ARHGAP45 controls naïve T‐ and B‐cell entry into lymph nodes and T‐cell progenitor thymus seeding

T and B cells continually recirculate between blood and secondary lymphoid organs. To promote their trans‐endothelial migration (TEM), chemokine receptors control the activity of RHO family small GTPases in part via GTPase‐activating proteins (GAPs). T and B cells express several RHO‐GAPs, the function of most of which remains unknown. The ARHGAP45 GAP is predominantly expressed in hematopoietic cells. To define its in vivo function, we describe two mouse models where ARHGAP45 is ablated systemically or selectively in T cells. We combine their analysis with affinity purification coupled to mass spectrometry to determine the ARHGAP45 interactome in T cells and with time‐lapse and reflection interference contrast microscopy to assess the role of ARGHAP45 in T‐cell polarization and motility. We demonstrate that ARHGAP45 regulates naïve T‐cell deformability and motility. Under physiological conditions, ARHGAP45 controls the entry of naïve T and B cells into lymph nodes whereas under competitive repopulation it further regulates hematopoietic progenitor cell engraftment in the bone marrow, and T‐cell progenitor thymus seeding. Therefore, the ARGHAP45 GAP controls multiple key steps in the life of T and B cells.     

Systemic Sclerosis Sera Impair Angiogenic Performance of Dermal Microvascular Endothelial Cells: Therapeutic Implications of Cyclophosphamide

In systemic sclerosis (SSc), dermal capillaries are progressively lost with consequent chronic tissue hypoxia insufficiently compensated by angiogenesis. Clinical studies reported that intravenous cyclophosphamide (CYC) may improve SSc-related peripheral microvascular damage. Recently, we showed that CYC treatment may normalize SSc sera-induced abnormalities in endothelial cell-matrix interactions. Our objective was to evaluate in vitro the effects of sera from treatment-naïve or CYC-treated SSc patients on dermal blood microvascular endothelial cell (dMVEC) angiogenesis, migration, proliferation and apoptosis. dMVECs were challenged with sera from 21 SSc patients, treatment-naïve (n = 8) or under CYC treatment (n = 13), and 8 healthy controls. Capillary morphogenesis on Geltrex matrix was significantly reduced upon challenge with sera from naïve SSc patients compared with healthy controls. When dMVECs were challenged with sera from CYC-treated SSc patients, their angiogenic capacity was comparable to that of cells treated with healthy sera. Wound healing capacity and chemotaxis in Boyden chamber were both significantly decreased in the presence either of naïve or CYC-treated SSc sera compared with healthy sera. WST-1 assay revealed that cell proliferation was significantly decreased in dMVECs challenged with sera from naïve SSc patients compared with healthy sera. Conversely, dMVEC proliferation was not impaired in the presence of sera from CYC-treated SSc patients. Accordingly, the percentage of TUNEL-positive apoptotic dMVECs was significantly higher in the presence of sera from naïve SSc patients than healthy controls, while CYC-treated SSc sera did not induce dMVEC apoptosis. Levels of the angiostatic mediators endostatin, pentraxin 3, angiostatin and matrix metalloproteinase-12 were all significantly elevated in sera from naïve SSc patients compared with sera from both healthy controls and CYC-treated SSc patients. In SSc, CYC treatment might boost angiogenesis and consequently improve peripheral microangiopathy through the normalization of the endothelial cell-matrix interactions, reduction of endothelial cell apoptosis and rebalance of dysregulated angiostatic factors.     

Different genome stability proteins underpin primed and na?ve adaptation in E. coli CRISPR-Cas immunity

CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration.     

