Ipsilateral immunization after a prior SARS-CoV-2 mRNA vaccination elicits superior B cell responses compared to contralateral immunization.

mRNA vaccines have proven to be pivotal in the fight against COVID-19. A recommended booster, given 3 to 4 weeks post the initial vaccination, can substantially amplify protective antibody levels. Here, we show that, compared to contralateral boost, ipsilateral boost of the SARS-CoV-2 mRNA vaccine induces more germinal center B cells (GCBCs) specific to the receptor binding domain (RBD) and generates more bone marrow plasma cells. Ipsilateral boost can more rapidly generate high-affinity RBD-specific antibodies with improved cross-reactivity to the Omicron variant. Mechanistically, the ipsilateral boost promotes the positive selection and plasma cell differentiation of pre-existing GCBCs from the prior vaccination, associated with the expansion of T follicular helper cells. Furthermore, we show that ipsilateral immunization with an unrelated antigen after a prior mRNA vaccination enhances the germinal center and antibody responses to the new antigen compared to contralateral immunization. These findings propose feasible approaches to optimize vaccine effectiveness.

A viral-specific CD4 + T cell response protects female mice from Coxsackievirus B3 infection.

Biological sex plays an integral role in the immune response to various pathogens. The underlying basis for these sex differences is still not well defined. Here, we show that Coxsackievirus B3 (CVB3) induces a viral-specific CD4 + T cell response that can protect female mice from mortality. We found that CVB3 can induce expansion of CD62L lo CD4 + T cells in the mesenteric lymph node and spleen of female but not male mice as early as 5 days post-inoculation, indicative of activation. Using a recombinant CVB3 virus expressing a model CD4 + T cell epitope, we found that this response is due to viral antigen and not bystander activation. Finally, the depletion of CD4 + T cells before infection increased mortality in female mice, indicating that CD4 + T cells play a protective role against CVB3 in our model. Overall, these data demonstrated that CVB3 can induce an early CD4 response in female but not male mice and further emphasize how sex differences in immune responses to pathogens affect disease outcomes.

Coxsackievirus B3 elicits a sex-specific CD8+ T cell response which protects female mice.

Sex is a significant contributor to the outcome of human infections. Males are frequently more susceptible to viral, bacterial, and fungal infections, often attributed to weaker immune responses. In contrast, a heightened immune response in females enables better pathogen elimination but leaves females more predisposed to autoimmune diseases. Unfortunately, the underlying basis for sex-specific immune responses remains poorly understood. Here, we show a sex difference in the CD8+ T cell response to an enteric virus, Coxsackievirus B3 (CVB3). We found that CVB3 induced expansion of CD8+ T cells in female mice but not in male mice. CVB3 also increased the proportion and number of CD11ahiCD62Llo CD8+ T cells in female mice, indicative of activation. This response was independent of the inoculation route and type I interferon. Using a recombinant CVB3 virus expressing a model CD8+ T cell epitope, we found that the expansion of CD8+ T cells in females is viral-specific and not due to bystander activation. Finally, the depletion of CD8+ T cells, prior to infection, led to enhanced mortality, indicating that CD8+ T cells are protective against CVB3 in female mice. These data demonstrate that CVB3 induces a CD8+ T cell response in female mice and highlight the importance of sex-specific immune responses to viral pathogens.

A viral-specific CD4+ T cell response protects female mice from Coxsackievirus B3 infection.

Background: Biological sex plays an integral role in the immune response to various pathogens. The underlying basis for these sex differences is still not well defined. Here, we show that Coxsackievirus B3 (CVB3) induces a viral-specific CD4+ T cell response that can protect female mice from mortality. Methods: We inoculated C57BL/6 Ifnar-/- mice with CVB3. We investigated the T cell response in the spleen and mesenteric lymph nodes in male and female mice following infection. Results: We found that CVB3 can induce expansion of CD62Llo CD4+ T cells in the mesenteric lymph node and spleen of female but not male mice as early as 5 days post-inoculation, indicative of activation. Using a recombinant CVB3 virus expressing a model CD4+ T cell epitope, we found that this response is due to viral antigen and not bystander activation. Finally, the depletion of CD4+ T cells before infection increased mortality in female mice, indicating that CD4+ T cells play a protective role against CVB3 in our model. Conclusions: Overall, these data demonstrated that CVB3 can induce an early CD4 response in female but not male mice and further emphasize how sex differences in immune responses to pathogens affect disease.

