical MHC-I (MHC-Ia), have been investigated and the interaction with non-classical MHC (MHC-Ib) has not been explored. Here, we show that ADP-ribosylation of CD8 facilitates the binding of the liver-restricted nonclassical MHC, H2-Q10, independent of the associated TCR or presented peptide, and propose that this highly regulated binding imposes an additional inhibitory leash on the activation of CD8-expressing cells in the presence of NAD+. These findings highlight additional important roles for nonclassical MHC-I in the regulation of immune responses.

Gut epithelial IL-27 confers intestinal immunity through the induction of intraepithelial lymphocytes.

IL-27 controls a diverse range of immune responses in many disease settings. Here, we identify intestinal epithelial cells (IECs) as one of the major IL-27 cellular sources in the gut-associated tissue. Unlike IL-27 secreted by innate immune cells, gut epithelial IL-27 is dispensable for T-bet+ regulatory T cell (T reg cell) differentiation or IL-10 induction. Rather, IEC-derived IL-27 specifically promotes a distinct CD8αα+CD4+ intraepithelial lymphocyte (IEL) population that acquires their functional differentiation at the intestinal epithelium. Loss of IL-27 in IECs leads to a selective defect in CD8αα+CD4+ IELs over time. Consequently, mice with IEC-specific IL-27 ablation exhibited elevated pathogen burden during parasitic infection, and this could be rescued by transfer of exogenous CD8αα+CD4+ IELs. Collectively, our data reveal that in addition to its known regulatory properties in preventing immune hyperactivity, gut epithelial IL-27 confers barrier immunity by inducing a specific IEL subset and further suggest that IL-27 produced by different cell types plays distinct roles in maintaining intestinal homeostasis.

Persistent High Percentage of HLA-DR+CD38high CD8+ T Cells Associated With Immune Disorder and Disease Severity of COVID-19.

Background: The global outbreak of coronavirus disease 2019 (COVID-19) has turned into a worldwide public health crisis and caused more than 100,000,000 severe cases. Progressive lymphopenia, especially in T cells, was a prominent clinical feature of severe COVID-19. Activated HLA-DR+CD38+ CD8+ T cells were enriched over a prolonged period from the lymphopenia patients who died from Ebola and influenza infection and in severe patients infected with SARS-CoV-2. However, the CD38+HLA-DR+ CD8+ T population was reported to play contradictory roles in SARS-CoV-2 infection. Methods: A total of 42 COVID-19 patients, including 32 mild or moderate and 10 severe or critical cases, who received care at Beijing Ditan Hospital were recruited into this retrospective study. Blood samples were first collected within 3 days of the hospital admission and once every 3-7 days during hospitalization. The longitudinal flow cytometric data were examined during hospitalization. Moreover, we evaluated serum levels of 45 cytokines/chemokines/growth factors and 14 soluble checkpoints using Luminex multiplex assay longitudinally. Results: We revealed that the HLA-DR+CD38+ CD8+ T population was heterogeneous, and could be divided into two subsets with distinct characteristics: HLA-DR+CD38dim and HLA-DR+CD38hi. We observed a persistent accumulation of HLA-DR+CD38hi CD8+ T cells in severe COVID-19 patients. These HLA-DR+CD38hi CD8+ T cells were in a state of overactivation and consequent dysregulation manifested by expression of multiple inhibitory and stimulatory checkpoints, higher apoptotic sensitivity, impaired killing potential, and more exhausted transcriptional regulation compared to HLA-DR+CD38dim CD8+ T cells. Moreover, the clinical and laboratory data supported that only HLA-DR+CD38hi CD8+ T cells were associated with systemic inflammation, tissue injury, and immune disorders of severe COVID-19 patients. Conclusions: Our findings indicated that HLA-DR+CD38hi CD8+ T cells were correlated with disease severity of COVID-19 rather than HLA-DR+CD38dim population.

Cdx2 regulates immune cell infiltration in the intestine.

