Natural Secretory Immunoglobulins Facilitate Enteric Viral Infection

Noroviruses are enteric pathogens causing significant morbidity, mortality and economic losses worldwide. Secretory immunoglobulins (SIg) are a first line of mucosal defense against enteric pathogens. They are secreted into the intestinal lumen via the polymeric immunoglobulin receptor (pIgR), where they bind to antigens. However, whether natural SIg protect against norovirus infection remains unknown. To determine if natural SIg alter murine norovirus (MNV) pathogenesis, I infected pIgR knockout (KO) mice, which lack SIg in mucosal secretions. Acute MNV infection was significantly reduced in pIgR KO mice compared to controls, despite increased MNV target cells in the Peyer's patch. Natural SIg did not alter MNV binding to, or crossing, of the follicle-associated epithelium (FAE) into the lymphoid follicle. Instead, naïve pIgR KO mice have enhanced levels of the antiviral inflammatory molecules interferon gamma (IFN-g) and inducible nitric oxide synthase (iNOS) in the ileum compared to controls. Strikingly, depletion of the intestinal microbiota in pIgR KO and control mice resulted in comparable IFN-g and iNOS levels, as well as MNV infectious titers. IFN-g treatment of WT mice and neutralization of IFN-g in pIgR KO mice modulated MNV titers, implicating this antiviral cytokine in the phenotype. Reduced gastrointestinal infection in pIgR KO mice was also observed with another enteric virus, reovirus. Collectively, my findings suggest that natural SIg are not protective during enteric virus infection, but rather that SIg promote enteric viral infection through alterations in microbial immune responses.PHDImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/149995/1/hturula_1.pd

Investigation of the impact of the common animal facility contaminant murine norovirus on experimental murine cytomegalovirus infection

AbstractMurine norovirus (MNV) is a recently discovered pathogen that has become a common contaminant of specific pathogen-free mouse colonies. MNV-1 induces a robust interferon-β response and causes histopathology in some mouse strains, suggesting that it may impact other mouse models of infection. Despite many concerns about MNV-1 contamination, there is little information about its impact on immune responses to other infections. This study addresses whether MNV-1 infection has an effect on a model of murine cytomegalovirus (MCMV) infection. Exposure to MNV-1 resulted in a decreased CD8 T cell response to immunodominant MCMV epitopes in both BALB/c and C57BL/6 mice. However, MNV-1 did not impact MCMV titers in either mouse strain, nor did it stimulate reactivation of latent MCMV. These data suggest that while MNV-1 has a mild impact on the immune response to MCMV, it is not likely to affect most experimental outcomes in immunocompetent mice in the MCMV model

Memory T Cells Specific for Murine Cytomegalovirus Re-Emerge after Multiple Challenges and Recapitulate Immunity in Various Adoptive Transfer Scenarios.

Reconstitution of CMV-specific immunity after transplant remains a primary clinical objective to prevent CMV disease, and adoptive immunotherapy of CMV-specific T cells can be an effective therapeutic approach. Because of viral persistence, most CMV-specific CD8(+) T cells become terminally differentiated effector phenotype CD8(+) T cells (TEFF). A minor subset retains a memory-like phenotype (memory phenotype CD8(+) T cells [TM]), but it is unknown whether these cells retain memory function or persist over time. Interestingly, recent studies suggest that CMV-specific CD8(+) T cells with different phenotypes have different abilities to reconstitute sustained immunity after transfer. The immunology of human CMV infections is reflected in the murine CMV (MCMV) model. We found that human CMV- and MCMV-specific T cells displayed shared genetic programs, validating the MCMV model for studies of CMV-specific T cells in vivo. The MCMV-specific TM population was stable over time and retained a proliferative capacity that was vastly superior to TEFF. Strikingly, after transfer, TM established sustained and diverse T cell populations even after multiple challenges. Although both TEFF and TM could protect Rag(-/-) mice, only TM persisted after transfer into immune replete, latently infected recipients and responded if recipient immunity was lost. Interestingly, transferred TM did not expand until recipient immunity was lost, supporting that competition limits the Ag stimulation of TM. Ultimately, these data show that CMV-specific TM retain memory function during MCMV infection and can re-establish CMV immunity when necessary. Thus, TM may be a critical component for consistent, long-term adoptive immunotherapy success

Stochastic expansions maintain the clonal stability of CD8+ T cell populations undergoing memory inflation driven by murine cytomegalovirus

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an Ag-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of Ag during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate Ag OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent Ag-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with Ag occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV

Systemic hematogenous maintenance of memory inflation by MCMV infection.

