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Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T B NK SCID pigs through site directed CRISPR/Cas9 mutagenesis of within a naturally occurring genetic background. We confirmed pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed injections of cultured human CD34 selected cord blood cells into the fetal SCID pigs. At birth, human CD45 CD3ε cells were detected in cord and peripheral blood of injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.

Severe combined immunodeficiency (SCID) is described as the lack of functional T and B cells. In some cases, mutant genes encoding proteins involved in the process of VDJ recombination retain partial activity and are classified as hypomorphs. Hypomorphic activity in the products from these genes can function in the development of T and B cells and is referred to as a leaky phenotype in patients and animals diagnosed with SCID. We previously described two natural, single nucleotide variants in ( ) in a line of Yorkshire pigs that resulted in SCID. One allele contains a splice site mutation within intron 8 of the gene ( ), while the other mutation is within exon 10 that results in a premature stop codon ( ). While initially characterized as SCID and lacking normal levels of circulating lymphoid cells, low levels of CD3ε cells can be detected in most SCID animals. Upon further assessment, we found that , and SCID pigs had abnormally small populations of CD3ε cells, but not CD79α cells, in circulation and lymph nodes. Newborn pigs (0 days of age) had CD3ε cells within lymph nodes prior to any environmental exposure. CD3ε cells in SCID pigs appeared to have a skewed CD4α CD8α CD8β T helper memory phenotype. Additionally, in some pigs, rearranged VDJ joints were detected in lymph node cells as probed by PCR amplification of TCRδ V5 and J1 genomic loci, as well as TCRβ V20 and J1.1, providing molecular evidence of residual Artemis activity. We additionally confirmed that TCRα and TCRδ constant region transcripts were expressed in the thymic and lymph node tissues of SCID pigs; although the expression pattern was abnormal compared to carrier animals. The leaky phenotype is important to characterize, as SCID pigs are an important tool for biomedical research and this additional phenotype may need to be considered. The pig model also provides a relevant model for hypomorphic human SCID patients.

A wide diversity of free-living organisms show increases in mortality rates and/or decreases in reproductive success with advancing age. However, the physiological mechanisms underlying these demographic patterns of senescence are poorly understood. Immunosenescence, the age-related deterioration of immune function, is well documented in humans and laboratory models, and often leads to increased morbidity and mortality due to disease. However, we know very little about immunosenescence in free-living organisms. Here, we studied immunosenescence in a free-living population of tree swallows, Tachycineta bicolor, assessing three components of the immune system and using both in vivo and in vitro immunological tests. Immune function in tree swallow females showed a complex pattern with age; acquired T-cell mediated immunity declined with age, but neither acquired nor innate humoral immunity did. In vitro lymphocyte proliferation stimulated by T-cell mitogens decreased with age, suggesting that reduced T-cell function might be one mechanism underlying the immunosenescence pattern of in vivo cell-mediated response recently described for this same population. Our results provide the most thorough description of immunosenescence patterns and mechanisms in a free-living vertebrate population to date. Future research should focus on the ecological implications of immunosenescence and the potential causes of variation in patterns among species.

The immunocompetence “pace-of-life” hypothesis proposes that fast-living organisms should invest more in innate immune defenses and less in adaptive defenses compared to slow-living ones. We found some support for this hypothesis in two life-history ecotypes of the snakeThamnophis elegans; fast-living individuals show higher levels of innate immunity compared to slow-living ones. Here, we optimized a lymphocyte proliferation assay to assess the complementary prediction that slow-living snakes should in turn show stronger adaptive defenses. We also assessed the “environmental” hypothesis that predicts that slow-living snakes should show lower levels of immune defenses (both innate and adaptive) given the harsher environment they live in. Proliferation of B- and T-lymphocytes of free-living individuals was on average higher in fast-living than slow-living snakes, opposing the pace-of-life hypothesis and supporting the environmental hypothesis. Bactericidal capacity of plasma, an index of innate immunity, did not differ between fast-living and slow-living snakes in this study, contrasting the previously documented pattern and highlighting the importance of annual environmental conditions as determinants of immune profiles of free-living animals. Our results do not negate a link between life history and immunity, as indicated by ecotype-specific relationships between lymphocyte proliferation and body condition, but suggest more subtle nuances than those currently proposed.

