A Lower Dose of Infection Generates a Better Long-Term Immune Response against Toxoplasma gondii.

Toxoplasma gondii, an obligate intracellular pathogen, induces a strong immune response in the infected host. In the encephalitis model of infection, long-term protective immunity is mediated by CD8 T cells, with the CD4 T cell population providing important help. Most of the immune studies have used a 10- to 20-cyst dose of T. gondii, which leads to T cell dysfunctionality during the late phase of chronic infection and increases the chances of reactivation. In the current study, we compared the immune response of mice orally infected with either 2 or 10 cysts of T. gondii. During the acute phase, we demonstrate that the lower dose of infection generates a reduced number of CD4 and CD8 T cells, but the frequency of functional CD4 or CD8 T cells is similar in animals infected with two different doses. However, Ag-experienced T cells (both CD4 and CD8) are better maintained in lower dose-infected mice at 8 wk postinfection, with an increase number functional cells that exhibit lower multiple inhibitory receptor expression. In addition to better long-term T cell immunity, animals infected with a lower dose display reduced inflammation manifested by lesser Ag-specific T cell and cytokine responses during the very early stage of the acute infection. Our studies suggest a previously unappreciated role of dose-dependent early programming/imprinting of the long-term CD4/CD8 T cell response during T. gondii infection. These observations point to the need for an in-depth analysis of how early events shape long-term immunity against this pathogen.

Immune responses to Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular parasite that can cause severe complications in the newborn and immunocompromised individuals. The parasite evokes a strong innate immune response in the infected hosts which is followed by a robust adaptive immunity. In the last few years, importance of innate immune mechanisms dependent on the role of MyD-88 independent pathways, inflammatory monocytes and innate lymphocyte have been identified. However, notwithstanding the strong immune response to the parasite, the chronic infection persists in the host. The inability to prevent chronic infection can be attributed to aberration in the memory CD8 T cell response caused by an increased expression of inhibitory receptors that leads to their dysfunctionality.

Immune Response to Microsporidia.

Microsporidia are a group of pathogens, which can pose severe risks to the immunocompromised population, such as HIV-infected individuals or organ transplant recipients. Adaptive immunity has been reported to be critical for protection, and mice depleted of T cells are unable to control these infections. In a mouse model of infection, CD8 T cells have been found to be the primary effector cells and are responsible for protecting the infected host. Also, as infection is acquired via a peroral route, CD8 T cells in the gut compartment act as a first line of defense against these pathogens. Thus, generation of a robust CD8 T-cell response exhibiting polyfunctional ability is critical for host survival. In this chapter, we describe the effector CD8 T cells generated during microsporidia infection and the factors that may be essential for generating protective immunity against these understudied but significant pathogens. Overall, this chapter will highlight the necessity for a better understanding of the development of CD8 T-cell responses in gut-associated lymphoid tissue (GALT) and provide some insights into therapies that may be used to restore defective CD8 T-cell functionality in an immunocompromised situation.

Nfkbid-mediated humoral immunity during secondary toxoplasmosis.

Vaccine-mediated immunity to parasites has not been achieved. Immune C57BL/6 mice are susceptible to secondary Toxoplasma gondii infection. Using a forward genetics approach, Souza et al. identify Nfkbid as an important factor for the regulation of B cell immunity during secondary Toxoplasma infection and protection against rechallenge.

FROM SHARING TO SELLING: CHALLENGES AND OPPORTUNITIES OF ESTABLISHING A DIGITAL HEALTH DATA MARKETPLACE USING BLOCKCHAIN TECHNOLOGIES.

During the COVID-19 pandemic, we witnessed how sharing of biological and biomedical data facilitated researchers, medical practitioners, and policymakers to tackle the pandemic on a global scale. Despite the growing use of electronic health records (EHRs) by medical practitioners and wearable digital gadgets by individuals, 80% of health and medical data remain unused, adding little value to the work of researchers and medical practitioners. Legislative constraints related to health data sharing, centralized siloed design of traditional data management systems, and most importantly, lack of incentivization models are thought to be the underpinning bottlenecks for sharing health data. With the advent of the General Data Protection Regulation (GDPR) of the European Union (EU) and the development of technologies like blockchain and distributed ledger technologies (DLTs), it is now possible to create a new paradigm of data sharing by changing the incentivization model from current authoritative or altruistic form to a shared economic model where financial incentivization will be the main driver for data sharing. This can be achieved by setting up a digital health data marketplace (DHDM). Here, we review papers that proposed technical models or implemented frameworks that use blockchain-like technologies for health data. We seek to understand and compare different technical challenges associated with implementing and optimizing the DHDM operation outlined in these articles. We also examine legal limitations in the context of the EU and other countries such as the USA to accommodate any compliance requirement for such a marketplace. Last but not least, we review papers that investigated the short-, medium-, and long-term socioeconomic impact of such a marketplace on a wide range of stakeholders.

