Eva-Stina Edholm

Nonclassical MHC class I-dependent invariant T cells are evolutionarily conserved and prominent from early development in amphibians

Human and murine MHC nonclassical class Ib-restricted invariant T (iT) cell subsets, such as invariant natural killer T cells (iNKT) and mucosal-associated invariant T cells, have specialized functions early in immune responses, especially in modulating subsequent adaptive immune responses. Here, we characterize a prominent iT population in the amphibian Xenopus laevis and show the requirement of the class Ib molecule, Xenopus nonclassical gene 10, in its differentiation and function. Using Xenopus nonclassical gene 10 tetramers and RNAi loss of function by transgenesis, we identified a large class Ib-dependent CD8−/CD4− iT subset in unmanipulated frogs and tadpoles. This population is critical for antiviral immunity during early larval stages when classical MHC class Ia function is suboptimal. Furthermore, in young tadpoles with low class Ia expression, deep sequencing revealed additional preponderant invariant T cell receptor (TCR)α rearrangements, implying other iT cell subsets and a predominant selection process mediated by other class Ib molecules. The restriction and requirement of class Ib molecules for development and antiviral immunity of a mammalian iNKT or mucosal-associated invariant T cell counterpart in the amphibian Xenopus show the importance of iT cells in the emergence and evolution of the adaptive immune system.

Evolution of nonclassical MHC-dependent invariant T cells.

TCR-mediated specific recognition of antigenic peptides in the context of classical MHC molecules is a cornerstone of adaptive immunity of jawed vertebrate. Ancillary to these interactions, the T cell repertoire also includes unconventional T cells that recognize endogenous and/or exogenous antigens in a classical MHC-unrestricted manner. Among these, the mammalian nonclassical MHC class I-restricted invariant T cell (iT) subsets, such as iNKT and MAIT cells, are now believed to be integral to immune response initiation as well as in orchestrating subsequent adaptive immunity. Until recently the evolutionary origins of these cells were unknown. Here we review our current understanding of a nonclassical MHC class I-restricted iT cell population in the amphibian Xenopus laevis. Parallels with the mammalian iNKT and MAIT cells underline the crucial biological roles of these evolutionarily ancient immune subsets.

A prominent role for invariant T cells in the amphibian Xenopus laevis tadpoles

Invariant T (iT) cells expressing an invariant or semi-invariant T cell receptor (TCR) repertoire have gained attention in recent years because of their potential as specialized regulators of immune function. These iT cells are typically restricted by nonclassical MHC class I molecules (e.g., CD1d and MR1) and undergo differentiation pathways distinct from conventional T cells. While the benefit of a limited TCR repertoire may appear counterintuitive in regard to the advantage of the diversified repertoire of conventional T cells allowing for exquisite specificity to antigens, the full biological importance and evolutionary conservation of iT cells are just starting to emerge. It is generally considered that iT cells are specialized to recognize conserved antigens equivalent to pathogen-associated molecular pattern. Until recently, little was known about the evolution of iT cells. The identification of class Ib and class I-like genes in nonmammalian vertebrates, despite the heterogeneity and variable numbers of these genes among species, suggests that iT cells are also present in ectothermic vertebrates. Indeed, recent studies in the amphibian Xenopus have revealed a drastic overrepresentation of several invariant TCRs in tadpoles and identified a prominent nonclassical MHC class I-restricted iT cell subset critical for tadpole antiviral immunity. This suggests an important and perhaps even dominant role of multiple nonclassical MHC class I-restricted iT cell populations in tadpoles and, by extension, other aquatic vertebrates with rapid external development that are under pressure to produce a functional lymphocyte repertoire with small numbers of cells.

Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes

To identify ranavirus virulence genes, we engineered Frog Virus 3 (FV3) knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD) protein (Δ64R-FV3) and a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3). Compared to wild type (WT) FV3, infection of Xenopus tadpoles with Δ64R- or Δ52L-FV3 resulted in significantly lower levels of mortality and viral replication. We further characterized these and two earlier KO mutants lacking the immediate-early18kDa protein (FV3-Δ18K) or the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α). All KO mutants replicated as well as WT-FV3 in non-amphibian cell lines, whereas in Xenopus A6 kidney cells replication of ΔvCARD-, ΔvβHSD- and ΔvIF-2α-FV3 was markedly reduced. Furthermore, Δ64R- and ΔvIF-2α-FV3 were more sensitive to interferon than WT and Δ18-FV3. Notably, Δ64R-, Δ18K- and ΔvIF-2α- but not Δ52L-FV3 triggered more apoptosis than WT FV3. These data suggest that vCARD (64R) and vβ-HSD (52L) genes contribute to viral pathogenesis.

Evolutionary Aspects of Macrophages Polarization

Macrophages constitute a heterogeneous population of myeloid cells that are essential for maintaining homeostasis and as a first line of innate responders controlling and organizing host defenses against pathogens. Monocyte-macrophage lineage cells are among the most functionally diverse and plastic cells of the immune system. They undergo specific activation into functionally distinct phenotypes in response to immune signals and microbial products. In mammals, macrophage functional heterogeneity is defined by two activation states, M1 and M2, which represent two polar ends of a continuum exhibiting pro-inflammatory and tissue repair activities, respectively. While the ancient evolutionary origin of macrophages as phagocytic defenders is well established, the evolutionary roots of the specialized division of macrophages into subsets with polarized activation phenotypes is less well defined. Accordingly, this chapter focuses on recent advances in the understanding of the evolution of macrophage polarization and functional heterogeneity with a focus on ectothermic vertebrates.