Testosterone Promotes the Intestinal Replication and Dissemination of Coxsackievirus B3 in an Oral Inoculation Mouse Model.

Enteroviruses initiate infection in the gastrointestinal tract, and sex is often a biological variable that impacts pathogenesis. Previous data suggest that sex hormones can influence the intestinal replication of Coxsackievirus B3 (CVB3), an enterovirus in the Picornaviridae family. However, the specific sex hormone(s) that regulates intestinal CVB3 replication is poorly understood. To determine if testosterone promotes intestinal CVB3 replication, we orally inoculated male and female Ifnar-/- mice that were treated with either placebo or testosterone-filled capsules. Following oral inoculation, we found that the testosterone-treated male and female mice shed significantly more CVB3 in their feces than did the placebo-treated mice, indicating that testosterone enhances intestinal replication. Similarly, testosterone enhanced viral dissemination in both sexes, as we observed higher viral loads in peripheral tissues following infection. Further, the testosterone-treated male mice also had a higher mortality rate than did the testosterone-depleted male mice. Finally, we observed that testosterone significantly affected the immune response to CVB3. We found that testosterone broadly increased proinflammatory cytokines and chemokines while decreasing the number of splenic B cells and dendritic cells following CVB3 infection. Moreover, while testosterone did not affect the early CD4 T cell response to CVB3, testosterone reduced the activation of CD8 T cells. These data indicate that testosterone can promote intestinal CVB3 replication and dissemination while also impacting the subsequent viral immune response. IMPORTANCE Biological sex plays a significant role in the outcomes of various infections and diseases. The impact of sex hormones on the intestinal replication and dissemination of Coxsackievirus B3 remains poorly understood. Using an oral inoculation model, we found that testosterone enhances CVB3 shedding and dissemination in male and female mice. Further, testosterone can alter the immune response to CVB3. This work highlights the role of testosterone in CVB3 pathogenesis and suggests that sex hormones can impact the replication and dissemination of enteric viruses.

Spatially mapping the immune landscape of melanoma using imaging mass cytometry.

Melanoma is an immunogenic cancer with a high response rate to immune checkpoint inhibitors (ICIs). It harbors a high mutation burden compared with other cancers and, as a result, has abundant tumor-infiltrating lymphocytes (TILs) within its microenvironment. However, understanding the complex interplay between the stroma, tumor cells, and distinct TIL subsets remains a substantial challenge in immune oncology. To properly study this interplay, quantifying spatial relationships of multiple cell types within the tumor microenvironment is crucial. To address this, we used cytometry time-of-flight (CyTOF) imaging mass cytometry (IMC) to simultaneously quantify the expression of 35 protein markers, characterizing the microenvironment of 5 benign nevi and 67 melanomas. We profiled more than 220,000 individual cells to identify melanoma, lymphocyte subsets, macrophage/monocyte, and stromal cell populations, allowing for in-depth spatial quantification of the melanoma microenvironment. We found that within pretreatment melanomas, the abundance of proliferating antigen-experienced cytotoxic T cells (CD8+CD45RO+Ki67+) and the proximity of antigen-experienced cytotoxic T cells to melanoma cells were associated with positive response to ICIs. Our study highlights the potential of multiplexed single-cell technology to quantify spatial cell-cell interactions within the tumor microenvironment to understand immune therapy responses.

An Epidemic Zika Virus Isolate Drives Enhanced T Follicular Helper Cell and B Cell-Mediated Immunity.

