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The zebrafish has become a powerful model organism to study host-pathogen interactions. Here, we developed a zebrafish model to dissect the innate immune response to Legionella pneumophila during infection. We show that L. pneumophila cause zebrafish larvae death in a dose dependent manner. Additionally, we show that macrophages are the first line of defence and cooperate with neutrophils to clear the infection. Immunocompromised humans have an increased propensity to develop pneumonia, similarly, when either macrophages or neutrophils are depleted, these "immunocompromised" larvae become lethally sensitive to L. pneumophila. Also, as observed in human infections, the adaptor signalling molecule Myd88 is not required to control disease in the larvae. Furthermore, proinflammatory cytokine genes il1β and tnf-α were upregulated during infection, recapitulating key immune responses seen in human infection. Strikingly, we uncovered a previously undescribed infection phenotype in zebrafish larvae, whereby bloodborne, wild type L. pneumophila invade and grow in the larval yolk region, a phenotype not observed with a type IV secretion system deficient mutant that cannot translocate effectors into its host cell. Thus, zebrafish larva represents an innovative L. pneumophila infection model that mimics important aspects of the human immune response to L. pneumophila infection and will allow the elucidation of mechanisms by which type IV secretion effectors allow L. pneumophila to cross host cell membranes and obtain nutrients from nutrient rich environments., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Viana et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pten is one of the most frequently mutated tumour suppressor gene in cancer. PTEN is generally altered in invasive cancers such as glioblastomas, but its function in collective cell migration and invasion is not fully characterised. Herein, we report that the loss of PTEN increases cell speed during collective migration of non-tumourous cells both in vitro and in vivo. We further show that loss of PTEN promotes LKB1-dependent phosphorylation and activation of the major metabolic regulator AMPK. In turn AMPK increases VASP phosphorylation, reduces VASP localisation at cell-cell junctions and decreases the interjunctional transverse actin arcs at the leading front, provoking a weakening of cell-cell contacts and increasing migration speed. Targeting AMPK activity not only slows down PTEN-depleted cells, it also limits PTEN-null glioblastoma cell invasion, opening new opportunities to treat glioblastoma lethal invasiveness., (© 2022. The Author(s).)

Nicotinamide adenine dinucleotide phosphate (NADPH) is the primary electron donor for reductive reactions that are essential for the biosynthesis of major cell components in all organisms. Nicotinamide adenine dinucleotide kinase (NADK) is the only enzyme that catalyzes the synthesis of NADP(H) from NAD(H). While the enzymatic properties and physiological functions of NADK have been thoroughly studied, the role of NADK in bacterial pathogenesis remains unknown. Here, we used CRISPR interference to knock down NADK gene expression to address the role of this enzyme in Staphylococcus aureus pathogenic potential. We find that NADK inhibition drastically decreases mortality of zebrafish infected with S. aureus . Furthermore, we show that NADK promotes S. aureus survival in infected macrophages by protecting bacteria from antimicrobial defense mechanisms. Proteome-wide data analysis revealed that production of major virulence-associated factors is sustained by NADK. We demonstrate that NADK is required for expression of the quorum-sensing response regulator AgrA, which controls critical S. aureus virulence determinants. These findings support a key role for NADK in bacteria survival within innate immune cells and the host during infection., Competing Interests: CL, LB, MD, JP, MM, EC, OD No competing interests declared, (© 2022, Leseigneur et al.)

