Comparative analysis of the repertoire of insulin-reactive B cells in type 1 diabetes-prone and resistant mice.

Seropositivity for autoantibodies against multiple islet antigens is associated with development of autoimmune type 1 diabetes (T1D), suggesting a role for B cells in disease. The importance of B cells in T1D is indicated by the effectiveness of B cell-therapies in mouse models and patients. B cells contribute to T1D by presenting islet antigens, including insulin, to diabetogenic T cells that kill pancreatic beta cells. The role of B cell receptor (BCR) affinity in T1D development is unclear. Here, we employed single cell RNA sequencing to define the relationship between BCR affinity for insulin and B cell phenotype during disease development. We utilized immunoglobulin (Ig) heavy chain (VH125) mouse models in which high-affinity insulin-reactive B cells (IBCs) were previously shown to be anergic in diabetes-resistant VH125.C57BL/6-H2g7 and activated in VH125. NOD mice developing disease. Here, high-affinity IBCs were found in the spleen of prediabetic VH125. NOD mice and exhibited marginal zone or follicular phenotypes. Ig light chains expressed by these B cells are unmutated and biased toward Vκ4-74 and Vκ4-57 usage. Receptors expressed by anergic high-affinity IBCs of diabetes-resistant VH125.C57BL/6-H2g7 are also unmutated; however, in this genetic background light chains are polymorphic relative to those of NOD. Light chains derived from NOD and C57BL/6-H2g7 genetic backgrounds conferred divergent kinetics of binding to insulin when paired with the VH125 heavy chain. These findings suggest that relaxation of tolerance mechanisms in the NOD mouse leads to accumulation and partial activation of B cells expressing germline encoded high-affinity BCRs that support development of autoimmunity.

Impacts of the MHC class I-like XNC10 and innate-like T cells on tumor tolerance and rejection in the amphibian Xenopus.

The conditions that lead to antitumor or protumor functions of natural killer T (NKT) cells against mammalian tumors are only partially understood. Therefore, insights into the evolutionary conservation of NKT and their analogs-innate-like T (iT) cells-may reveal factors that contribute to tumor eradication. As such, we investigated the amphibian Xenopus laevis iT cells and interacting MHC class I-like (XNC or mhc1b.L) genes against ff-2 thymic lymphoid tumors. Upon ff-2 intraperitoneal transplantation into syngeneic tadpoles, two iT cell subsets iVα6 and iVα22, characterized by an invariant T-cell receptor α chain rearrangement (Vα6-Jα1.43 and Vα22-Jα1.32 respectively), were recruited to the peritoneum, concomitant with a decreased level of these transcripts in the spleen and thymus. To address the hypothesize that different iT cell subsets have distinct, possibly opposing, roles upon ff-2 tumor challenge, we determined whether ff-2 tumor growth could be manipulated by impairing Vα6 iT cells or by deleting their restricting element, the XNC gene, XNC10 (mhc1b10.1.L), on ff-2 tumors. Accordingly, the in vivo depletion of Vα6 iT cells using XNC10-tetramers enhanced tumor growth, indicating Vα6 iT cell-mediated antitumor activities. However, XNC10-deficient transgenic tadpoles that also lack Vα6 iT cells were resistant to ff-2 tumors, uncovering a potential new function of XNC10 besides Vα6 iT cell development. Furthermore, the CRISPR/Cas9-mediated knockout of XNC10 in ff-2 tumors broke the immune tolerance. Together, our findings demonstrate the relevance of XNC10/iT cell axis in controlling Xenopus tumor tolerance or rejection.

Evolutionary Underpinnings of Innate-Like T Cell Interactions with Cancer.