Several low-grade persistent viral infections induce and sustain very large numbers of virus-specific effector T cells. This was first described as a response to cytomegalovirus (CMV), a herpesvirus that establishes a life-long persistent/latent infection, and sustains the largest known effector T cell populations in healthy people. These T cells remain functional and traffic systemically, which has led to the recent exploration of CMV as a persistent vaccine vector. However, the maintenance of this remarkable response is not understood. Current models propose that reservoirs of viral antigen and/or latently infected cells in lymph nodes stimulate T cell proliferation and effector differentiation, followed by migration of progeny to non-lymphoid tissues where they control CMV reactivation. We tested this model using murine CMV (MCMV), a natural mouse pathogen and homologue of human CMV (HCMV). While T cells within draining lymph nodes divided at a higher rate than cells elsewhere, antigen-dependent proliferation of MCMV-specific effector T cells was observed systemically. Strikingly, inhibition of T cell egress from lymph nodes failed to eliminate systemic T cell division, and did not prevent the maintenance of the inflationary populations. In fact, we found that the vast majority of inflationary cells, including most cells undergoing antigen-driven division, had not migrated into the parenchyma of non-lymphoid tissues but were instead exposed to the blood supply. Indeed, the immunodominance and effector phenotype of inflationary cells, both of which are primary hallmarks of memory inflation, were largely confined to blood-localized T cells. Together these results support a new model of MCMV-driven memory inflation in which most immune surveillance occurs in circulation, and in which most inflationary effector T cells are produced in response to viral antigen presented by cells that are accessible to the blood supply

The Role of the Polymeric Immunoglobulin Receptor and Secretory Immunoglobulins during Mucosal Infection and Immunity

The gastrointestinal tract houses millions of microbes, and thus has evolved several host defense mechanisms to keep them at bay, and prevent their entry into the host. One such mucosal surface defense is the secretion of secretory immunoglobulins (SIg). Secretion of SIg depends on the polymeric immunoglobulin receptor (pIgR), which transports polymeric Ig (IgA or IgM) from the basolateral surface of the epithelium to the apical side. Upon reaching the luminal side, a portion of pIgR, called secretory component (SC) is cleaved off to release Ig, forming SIg. Through antigen-specific and non-specific binding, SIg can modulate microbial communities and pathogenic microbes via several mechanisms: agglutination and exclusion from the epithelial surface, neutralization, or via host immunity and complement activation. Given the crucial role of SIg as a microbial scavenger, some pathogens also evolved ways to modulate and utilize pIgR and SIg to facilitate infection. This review will cover the regulation of the pIgR/SIg cycle, mechanisms of SIg-mediated mucosal protection as well as pathogen utilization of SIg

CD27high/KLRG1low CD8+ T cells that persist throughout MCMV infection are highly expansive and have the ability to reestablish MCMV immunity

Abstract Cytomegalovirus (CMV) is a herpesvirus that establishes life-long latency in 60-80% of Americans. Constant immune surveillance is necessary to prevent viral reactivation from latency and results in the accumulation of functional CMV-specific CD8+ T cells (CD8s) over time, a process termed memory inflation. As such, CMV reactivations remain a clinical concern for immunosuppressed patients and reconstituting CMV immunity is critical for the long-term prevention of CMV disease. Understanding the maintenance of memory inflation may reveal novel approaches to restore CMV immunity. Previous work has shown that the majority of inflationary CD8s express a terminally-differentiated “effector” (TEFF) phenotype, have a short half-life and appear unable to sustain long-term CMV immunity. Interestingly, inflationary populations also include a minor subset of CD8s that express a “memory” phenotype (TM)

Murine CMV Infection Induces the Continuous Production of Mucosal Resident T Cells

Cytomegalovirus (CMV) is a herpesvirus that persists for life and maintains extremely large numbers of T cells with select specificities in circulation. However, it is unknown how viral persistence impacts T cell populations in mucosal sites. We found that many murine (M)CMV-specific CD8s in mucosal tissues became resident memory T cells (TRM). These cells adopted an intraepithelial localization in the salivary gland that correlated with, but did not depend on, expression of the integrin CD103. MCMV-specific TRM cells formed early after infection, and spleen-localized cells had reduced capacities to become TRM at late times. Surprisingly, however, small numbers of new TRM cells were formed from the circulating pool throughout infection, favoring populations maintained at high levels in the blood and shifting the immunodominance within the TRM populations over time. These data show that mucosal TRM populations can be dynamically maintained by a persistent infection

Long-term FTY720 treatment does not alter the maintenance of memory inflation.