The intestinal epithelium is a unique tissue, serving both as a barrier against pathogens and to conduct the end digestion and adsorption of nutrients. As regards the former, the intestinal epithelium contains a diverse repertoire of immune cells, including a variety of resident lymphocytes, macrophages and dendritic cells. These cells serve a number of roles including mitigation of infection and to stimulate regeneration in response to damage. The transcription factor Cdx2, and to a lesser extent Cdx1, plays essential roles in intestinal homeostasis, and acts as a context-dependent tumour suppressor in colorectal cancer. Deletion of Cdx2 from the murine intestinal epithelium leads to macrophage infiltration resulting in a chronic inflammatory response. However the mechanisms by which Cdx2 loss evokes this response are poorly understood. To better understand this relationship, we used a conditional mouse model lacking all intestinal Cdx function to identify potential target genes which may contribute to this inflammatory phenotype. One such candidate encodes the histocompatability complex protein H2-T3, which functions to regulate intestinal iCD8α lymphocyte activity. We found that Cdx2 occupies the H3-T3 promoter in vivo and directly regulates its expression via a Cdx response element. Loss of Cdx function leads to a rapid and pronounced attenuation of H2-T3, followed by a decrease in iCD8α cell number, an increase in macrophage infiltration and activation of pro-inflammatory cascades. These findings suggest a previously unrecognized role for Cdx in intestinal homeostasis through H2-T3-dependent regulation of iCD8α cells.

Positron emission tomography imaging with 89Zr-labeled anti-CD8 cys-diabody reveals CD8+ cell infiltration during oncolytic virus therapy in a glioma murine model.

Determination of treatment response to immunotherapy in glioblastoma multiforme (GBM) is a process which can take months. Detection of CD8+ T cell recruitment to the tumor with a noninvasive imaging modality such as positron emission tomography (PET) may allow for tumor characterization and early evaluation of therapeutic response to immunotherapy. In this study, we utilized 89Zr-labeled anti-CD8 cys-diabody-PET to provide proof-of-concept to detect CD8+ T cell immune response to oncolytic herpes simplex virus (oHSV) M002 immunotherapy in a syngeneic GBM model. Immunocompetent mice (n = 16) were implanted intracranially with GSC005 GBM tumors, and treated with intratumoral injection of oHSV M002 or saline control. An additional non-tumor bearing cohort (n = 4) receiving oHSV M002 treatment was also evaluated. Mice were injected with 89Zr-labeled anti-CD8 cys-diabody seven days post oHSV administration and imaged with a preclinical PET scanner. Standardized uptake value (SUV) was quantified. Ex vivo tissue analyses included autoradiography and immunohistochemistry. PET imaging showed significantly higher SUV in tumors which had been treated with M002 compared to those without M002 treatment (p = 0.0207) and the non-tumor bearing M002 treated group (p = 0.0021). Accumulation in target areas, especially the spleen, was significantly reduced by blocking with the non-labeled diabody (p < 0.001). Radioactive probe accumulation in brains was consistent with CD8+ cell trafficking patterns after oHSV treatment. This PET imaging strategy could aid in distinguishing responders from non-responders during immunotherapy of GBM.

CD8 coreceptor-mediated focusing can reorder the agonist hierarchy of peptide ligands recognized via the T cell receptor.

CD8+ T cells are inherently cross-reactive and recognize numerous peptide antigens in the context of a given major histocompatibility complex class I (MHCI) molecule via the clonotypically expressed T cell receptor (TCR). The lineally expressed coreceptor CD8 interacts coordinately with MHCI at a distinct and largely invariant site to slow the TCR/peptide-MHCI (pMHCI) dissociation rate and enhance antigen sensitivity. However, this biological effect is not necessarily uniform, and theoretical models suggest that antigen sensitivity can be modulated in a differential manner by CD8. We used two intrinsically controlled systems to determine how the relationship between the TCR/pMHCI interaction and the pMHCI/CD8 interaction affects the functional sensitivity of antigen recognition. Our data show that modulation of the pMHCI/CD8 interaction can reorder the agonist hierarchy of peptide ligands across a spectrum of affinities for the TCR.

NKG2D Enhances Double-Negative T Cell Regulation of B Cells.