Hemocyte Density Increases with Developmental Stage in an Immune-Challenged Forest Caterpillar

The cellular arm of the insect immune response is mediated by the activity of hemocytes. While hemocytes have been well-characterized morphologically and functionally in model insects, few studies have characterized the hemocytes of non-model insects. Further, the role of ontogeny in mediating immune response is not well understood in non-model invertebrate systems. The goals of the current study were to (1) determine the effects of caterpillar size (and age) on hemocyte density in naïve caterpillars and caterpillars challenged with non-pathogenic bacteria, and (2) characterize the hemocyte activity and diversity of cell types present in two forest caterpillars: Euclea delphinii and Lithacodes fasciola (Limacodidae). We found that although early and late instar (small and large size, respectively) naïve caterpillars had similar constitutive hemocyte densities in both species, late instar Lithacodes caterpillars injected with non-pathogenic E. coli produced more than a twofold greater density of hemocytes than those in early instars. We also found that both caterpillar species contained plasmatocytes, granulocytes and oenocytoids, all of which are found in other lepidopteran species, but lacked spherulocytes. Granulocytes and plasmatocytes were found to be strongly phagocytic in both species, but granulocytes exhibited a higher phagocytic activity than plasmatocytes. Our results strongly suggest that for at least one measure of immunological response, the production of hemocytes in response to infection, response magnitudes can increase over ontogeny. While the underlying raison d’ être for this improvement remains unclear, these findings may be useful in explaining natural patterns of stage-dependent parasitism and pathogen infection.     

Transmission and Infectious Dose of Escherichia coli O157:H7 in Swine

Escherichia coli O157:H7 is only occasionally isolated from healthy swine, but some experimentally infected animals will shed the organism in their feces for at least 2 months. Potential explanations for the paucity of naturally occurring infections in swine, as compared to cattle, include a lack of animal-to-animal transmission so that the organism cannot be maintained within a herd, a high infectious dose, or herd management practices that prevent the maintenance of the organism in the gastrointestinal tract. We hypothesized that donor pigs infected with E. coli O157:H7 would transmit the organism to naïve pigs. We also determined the infectious dose and whether housing pigs individually on grated floors would decrease the magnitude or duration of fecal shedding. Infected donor pigs shedding <10⁴ CFU of E. coli O157:H7 per g transmitted the organism to 6 of 12 naïve pigs exposed to them. The infectious dose of E. coli O157:H7 for 3-month-old pigs was approximately 6 × 10³ CFU. There was no difference in the magnitude and duration of fecal shedding by pigs housed individually on grates compared to those housed two per pen on cement floors. These results suggest that swine do not have an innate resistance to colonization by E. coli O157:H7 and that they could serve as a reservoir host under suitable conditions.     

Great tits (Parus major) flexibly learn that herbivore‐induced plant volatiles indicate prey location: An experimental evidence with two tree species

1. When searching for food, great tits (Parus major) can use herbivore‐induced plant volatiles (HIPVs) as an indicator of arthropod presence. Their ability to detect HIPVs was shown to be learned, and not innate, yet the flexibility and generalization of learning remain unclear.
2. We studied if, and if so how, naïve and trained great tits (Parus major) discriminate between herbivore‐induced and noninduced saplings of Scotch elm (Ulmus glabra) and cattley guava (Psidium cattleyanum). We chemically analyzed the used plants and showed that their HIPVs differed significantly and overlapped only in a few compounds.
3. Birds trained to discriminate between herbivore‐induced and noninduced saplings preferred the herbivore‐induced saplings of the plant species they were trained to. Naïve birds did not show any preferences. Our results indicate that the attraction of great tits to herbivore‐induced plants is not innate, rather it is a skill that can be acquired through learning, one tree species at a time.
4. We demonstrate that the ability to learn to associate HIPVs with food reward is flexible, expressed to both tested plant species, even if the plant species has not coevolved with the bird species (i.e., guava). Our results imply that the birds are not capable of generalizing HIPVs among tree species but suggest that they either learn to detect individual compounds or associate whole bouquets with food rewards.

