Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Antiviral Responses to Intestinal Frog Virus 3 Infections.

The global amphibian declines are compounded by ranavirus infections such as Frog Virus 3 (FV3), and amphibian tadpoles more frequently succumb to these pathogens than adult animals. Amphibian gastrointestinal tracts represent a major route of ranavirus entry, and viral pathogenesis often leads to hemorrhaging and necrosis within this tissue. Alas, the differences between tadpole and adult amphibian immune responses to intestinal ranavirus infections remain poorly defined. As interferon (IFN) cytokine responses represent a cornerstone of vertebrate antiviral immunity, it is pertinent that the tadpoles and adults of the anuran Xenopus laevis frog mount disparate IFN responses to FV3 infections. Presently, we compared the tadpole and adult X. laevis responses to intestinal FV3 infections. Our results indicate that FV3-challenged tadpoles mount more robust intestinal type I and III IFN responses than adult frogs. These tadpole antiviral responses appear to be mediated by myeloid cells, which are recruited into tadpole intestines in response to FV3 infections. Conversely, myeloid cells bearing similar cytology already reside within the intestines of healthy (uninfected) adult frogs, possibly accounting for some of the anti-FV3 resistance of these animals. Further insight into the differences between tadpole and adult frog responses to ranaviral infections is critical to understanding the facets of susceptibility and resistance to these pathogens.

Functional-genomic analysis reveals intraspecies diversification of antiviral receptor transporter proteins in Xenopus laevis.

The Receptor Transporter Protein (RTP) family is present in most, if not all jawed vertebrates. Most of our knowledge of this protein family comes from studies on mammalian RTPs, which are multi-function proteins that regulate cell-surface G-protein coupled receptor levels, influence olfactory system development, regulate immune signaling, and directly inhibit viral infection. However, mammals comprise less than one-tenth of extant vertebrate species, and our knowledge about the expression, function, and evolution of non-mammalian RTPs is limited. Here, we explore the evolutionary history of RTPs in vertebrates. We identify signatures of positive selection in many vertebrate RTP clades and characterize multiple, independent expansions of the RTP family outside of what has been described in mammals. We find a striking expansion of RTPs in the African clawed frog, Xenopus laevis, with 11 RTPs in this species as opposed to 1 to 4 in most other species. RNA sequencing revealed that most X. laevis RTPs are upregulated following immune stimulation. In functional assays, we demonstrate that at least three of these X. laevis RTPs inhibit infection by RNA viruses, suggesting that RTP homologs may serve as antiviral effectors outside of Mammalia.

TLR5-Mediated Reactivation of Quiescent Ranavirus FV3 in Xenopus Peritoneal Macrophages.

Ranaviruses such as frog virus 3 (FV3) are large double-stranded DNA (dsDNA) viruses causing emerging infectious diseases leading to extensive morbidity and mortality of amphibians and other ectothermic vertebrates worldwide. Among the hosts of FV3, some are highly susceptible, whereas others are resistant and asymptomatic carriers that can take part in disseminating the infectious virus. To date, the mechanisms involved in the processes of FV3 viral persistence associated with subclinical infection transitioning to lethal outbreaks remain unknown. Investigation in Xenopus laevis has revealed that in asymptomatic FV3 carrier animals, inflammation induced by heat-killed (HK) Escherichia coli stimulation can provoke the relapse of active infection. Since Toll-like receptors (TLRs) are critical for recognizing microbial molecular patterns, we investigated their possible involvement in inflammation-induced FV3 reactivation. Among the 10 different TLRs screened for changes in expression levels following FV3 infection and HK E. coli stimulation, only TLR5 and TLR22, both of which recognize bacterial products, showed differential expression, and only the TLR5 ligand flagellin was able to induce FV3 reactivation similarly to HK E. coli Furthermore, only the TLR5 ligand flagellin induced FV3 reactivation in peritoneal macrophages both in vitro and in vivo These data indicate that the TLR5 signaling pathway can trigger FV3 reactivation and suggest a role of secondary bacterial infections or microbiome alterations (stress or pollution) in initiating sudden deadly disease outbreaks in amphibian populations with detectable persistent asymptomatic ranavirus.IMPORTANCE This study in the amphibian Xenopus laevis provides new evidence of the critical role of macrophages in the persistence of ranaviruses in a quiescent state as well as in the reactivation of these pathogens into a virulent infection. Among the multiple microbial sensors expressed by macrophages, our data underscore the preponderant involvement of TLR5 stimulation in triggering the reactivation of quiescent FV3 in resident peritoneal macrophages, unveiling a mechanistic connection between the reactivation of persisting ranavirus infection and bacterial coinfection. This suggests a role for secondary bacterial infections or microbiome alterations (stress or pollution) in initiating sudden deadly disease outbreaks in amphibian populations with detectable persistent asymptomatic ranavirus.