Cancers impose a significant health and economic burden. By harnessing the immune system, current immunotherapies have revolutionized the treatment against human cancers and potentially offer a long-term cure. Among others, innate-like T (iT) cells, including natural killer T cells, are promising candidates for immunotherapies. Unlike conventional T cells, iT cells regulate multiple immune processes and express an invariant T cell receptor that is shared among different individuals. However, the conditions that activate the pro- and antitumor functions of iT cells are partially understood. These gaps in knowledge hamper the use of iT cell in clinics. It might be beneficial to examine the roles of iT cells in an alternative animal model - the amphibian Xenopus whose immune system shares many similarities to that of mammals. Here, we review the iT cell biology in the context of mammalian cancers and discuss the challenges currently found in the field. Next, we introduce the advantages of Xenopus as a model to investigate the role of iT cells and interacting major histocompatibility complex (MHC) class I-like molecules in tumor immunity. In Xenopus, 2 specific iT cell subsets, Vα6 and Vα22 iT cells, recognize and fight tumor cells. Furthermore, our recent data reveal the complex functions of the Xenopus MHC class I-like (XNC) gene XNC10 in tumor immune responses. By utilizing reverse genetics, transgenesis, and MHC tetramers, we have a unique opportunity to uncover the relevance of XNC genes and iT cell in Xenopus tumor immunity.

Distinct MHC class I-like interacting invariant T cell lineage at the forefront of mycobacterial immunity uncovered in Xenopus.

The amphibian Xenopus laevis is to date the only species outside of mammals where a MHC class I-like (MHC-like) restricted innate-like (i) T cell subset (iVα6 T cells) reminiscent of CD1d-restricted iNKT cells has been identified and functionally characterized. This provides an attractive in vivo model to study the biological analogies and differences between mammalian iT cells and the evolutionarily antecedent Xenopus iT cell defense system. Here, we report the identification of a unique iT cell subset (Vα45-Jα1.14) requiring a distinct MHC-like molecule (mhc1b4.L or XNC4) for its development and function. We used two complementary reverse genetic approaches: RNA interference by transgenesis to impair expression of either XNC4 or the Vα45-Jα1.14 rearrangement, and CRISPR/Cas9-mediated disruption of the Jα1.14 gene segment. Both XNC4 deficiency that ablates iVα45T cell development and the direct disruption of the iVα45-Jα1.14 T cell receptor dramatically impairs tadpole resistance to Mycobacterium marinum (Mm) infection. The higher mortality of Mm-infected tadpoles deficient for iVα45T cells correlates with dysregulated expression responses of several immune genes. In contrast, iVα45-Jα1.14-deficient tadpoles remain fully competent against infection by the ranavirus FV3, which indicates a specialization of this unique iT cell subset toward mycobacterial rather than viral pathogens that involve iVα6 T cells. These data suggest that amphibians, which are evolutionarily separated from mammals by more than 350 My, have independently diversified a prominent and convergent immune surveillance system based on MHC-like interacting innate-like T cells.

Tumor immunology viewed from alternative animal models-the Xenopus story.

A PURPOSE OF REVIEW: Nonmammalian comparative animal models are important not only to gain fundamental evolutionary understanding of the complex interactions of tumors with the immune system, but also to better predict the applicability of novel immunotherapeutic approaches to humans. After reviewing recent advances in developing alternative models, we focus on the amphibian Xenopus laevis and its usefulness in deciphering the perplexing roles of MHC class I-like molecules and innate (i)T cells in tumor immunity. B RECENT FINDINGS: Experiments using MHC-defined inbred and cloned animals, tumor cell lines, effective reagents, sequenced genomes, and adapted gene editing techniques in Xenopus, have revealed that the critical involvement of class I-like molecules and iT cells in tumor immunity has been conserved during evolution. C SUMMARY: Comparative studies with the X. laevis tumor immunity model can contribute to the development of better and more efficient cancer immunotherapies.

Collagen-Embedded Tumor Transplantations in Xenopus laevis Tadpoles.

The Xenopus laevis tadpole provides a valuable model for studying tumorigenesis and tumor immunity by intravital real-time microscopy. Using well-characterized thymic lymphoid tumor lines (15/0 and ff-2) that are transplantable into their compatible hosts (LG-15 isogenic clones and the F inbred strain, respectively), a system of semisolid tumor engraftment has been designed. Because these lymphoid tumor cell lines are not adherent and grow in suspension, they are first immobilized in a matrix of type I rat tail collagen before transplantation as a semisolid tumor graft under the transparent dorsal skin in the head region of a tadpole. This semisolid tumor engraftment is amenable to manipulation and permits real-time visualization of tumor growth, neovascularization, collagen rearrangements, immune cell infiltration, and formation of the tumor microenvironment.

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.

Evolution of innate-like T cells and their selection by MHC class I-like molecules.