Animal models are essential to understanding COVID-19 pathophysiology and for preclinical assessment of drugs and other therapeutic or prophylactic interventions. We explored the small, cheap, and transparent zebrafish larva as a potential host for SARS-CoV-2. Bath exposure, as well as microinjection in the coelom, pericardium, brain ventricle, or bloodstream, resulted in a rapid decrease of SARS-CoV-2 RNA in wild-type larvae. However, when the virus was inoculated in the swim bladder, viral RNA stabilized after 24 h. By immunohistochemistry, epithelial cells containing SARS-CoV-2 nucleoprotein were observed in the swim bladder wall. Our data suggest an abortive infection of the swim bladder. In some animals, several variants of concern were also tested with no evidence of increased infectivity in our model. Low infectivity of SARS-CoV-2 in zebrafish larvae was not due to the host type I interferon response, as comparable viral loads were detected in type I interferon-deficient animals. A mosaic overexpression of human ACE2 was not sufficient to increase SARS-CoV-2 infectivity in zebrafish embryos or in fish cells in vitro . In conclusion, wild-type zebrafish larvae appear mostly non-permissive to SARS-CoV-2, except in the swim bladder, an aerial organ sharing similarities with the mammalian lung., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Laghi, Rezelj, Boucontet, Frétaud, Da Costa, Boudinot, Salinas, Lutfalla, Vignuzzi and Levraud.)

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is unique amongst human pathogens in its capacity to produce a lipid toxin called mycolactone. While previous studies have demonstrated that bacterially-released mycolactone diffuses beyond infection foci, the spatiotemporal distribution of mycolactone remained largely unknown. Here, we used the zebrafish model to provide the first global kinetic analysis of mycolactone's diffusion in vivo, and multicellular co-culture systems to address the critical question of the toxin's access to the brain. Zebrafish larvae were injected with a fluorescent-derivative of mycolactone to visualize the in vivo diffusion of the toxin from the peripheral circulation. A rapid, body-wide distribution of mycolactone was observed, with selective accumulation in tissues near the injection site and brain, together with an important excretion through the gastro-intestinal tract. Our conclusion that mycolactone reached the central nervous system was reinforced by an in cellulo model of human blood brain barrier and a mouse model of M. ulcerans-infection. Here we show that mycolactone has a broad but heterogenous profile of distribution in vivo. Our investigations in vitro and in vivo support the view that a fraction of bacterially-produced mycolactone gains access to the central nervous system. The relative persistence of mycolactone in the bloodstream suggests that assays of circulating mycolactone are relevant for BU disease monitoring and treatment optimization., Competing Interests: The authors have declared that no competing interests exist.

Enhanced susceptibility to bacterial infection in the days following an acute virus infection such as flu is a major clinical problem. Mouse models have provided major advances in understanding viral-bacterial superinfections, yet interactions of the anti-viral and anti-bacterial responses remain elusive. Here, we have exploited the transparency of zebrafish to study how viral infections can pave the way for bacterial co-infections. We have set up a zebrafish model of sequential viral and bacterial infection, using sublethal doses of Sindbis virus and Shigella flexneri bacteria. This virus induces a strong type I interferons (IFN) response, while the bacterium induces a strong IL1β and TNFα-mediated inflammatory response. We found that virus-infected zebrafish larvae showed an increased susceptibility to bacterial infection. This resulted in the death with concomitant higher bacterial burden of the co-infected fish compared to the ones infected with bacteria only. By contrast, infecting with bacteria first and virus second did not lead to increased mortality or microbial burden. By high-resolution live imaging, we showed that neutrophil survival was impaired in Sindbis-then- Shigella co-infected fish. The two types of cytokine responses were strongly induced in co-infected fish. In addition to type I IFN, expression of the anti-inflammatory cytokine IL10 was induced by viral infection before bacterial superinfection. Collectively, these observations suggest the zebrafish larva as a useful animal model to address mechanisms underlying increased bacterial susceptibility upon viral infection.

Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. Infection by S. flexneri has been well-studied in vitro and is a paradigm for bacterial interactions with the host immune system. Recent work has revealed that components of the cytoskeleton have important functions in innate immunity and inflammation control. Septins, highly conserved cytoskeletal proteins, have emerged as key players in innate immunity to bacterial infection, yet septin function in vivo is poorly understood. Here, we use S. flexneri infection of zebrafish (Danio rerio) larvae to study in vivo the role of septins in inflammation and infection control. We found that depletion of Sept15 or Sept7b, zebrafish orthologs of human SEPT7, significantly increased host susceptibility to bacterial infection. Live-cell imaging of Sept15-depleted larvae revealed increasing bacterial burdens and a failure of neutrophils to control infection. Strikingly, Sept15-depleted larvae present significantly increased activity of Caspase-1 and more cell death upon S. flexneri infection. Dampening of the inflammatory response with anakinra, an antagonist of interleukin-1 receptor (IL-1R), counteracts Sept15 deficiency in vivo by protecting zebrafish from hyper-inflammation and S. flexneri infection. These findings highlight a new role for septins in host defence against bacterial infection, and suggest that septin dysfunction may be an underlying factor in cases of hyper-inflammation.

A large fraction of innate NKTγδ T cells uses TCRs composed of a semi-invariant Vδ6.3/6.4-Dδ2-Jδ1 chain together with more diverse Vγ1-Jγ4 chains. To address the role of γδTCR specificity in their generation, we analyzed their development in mice transgenic (Tg) for a Vγ1-Jγ4 chain frequently expressed by NKTγδ cells (Tg-γ) and in mice Tg for the same Vγ1-Jγ4 chain together with a Vδ6BDδ2Jδ1 chain not usually found among NKTγδ cells (Tg-γδ). Surprisingly, both promyelocytic leukemia zinc finger (PLZF)(+) and NK1.1(+) NKTγδ cells were found in the thymus of Tg-γδ albeit at lower numbers than in Tg-γ mice, and virtually all of them expressed the Tg TCR. However, the PLZF(+) subset, but not the NK1.1(+) subset, also expressed an endogenous Vδ6.3/6.4 chain, and its size was severely reduced in TCRδ(-/-) Tg-γδ mice. These results could suggest that the PLZF(+) and the NK1.1(+) subsets are developmentally unrelated. However, PLZF(+) and NK1.1(+) NKTγδ cells express identical Vδ6.3/6.4 chains, and NK1.1(+) cells can be obtained upon intrathymic injection of sorted PLZF(+) cells, thus indicating their developmental relationship. In fact, the NK1.1(+) γδ thymocytes present in Tg-γδ mice correspond to a small subset of NK1.1(+) γδ thymocytes in wild-type animals, which express a more diverse repertoire of TCRs and can be recognized by the expression of the CD62L Ag. Collectively, our data demonstrated that TCR specificity is essential for the development of most NKTγδ T cells and revealed a developmental heterogeneity in γδ T cells expressing the NK1.1 marker.

Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo.

The transcriptional regulator promyelocytic leukemia zinc finger (PLZF) is highly expressed during the differentiation of natural killer T (NKT) cells and is essential for the acquisition of their effector/memory innate-like phenotype. Staining with anti-PLZF and anti-NK1.1 Abs allows the definition of two subsets of NKTαβ and NKTγδ thymocytes that differ phenotypically and functionally: a PLZF(+) NK1.1(-) subset composed of mostly quiescent cells that secrete more IL-4 than IFN-γ upon activation and a PLZF(+/-) NK1.1(+) subset that expresses CD127, NK1.1, and other NK-cell markers, secrete more IFN-γ than IL-4 upon activation and contains a sizable fraction of dividing cells. The size of the NK1.1(+) population is very tightly regulated and NK1.1(+) αβ and γδ thymocytes compete for a thymic niche. Furthermore, the relative representation of the PLZF(+) and NK1.1(+) subsets varies in a strain-specific manner with C57BL/6 (B6) mice containing more NK1.1(+) cells and (B6 × DBA/2)F1 (B6D2F1) mice more PLZF(+) cells. Consequently, activation of NKT cells in vivo is expected to result in higher levels of IL-4 secreted in B6D2F1 mice than in B6 mice. Consistent with this possibility, B6D2F1 mice, when compared with B6 mice, contain more "innate" CD8(+) thymocytes, the generation of which depends on IL-4 secreted by NKT cells., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