Zika virus (ZIKV) is a mosquito-borne pathogen that recently caused a series of increasingly severe outbreaks. We previously demonstrated that, compared with a pre-epidemic isolate (ZIKVCDN), a Brazilian ZIKV isolate (ZIKVBR) possesses a novel capacity to suppress host immunity, resulting in delayed viral clearance. However, whether ZIKVBR modulates CD4 T cell responses remains unknown. In this study, we show that, in comparison with ZIKVCDN infection, CD4 T cells are less polarized to the Th1 subtype following ZIKVBR challenge in mice. In contrast, we observed an enhanced accumulation of T follicular helper cells 10, 14, and 21 d postinfection with ZIKVBR This response correlated with an enhanced germinal center B cell response and robust production of higher avidity-neutralizing Abs following ZIKVBR infection. Taken together, our data suggest that contemporary ZIKV strains have evolved to differentially induce CD4 T cell, B cell, and Ab responses and this could provide a model to further define the signals required for T follicular helper cell development.

Pre-existing chromatin accessibility and gene expression differences among naive CD4+ T cells influence effector potential.

CD4+ T cells have a remarkable potential to differentiate into diverse effector lineages following activation. Here, we probe the heterogeneity present among naive CD4+ T cells before encountering their cognate antigen to ask whether their effector potential is modulated by pre-existing transcriptional and chromatin landscape differences. Single-cell RNA sequencing shows that key drivers of variability are genes involved in T cell receptor (TCR) signaling. Using CD5 expression as a readout of the strength of tonic TCR interactions with self-peptide MHC, and sorting on the ends of this self-reactivity spectrum, we find that pre-existing transcriptional differences among naive CD4+ T cells impact follicular helper T (TFH) cell versus non-TFH effector lineage choice. Moreover, our data implicate TCR signal strength during thymic development in establishing differences in naive CD4+ T cell chromatin landscapes that ultimately shape their effector potential.

An epidemic Zika virus isolate suppresses antiviral immunity by disrupting antigen presentation pathways.

Zika virus (ZIKV) has emerged as an important global health threat, with the recently acquired capacity to cause severe neurological symptoms and to persist within host tissues. We previously demonstrated that an early Asian lineage ZIKV isolate induces a highly activated CD8 T cell response specific for an immunodominant epitope in the ZIKV envelope protein in wild-type mice. Here we show that a contemporary ZIKV isolate from the Brazilian outbreak severely limits CD8 T cell immunity in mice and blocks generation of the immunodominant CD8 T cell response. This is associated with a more sustained infection that is cleared between 7- and 14-days post-infection. Mechanistically, we demonstrate that infection with the Brazilian ZIKV isolate reduces the cross-presentation capacity of dendritic cells and fails to fully activate the immunoproteasome. Thus, our study provides an isolate-specific mechanism of host immune evasion by one Brazilian ZIKV isolate, which differs from the early Asian lineage isolate and provides potential insight into viral persistence associated with recent ZIKV outbreaks.

Point-activated ESR1Y541S has a dramatic effect on the development of sexually dimorphic organs.

Mutations in the estrogen receptor α (ERα) occur in endocrine-resistant metastatic breast cancer. However, a major gap persists with the lack of genetically tractable immune competent mouse models to study disease. Hence, we developed a Cre-inducible murine model expressing a point-activated ESR1Y541S (ESR1Y537S in humans) driven by its endogenous promoter. Germline expression of mutant ESR1Y541S reveals dramatic developmental defects in the reproductive organs, mammary glands, and bones of the mice. These observations provide critical insights into the tissue-specific roles of ERα during development and highlights the potential use of our model in further developmental and cancer studies.

Cyclophilin D Regulates Antiviral CD8+ T Cell Survival in a Cell-Extrinsic Manner.