Developing Tadpole Xenopus laevis as a Comparative Animal Model to Study Mycobacterium abscessus Pathogenicity.

Mycobacterium abscessus (Mab) is an emerging, nontuberculosis mycobacterium (NTM) that infects humans. Mab has two morphotypes, smooth (S) and rough (R), related to the production of glycopeptidolipid (GPL), that differ in pathogenesis. To further understand the pathogenicity of these morphotypes in vivo, the amphibian Xenopus laevis was used as an alternative animal model. Mab infections have been previously modeled in zebrafish embryos and mice, but Mab are cleared early from immunocompetent mice, preventing the study of chronic infection, and the zebrafish model cannot be used to model a pulmonary infection and T cell involvement. Here, we show that X. laevis tadpoles, which have lungs and T cells, can be used as a complementary model for persistent Mab infection and pathogenesis. Intraperitoneal (IP) inoculation of S and R Mab morphotypes disseminated to tadpole tissues including liver and lungs, persisting for up to 40 days without significant mortality. Furthermore, the R morphotype was more persistent, maintaining a higher bacterial load at 40 days postinoculation. In contrast, the intracardiac (IC) inoculation with S Mab induced significantly greater mortality than inoculation with the R Mab form. These data suggest that X. laevis tadpoles can serve as a useful comparative experimental organism to investigate pathogenesis and host resistance to M. abscessus.

Exploring the relationships between amphibian (Xenopus laevis) myeloid cell subsets.

The differentiation of distinct leukocyte subsets is governed by lineage-specific growth factors that elicit disparate expression of transcription factors and markers by the developing cell populations. For example, macrophages (Mφs) and granulocytes (Grns) arise from common granulocyte-macrophage progenitors in response to distinct myeloid growth factors. In turn, myelopoiesis of the Xenopus laevis anuran amphibian appears to be unique to other studied vertebrates in several respects while the functional differentiation of amphibian Mφs and Grns from their progenitor cells remains poorly understood. Notably, the expression of colony stimulating factor-1 receptor (CSF-1R) or CSF-3R on granulocyte-macrophage progenitors marks their commitment to Mφ- or Grn-lineages, respectively. CSF-1R is activated by the colony stimulating factor-1 (CSF-1) and interleukin (IL-34) cytokines, resulting in morphologically and functionally distinct Mφ cell types. Conversely, CSF-3R is ligated by CSF-3 in a process indispensable for granulopoiesis. Presently, we explore the relationships between X. laevis CSF-1-Mφs, IL-34-Mφs and CSF-3-Grns by examining their expression of key lineage-specific transcription factor and myeloid marker genes as well as their enzymology. Our findings suggest that while the CSF-1- and IL-34-Mφs share some commonalities, the IL-34-Mφs possess transcriptional patterns more akin to the CSF-3-Grns. IL-34-Mφs also possess robust expression of dendritic cell-associated transcription factors and surface marker genes, further underlining the difference between this cell type and the CSF-1-derived frog Mφ subset. Moreover, the three myeloid populations differ in their respective tartrate-resistant acid phosphatase, specific- and non-specific esterase activity. Together, this work grants new insights into the developmental relatedness of these three frog myeloid subsets.

A Glimpse of the Peptide Profile Presentation by Xenopus laevis MHC Class I: Crystal Structure of pXela-UAA Reveals a Distinct Peptide-Binding Groove.