How T cell progenitors engage into the γδ or αβ T cell lineages is a matter of intense debate. In this study, we analyzed the differentiation potential of single thymocytes from wild-type and TCRγδ-transgenic mice at two sequential early developmental stages. Double-negative (DN) 3 progenitors from both wild-type and transgenic mice retain the capacity to engage into both pathways, indicating that full commitment is only completed after this stage. More importantly, DN2 and DN3 progenitors from TCRγδ transgenic mice have strong biases for opposite fates, indicating that developmentally regulated changes, other than the production of a functional TCR, altered their likelihood to become a γδ or an αβ T cell. Thus, unlike the differentiation in other hematopoietic lineages, T cell progenitors did not restrict, but rather switch their differentiation potential as they developed.

The transcription factor RORγt is required for the development of several innate lymphoid populations, such as lymphoid tissue-inducer cells (LTi cells) and cells that secrete interleukin 17 (IL-17) or IL-22. The progenitor cells as well as the developmental stages that lead to the emergence of RORγt(+) innate lymphoid cells (ILCs) remain undefined. Here we identify the chemokine receptor CXCR6 as an additional marker of the development of ILCs and show that common lymphoid progenitors lost B cell and T cell potential as they successively acquired expression of the integrin α(4)β(7) and CXCR6. Whereas fetal RORγt(+) cells matured in the fetal liver environment, adult bone marrow-derived RORγt(+) ILCs matured outside the bone marrow, in a Notch2-dependent manner. Therefore, fetal and adult environments influence the differentiation of RORγt(+) cells differently.

By triggering immunogenic cell death, some anticancer compounds, including anthracyclines and oxaliplatin, elicit tumor-specific, interferon-γ-producing CD8(+) αβ T lymphocytes (Tc1 CTLs) that are pivotal for an optimal therapeutic outcome. Here, we demonstrate that chemotherapy induces a rapid and prominent invasion of interleukin (IL)-17-producing γδ (Vγ4(+) and Vγ6(+)) T lymphocytes (γδ T17 cells) that precedes the accumulation of Tc1 CTLs within the tumor bed. In T cell receptor δ(-/-) or Vγ4/6(-/-) mice, the therapeutic efficacy of chemotherapy was compromised, no IL-17 was produced by tumor-infiltrating T cells, and Tc1 CTLs failed to invade the tumor after treatment. Although γδ T17 cells could produce both IL-17A and IL-22, the absence of a functional IL-17A-IL-17R pathway significantly reduced tumor-specific T cell responses elicited by tumor cell death, and the efficacy of chemotherapy in four independent transplantable tumor models. Adoptive transfer of γδ T cells restored the efficacy of chemotherapy in IL-17A(-/-) hosts. The anticancer effect of infused γδ T cells was lost when they lacked either IL-1R1 or IL-17A. Conventional helper CD4(+) αβ T cells failed to produce IL-17 after chemotherapy. We conclude that γδ T17 cells play a decisive role in chemotherapy-induced anticancer immune responses.

Although NK cells in the mouse are thought to develop in the bone marrow, a small population of NK cells in the thymus has been shown to derive from a GATA3-dependent pathway. Characteristically, thymic NK cells express CD127 and few Ly49 molecules and lack CD11b. Because these NK cells develop in the thymus, the question of their relationship to the T cell lineage has been raised. Using several different mouse models, we find that unlike T cells, thymic NK cells are not the progeny of Rorc-expressing progenitors and do not express Rag2 or rearrange the TCRγ locus. We further demonstrate that thymic NK cells develop independently of the Notch signaling pathway, supporting the idea that thymic NK cells represent bona fide NK cells that can develop independently of all T cell precursors.