Numerous studies reported a small subpopulation of TCRαß+CD4-CD8- (double-negative) T cells that exert regulatory functions in the peripheral lymphocyte population. However, the origin of these double-negative T (DNT) cells is controversial. Some researchers reported that DNT cells originated from the thymus, and others argued that these cells are derived from peripheral immune induction. We report a possible mechanism for the induction of nonregulatory CD4+ T cells to become regulatory double-negative T (iDNT) cells in vitro. We found that immature bone marrow dendritic cells (CD86+MHC-II- DCs), rather than mature DCs (CD86+MHC-II+), induced high levels of iDNT cells. The addition of an anti-MHC-II antibody to the CD86+MHC-II+ DC group significantly increased induction. These iDNT cells promoted B cell apoptosis and inhibited B cell proliferation and plasma cell formation. A subgroup of iDNT cells expressed NKG2D. Compared to NKG2D- iDNT cells, NKG2D+ iDNT cells released more granzyme B to enhance B cell regulation. This enhancement may function via NKG2D ligands expressed on B cells following lipopolysaccharide stimulation. These results demonstrate that MHC-II impedes induction, and iDNT cells may be MHC independent. NKG2D expression on iDNT cells enhanced the regulatory function of these cells. Our findings elucidate one possible mechanism of the induction of peripheral immune tolerance and provide a potential treatment for chronic allograft rejection in the future.

Cell-mediated immune response against mycolic acids of Mycobacteroides salmoniphilum in rainbow trout Oncorhynchus mykiss.

Mycobacteriosis caused by Mycobacterium spp. causes economic damages to the world aquaculture industry. In mammals, mycolic acids contained in the cell wall of Mycobacterium spp. are presented by CD1b molecule as lipid antigens and induce cell-mediated immunity (CMI). Here, we investigated CMI responses against the mycolic acids of Mycobacterioides salmoniphilum in a CD1-lacking teleost fish, rainbow trout. After stimulation of trout leukocytes with mycolic acids, the number and percentage of CD8α+ T cells increased. Fish immunized with mycolic acids showed an up-regulation of IFN-γ. Further, in vitro re-stimulation of leukocytes derived from immunized fish resulted in proliferation of CD8α+ cells. These data suggest that mycolic acids are recognized as lipid antigens resulting in an activation of rainbow trout CD8α+ cells and up-regulation of the Th1 cytokine IFN-γ. The mycolic acids are promising candidates for vaccines to activate CD8α+ T cells against fish mycobacteriosis.

CD8+PD-L1+CXCR3+ polyfunctional T cell abundances are associated with survival in critical SARS-CoV-2-infected patients.

The importance of the adaptive T cell response in the control and resolution of viral infection has been well established. However, the nature of T cell-mediated viral control mechanisms in life-threatening stages of COVID-19 has yet to be determined. The aim of the present study was to determine the function and phenotype of T cell populations associated with survival or death of patients with COVID-19 in intensive care as a result of phenotypic and functional profiling by mass cytometry. Increased frequencies of circulating, polyfunctional CD4+CXCR5+HLA-DR+ stem cell memory T cells (Tscms) and decreased proportions of granzyme B-expressing and perforin-expressing effector memory T cells were detected in recovered and deceased patients, respectively. The higher abundance of polyfunctional PD-L1+CXCR3+CD8+ effector T cells (Teffs), CXCR5+HLA-DR+ Tscms, and anti-nucleocapsid (anti-NC) cytokine-producing T cells permitted us to differentiate between recovered and deceased patients. The results from a principal component analysis show an imbalance in the T cell compartment that allowed for the separation of recovered and deceased patients. The paucity of circulating PD-L1+CXCR3+CD8+ Teffs and NC-specific CD8+ T cells accurately forecasts fatal disease outcome. This study provides insight into the nature of the T cell populations involved in the control of COVID-19 and therefore might impact T cell-based vaccine designs for this infectious disease.

Mucosal and Systemic Immune Responses to Salmon Gill Poxvirus Infection in Atlantic Salmon Are Modulated Upon Hydrocortisone Injection.