Background: Transgenic maize, which produces the nontoxic B subunit of the Escherichia coli heat-labile toxin (LT-B) in seed, has proven to be an effective oral immunogen in mice. Currently, there is considerable concern over accidental consumption of transgenic maize expressing LT-B by humans and domestic animals. We have yet to define nonimmunogenic levels of transgenic LT-B when ingested. Objectives: Our goal in this study was to determine the highest dose of LT-B orally administered in mice that does not result in a measurable immune response. We defined an immune response as specific serum or mucosal IgG or IgA significantly greater than background after three feedings (0.0002-20 µg) or a priming response induced by the intermittent feeding. Methods: We fed transgenic maize pellets on days 0, 7, 21, and 49 and collected serum and fecal samples weekly. Serum was analyzed for LT-B-specific IgG and IgA, and feces was analyzed for LT-B-specific IgA. Results: We observed a dose-dependent anti-LT-B antibody response with high specific antibody concentrations in groups fed high doses (0.2, 2, 20 µg) of LT-B maize. Mice fed 0.02 µg LT-B demonstrated immune priming in 62.5% of the animals. Mice that were fed ≤ 0.002 µg LT-B showed no increase in specific antibody nor did they demonstrate immune priming, indicating that 0.002 µg LT-B was the highest nonimmunogenic dose tested. Conclusion: Our results demonstrate that LT-B derived from transgenic maize is immunogenic at nanogram levels when orally administered to mice.

Influenza vaccine efficacy is reduced among adults over age 65 and a significant number of vaccinated elderly may remain susceptible to influenza virus infection. The effect of moderate exercise training on the immune response to influenza immunization was evaluated in this study. Twenty-seven adults ≥age 64 were assigned to an exercise group ( n=14) or a control group ( n=13). The subjects exercised at 65–75% heart rate reserve (HRR), 25–30 min, 3 days per week, for 10 months. Controls did not change activity. Subjects were immunized with trivalent influenza vaccine before and after the exercise intervention. After the exercise intervention, exercisers exhibited a greater mean fold increase (MFI) in antibody titer to influenza A/New Caledonia/20/99 (H1N1) and A/Panama/2007/99 (H3N2) than controls, and a greater Granzyme B activity to A/Panama/2007/99 than controls. These findings suggest that exercise may enhance the mean fold increase in antibody titer in response to influenza immunization if the influenza antigen was contained in the previous year’s vaccine.

Understanding the relationships among immune components in free-living animals is a challenge in ecoimmunology, and it is important not only for selecting the immune assays to be used but also for more knowledgeable interpretation of results. In this study, we investigated the relationships among six immune defense indexes commonly used by ecoimmunologists and measured simultaneously in individual free-living tree swallows. Three main axes of variation in immune function were identified using a principal components analysis, representing variation in T-cell, B-cell, and innate immunity. Measures within each axis tended to be positively correlated among individuals, while measures in different axes were uncorrelated. A trade-off between T-cell function and B-cell function became apparent only when variation among individuals in body condition, age, and general quality was taken into account. Interestingly, the level of natural antibodies, a component of innate immunity, showed the strongest association with components of acquired B-cell function, possibly reflecting a common underlying genetic mechanism, as has been documented in poultry. Our results indicate that despite the complexity of the immune system, important insights can be gained by using the currently available assays but in a more comprehensive approach than has generally been used in the field of ecoimmunology.

We report the first experimental study of the effect of long-term (over 2 years) exposure to a stressor on cellular immune response. Forty-three male cynomolgus monkeys were randomly assigned to stable or unstable social conditions for 26 months. The proportion of time spent in affiliative behaviors was assessed by observations made twice weekly. T-cell immune response (mitogen-stimulated cell proliferation) was assessed weekly for 3 weeks immediately following the 26-month manipulation. The possibility that affiliative behavior represents an attempt to cope with social stress was supported by greater affiliation among animals in the unstable condition than in the stable condition. Animals in the unstable condition also demonstrated relatively suppressed immune response. More affiliative animals showed enhanced immune response, with the beneficial effects of affiliation occurring primarily among unstable animals. The data are interpreted as consistent with the stress-buffering hypothesis; that is, affiliation protects animals from the potentially pathogenic influences of chronic social stress.

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (Artemis)-/- genetic background. We confirmed Art-/- IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we and successfully performed a bone marrow transplant on one Art-/- IL2RG-/Y male SCID pig with a bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal Art-/- IL2RG-/Y SCID pigs. At birth, human CD45+ CD3ε+ cells were detected in peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.