Recombination activating gene (RAG)-deficient TCR (T Cell Receptor) Tg (transgenic) mice are routinely used as sources of monoclonal T cells. We found that after the transfer of T cells from a RAG-2-deficient 5CC7 TCR Tg mice into allogeneic hosts we recovered a population of T cells expressing diverse alphabeta-TCRs. In fact, in the thymus and spleen of the 5CC7 RAG-2-deficient donor mice, we detected rare T cells expressing non-Tg TCR chains. Similar observations were obtained using T cells from two other TCR transgenic strains, namely RAG-2-deficient aHY and RAG-1-deficient OT-1 mice. The sequences of the endogenous TCR transcripts suggested that gene recombination could occur, albeit quite inefficiently, in the RAG-deficient mice we used. In agreement, we evidenced rare TCR Valpha and Vbeta-chain transcripts in non-Tg RAG-2-deficient mice. Since in these non-Tg RAG-deficient mice no mature T cells could ever be found, our findings suggested a role for the TCR Tg in rescuing rare recombined endogenous chains. Robust T-cell activation by the allogeneic environment favored the selection and expansion of the rare cells expressing endogenous TCRs. Potential mechanisms involved in the recombination of the endogenous TCR chains in the different strains of RAG-deficient mice used, and in particular the possibility of RAG-1 hypomorphism due to an incomplete knocking out procedure, are discussed. Our findings have important experimental implications for studies using TCR-Tg RAG-deficient cells as monoclonal T cell populations.

We investigated the ability of the most common TCR-gamma and delta chains to express on the cell surface. Vgamma1Cgamma4 and Vgamma7Cgamma1 chains paired with all TCR-delta chains tested, whereas Vgamma4Cgamma1 chains were found with Vdelta4 and Vdelta5, but not with Vdelta2 or Vdelta6 chains, and Vgamma2Cgamma2 chains were expressed only with Vdelta5. Mapping studies showed that up to four polymorphic residues influence the different co-expressions of Vgamma1 and Vgamma2 chains with Vdelta chains. Unexpectedly, these residues are not located in the canonical gamma/delta interface, but in the outer part of the gammadelta TCR complex exposed to the solvent. Expression of functional Vdelta4 or Vdelta6 chains in Vgamma2/Vdelta5(+) cells or of functional Vgamma2Cgamma2 in Vgamma1(+) cells reduced cell-surface expression of the gammadelta TCR. Taken together, these data show that (i) the Vgamma/Vdelta repertoire of mouse gammadelta T cells is reduced by physical constraints in their associations. (ii) Lack of Vgamma2/Vdelta expression is due to the formation of aberrant TCR complexes, rather than to an intrinsic inability of the chains to pair and (iii) despite not being expressed at the cell surface, the presence of a functionally rearranged Vgamma2 chain in gammadelta T cells results in reduced TCR levels.

The mechanisms controlling the recombination process of the gamma genes that encode the gamma chain of the antigen receptor of the gammadelta T lymphocytes are unclear. Based on experimental data on the recombination status of the two major TCR gamma genes expressed in V(gamma)4+ and V(gamma)1+ thymocytes, we tested the plausibility of three possible rearrangement mechanisms: (1) a time window mechanism according to which the two chromosomes are accessible to the recombination machinery during a defined period of time; (2) a feedback mechanism in which recombination stops shortly after the first in-frame rearrangement event anywhere in both chromosomes; and (3) a feedback mechanism with asynchronous chromosome accessibility, in which there is a first period when only one chromosome is accessible for recombination, followed by a second period when both chromosomes are accessible; shortly after the first in-frame rearrangement event, during any of these two periods, recombination will definitely stop. We model the time window mechanism using a pure probabilistic approach and the two feedback mechanisms using a continuous-time Markov chain formalism. We used maximum likelihood methodology to infer the goodness-of-fit of the models showing evidence for the last model, which best fits the data. Further analysis of this model suggests an evolutionary tradeoff between allelic and isotypic exclusion and the probability that a precursor differentiates into a mature gammadelta T lymphocyte.