Salmon Gill Poxvirus Disease (SGPVD) has emerged as a cause of acute mortality in Atlantic salmon (Salmo salar L.) presmolts in Norwegian aquaculture. The clinical phase of the disease is associated with apoptotic cell death in the gill epithelium causing acute respiratory distress, followed by proliferative changes in the regenerating gill in the period after the disease outbreak. In an experimental SGPV challenge trial published in 2020, acute disease was only seen in fish injected with hydrocortisone 24 h prior to infection. SGPV-mediated mortality in the hydrocortisone-injected group was associated with more extensive gill pathology and higher SGPV levels compared to the group infected with SGPV only. In this study based on the same trial, SGPV gene expression and the innate and adaptive antiviral immune response was monitored in gills and spleen in the presence and absence of hydrocortisone. Whereas most SGPV genes were induced from day 3 along with the interferon-regulated innate immune response in gills, the putative SGPV virulence genes of the B22R family were expressed already one day after SGPV exposure, indicating a potential role as early markers of SGPV infection. In gills of the hydrocortisone-injected fish infected with SGPV, MX expression was delayed until day 10, and then expression skyrocketed along with the viral peak, gill pathology and mortality occurring from day 14. A similar expression pattern was observed for Interferon gamma (IFNγ) and granzyme A (GzmA) in the gills, indicating a role of acute cytotoxic cell activity in SGPVD. Duplex in situ hybridization demonstrated effects of hydrocortisone on the number and localization of GzmA-containing cells, and colocalization with SGPV infected cells in the gill. SGPV was generally not detected in spleen, and gill infection did not induce any corresponding systemic immune activity in the absence of stress hormone injection. However, in fish injected with hydrocortisone, IFNγ and GzmA gene expression was induced in spleen in the days prior to acute mortality. These data indicate that suppressed mucosal immune response in the gills and the late triggered systemic immune response in the spleen following hormonal stress induction may be the key to the onset of clinical SGPVD.

Canonical T cell receptor docking on peptide-MHC is essential for T cell signaling.

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRß-variable (TRBV) 17+ TCRs from the naïve mouse CD8+ T cell repertoire that recognizes the H-2Db-NP366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.

PD-1 suppresses TCR-CD8 cooperativity during T-cell antigen recognition.

Despite the clinical success of blocking its interactions, how PD-1 inhibits T-cell activation is incompletely understood, as exemplified by its potency far exceeding what might be predicted from its affinity for PD-1 ligand-1 (PD-L1). This may be partially attributed to PD-1's targeting the proximal signaling of the T-cell receptor (TCR) and co-stimulatory receptor CD28 via activating Src homology region 2 domain-containing phosphatases (SHPs). Here, we report PD-1 signaling regulates the initial TCR antigen recognition manifested in a smaller spreading area, fewer molecular bonds formed, and shorter bond lifetime of T cell interaction with peptide-major histocompatibility complex (pMHC) in the presence than absence of PD-L1 in a manner dependent on SHPs and Leukocyte C-terminal Src kinase. Our results identify a PD-1 inhibitory mechanism that disrupts the cooperative TCR-pMHC-CD8 trimolecular interaction, which prevents CD8 from augmenting antigen recognition, explaining PD-1's potent inhibitory function and its value as a target for clinical intervention.

IL-4Rα signaling by CD8α+ dendritic cells contributes to cerebral malaria by enhancing inflammatory, Th1, and cytotoxic CD8+ T cell responses.

Persistent high levels of proinflammatory and Th1 responses contribute to cerebral malaria (CM). Suppression of inflammatory responses and promotion of Th2 responses prevent pathogenesis. IL-4 commonly promotes Th2 responses and inhibits inflammatory and Th1 responses. Therefore, IL-4 is widely considered as a beneficial cytokine via its Th2-promoting role that is predicted to provide protection against severe malaria by inhibiting inflammatory responses. However, IL-4 may also induce inflammatory responses, as the result of IL-4 action depends on the timing and levels of its production and the tissue environment in which it is produced. Recently, we showed that dendritic cells (DCs) produce IL-4 early during malaria infection in response to a parasite protein and that this IL-4 response may contribute to severe malaria. However, the mechanism by which IL-4 produced by DCs contributing to lethal malaria is unknown. Using Plasmodium berghei ANKA-infected C57BL/6 mice, a CM model, we show here that mice lacking IL-4Rα only in CD8α+ DCs are protected against CM pathogenesis and survive, whereas WT mice develop CM and die. Compared with WT mice, mice lacking IL-4Rα in CD11c+ or CD8α+ DCs showed reduced inflammatory responses leading to decreased Th1 and cytotoxic CD8+ T cell responses, lower infiltration of CD8+ T cells to the brain, and negligible brain pathology. The novel results presented here reveal a paradoxical role of IL-4Rα signaling in CM pathogenesis that promotes CD8α+ DC-mediated inflammatory responses that generate damaging Th1 and cytotoxic CD8+ T cell responses.