CD8+ T cell-mediated immunity is critical for host defense against viruses and requires mitochondria-mediated type I IFN (IFN-I) signaling for optimal protection. Cyclophilin D (CypD) is a mitochondrial matrix protein that modulates the mitochondrial permeability transition pore, but its role in IFN-I signaling and CD8+ T cell responses to viral infection has not been previously explored. In this study, we demonstrate that CypD plays a critical extrinsic role in the survival of Ag-specific CD8+ T cell following acute viral infection with lymphocytic choriomeningitis virus in mice. CypD deficiency resulted in reduced IFN-I and increased CD8+ T cell death, resulting in a reduced antiviral CD8+ T cell response. In addition, CypD deficiency was associated with an increase in pathogen burden at an early time-point following infection. Furthermore, our data demonstrate that transfer of wild-type macrophages (expressing CypD) to CypD-deficient mice can partially restore CD8+ T cell responses. These results establish that CypD plays an extrinsic role in regulating optimal effector CD8+ T cell responses to viral infection. Furthermore, this suggests that, under certain circumstances, inhibition of CypD function may have a detrimental impact on the host's ability to respond to viral infection.

Control of memory CD8+ T cell longevity and effector functions by IL-15.

IL-15 is a member of the common gamma chain family of cytokines and plays important roles in regulating several aspects of innate and adaptive immunity. Besides its established role in controlling homeostatic proliferation and survival of memory CD8+ T cells and natural killer cells, recent findings demonstrate that inflammatory IL-15 can also stimulate a variety of effector functions, such as enhanced cytotoxicity, entry into the cell cycle, and trafficking into non-lymphoid tissues. Here, we discuss how IL-15 is critical in regulating many functions of memory CD8+ T cells and how these processes act collectively to ensure optimal protective cellular immunity against re-infections.

Metabolic Profiling Using Stable Isotope Tracing Reveals Distinct Patterns of Glucose Utilization by Physiologically Activated CD8+ T Cells.

Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.

Zika Virus Pathogenesis: From Early Case Reports to Epidemics.

For the first 60 years following its isolation, Zika virus (ZIKV) remained a relatively poorly described member of the Flaviviridae family. However, since 2007, it has caused a series of increasingly severe outbreaks and is now associated with neurological symptoms such as Guillain-Barré syndrome and congenital Zika syndrome (CZS). A number of reports have improved our understanding of rare complications that may be associated with ZIKV infection in adults, the areas of the body to which it spreads, and viral persistence in various tissues. Likewise, studies on the effect of ZIKV infection during pregnancy have identified risk factors for CZS and the impact this syndrome has on early childhood. Understanding these outcomes and the factors that drive ZIKV pathogenesis are key to developing vaccination and therapeutic approaches to avoid these severe and potentially debilitating symptoms.

Protective to a T: The Role of T Cells during Zika Virus Infection.

CD4 and CD8 T cells are an important part of the host's capacity to defend itself against viral infections. During flavivirus infections, T cells have been implicated in both protective and pathogenic responses. Given the recent emergence of Zika virus (ZIKV) as a prominent global health threat, the question remains as to how T cells contribute to anti-ZIKV immunity. Furthermore, high homology between ZIKV and other, co-circulating flaviviruses opens the possibility of positive or negative effects of cross-reactivity due to pre-existing immunity. In this review, we will discuss the CD4 and CD8 T cell responses to ZIKV, and the lessons we have learned from both mouse and human infections. In addition, we will consider the possibility of whether T cells, in the context of flavivirus-naïve and flavivirus-immune subjects, play a role in promoting ZIKV pathogenesis during infection.

Running interference: Interplay between Zika virus and the host interferon response.

The interferon (IFN) family of cytokines is a crucial part of the host's ability to mount an effective immune response against viral infections. In addition to establishing an antiviral state within cells, IFNs also support the optimal activation of other key immune cell types. The ability of members of the Flaviviridae family to suppress type I IFN responses has been well-described. Of these viruses, Zika virus (ZIKV) has recently attracted international attention due to a series of major outbreaks that featured the novel association of neurological symptoms with infection. Researchers have begun to investigate the strategies ZIKV uses to evade type I IFNs, and the impact this has on the host. However, a unique feature of ZIKV infection compared to other flaviviruses is its capacity to be transmitted sexually, as well as its ability to infect and persist within reproductive tissues. As such, this raises the question of a potential role for type III IFN during ZIKV infection. In this review, we will discuss the interplay between these two classes of IFN with ZIKV, models that have been used to interrogate these interactions, and the effect this interplay has on infection and infection outcomes. We will also consider the intriguing possibility of whether ZIKV has evolved improved evasion mechanisms to suppress the IFN response in recent outbreaks.