Analyses of Vgamma-Jgamma rearrangements producing the most commonly expressed TCRgamma chains in over 200 gammadelta TCR(+) thymocytes showed that assembly of TCRgamma V-region genes display properties of allelic exclusion. Moreover, introduction of functionally rearranged TCRgamma and delta transgenes results in a profound inhibition of endogenous TCRgamma rearrangements in progenitor cells. The extent of TCRgamma rearrangements in these cells is best explained by a model in which initiation of TCRgamma rearrangements at both alleles is asymmetric, occurs at different frequencies depending on the V or J segments involved, and is terminated upon production of a functional gammadelta TCR. Approximately 10% of the cells studied contained two functional TCRgamma chains involving different V and Jgamma gene segments, thus defining a certain degree of isotypic inclusion. However, these cells are isotypically excluded at the level of cell surface expression possibly due to pairing restrictions between different TCRgamma and delta chains.

Little is known about hematopoietic stem cell (HSC) development from mesoderm. To gain more information on the intraembryonic HSC site of origin, we purified multipotent hematopoietic progenitors from the aorta-gonads-mesonephros (AGM) of mice. This population, expressing c-Kit, AA4.1, CD31, and CD41, but not Flk1, and mainly negative for CD45, proved capable of long-term reconstitution in sublethally irradiated Rag2gammac(-/-) recipients. We assigned the expression of GATA-2, GATA-3, and lmo2 to AGM-HSC, whereas erythromyeloid progenitors express only GATA-2. This unique combination of surface markers and transcription factors could be allocated in the AGM to the intraaortic clusters and the subaortic patches underlying aortic endothelial cells. Taken together, those data indicate that embryonic HSCs (i) differ from their fetal liver and adult counterpart by the low expression of CD45, (ii) do not colocalize with aortic endothelial cells as previously thought, and (iii) are localized, at 10.5 days postcoitum, in the splanchnic mesoderm underlying aortic endothelial cells, within GATA-3(+)CD31(+) cell clusters.

Thymectomy at day 3 after birth leads to autoimmune disease in some genetic backgrounds. Disease is thought to be caused by the lack/paucity of regulatory T cells. We show that 3-day-old mice already contain a significant compartment of Foxp3-expressing CD25(+)CD4(+) splenocytes. Whereas, in adult spleen, the subsets of regulatory T cells (CD25(+) and/or CD103(+)) express high amounts of Foxp3 mRNA, in 3-day-old mice, both thymic and splenic CD25(+)CD4(+) T cell subsets express lower amounts of Foxp3 mRNA, and CD103(+) cells are barely detected. In adult day 3-thymectomized mice, the CD25(+)CD4(+) T cell subset is overrepresented (most of the cells being CD103(+)) and expresses high amounts of Foxp3 mRNA, independent of the development of autoimmune gastritis. These cells control inflammatory bowel disease and the homeostatic expansion of lymphocytes. This study demonstrates that the peripheral immune system of newborn mice is endowed of a remarkable regulatory potential, which develops considerably in the absence of thymic supply.

Analyses of the rearrangement status of the TCRgamma and TCRdelta chain loci in progenies of individual gammadelta thymocytes showed a hierarchy of the different Vgamma and Vdelta gene segments to participate in a recombination reaction. Moreover, individual TCRgamma chains only pair efficiently with a variable number of TCRdelta chains. Interestingly, these two parameters are inversely correlated such that the TCRgamma and TCRdelta chains that rearrange more often show a higher level of restriction in their pairing capabilities. Our data suggest that these mechanisms, together with a natural variation affecting the expected frequencies at which rearrangement of different Vgamma gene segments give raise to functional TCRgamma chains, have coevolved to maximize the diversity of the gammadelta TCR repertoire minimizing the risk that a gammadelta T cell will express more than one TCR specificity at the cell surface, despite the fact that multiple TCRgamma rearrangements take place in the same progenitor cell.