The African clawed frog, Xenopus laevis, is a model species for amphibians. Before metamorphosis, tadpoles do not efficiently express the single classical MHC class I (MHC-I) molecule Xela-UAA, but after metamorphosis, adults express this molecule in abundance. To elucidate the Ag-presenting mechanism of Xela-UAA, in this study, the Xela-UAA structure complex (pXela-UAAg) bound with a peptide from a synthetic random peptide library was determined. The amino acid homology between the Xela-UAA and MHC-I sequences of different species is <45%, and these differences are fully reflected in the three-dimensional structure of pXela-UAAg. Because of polymorphisms and interspecific differences in amino acid sequences, pXela-UAAg forms a distinct peptide-binding groove and presents a unique peptide profile. The most important feature of pXela-UAAg is the two-amino acid insertion in the α2-helical region, which forms a protrusion of ∼3.8 Å that is involved in TCR docking. Comparison of peptide-MHC-I complex (pMHC-I) structures showed that only four amino acids in ß2-microglobulin that were bound to MHC-I are conserved in almost all jawed vertebrates, and the most unique feature in nonmammalian pMHC-I molecules is that the AB loop bound ß2-microglobulin. Additionally, the binding distance between pMHC-I and CD8 molecules in nonmammals is different from that in mammals. These unique features of pXela-UAAg provide enhanced knowledge of T cell immunity and bridge the knowledge gap regarding the coevolutionary progression of the MHC-I complex from aquatic to terrestrial species.

Complement C1q subunit molecules from Xenopus laevis possess conserved function in C1q-immunoglobulin interaction.

Complement component 1q (C1q), together with C1r and C1s to form C1, recognize and bind immune complex to initiate the classical complement pathway. In this study, C1q subunit molecules (XlC1qA, XlC1qB, XlC1qC) were cloned and analyzed from Xenopus laevis (X. laevis). The open reading frame (ORF) of XlC1qA is 819 bp of nucleotide sequence encoding 272 amino acids, the ORF of XlC1qB is 711 bp encoding 236 aa, and the XlC1qC is consists of 732 bp encoding 243 aa. The deduced amino acid sequences contain a collagen-like region (CLR), Gly-X-Y repeats in the N-terminus and a C1q family domain at the C-terminus. Phylogenetic analysis revealed that the XlC1qs are clustered with the amphibian clade. Expression analysis indicated that the XlC1qs exhibited constitutive expression in all examined tissues, with the highest expression in liver. Additionally, XlC1q could interact with heat-aggregated mouse IgG and IgM, Xenopus IgM and Nile tilapia IgM, respectively, indicating the functional conservation of XlC1q binding to immunoglobulins. Further, XlC1qs can inhibit C1q-dependent hemolysis of sensitized sheep red blood cells with concentration-dependent manner. These data collectively suggest that the function of C1qs in X. laevis may be conserved in interaction with immunoglobulins, as that of mammals and teleosts.

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.

Isolation of nanobodies against Xenopus embryonic antigens using immune and non-immune phage display libraries.

The use of Xenopus laevis as a model for vertebrate developmental biology is limited by a lack of antibodies specific for embryonic antigens. This study evaluated the use of immune and non-immune phage display libraries for the isolation of single domain antibodies, or nanobodies, with specificities for Xenopus embryonic antigens. The immune nanobody library was derived from peripheral blood lymphocyte RNA obtained from a llama immunized with Xenopus gastrula homogenates. Screening this library by immunostaining of embryonic tissues with pooled periplasmic material and sib-selection led to the isolation of several monoclonal phages reactive with the cytoplasm and nuclei of gastrula cells. One antigen recognized by a group of nanobodies was identified using a reverse proteomics approach as nucleoplasmin, an abundant histone chaperone. As an alternative strategy, a semi-synthetic non-immune llama nanobody phage display library was panned on highly purified Xenopus proteins. This proof-of-principle approach isolated monoclonal nanobodies that specifically bind Nuclear distribution element-like 1 (Ndel1) in multiple immunoassays. Our results suggest that immune and non-immune phage display screens on crude and purified embryonic antigens can efficiently identify nanobodies useful to the Xenopus developmental biology community.