     

Prior Infection of Chickens with H1N1 or H1N2 Avian Influenza Elicits Partial Heterologous Protection against Highly Pathogenic H5N1

There is a critical need to have vaccines that can protect against emerging pandemic influenza viruses. Commonly used influenza vaccines are killed whole virus that protect against homologous and not heterologous virus. Using chickens we have explored the possibility of using live low pathogenic avian influenza (LPAI) A/goose/AB/223/2005 H1N1 or A/WBS/MB/325/2006 H1N2 to induce immunity against heterologous highly pathogenic avian influenza (HPAI) A/chicken/Vietnam/14/2005 H5N1. H1N1 and H1N2 replicated in chickens but did not cause clinical disease. Following infection, chickens developed nucleoprotein and H1 specific antibodies, and reduced H5N1 plaque size in vitro in the absence of H5 neutralizing antibodies at 21 days post infection (DPI). In addition, heterologous cell mediated immunity (CMI) was demonstrated by antigen-specific proliferation and IFN-γ secretion in PBMCs re-stimulated with H5N1 antigen. Following H5N1 challenge of both pre-infected and naïve controls chickens housed together, all naïve chickens developed acute disease and died while H1N1 or H1N2 pre-infected chickens had reduced clinical disease and 70–80% survived. H1N1 or H1N2 pre-infected chickens were also challenged with H5N1 and naïve chickens placed in the same room one day later. All pre-infected birds were protected from H5N1 challenge but shed infectious virus to naïve contact chickens. However, disease onset, severity and mortality was reduced and delayed in the naïve contacts compared to directly inoculated naïve controls. These results indicate that prior infection with LPAI virus can generate heterologous protection against HPAI H5N1 in the absence of specific H5 antibody.     

Timing of Immune Escape Linked to Success or Failure of Vaccination

Successful vaccination against HIV should limit viral replication sufficiently to prevent the emergence of viral immune escape mutations. Broadly directed immunity is likely to be required to limit opportunities for immune escape variants to flourish. We studied the emergence of an SIV Gag cytotoxic T cell immune escape variant in pigtail macaques expressing the Mane-A*10 MHC I allele using a quantitative RT-PCR to measure viral loads of escape and wild type variants. Animals receiving whole Gag expressing vaccines completely controlled an SIV_mac251 challenge, had broader CTL responses and exhibited minimal CTL escape. In contrast, animals vaccinated with only a single CTL epitope and challenged with the same SIV_mac251 stock had high levels of viral replication and rapid CTL escape. Unvaccinated naïve animals exhibited a slower emergence of immune escape variants. Thus narrowly directed vaccination against a single epitope resulted in rapid immune escape and viral levels equivalent to that of naïve unvaccinated animals. These results emphasize the importance of inducing broadly directed HIV-specific immunity that effectively quashes early viral replication and limits the generation of immune escape variants. This has important implications for the selection of HIV vaccines for expanded human trials.     

Natural variation in colony inbreeding does not influence susceptibility to a fungal pathogen in a termite

Reduced genetic diversity through inbreeding can negatively affect pathogen resistance. This relationship becomes more complicated in social species, such as social insects, since the chance of disease transmission increases with the frequency of interactions among individuals. However, social insects may benefit from social immunity, whereby individual physiological defenses may be bolstered by collective‐level immune responses, such as grooming or sharing of antimicrobial substance through trophallaxis. We set out to determine whether differences in genetic diversity between colonies of the subterranean termite, Reticulitermes flavipes, accounts for colony survival against pathogens. We sampled colonies throughout the United States (Texas, North Carolina, Maryland, and Massachusetts) and determined the level of inbreeding of each colony. To assess whether genetically diverse colonies were better able to survive exposure to diverse pathogens, we challenged groups of termite workers with two strains of a pathogenic fungus, one local strain present in the soil surrounding sampled colonies and another naïve strain, collected outside the range of this species. We found natural variation in the level of inbreeding between colonies, but this variation did not explain differences in susceptibility to either pathogen. Although the naïve strain was found to be more hazardous than the local strain, colony resistance was correlated between two strains, meaning that colonies had either relatively high or low susceptibility to both strains regardless of their inbreeding coefficient. Overall, our findings may reflect differential virulence between the strains, immune priming of the colonies via prior exposure to the local strain, or a coevolved resistance toward this strain. They also suggest that colony survival may rely more upon additional factors, such as different behavioral response thresholds or the influence of a specific genetic background, rather than the overall genetic diversity of the colony.     