To investigate the consequences of the simultaneous expression in progenitor cells of a TCRgammadelta and a pre-TCR on alphabeta/gammadelta lineage commitment, we have forced expression of functionally rearranged TCRbeta, TCRgamma, and TCRdelta chains by means of transgenes. Mice transgenic for the three TCR chains contain numbers of gammadelta thymocytes comparable to those of mice transgenic for both TCRgamma and TCRdelta chains, and numbers of alphabeta thymocytes similar to those found in mice solely transgenic for a rearranged TCRbeta chain gene. gammadelta T cells from the triple transgenic mice express the transgenic TCRbeta chain, but do not express a TCRalpha chain, and, by a number of phenotypic and molecular parameters, appear to be bona fide gammadelta thymocytes. Our results reveal a remarkable degree of independence in the generation of alphabeta and gammadelta lineage cells from progenitor cells that, in theory, could simultaneously express a TCRgammadelta and a pre-TCR.

Thy-1(dull) gammadelta T cells constitute a distinct adult gammadelta T cell subset characterized by the expression of a TCR composed of Vgamma1Cgamma4 and Vdelta6Cdelta chains with limited junctional sequence diversity. However, several features of the expressed Thy-1(dull) TCR-gammadelta genes, in particular the absence or minimal presence of N region diversity and the almost invariable Ddelta2-Jdelta1 junction, are typical of rearrangements often found in the fetal thymus. In this study, we have investigated the origin of these cells. Few Thy-1(dull) gammadelta thymocytes developed in syngeneic radiation adult chimeras, regardless of whether the recipient mice were given adult bone marrow or fetal liver cells as a source of hemopoietic precursors. In contrast, normal numbers of Thy-1(dull) gammadelta T cells developed in fetal thymi grafted into adult syngeneic recipients. Interestingly, the majority of Thy-1(dull) gammadelta thymocytes present in the grafts were of graft origin, even when most conventional gammadelta and alphabeta thymocytes in the grafted thymi originated from T cell precursors of recipient origin. Single-cell PCR analyses of the nonselected TCR-gamma rearrangements present in adult Thy-1(dull) gammadelta thymocytes revealed that more than one-half of these cells represent the progenies of a limited number of clones that greatly expanded possibly during the first weeks of life. Finally, the second TCR-delta allele of a large number of Thy-1(dull) gammadelta T cells contained incomplete TCR-delta rearrangements, thus providing an explanation for the adult-type rearrangements previously found among nonfunctional V(D)J rearrangements present in Thy-1(dull) gammadelta thymocytes.

Previous genetic analyses have shown that the relative representation of subsets of gammadelta intestinal intraepithelial lymphocytes (i-IELs) is influenced by genes linked to the TCRgamma, TCRdelta, and MHC loci. Here, we have analyzed V-gene use in gammadelta i-IELs from C57BL/6 (B6) and C57BL/10 (B10) mice and from their F(1) and F(2) progenies with a larger panel of Vgamma- and Vdelta-specific mAbs and have shown that the influence of TCRgamma-linked genes operates at two levels: one influencing the representation of Vgamma1 (or Vgamma7) i-IELs and other acting specifically on the Vgamma1/Vdelta4 i-IEL subset, which represents 3% and 15% of the gammadelta i-IELs in B6 and B10 mice, respectively. Analysis of mice transgenic for a rearranged Vgamma1Jgamma4Cgamma4 chain of B6 origin demonstrated that the TCRgamma-linked genes influencing the representation of the Vgamma1/Vdelta4 i-IEL subset are the structural genes of TCRgamma chains. This influence is allele specific and cell autonomous, as evidenced by the different behavior of Vgamma1/Vdelta4 cells bearing either parental allele in F(1) mice. The representation of Vgamma1/Vdelta4 cells among gammadelta thymocytes is similar in B6 and B10 mice, demonstrating that the Vdelta4 chain can pair well with both alleles of the Vgamma1Jgamma4Cgamma4 chain and strongly suggesting that a cellular selection mechanism is responsible for the observed differences. The Vgamma1-Jgamma4 junctional amino acid sequences of B6 Vgamma1/Vdelta4 i-IELs are diverse but display less variation in length than those found in similar cells from B10 mice, indicating that B6 Vgamma1/Vdelta4 cells are the target of this cellular selection event.