Ranavirus Host Immunity and Immune Evasion

Ranaviruses (RV, Iridoviridae) are now known to infect fish, amphibians, and reptiles, raising considerable ecological and commercial concerns due to the escalating infection prevalence and the resulting die-offs of wild and aquacultural species. Notably, ranaviruses exhibit uncanny capacities to cross host species barriers of their poikilothermic hosts, likely owing to their potent immune evasion mechanisms. In turn, the species infected by these pathogens possess immune systems that are less well understood than those of mammals, and are often comprised of unique immune genes or multiple copy orthologs of the single hallmark mammalian immune factors. Thus, garnering greater insight into ranavirus infection strategies is largely contingent on gaining further insights into host immune barriers faced by these emerging infectious agents. Accordingly, here we coalesce the current state of understanding of the distinct facets of lower vertebrate immune responses to ranaviral infections and underline some of the evasion strategies by which these pathogens circumvent these host defenses.

Nonclassical MHC-Restricted Invariant Vα6 T Cells Are Critical for Efficient Early Innate Antiviral Immunity in the Amphibian Xenopus laevis

Nonclassical MHC class Ib–restricted invariant T (iT) cell subsets are attracting interest because of their potential to regulate immune responses against various pathogens. The biological relevance and evolutionary conservation of iT cells have recently been strengthened by the identification of iT cells (invariant Vα6 [iVα6]) restricted by the nonclassical MHC class Ib molecule XNC10 in the amphibian Xenopus laevis. These iVα6 T cells are functionally similar to mammalian CD1d-restricted invariant NKT cells. Using the amphibian pathogen frog virus 3 (FV3) in combination with XNC10 tetramers and RNA interference loss of function by transgenesis, we show that XNC10-restricted iVα6 T cells are critical for early antiviral immunity in adult X. laevis. Within hours following i.p. FV3 infection, iVα6 T cells were specifically recruited from the spleen into the peritoneum. XNC10 deficiency and concomitant lack of iVα6 T cells resulted in less effective antiviral and macrophage antimicrobial responses, which led to impaired viral clearance, increased viral dissemination, and more pronounced FV3-induced kidney damage. Together, these findings imply that X. laevis XNC10-restricted iVα6 T cells play important roles in the early anti-FV3 response and that, as has been suggested for mammalian invariant NKT cells, they may serve as immune regulators polarizing macrophage effector functions toward more effective antiviral states.

Inflammation-Induced Reactivation of the Ranavirus Frog Virus 3 in Asymptomatic Xenopus laevis

Natural infections of ectothermic vertebrates by ranaviruses (RV, family Iridoviridae) are rapidly increasing, with an alarming expansion of RV tropism and resulting die-offs of numerous animal populations. Notably, infection studies of the amphibian Xenopus laevis with the ranavirus Frog Virus 3 (FV3) have revealed that although the adult frog immune system is efficient at controlling RV infections, residual quiescent virus can be detected in mononuclear phagocytes of otherwise asymptomatic animals following the resolution of RV infections. It is noteworthy that macrophage-lineage cells are now believed to be a critical element in the RV infection strategy. In the present work, we report that inflammation induced by peritoneal injection of heat-killed bacteria in asymptomatic frogs one month after infection with FV3 resulted in viral reactivation including detectable viral DNA and viral gene expression in otherwise asymptomatic frogs. FV3 reactivation was most prominently detected in kidneys and in peritoneal HAM56+ mononuclear phagocytes. Notably, unlike adult frogs that typically clear primary FV3 infections, a proportion of the animals succumbed to the reactivated FV3 infection, indicating that previous exposure does not provide protection against subsequent reactivation in these animals.

Effective RNAi-mediated β2-microglobulin loss of function by transgenesis in Xenopus laevis

Summary To impair MHC class I (class I) function in vivo in the amphibian Xenopus, we developed an effective reverse genetic loss of function approach by combining I-SceI meganuclease-mediated transgenesis with RNAi technology. We generated transgenic outbred X. laevis and isogenetic laevis/gilli cloned lines with stably silenced expression of &bgr;2-microglobulin (b2m) critical for class I function. Transgenic F1 frogs exhibited decreased surface class I expression on erythrocytes and lymphocytes, decreased frequency of peripheral CD8 T cells and impaired CD8 T cell-mediated skin allograft rejection. Additionally, b2m knockdown increased susceptibility to viral infection of F0 transgenic larvae. This loss of function strategy offers new avenues for studying ontogeny of immunity and other developmental processes in Xenopus.

Exploring the functions of nonclassical MHC class Ib genes in Xenopus laevis by the CRISPR/Cas9 system.

A large family of highly related and clustered Xenopus nonclassical MHC class Ib (XNC) genes influences Xenopus laevis immunity and potentially other physiological functions. Using RNA interference (RNAi) technology, we previously demonstrated that one of XNC genes, XNC10.1, is critical for the development and function of a specialized innate T (iT) cell population. However, RNAi limitation such as a variable and unstable degree of gene silencing in F0 and F1 generations is hampering a thorough functional analysis of XNC10.1 and other XNC genes. To overcome this obstacle, we adapted the CRISPR/Cas9-mediated gene editing technique for XNC genes. We efficiently and specifically generated single gene knockouts of XNC10.1, XNC11, and XNC1 as well as double gene knockouts of XNC10.1 and XNC11 in X. laevis. In single XNC10.1 knockout X. laevis tadpoles, the absence of XNC10.1 and Vα6-Jα1.43 invariant T cell receptor rearrangement transcripts indicated XNC10.1 loss-of-function and deficiency in Vα6-Jα1.43 iT cells. Notably, targeting XNC10.1 did not affect neighboring XNC genes exhibiting high sequence similarity. Furthermore, XNC1 gene disruption induced mortality during developmental stage 47, suggesting some non-immune but essential function of this gene. These data demonstrate that the CRISPR/Cas9 system can be successfully adapted for genetic analysis in F0 generation of X. laevis.