Evaluation of the Murine Immune Response to Xenopsylla cheopis Flea Saliva and Its Effect on Transmission of Yersinia pestis

Background/Aims

Arthropod-borne pathogens are transmitted into a unique intradermal microenvironment that includes the saliva of their vectors. Immunomodulatory factors in the saliva can enhance infectivity; however, in some cases the immune response that develops to saliva from prior uninfected bites can inhibit infectivity. Most rodent reservoirs of Yersinia pestis experience fleabites regularly, but the effect this has on the dynamics of flea-borne transmission of plague has never been investigated. We examined the innate and acquired immune response of mice to bites of Xenopsylla cheopis and its effects on Y. pestis transmission and disease progression in both naïve mice and mice chronically exposed to flea bites.

Methods/Principal Findings

The immune response of C57BL/6 mice to uninfected flea bites was characterized by flow cytometry, histology, and antibody detection methods. In naïve mice, flea bites induced mild inflammation with limited recruitment of neutrophils and macrophages to the bite site. Infectivity and host response in naïve mice exposed to flea bites followed immediately by intradermal injection of Y. pestis did not differ from that of mice infected with Y. pestis without prior flea feeding. With prolonged exposure, an IgG1 antibody response primarily directed to the predominant component of flea saliva, a family of 36–45 kDa phosphatase-like proteins, occurred in both laboratory mice and wild rats naturally exposed to X. cheopis, but a hypersensitivity response never developed. The incidence and progression of terminal plague following challenge by infective blocked fleas were equivalent in naïve mice and mice sensitized to flea saliva by repeated exposure to flea bites over a 10-week period.

Conclusions

Unlike what is observed with many other blood-feeding arthropods, the murine immune response to X. cheopis saliva is mild and continued exposure to flea bites leads more to tolerance than to hypersensitivity. The immune response to flea saliva had no detectable effect on Y. pestis transmission or plague pathogenesis in mice.     

Self DNA from Lymphocytes That Have Undergone Activation-Induced Cell Death Enhances Murine B Cell Proliferation and Antibody Production

Systemic lupus erythematosus (SLE) is characterized by prominent autoinflammatory tissue damage associated with impaired removal of dying cells and DNA. Self DNA-containing immune complexes are able to activate both innate and adaptive immune responses and play an important role in the maintenance and exacerbation of autoimmunity in SLE. In this study, we used DNA from lymphocytes that have undergone activation-induced cell death (ALD-DNA) and analyzed its role on the activation and differentiation of B cells from normal BALB/c mice as well as lupus-prone MRL^+/+ and MRL/lpr mice. We found that ALD-DNA directly increased the expression of costimulatory molecules and the survival of naïve B cells in vitro. Although ALD-DNA alone had little effect on the proliferation of naïve B cells, it enhanced LPS-activated B cell proliferation in vitro and in vivo. In addition, ALD-DNA increased plasma cell numbers and IgG production in LPS-stimulated cultures of naïve B cells, in part via enhancing IL-6 production. Importantly, B cells from lupus mice were hyperresponsive to ALD-DNA and/or LPS relative to normal control B cells in terminal plasma cell differentiation, as evidenced by increases in CD138⁺ cell numbers, IgM production, and mRNA levels of B lymphocyte-induced maturation protein-1 (Blimp-1) and the X-box binding protein 1 (XBP1). Furthermore, ALD-DNA enhanced CD40-activated naïve B cell proliferation. Collectively, these data indicate that self DNA can serve as a DAMP (damage-associated molecular pattern) that cooperates with signals from both innate and adaptive immunity to promote polyclonal B cell activation, a common characteristic of autoimmune diseases.     