Complex and Multilayered Role of IL-21 Signaling during Thymic Development.

Unlike IL-7, which is known to be critical for T cell thymic development, the role of IL-21 in this process is still controversial. IL-21 has been shown to accelerate thymic recovery in mice treated with glucocorticoids and revives the peripheral T cell pool in aged animals. However, mice with a defect in IL-21 signaling exhibit normal thymic cellularity, challenging the importance of this cytokine in the thymic developmental process. Using mixed bone marrow chimeric mice, our studies describe a multilayered role for IL-21 in thymopoiesis. In this system, IL-21R-deficient cells are unable to compete with wild-type populations at different stages of the thymic development. Using a mixed bone marrow chimeric animal model, IL-21 seems to be involved as early as the double-negative 1 stage, and the cells from the knockout compartment have problems transitioning to subsequent double-negative stages. Also, similar to IL-7, IL-21 seems to be involved in the positive selection of double-positive lymphocytes and appears to play a role in the migration of single-positive T cells to the periphery. Although not as critical as IL-7, based on our studies, IL-21 plays an important complementary role in thymic T cell development, which, to date, has been underrecognized.

Toxoplasma: Immunity and Pathogenesis.

Toxoplasma gondii infection induces a strong immunity in the host. Although the response is manifested by innate response during early infection, adaptive immunity is critical for long-term protection. Amongst the adaptive immune response CD4 T cells play an important helper role for CD8 T cells which are the primary effector cells responsible for controlling the infection. Notwithstanding the induction of robust CD8 T immunity during acute infection, the parasite is not eradicated. One of the reasons for this is the functional exhaustion of CD8 T cells during latent infection. Recent studies from our laboratory have reported that primary cause of CD8 T cell exhaustion is compromised CD4 T cell help during latent toxoplasmosis. CD8 T cell dysfunctionality is preceded by CD4 exhaustion and effector immunity is severely compromised.

Toxoplasma gondii: CD8 T Cells Cry for CD4 Help.

Toxoplasma gondii, an apicomplexan parasite, is a pathogenic protozoan that can infect the central nervous system. In pregnant women, infection can result in congenital problems of the fetus, while in immunocompromised individual it can lead to severe neurological consequences. Although CD8 T cells play an important effector role in controlling the chronic infection, their maintenance is dependent on the critical help provided by CD4 T cells. In a recent study, we demonstrated that reactivation of the infection in chronically infected host is a consequence of CD8 T dysfunction caused by CD4 T cell exhaustion. Furthermore, treatment of chronically infected host with antigen-specific non-exhausted CD4 T cells can restore CD8 T cell functionality and prevent reactivation of the latent infection. The exhaustion status of CD4 T cells is mediated by the increased expression of the transcription factor BLIMP-1, and deletion of this molecule led to the restoration of CD4 T cell function, reversal of CD8 exhaustion and prevention of reactivation of the latent infection. In a recent study from our laboratory, we also observed an increased expression of miR146a levels by CD4 T cells from the chronically infected animals. Recent reports have demonstrated that microRNAs (especially miR146a) has a strong impact on the immune system of T. gondii infected host. Whether these molecules have any role in the BLIMP-1 up-regulation and dysfunctionality of these cells needs to be investigated.

Downregulated IL-21 Response and T Follicular Helper Cell Exhaustion Correlate with Compromised CD8 T Cell Immunity during Chronic Toxoplasmosis.