Critical Role of an MHC Class I-Like/Innate-Like T Cell Immune Surveillance System in Host Defense against Ranavirus (Frog Virus 3) Infection.

Besides the central role of classical Major Histocompatibility Complex (MHC) class Ia-restricted conventional Cluster of Differentiation 8 (CD8) T cells in antiviral host immune response, the amphibian Xenopuslaevis critically rely on MHC class I-like (mhc1b10.1.L or XNC10)-restricted innate-like (i)T cells (iVα6 T cells) to control infection by the ranavirus Frog virus 3 (FV3). To complement and extend our previous reverse genetic studies showing that iVα6 T cells are required for tadpole survival, as well as for timely and effective adult viral clearance, we examined the conditions and kinetics of iVα6 T cell response against FV3. Using a FV3 knock-out (KO) growth-defective mutant, we found that upregulation of the XNC10 restricting class I-like gene and the rapid recruitment of iVα6 T cells depend on detectable viral replication and productive FV3 infection. In addition, by in vivo depletion with XNC10 tetramers, we demonstrated the direct antiviral effector function of iVα6 T cells. Notably, the transitory iV6 T cell defect delayed innate interferon and cytokine gene response, resulting in long-lasting negative inability to control FV3 infection. These findings suggest that in Xenopus and likely other amphibians, an immune surveillance system based on the early activation of iT cells by non-polymorphic MHC class-I like molecules is important for efficient antiviral immune response.

Developmental exposure to chemicals associated with unconventional oil and gas extraction alters immune homeostasis and viral immunity of the amphibian Xenopus.

Although aquatic vertebrates and humans are increasingly exposed to water pollutants associated with unconventional oil and gas extraction (UOG), the long-term effects of these pollutants on immunity remains unclear. We have established the amphibian Xenopus laevis and the ranavirus Frog Virus 3 (FV3) as a reliable and sensitive model for evaluating the effects of waterborne pollutants. X. laevis tadpoles were exposed to a mixture of equimass amount of UOG chemicals with endocrine disrupting activity (0.1 and 1.0 µg/L) for 3 weeks, and then long-term effects on immune function at steady state and following viral (FV3) infection was assessed after metamorphosis. Notably, developmental exposure to the mixture of UOG chemicals at the tadpole stage affected metamorphic development and fitness by significantly decreasing body mass after metamorphosis completion. Furthermore, developmental exposure to UOGs resulted in perturbation of immune homeostasis in adult frogs, as indicated by significantly decreased number of splenic innate leukocytes, B and T lymphocytes; and a weakened antiviral immune response leading to increased viral load during infection by the ranavirus FV3. These findings suggest that mixture of UOG-associated waterborne endocrine disruptors at low but environmentally-relevant levels have the potential to induce long-lasting alterations of immune function and antiviral immunity in aquatic vertebrates and ultimately human populations.

Expression of TCR genes in adult and larval Xenopus laevis.

In order to better understand the development and function of γδ T cells in Xenopus frogs, it is necessary to determine where and when γδ T cells are found in Xenopus tissues. This study examined the expression of TCR genes, focused primarily on TCR γ, in tissues of adult and larval Xenopus laevis and provide new data about the expression pattern of these different TCR genes in this anuran amphibian. TCR gene expression was detected by RT-PCR in adult frog tissues including the thymus, spleen, skin, intestine, lung, and liver, but not the testes. TCR γ and ß genes were detected in the larval (tadpole) tail and intestine. The absence of RAG-1 expression in these larval tissues is consistent with differentiation of the T cells in the thymus. Together, these data provide evidence that migration of these cells from the thymus likely occurs relatively early in larval development. These studies provide a necessary foundation for future studies of the functions of γδ T cells in amphibians, which are placed at an intermediate position flanked by fishes on one end and mammals and chickens on the other.

Skin Grafting in Xenopus laevis: A Technique for Assessing Development and Immunological Disparity.