Superior Cervical Ganglia Neurons Induce Foxp3+ Regulatory T Cells via Calcitonin Gene-Related Peptide

The nervous and immune systems communicate bidirectionally, utilizing diverse molecular signals including cytokines and neurotransmitters to provide an integrated response to changes in the body’s internal and external environment. Although, neuro-immune interactions are becoming better understood under inflammatory circumstances and it has been evidenced that interaction between neurons and T cells results in the conversion of encephalitogenic T cells to T regulatory cells, relatively little is known about the communication between neurons and naïve T cells. Here, we demonstrate that following co-culture of naïve CD4⁺ T cells with superior cervical ganglion neurons, the percentage of Foxp3 expressing CD4⁺CD25⁺ cells significantly increased. This was mediated in part by immune-regulatory cytokines TGF-β and IL-10, as well as the neuropeptide calcitonin gene-related peptide while vasoactive intestinal peptide was shown to play no role in generation of T regulatory cells. Additionally, T cells co-cultured with neurons showed a decrease in the levels of pro-inflammatory cytokine IFN-γ released upon in vitro stimulation. These findings suggest that the generation of Tregs may be promoted by naïve CD4⁺ T cell: neuron interaction through the release of neuropeptide CGRP.     

CD4+ T cells in aged or thymectomized recipients of allogeneic stem cell transplantations

Background

CD4+CD25highFOXP3+ regulatory T (Treg) cells, which include thymus-derived and peripherally induced cells, play a central role in immune regulation, and are therefore crucial to prevent graft-versus-host disease (GVHD). The increasing use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for elderly patients with thymus regression, and our case of allo-HSCT shortly after total thymectomy, raised questions about the activity of thymus-derived Treg cells and peripherally induced Treg cells, which are otherwise indistinguishable.

Results

We found that despite pre-transplant thymectomy or older age, both naïve and effector Treg cells, as well as naïve and effector conventional T cells, proliferated in allo-HSCT recipients. Higher proportions of total Treg cells 1 month post allo-HSCT, and naïve Treg cells 1 year post allo-HSCT, appeared in patients achieving complete chimera without developing significant chronic GVHD, including our thymectomized patient, compared with patients who developed chronic GVHD.

Conclusions

Treg cells that modulate human allogeneic immunity may arise peripherally as well as in the thymus of allo-HSCT recipients.     

Making the most of the old age: Autumn breeding as an extra reproductive investment in older seabirds

The extrinsic and intrinsic factors affecting differing reproductive strategies among populations are central to understanding population and evolutionary ecology. To evaluate whether individual reproductive strategies responded to annual patterns in marine productivity and age‐related processes in a seabird we used a long term (2003–2013), a continuous dataset on nest occupancy and attendance at the colony by little penguins (Eudyptula minor) at Phillip Island (Victoria, Australia). We found that concurrent with a secondary annual peak of marine productivity, a secondary peak in colony attendance and nest occupancy was observed in Autumn (out of the regular breeding season in spring/summer) with individuals showing mating‐like behavior. Individuals attending this autumn peak averaged 2.5 years older than those individuals that exclusively bred during spring/summer. Rather than being a naïve response by younger and inexperienced birds misreading environmental cues, our data indicate that the autumn peak attendance is an earlier attempt to breed by older and more experienced penguins. Therefore, we provide strong support for the fundamental prediction of the life‐history theory of increasing investment in reproduction with age to maximize lifetime fitness as future survival prospects diminish and experience increases.     

Discussions on the quality of antibodies are no reason to ban animal immunization

Debates about the source of antibodies and their use are confusing two different issues. A ban on life immunization would have no repercussions on the quality of antibodies. Subject Categories: S&S: Economics & Business, Methods & Resources, Chemical Biology