CD8 T cells are important for maintaining the chronicity of Toxoplasma gondii infection. In a T. gondii encephalitis susceptible model, we recently demonstrated that CD4 T cells play an essential helper role in the maintenance of the effector response and CD8 T cell dysfunctionality was linked to CD4 T cell exhaustion. However, CD4 T cells are constituted of different subsets with various functions and the population(s) providing help to the CD8 T cells has not yet been determined. In the present study, T follicular helper cells (Tfh), which are known to be essential for B cell maturation and are one of the main sources of IL-21, were significantly increased during chronic toxoplasmosis. However, at week 7 p.i., when CD8 T cells are exhausted, the Tfh population exhibited increased expression of several inhibitory receptors and levels of IL-21 in the serum were decreased. The importance of IL-21 in the maintenance of CD8 T cells function after T. gondii infection was further demonstrated in IL-21R KO mouse model. Interestingly, while CD8 T cells from both knockout (KO) and wild-type mice expressed similar levels of PD-1, animals with defective IL-21 signaling exhibited lower polyfunctionality than wild-type controls. This reduced polyfunctional ability observed in CD8 T cells from KO mice was associated with a significant increase in other inhibitory receptors like Tim-3, LAG-3, and 2B4. Furthermore, the animals exhibited greater signs of Toxoplasma reactivation manifested by the reduced number of cysts and increased expression of tachyzoite (replicative form of the parasite) specific genes (SAG1 and ENO2) in the brain. Also, IL-21R KO mice displayed a higher frequency of tachyzoite-infected monocytes in the blood and spleen. Our findings suggest the importance of Tfh and IL-21 during chronic toxoplasmosis and establish a critical role for this cytokine in regulating CD8 T cell dysfunction by preventing the co-expression of multiple inhibitory receptors during chronic parasitic infection.

Investing in CenteringPregnancy™ Group Prenatal Care Reduces Newborn Hospitalization Costs.

OBJECTIVES: CenteringPregnancy™ group prenatal care is an innovative model with promising evidence of reducing preterm birth. The outpatient costs of offering CenteringPregnancy pose barriers to model adoption. Enhanced provider reimbursement for group prenatal care may improve birth outcomes and generate newborn hospitalization cost savings for insurers. To investigate potential cost savings for investment in CenteringPregnancy, we evaluated the impact on newborn hospital admission costs of a pilot incentive project, where BlueChoice Health Plan South Carolina Medicaid managed care organization paid an obstetric practice offering CenteringPregnancy $175 for each patient who participated in at least five group prenatal care sessions. METHODS: Using a one to many case-control matching without replacement, each CenteringPregnancy participant was matched retrospectively on propensity score, age, race, and clinical risk factors with five individual care participants. We estimated the odds of newborn hospital admission type (neonatal intensive care unit [NICU] or well-baby admission) for matched CenteringPregnancy and individual care cohorts with four or more visits using multivariate logistic regression. Cost savings were calculated using mean costs per admission type at the delivery hospital. RESULTS: Of the CenteringPregnancy newborns, 3.5% had a NICU admission compared with 12.0% of individual care newborns (p < .001). Investing in CenteringPregnancy for 85 patients ($14,875) led to an estimated net savings for the managed care organization of $67,293 in NICU costs. CONCLUSIONS: CenteringPregnancy may reduce costs through fewer NICU admissions. Enhanced reimbursement from payers to obstetric practices supporting CenteringPregnancy sustainability may improve birth outcomes and reduce associated NICU costs.

Blimp-1-mediated CD4 T cell exhaustion causes CD8 T cell dysfunction during chronic toxoplasmosis.

CD8, but not CD4, T cells are considered critical for control of chronic toxoplasmosis. Although CD8 exhaustion has been previously reported in Toxoplasma encephalitis (TE)-susceptible model, our current work demonstrates that CD4 not only become exhausted during chronic toxoplasmosis but this dysfunction is more pronounced than CD8 T cells. Exhausted CD4 population expressed elevated levels of multiple inhibitory receptors concomitant with the reduced functionality and up-regulation of Blimp-1, a transcription factor. Our data demonstrates for the first time that Blimp-1 is a critical regulator for CD4 T cell exhaustion especially in the CD4 central memory cell subset. Using a tamoxifen-dependent conditional Blimp-1 knockout mixed bone marrow chimera as well as an adoptive transfer approach, we show that CD4 T cell-intrinsic deletion of Blimp-1 reversed CD8 T cell dysfunction and resulted in improved pathogen control. To the best of our knowledge, this is a novel finding, which demonstrates the role of Blimp-1 as a critical regulator of CD4 dysfunction and links it to the CD8 T cell dysfunctionality observed in infected mice. The critical role of CD4-intrinsic Blimp-1 expression in mediating CD4 and CD8 T cell exhaustion may provide a rational basis for designing novel therapeutic approaches.