Skin grafting in the amphibian Xenopus laevis has been used to detect not only allogeneic antigens that differ by minor H antigens or by one MHC haplotype, but also to detect ontogeny-specific antigens (including both emerging adult- and disappearing larval-specific) during metamorphosis. To understand the mechanisms underlying allogeneic tolerance or immune responses against larval- and/or adult-specific antigens, a complete MHC homozygous, inbred strain is the most appropriate experimental model. The inbred J strain established in Japan is used here. Owing to complete histocompatibility, the inbred J strain shows no grafted skin rejection among the same strain of adult frogs, and its genuine homozygosity was reconfirmed by genomic sequence analysis in 2016. Therefore, the J strain enables immunologists and embryologists to understand evolutionary processes as well as immunological events and tissue remodeling mechanisms present during development. Furthermore, an F1 hybrid between the J strain and a GFP-labeled transgenic line is available from our laboratory and can be used as a model for long-term cell tracking. This protocol explains the methodology for skin grafting in X. laevis to determine immunological discrepancies between the host and donor. It is also possible to trace cell and tissue fates in the hosts during early embryogenesis and during complete development from larvae to adults, which is extremely difficult to perform using other species.

Assessing Antibody Responses to Pathogens or Model Antigens in Xenopus by Enzyme-Linked Immunosorbent Assay (ELISA).

Xenopus laevis-specific monoclonal antibodies recognize IgM and IgY antibodies not only from X. laevis but also X. tropicalis as well as a variety of amphibian species including Ranidae, Bufonidae, and even some salamanders. These reagents are very useful to assess antibody responses from the serum or other animal secretions (e.g., peritoneal fluid). We present here an enzyme-linked immunosorbent assay (ELISA) optimized for amphibians that permits users to detect and titrate the presence of each type of antibody (IgM and IgY) produced against particular pathogens (e.g., virus, bacteria, or fungus) or antigens (e.g., DNP-KLH).

Adoptive Transfer of Fluorescently Labeled Immune Cells in Xenopus.

Adoptive cell transfer from inbred adult Xenopus to inbred tadpoles is a useful way to study the dissemination of immune cells or pathogen-infected immune cells in tadpoles. For example, Xenopus peritoneal leukocytes (PLs) can be readily infected by pathogens such as Frog virus 3 (FV3) and Mycobacterium marinum (M. marinum). By transferring fluorescently labeled, FV3-infected PLs into tadpoles, we observed infiltration of these cells into the tadpole's brain, which indicates that FV3-infected PLs can cross blood brain barrier. Taking advantage of tadpoles' transparency, fluorescently labeled immune cells can be tracked in real time using fluorescence microscopy.

Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Use of Expanded Repertoires of Type I and Type III Interferon Cytokines.

While amphibians around the globe are facing catastrophic declines, in part because of infections with pathogens such as the Frog Virus 3 (FV3) ranavirus; the mechanisms governing amphibian susceptibility and resistance to such pathogens remain poorly understood. The type I and type III interferon (IFN) cytokines represent a cornerstone of vertebrate antiviral immunity, while our recent work indicates that tadpoles and adult frogs of the amphibian Xenopus laevis may differ in their IFN responses to FV3. In this respect, it is notable that anuran (frogs and toads) tadpoles are significantly more susceptible to FV3 than adult frogs, and thus, gaining greater insight into the differences in the tadpole and adult frog antiviral immunity would be invaluable. Accordingly, we examined the FV3-elicited expression of a panel of type I and type III IFN genes in the skin (site of FV3 infection) and kidney (principal FV3 target) tissues and isolated cells of X. laevis tadpoles and adult frogs. We also examined the consequence of tadpole and adult frog skin and kidney cell stimulation with hallmark pathogen-associated molecular patterns (PAMPs) on the IFN responses of these cells. Together, our findings indicate that tadpoles and adult frogs mount drastically distinct IFN responses to FV3 as well as to viral and non-viral PAMPs, while these expression differences do not appear to be the result of a distinct pattern recognition receptor expression by tadpoles and adults.

Xenopus laevis macrophage-like cells produce XCL-1, an intelectin family serum lectin that recognizes bacteria.