There is an ongoing debate on how antibodies are being generated, produced and used (Gray, 2020; Marx, 2020). Or rather, there are two debates, which are not necessarily related to each other. The first one concerns the quality of antibodies used in scientific research and the repercussions for the validity of results (Bradbury & Pluckthun, 2015). The second debate is about the use of animals to generate and produce antibodies. Although these are two different issues, we observe that the debates have become entangled with arguments for one topic incorrectly being used to motivate the other and vice versa. This is not helpful, and we should disentangle the knot.Polyclonal antibodies are being criticized because they suffer from cross‐reactivity, high background and batch‐to‐batch variation (Bradbury & Pluckthun, 2015). Monoclonal antibodies produced from hybridomas are criticized because they often lack specificity owing to genetic heterogeneity introduced during hybridoma generation that impairs the quality of the monoclonals (Bradbury et al, 2018). These are valid criticisms and producing antibodies in a recombinant manner will, indeed, help to improve quality and specificity. But a mediocre antibody will remain a mediocre antibody, no matter how it is produced. Recombinant methods will just produce a mediocre antibody more consistently.Getting a good antibody is not easy and much depends on the nature and complexity of the antigen. And low‐quality antibodies are often the result of poor screening, poor quality control, incomplete characterization and the lack of international standards. Nevertheless, the technologies to ensure good selection and to guarantee consistent quality are much more advanced than a decade ago, and scientists and antibody producers should implement these to deliver high‐quality antibodies. Whether antibodies are generated by animal immunization or from naïve or synthetic antibody libraries is less relevant; they can all be produced recombinantly, and screening and characterization are needed in all cases to determine quality, and if the antibody is fit for purpose.But criticisms on the quality of many antibodies and pleas for switching to recombinant production of antibodies cannot be mixed up with a call to ban animal immunization. The EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) recently published a recommendation to stop using animals for generating and producing antibodies for scientific, diagnostic and even therapeutic applications (EURL ECVAM, 2020). This recommendation is mainly supported by scientists who seem to be biased towards synthetic antibody technology for various reasons. Their main argument is that antibodies derived from naïve or synthetic libraries are a valid (and exclusive) alternative. But are they?One can certainly select antibodies from non‐immune libraries, and, depending on the antigen and the type of application, these antibodies can be fit for purpose. In fact, a few of such antibodies have made it to the market as therapeutics, Adalimumab (Humira®) being a well‐known example. But up to now, the vast majority of antibodies continues to come from animal immunization (Lu et al, 2020). And there is a good reason for that. It is generally possible to generate a few positive hits in a naïve/synthetic library; and the more diverse the library, the more hits one is likely to get. But many decades of experience with immunization of animals—especially when they are outbred—shows that they generate larger amounts of antibodies with superior properties. And the more complex your antigen is, the more the balance swings towards animal immunization if you want to have a guarantee for success.There are different factors at work here. First, the immune system of mammals has evolved over millions of years to efficiently produce excellent antibodies against a very diverse range of antigens. Second, presenting the antigen multiple times in its desired (native) conformation to the animal immune system exploits the natural maturation process to fine‐tune the immune response against particular qualities. Another factor is that in vivo maturation seems to select against negative properties such as self‐recognition and aggregation. It also helps to select for important properties that go beyond mere molecular recognition (Jain et al, 2017). In industrial parlance, antibodies from animal immunization are more “developable” and have favourable biophysical properties (Lonberg, 2005). Indeed, the failure rate for antibodies selected from naïve or synthetic libraries is significantly higher.Of course, the properties of synthetic antibodies selected from non‐immune libraries can be further matured in vitro, for example by light chain shuffling or targeted mutagenesis of the complementarity determining region (CDR). While this method has become more sophisticated over the years, it remains a very complex and iterative process without guarantee that it produces a high‐quality antibody.Antibodies are an ever more important tool in scientific research and a growing area in human and veterinary therapeutics. Major therapeutic breakthroughs in immunology and oncology in the past decades are based on antibodies (Lu et al, 2020). The vast majority of these therapeutic antibodies were derived from animals. An identical picture appears when you look at the antibodies in fast‐track development to combat the current COVID‐19 crisis: again, the vast majority are either derived from patients or from animal immunizations. The same holds true for antibodies that are used in diagnostics and epidemiologic studies for COVID‐19.It is for that reason that we need the tools and methods that guarantee antibodies of the highest quality and provide the best chance for success. The COVID‐19 pandemic is only one illustration of this need. If we block access to these tools, both scientific research and society at large will be negatively impacted. We therefore should not limit ourselves to naïve and synthetic libraries. Animal immunization remains an inevitable method that needs to stay. But we all agree that these immunizations must be performed under best practice to further reduce the harm to animals.