Interleukin-10 Directly Inhibits CD8+ T Cell Function by Enhancing N-Glycan Branching to Decrease Antigen Sensitivity.

Chronic viral infections remain a global health concern. The early events that facilitate viral persistence have been linked to the activity of the immunoregulatory cytokine IL-10. However, the mechanisms by which IL-10 facilitates the establishment of chronic infection are not fully understood. Herein, we demonstrated that the antigen sensitivity of CD8+ T cells was decreased during chronic infection and that this was directly mediated by IL-10. Mechanistically, we showed that IL-10 induced the expression of Mgat5, a glycosyltransferase that enhances N-glycan branching on surface glycoproteins. Increased N-glycan branching on CD8+ T cells promoted the formation of a galectin 3-mediated membrane lattice, which restricted the interaction of key glycoproteins, ultimately increasing the antigenic threshold required for T cell activation. Our study identified a regulatory loop in which IL-10 directly restricts CD8+ T cell activation and function through modification of cell surface glycosylation allowing the establishment of chronic infection.

Protein Tyrosine Phosphatase Inhibition Prevents Experimental Cerebral Malaria by Precluding CXCR3 Expression on T Cells.

Cerebral malaria induced by Plasmodium berghei ANKA infection is dependent on the sequestration of cytotoxic T cells within the brain and augmentation of the inflammatory response. Herein, we demonstrate that inhibition of protein tyrosine phosphatase (PTP) activity significantly attenuates T cell sequestration within the brain and prevents the development of neuropathology. Mechanistically, the initial upregulation of CXCR3 on splenic T cells upon T cell receptor stimulation was critically decreased through the reduction of T cell-intrinsic PTP activity. Furthermore, PTP inhibition markedly increased IL-10 production by splenic CD4+ T cells by enhancing the frequency of LAG3+CD49b+ type 1 regulatory cells. Overall, these findings demonstrate that modulation of PTP activity could possibly be utilized in the treatment of cerebral malaria and other CXCR3-mediated diseases.

Analysis of the T Cell Response to Zika Virus and Identification of a Novel CD8+ T Cell Epitope in Immunocompetent Mice.

Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although ZIKV infection is typically mild and self-limiting in healthy adults, infection has been associated with neurological symptoms such as Guillain-Barré syndrome, and a causal link has been established between fetal microcephaly and ZIKV infection during pregnancy. These risks, and the magnitude of the ongoing ZIKV pandemic, have created an urgent need for the development of animal models to study the immune response to ZIKV infection. Previous animal models have primarily focused on pathogenesis in immunocompromised mice. In this study, we provide a model of ZIKV infection in wild-type immunocompetent C57BL/6 mice, and have provided an analysis of the immune response to infection. We evaluated the activation of several innate immune cell types, and studied the kinetics, phenotype, and functionality of T cell responses to ZIKV infection. Our results demonstrate that ZIKV infection is mild in wild-type immunocompetent C57BL/6 mice, resulting in minimal morbidity. Our data establish that at the peak of the adaptive response, antigen-experienced CD4+ T cells polarize to a Th1 phenotype, and antigen-experienced CD8+ T cells exhibit an activated effector phenotype, producing both effector cytokines and cytolytic molecules. Furthermore, we have identified a novel ZIKV CD8+ T cell epitope in the envelope protein that is recognized by the majority of responding cells. Our model provides an important reference point that will help dissect the impact of polymorphisms in the circulating ZIKV strains on the immune response and ZIKV pathogenesis. In addition, the identification of a ZIKV epitope will allow for the design of tetramers to study epitope-specific T cell responses, and will have important implications for the design and development of ZIKV vaccine strategies.