IL-21 Is Important for Induction of KLRG1+ Effector CD8 T Cells during Acute Intracellular Infection.

Microsporidia, a latent opportunistic infection associated with mild inflammation, is characterized by a strong CD8 T cell response, which has been shown to be CD4 T cell dependent. In this manuscript, we demonstrate that CD4 help is provided via IL-21 production, a common γ-chain cytokine closely related to IL-2. The peak of IL-21 expression, observed during the acute infection, is associated with an elevated IL-21(+) CD4 T subset, and these cells bear a phenotypic resemblance to T follicular helper cells. We observed that, during per-oral microsporidial infection, IL-21 was critical for the generation of an optimal effector CD8 T cell immunity. Sharply decreased effector KLRG1(+) CD8 response was observed in IL-21R knockout mice, and although these cells exhibited reduced functional properties, they retained the ability to proliferate. The role of IL-21 in the generation of CD8 effectors was cell intrinsic, as stronger defects were observed in the IL-21-deficient compartment from the bone marrow chimeric mice (IL-21R knockout/wild-type). These findings are different from those reported for viral infections in which IL-21 has been primarily associated with the generation and maintenance of CD8 memory response. To the best of our knowledge, this report demonstrates a critical role for IL-21 in the generation of a primary effector CD8 T cell response to an infectious disease model.

Interleukin-12-producing CD103+ CD11b- CD8+ dendritic cells are responsible for eliciting gut intraepithelial lymphocyte response against Encephalitozoon cuniculi.

Microsporidia, which belong to the kingdom Fungi, are important opportunistic pathogens in HIV-infected populations and organ transplant recipients that are often associated with a broad range of symptoms, such as diarrhea, nephritis, and encephalitis. Natural infection occurs via the oral route, and as a consequence, gut immunity plays an important role in restricting the dissemination of these pathogens. Studies from our laboratory have reported that the pathogens induce a rapid intraepithelial lymphocyte (IEL) response important for host protection. Although mucosal dendritic cells (DC) are likely involved in triggering an antigen-specific IEL response, the specific subset(s) responsible has yet to be identified. Toward this goal, we demonstrate a very important role for mucosal CD11b(-) CD8(+) DC in the initiation of an antigen-specific IEL in vivo. Effectively, after Encephalitozoon cuniculi infection, CD11b(-) CD8(+) DC were activated in the lamina propria (LP) and acquired the ability to process retinoic acid (RA). However, this subset did not produce interleukin 12 (IL-12) but upregulated CD103, which is essential for migration to the mesenteric lymph nodes (MLN). Interestingly, CD103(+) CD11b(-) CD8(+) DC in the MLN, in addition to processing RA, also secreted IL-12 and were responsible for gut imprinting specificity on mucosal CD8 T cells. To the best of our knowledge, this is the first report describing the importance of MLN CD103(+) CD11b(-) CD8(+) DC isolated from infected animals in the generation of an IEL response against a live pathogen.

CD8+ T cell immunity in an encephalitis model of Toxoplasma gondii infection.

Toxoplasma gondii infection induces a robust CD8 T cell immunity in the infected host, which is critical for keeping chronic infection under control. IFNγ production and cytolytic activity exhibited by CD8 T cells are critical functions needed to prevent the reactivation of latent infection. Paradoxically, the susceptible mice infected with the parasite develop encephalitis irrespective of the presence of vigorous CD8 T cell immunity. Recent studies from our laboratory have demonstrated that these animals have defect in the memory CD8 T cell population, which become dysfunctional due to exhibition of inhibitory receptors like PD-1. Although the blockade of PD-1-PDL-1 pathway rescues the CD8 response, PD-1(hi) expressing cells are refractory to the treatment. In this review, we discuss the development of CD8 memory response during chronic infection, mechanism responsible for their dysfunctionality, and possible therapeutic measures that can be taken to reverse the process.

Effector CD8 T cell immunity in microsporidial infection: a lone defense mechanism.

