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Most West Nile virus (WNV) infections are asymptomatic, but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death. Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection. However, other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage. We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen (HLA) class II tetramers. Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections. Independent of this, we also observed age related increases in WNV-specific T cell responses. Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells, whose presence was highly correlated with neuroinvasive disease. Moreover, a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects. More globally, subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections. Thus, subjects with neuroinvasive WNV infections exhibited elevated, dysregulated, and atypically polarized responses, suggesting that immune mediated damage may indeed contribute to pathogenic outcomes.

Monitoring the frequency of circulatory CXCR5+ (cCXCR5+) CD4+ T cells in periphery blood provides a potential biomarker to draw inferences about T follicular helper (TFH) activity within germinal center. However, cCXCR5+ T cells are highly heterogeneous in their expression of ICOS, PD1 and CD38 and the relationship between different cCXCR5 subsets as delineated by these markers remains unclear. We applied class II tetramer reagents and mass cytometry to investigate the ontogeny of different subsets of cCXCR5+ T cell following yellow fever immunization. Through unsupervised analyses of mass cytometry data, we show yellow fever virus-specific cCXCR5 T cells elicited by vaccination were initially CD38+ICOS+PD1+, but then transitioned to become CD38+ICOS−PD1+ and CD38−ICOS−PD1+ before coming to rest as a CD38−ICOS−PD1− subset. These results imply that most antigen-specific cCXCR5+ T cells, including the CD38−ICOS−PD1− CXCR5+ T cells are derived from the CXCR5+CD38+ICOS+PD1+ subset, the subset that most resembles preTFH/TFH in the germinal center. [ABSTRACT FROM AUTHOR]

Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8+ T cell responses, less is known about YFV-specific CD4+ T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4+ T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4+ T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4+ T cell responses that contract, forming a detectable memory population. [ABSTRACT FROM AUTHOR]

DRB1*08:01 (DR0801) and DRB1*11:01 (DR1101) are highly homologous alleles that have opposing effects on susceptibility to primary biliary cirrhosis (PBC). DR0801 confers risk and shares a key feature with other HLA class II alleles that predispose to autoimmunity: a nonaspartic acid at beta57. DR1101 is associated with protection from PBC, and its sequence includes an aspartic acid at beta57. To elucidate a mechanism for the opposing effects of these HLA alleles on PBC susceptibility, we compared the features of epitopes presented by DR0801 and DR1101. First, we identified DR0801- and DR1101-restricted epitopes within multiple viral Ags, observing both shared and distinct epitopes. Because DR0801 is not well characterized, we deduced its motif by measuring binding affinities for a library of peptides, confirming its key features through structural modeling. DR0801 was distinct from DR1101 in its ability to accommodate charged residues within all but one of its binding pockets. In particular, DR0801 strongly preferred acidic residues in pocket 9. These findings were used to identify potentially antigenic sequences within PDC-E2 (an important hepatic autoantigen) that contain a DR0801 motif. Four peptides bound to DR0801 with reasonable affinity, but only one of these bound to DR1101. Three peptides, PDC-E2145-159, PDC-E2249-263, and PDC-E2629-643, elicited high-affinity T cell responses in DR0801 subjects, implicating these as likely autoreactive specificities. Therefore, the unique molecular features of DR0801 may lead to the selection of a distinct T cell repertoire that contributes to breakdown of self-tolerance in primary biliary cirrhosis, whereas those of DR1101 promote tolerance. [ABSTRACT FROM AUTHOR]

Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8(+) T cell responses, less is known about YFV-specific CD4(+) T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4(+) T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4(+) T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4(+) T cell responses that contract, forming a detectable memory population.

We recently found that normal human brain and low-grade astrocytomas express the receptor protein tyrosine phosphatase mu (PTPmu) and that the more invasive astrocytomas, glioblastoma multiforme (GBM), downregulate full-length PTPmu expression. Loss of PTPmu expression in GBMs is due to proteolytic cleavage that generates an intracellular and potentially a cleaved and released extracellular fragment of PTPmicro. Here, we identify that a cleaved extracellular fragment containing the domains required for PTPmicro-mediated adhesion remains associated with GBM tumor tissue. We hypothesized that detection of this fragment would make an excellent diagnostic tool for the localization of tumor tissue within the brain. To this end, we generated a series of fluorescently tagged peptide probes that bind the PTPmu fragment. The peptide probes specifically recognize GBM cells in tissue sections of surgically resected human tumors. To test whether the peptide probes are able to detect GBM tumors in vivo, the PTPmu peptide probes were tested in both mouse flank and intracranial xenograft human glioblastoma tumor model systems. The glial tumors were molecularly labeled with the PTPmu peptide probes within minutes of tail vein injection using the Maestro FLEX In Vivo Imaging System. The label was stable for at least 3 hours. Together, these results indicate that peptide recognition of the PTPmu extracellular fragment provides a novel molecular diagnostic tool for detection of human glioblastomas. Such a tool has clear translational applications and may lead to improved surgical resections and prognosis for patients with this devastating disease.