Xenopus laevis Ca2+ -dependent lectin-1 (XCL-1) is an intelectin family serum lectin that selectively recognizes carbohydrate chains on the bacterial cell surface. Immunofluorescence examination of control spleen tissues from normal X. laevis revealed cells producing XCL-1 (XCL-1+ cells) exclusively in red pulps. Intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) caused a marked increase in the number of XCL-1+ cells in red pulps on day 3, followed by a rapid decrease to near control levels by day 7. XCL-1+ cells were also detected in peripheral blood leukocytes (PBLs) and peritoneal exudate cells (PECs), and their numbers increased upon LPS injection until day 7. The XCL-1+ cells exhibited the morphological characteristics of macrophages, with a large oval or lobulated nucleus and abundant cytoplasm with vacuoles and dendritic projections. Western blot analyses revealed concurrent increases in XCL-1 levels in the spleen, PBLs, and PECs. When LPS-stimulated frogs were intraperitoneally injected with paraformaldehyde-fixed, green fluorescent protein-labeled E. coli cells (GFP-Eco), these were phagocytosed by XCL-1+ PECs. The purified XCL-1 protein agglutinated GFP-Eco in a Ca2+ -dependent manner, which was blocked effectively by xylose and partly by LPS and Staphylococcus aureus peptidoglycan, but not by sucrose. These results indicate that X. laevis macrophage-like cells produce XCL-1 and suggest that XCL-1 promotes the clearance of invaded bacteria by facilitating phagocytosis.

Innate Immune Response and Off-Target Mis-splicing Are Common Morpholino-Induced Side Effects in Xenopus.

Antisense morpholino oligomers (MOs) have been indispensable tools for developmental biologists to transiently knock down (KD) genes rather than to knock them out (KO). Here we report on the implications of genetic KO versus MO-mediated KD of the mesoderm-specifying Brachyury paralogs in the frog Xenopus tropicalis. While both KO and KD embryos fail to activate the same core gene regulatory network, resulting in virtually identical morphological defects, embryos injected with control or target MOs also show a systemic GC content-dependent immune response and many off-target splicing defects. Optimization of MO dosage and increasing incubation temperatures can mitigate, but not eliminate, these MO side effects, which are consistent with the high affinity measured between MO and off-target sequence in vitro. We conclude that while MOs can be useful to profile loss-of-function phenotypes at a molecular level, careful attention must be paid to their immunogenic and off-target side effects.

"Double-duty" conventional dendritic cells in the amphibian Xenopus as the prototype for antigen presentation to B cells.

Two populations of dendritic cells (DCs) are found in mammals, one derived from hematopoietic precursors (conventional/cDC), and another derived from mesenchymal precursors, the follicular DC (FDC); the latter is specialized for antigen presentation to B cells, and has only been definitively demonstrated in mammals. Both cDC and FDC are necessary for induction of germinal centers (GC) and GC-dependent class switch recombination (CSR) and somatic hypermutation (SHM). We demonstrate that in Xenopus, an amphibian in which immunoglobulin CSR and SHM occur without GC formation, a single type of DC has properties of both cDC and FDC, including high expression of MHC class II for the former and display of native antigen at the cell surface for the latter. Our data confirm that the advent of FDC functionality preceded emergence of bona fide FDC, which was in turn crucial for the development of GC formation and efficient affinity maturation in mammals.

Elicitation of Xenopus laevis Tadpole and Adult Frog Peritoneal Leukocytes.

Peritoneal lavage of Xenopus laevis tadpoles and adult frogs is a reliable way of isolating resident and/or recruited innate immune populations. This protocol details the isolation of tadpole and adult amphibian (Xenopus laevis) peritoneal leukocytes. The isolated cells are comprised predominantly of innate immune populations and chiefly of mononuclear and polymorphonuclear granulocytes. As described here, these cells are typically elicited by peritoneal injections of animals with heat-killed Escherichia coli, causing peritoneal accumulation of inflammatory cell populations, which are then isolated from the stimulated animals by lavage. E. coli-mediated elicitation of tadpole and adult peritoneal leukocytes greatly enhances the total numbers of recovered cells, at the cost of their inflammatory activation. Conversely, lavage may be performed on naïve, unstimulated animals to isolate nonactivated cells with much lower yield. This protocol represents a reliable means of deriving tadpole and adult frog innate immune cell populations, and the conditions of the stimulation may be amended to suit the specifics of a given experimental design.