<p>Mice infected with K181 MCMV for more than 3 months were treated with FTY720 in their drinking water for five weeks. Mice were injected with BrdU 16(A–D) The M38-specific T cell population is stable in the blood of mice during prolonged FTY720 treatment. Shown is (A) the number of naïve CD8s per milliliter of blood, (B) the number of M38-specific CD8s per milliliter of blood, (C) the percentage of blood-localized M38-specific CD8s expressing the effector phenotype (KLRG1^pos, CD127^low), and (D) the frequency of Ki67-positive M38-specific CD8s in the blood before and during the time course in treated mice (top) and controls (bottom). (E–G) Prolonged FTY720 treatment does not alter the number, division, or phenotype of dividing M38-specific T cells associated with the organs. Shown are (E) the absolute numbers of M38-specific CD8s and (F) the frequency of M38-specific T cells that were Ki67-positive and labeled with BrdU in control (white) and FTY720 treated (grey) mice. The representative FACS plots (G) show the KLRG1 and CD127 expression of M38-specific T cells (left panels) and dividing Ki67-positive M38-specific population (right panels) in the blood of FTY720 treated (top panels) and control mice (bottom panels) at the end of the treatment period. In all cases, error bars represent the standard error of the mean (n = 5 per group). Statistical significance was measured by paired (A–D) and unpaired (E–F) student's t-tests (*p<.05, **p<.01, ***p<.001).</p

Effector-phenotype CD8s turnover continuously and undergo antigen-dependent division during MCMV-induced memory inflation.

<p>(A) In C57BL/6 mice infected with K181 MCMV, M38-specific T cells accumulate over time while M45-specific T cells contract. Shown is the frequency of MCMV-specific CD8s in the blood over time, as measured by tetramer staining. (B) Inflationary T cells are also found in non-lymphoid organs. Shown is the frequency of M38-specific CD8s in the indicated organs of mice infected as in A, more than 3 months post infection. Each symbol represents an individual mouse. (C) Most inflationary T cells express an effector phenotype. Mice were infected as in A. The representative FACS plot (left) shows the KLRG1 and CD127 expression of M38-specific CD8s in the blood. The graph (right) shows the KLRG1 and CD127 expression of M38-specific T cells in organs more than 3 months post infection (n = 12, CLN  =  cervical lymph nodes, MLN  =  mediastinal lymph nodes). (D) Recently divided inflationary T cells are lost over time. B6 mice infected with K181 MCMV or WT-BAC MCMV for more than 3 months were pulsed with BrdU for 3 (n = 6) or 7 (n = 5) days. To account for mouse-to-mouse variation in the incorporation of BrdU, the data was normalized to the frequency of inflationary T cells that were BrdU-positive 7 days post-pulse. Shown is the proportion of M38- and IE3-specific CD8s that retained BrdU in the blood over time. Statistical significance was determined by comparing the proportion of cells retaining BrdU relative to the week 1 time point, using a paired student's t-test (*p<.05, **p<.01, ***p<.001). As a comparison, the same analysis was performed on total CD8 T cells (not antigen-specific) expressing CD127 and lacking KLRG1. Combined data from two independent experiments is displayed. (E) Recently divided inflationary T cells with an effector phenotype are lost over time. Shown is the phenotype of BrdU-positive M38-specific T cells at the indicated time points after BrdU pulse. Any data points with fewer than 25 labeled M38-specific T cells were excluded from the analysis at that time point. Statistical significance was determined as in “D” except that the proportion of BrdU-labeled cells expressing an effector phenotype was compared to the week 0 time point. (F) Adoptive transfer schematic. The representative FACS plot shows donor OT-Is, as a frequency of all CD8s, in the spleen of recipients 28 days after transfer. (G) Donor OT-Is with an effector phenotype only express Ki67 in the presence of cognate antigen. Shown is the Ki67 expression of effector-phenotype (KLRG1^pos, CD127^low) and memory-phenotype (KLRG-1^neg, CD127^pos) OT-Is in the spleen 28 days post transfer. Results are a combination of two independent experiments (n = 6 per group). Statistical significance was measured by an unpaired student's t-test (*p<.05). In all cases above, error bars represent the standard error of the mean.</p