Microsporidia is a group of pathogens, which can pose severe risks to the immunocompromised population such as HIV-infected individuals. The expertise to diagnose these pathogens is limited and therefore their prevalence is believed to be much higher than what is currently known. In a mouse model of infections, it has been reported that CD8 T cells are the primary effector cells responsible for protecting the infected host. As the infection is acquired via per-oral route, CD8 T cells in the gut compartment apparently act as a first line of defense against the pathogens. Thus, generation of a robust CD8 T cell response that exhibits polyfunctional ability is critical for host survival. In this review, we describe the effector CD8 T cells generated during microsporidial infection and underline the factors that may be essential for the elicitation of protective immunity against this understudied but significant pathogen. Overall, this review will highlight the necessity for a better understanding of the development of the CD8 T cell response in gut associated lymphoid tissue (GALT) and provide some insights into therapies that may be used to restore defective CD8 T cell functionality in an immunocompromised situation.

ProtocolNavigator: emulation-based software for the design, documentation and reproduction biological experiments.

MOTIVATION: Experimental reproducibility is fundamental to the progress of science. Irreproducible research decreases the efficiency of basic biological research and drug discovery and impedes experimental data reuse. A major contributing factor to irreproducibility is difficulty in interpreting complex experimental methodologies and designs from written text and in assessing variations among different experiments. Current bioinformatics initiatives either are focused on computational research reproducibility (i.e. data analysis) or laboratory information management systems. Here, we present a software tool, ProtocolNavigator, which addresses the largely overlooked challenges of interpretation and assessment. It provides a biologist-friendly open-source emulation-based tool for designing, documenting and reproducing biological experiments. AVAILABILITY AND IMPLEMENTATION: ProtocolNavigator was implemented in Python 2.7, using the wx module to build the graphical user interface. It is a platform-independent software and freely available from http://protocolnavigator.org/index.html under the GPL v2 license.

Intrinsic TGF-ß signaling promotes age-dependent CD8+ T cell polyfunctionality attrition.

Advanced age is associated with immune system deficits that result in an increased susceptibility to infectious diseases; however, specific mediators of age-dependent immune dysfunction have not been fully elucidated. Here we demonstrated that aged mice exhibit poor effector CD8+ T cell polyfunctionality, primarily due to CD8+ T cell-extrinsic deficits, and that reduced CD8+ T cell polyfunctionality correlates with increased susceptibility to pathogenic diseases. In aged animals challenged with the parasite Encephalitozoon cuniculi, effector CD8+ T cell survival and polyfunctionality were suppressed by highly elevated TGF-ß1. Furthermore, TGF-ß depletion reduced effector CD8+ T cell apoptosis in both young and aged mice and enhanced effector CD8+ T cell polyfunctionality in aged mice. Surprisingly, intrinsic blockade of TGF-ß signaling in CD8+ T cells was sufficient to rescue polyfunctionality in aged animals. Together, these data demonstrate that low levels of TGF-ß1 promote apoptosis of CD8+ effector T cells and high TGF-ß1 levels associated with age result in both CD8+ T cell apoptosis and an altered transcriptional profile, which correlates with loss of polyfunctionality. Furthermore, elevated TGF-ß levels are observed in the elderly human population and in aged Drosophila, suggesting that TGF-ß represents an evolutionarily conserved negative regulator of the immune response in aging organisms.

CD40 signaling to the rescue: A CD8 exhaustion perspective in chronic infectious diseases.

Chronic infectious diseases such as HIV, HBV, and HCV, among others, cause severe morbidity and mortality globally. Progressive decline in CD8 functionality, survival, and proliferative potential-a phenomenon referred to as CD8 exhaustion-is believed to be responsible for poor pathogen control in chronic infectious diseases. While the role of negative inhibitory receptors such as PD-1 in augmenting CD8 exhaustion has been extensively studied, the role of positive costimulatory receptors remains poorly understood. In this review, we discuss how one such costimulatory pathway, CD40-CD40L, regulates CD8 dysfunction and rescue. While the significance of this pathway has been extensively investigated in models of autoimmunity, acute infectious diseases, and tumor models, the role played by CD40-CD40L in regulating CD8 exhaustion in chronic infectious diseases is just beginning to be understood. Considering that monotherapy with blocking antibodies targeting inhibitory PD-1-PD-L1 pathway is only partially effective at ameliorating CD8 exhaustion and that humanized CD40 agonist antibodies are currently available, a better understanding of the role of the CD40-CD40L pathway in chronic infectious diseases will pave the way for the development of more robust immunotherapeutic and prophylactic vaccination strategies.