To quantitate the frequency of Valpha/delta gene utilization by TCRgammadelta T cells we have generated a large panel of gammadelta T cell hybridomas and characterized their productive VDJ rearrangements. Using three novel mAb specific for the Vdelta5 chain and for several members of the Vdelta6 subfamily together with previously described Valpha- and Vdelta-specific mAb we have also quantitated the frequency of gammadelta and alphabeta cells expressing those Valpha/delta gene segments and located in different anatomical sites. We have also characterized the members of the Vdelta7/ADV10 subfamily expressed in C57BL/6 mice and analyzed the representation of individual ADV10 gene segments in alphabeta and gammadelta cells, as well as in precursor cells, in a situation in which TCR-dependent selection is negligible. Our results show that (i) although many Valpha/delta gene segments have the potential to rearrange to either Ddelta and Jdelta segments or to Jalpha segments, only a limited number of Valpha/delta gene segments are expressed by a quantitatively important fraction of gammadelta cells; (ii) such restricted usage of a limited number of Vdelta gene segments by gammadelta cells is mainly established at the level of V(D)J rearrangement, and (iii) there is very little overlap between Valpha/delta gene segments expressed by gammadelta and alphabeta cells.

This study examines the relationships between metabolic status and behavior in spontaneously fasting birds in the context of long-term regulation of body mass and feeding. Locomotor activity, escape behavior, display songs, body mass, and metabolic and endocrine status of captive male emperor penguins were recorded during a breeding fast. We also examined whether body mass at the end of the fast affected further survival. The major part of the fast (phase II) was characterized by the maintenance of a very low level of locomotor activity, with almost no attempt to escape, by an almost constant rate of body mass loss, and by steady plasma levels of uric acid, beta-hydroxybutyrate, and corticosterone. This indicates behavioral and metabolic adjustments directed toward sparing energy and body protein. Below a body mass of approximately 24 kg (phase III), spontaneous locomotor activity and attempts to escape increased by up to 8- and 15-fold, respectively, and display songs were resumed. This probably reflected an increase in the drive to refeed. Simultaneously, daily body mass loss and plasma levels of uric acid and corticosterone increased, whereas plasma levels of beta-hydroxybutyrate decreased. Some experimental birds were seen again in following years. These findings suggest that at a threshold of body mass, a metabolic and endocrine shift, possibly related to a limited availability of fat stores, acts as a "refeeding signal" that improves the survival of penguins to fasting.

Hyperglycaemia and hyperinsulinaemia were induced in rats by a continuous 48-h infusion with glucose. Discontinuation of glucose infusion resulted in marked, persistent hypoglycaemia. To further delineate the mechanism underlying this condition, we measured counterregulatory hormone levels, in vivo glucose kinetics (glucose production = rate of appearance = Ra; glucose utilization = rate of disappearance = Rd), and in vitro gluconeogenesis during the 48-h postinfusion period. Prior to cessation of glucose infusion, Rd was increased 6-fold when compared to control rats, whereas Ra was totally abolished. During the first hour after the end of glucose infusion, Ra increased and Rd decreased (but was still higher than Ra), inducing hypoglycaemia which stabilized after 1 h at ¿¿126¿¿3.5 mmol/l when both Ra and Rd became equal. Despite hypoglycaemia, plasma glucagon and catecholamine levels did not increase during the 3-to 36-h time interval. The increase in Ra during the first hour post-infusion was not related to changes in counterregulatory hormone response. The increase in glucose production was accounted for by glycogenolysis, as shown by total depletion in liver glycogen within 6 h and thereafter by gluconeogenesis. In vitro experiments using isolated hepatocytes suggested that gluconeogenesis was supported during the first 24 h by substrates entering the pathway beyond the step catalysed by the PEPCK enzyme. Thereafter, lactate became the major substrate, and this condition was associated with a progressive rise in glucagon concentration. It is concluded that 48 h of hyperglycaemia/hyperinsulinaemia resulted in a failure of counterregulatory hormonal response to hypoglycaemia. Yet, despite this lack of counterregulatory response, hepatic gluconeogenesis was stimulated in response to hypoglycaemia.