Until recently, major histocompatibility complex (MHC) class I-like-restricted innate-like αßT (iT) cells expressing an invariant or semi-invariant T cell receptor (TCR) repertoire were thought to be a recent evolutionary acquisition restricted to mammals. However, molecular and functional studies in Xenopus laevis have demonstrated that iT cells, defined as MHC class I-like-restricted innate-like αßT cells with a semi-invariant TCR, are evolutionarily conserved and prominent from early development in amphibians. As these iT cells lack the specificity conferred by conventional αß TCRs, it is generally considered that they are specialized to recognize conserved antigens equivalent to pathogen-associated molecular patterns. Thus, one advantage offered by the MHC class I-like iT cell-based recognition system is that it can be adapted to a common pathogen and function on the basis of a relatively small number of T cells. Although iT cells have only been functionally described in mammals and amphibians, the identification of non-classical MHC/MHC class I-like genes in other groups of endothermic and ectothermic vertebrates suggests that iT cells have a broader phylogenetic distribution than previously envisioned. In this review, we discuss the possible role of iT cells during the emergence of the jawed vertebrate adaptive immune system.

Semi-solid tumor model in Xenopus laevis/gilli cloned tadpoles for intravital study of neovascularization, immune cells and melanophore infiltration.

Tumors have the ability to grow as a self-sustaining entity within the body. This autonomy is in part accomplished by the tumor cells ability to induce the formation of new blood vessels (angiogenesis) and by controlling cell trafficking inside the tumor mass. These abilities greatly reduce the efficacy of many cancer therapies and pose challenges for the development of more effective cancer treatments. Hence, there is a need for animal models suitable for direct microscopy observation of blood vessel formation and cell trafficking, especially during early stages of tumor establishment. Here, we have developed a reliable and cost effective tumor model system in tadpoles of the amphibian Xenopus laevis. Tadpoles are ideally suited for direct microscopy observation because of their small size and transparency. Using the thymic lymphoid tumor line 15/0 derived from, and transplantable into, the X. laevis/gilli isogenic clone LG-15, we have adapted a system that consists in transplanting 15/0 tumor cells embedded into rat collagen under the dorsal skin of LG-15 tadpole recipients. This system recapitulates many facets of mammalian tumorigenesis and permits real time visualization of the active formation of the tumor microenvironment induced by 15/0 tumor cells including neovascularization, collagen rearrangements as well as infiltration of immune cells and melanophores.

A critical role of non-classical MHC in tumor immune evasion in the amphibian Xenopus model.

Non-classical class Ib (class Ib) genes are found in all jawed vertebrates, including the amphibian Xenopus, which possesses at least 20 distinct Xenopus non-classical class Ib genes (XNCs). As an immune evasion strategy, tumors often downregulate surface expression of classical major histocompatibility complex class Ia molecules. In contrast, cancers commonly express class Ib molecules, presenting an alternative for tumor immune recognition. We characterized a novel XNC, XNC10, functionally similar to CD1d from a class Ia-deficient thymic lymphoid tumor (15/0), which grows aggressively in Xenopus LG-15 cloned animals. To investigate the roles of XNC10 in antitumor immunity, we generated stable 15/0-transfectants with silenced XNC10 mRNA and protein expression. Notably, XNC10 silencing resulted in acute tumor rejection by naturally class Ia-deficient syngeneic tadpoles, with greater potency of rejection in tumors with more efficient XNC10 knockdown. In vivo killing assays shows that the rejection of XNC10-deficient tumors is due to a cell-mediated cytotoxic immune response elicited by the tadpole host. Importantly, priming enhances XNC10-deficient tumor rejection. Flow cytometry reveals that XNC10-deficient tumor rejection is associated with an accumulation of XNC10-restricted invariant T cells and conventional CD8 T cells as well as other leukocytes. Similarly, semisolid tumor grafts in tadpoles also exhibit leukocytes infiltration. These findings suggest that XNC10 allows the 15/0-tumor to escape immune recognition and class Ia-independent cytotoxicity, thus emphasizing the critical roles of class Ibs in tumor immunity.

Pro-oxidative synergic bactericidal effect of NO: kinetics and inhibition by nitroxides.

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H2O2. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a "shoulder" sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H2O2-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-µM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H2O2. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H2O2, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as (•)NO2 or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed.