The TYK2-P1104A Autoimmune Protective Variant Limits Coordinate Signals Required to Generate Specialized T Cell Subsets

TYK2 is a JAK family member that functions downstream of multiple cytokine receptors. Genome wide association studies have linked a SNP (rs34536443) within TYK2 encoding a Proline to Alanine substitution at amino acid 1104, to protection from multiple autoimmune diseases including systemic lupus erythematosus (SLE) and multiple sclerosis (MS). The protective role of this SNP in autoimmune pathogenesis, however, remains incompletely understood. Here we found that T follicular helper (Tfh) cells, switched memory B cells, and IFNAR signaling were decreased in healthy individuals that expressed the protective variant TYK2A1104 (TYK2P). To study this variant in vivo, we developed a knock-in murine model of this allele. Murine Tyk2P expressing T cells homozygous for the protective allele, but not cells heterozygous for this change, manifest decreased IL-12 receptor signaling, important for Tfh lineage commitment. Further, homozygous Tyk2P T cells exhibited diminished in vitro Th1 skewing. Surprisingly, despite these signaling changes, in vivo formation of Tfh and GC B cells was unaffected in two models of T cell dependent immune responses and in two alternative SLE models. TYK2 is also activated downstream of IL-23 receptor engagement. Here, we found that Tyk2P expressing T cells had reduced IL-23 dependent signaling as well as a diminished ability to skew toward Th17 in vitro. Consistent with these findings, homozygous, but not heterozygous, Tyk2P mice were fully protected in a murine model of MS. Homozygous Tyk2P mice had fewer infiltrating CD4+ T cells within the CNS. Most strikingly, homozygous mice had a decreased proportion of IL-17+/IFNγ+, double positive, pathogenic CD4+ T cells in both the draining lymph nodes (LN) and CNS. Thus, in an autoimmune model, such as EAE, impacted by both altered Th1 and Th17 signaling, the Tyk2P allele can effectively shield animals from disease. Taken together, our findings suggest that TYK2P diminishes IL-12, IL-23, and IFN I signaling and that its protective effect is most likely manifest in the setting of autoimmune triggers that concurrently dysregulate at least two of these important signaling cascades

Reviewing the use of resilience concepts in forest sciences

Purpose of the review Resilience is a key concept to deal with an uncertain future in forestry. In recent years, it has received increasing attention from both research and practice. However, a common understanding of what resilience means in a forestry context, and how to operationalise it is lacking. Here, we conducted a systematic review of the recent forest science literature on resilience in the forestry context, synthesising how resilience is defined and assessed. Recent findings Based on a detailed review of 255 studies, we analysed how the concepts of engineering resilience, ecological resilience, and social-ecological resilience are used in forest sciences. A clear majority of the studies applied the concept of engineering resilience, quantifying resilience as the recovery time after a disturbance. The two most used indicators for engineering resilience were basal area increment and vegetation cover, whereas ecological resilience studies frequently focus on vegetation cover and tree density. In contrast, important social-ecological resilience indicators used in the literature are socio-economic diversity and stock of natural resources. In the context of global change, we expected an increase in studies adopting the more holistic social-ecological resilience concept, but this was not the observed trend. Summary Our analysis points to the nestedness of these three resilience concepts, suggesting that they are complementary rather than contradictory. It also means that the variety of resilience approaches does not need to be an obstacle for operationalisation of the concept. We provide guidance for choosing the most suitable resilience concept and indicators based on the management, disturbance and application context

Building isotype-optimized pathogen-specific mAbs from memory B cells

Thesis (Ph.D.)--University of Washington, 2023Emerging viruses and antibiotic-resistant bacteria are major threats to human health. Despite well-established benefits of the passive transfer of immunity in animal models, engineered monoclonal antibodies (mAbs) have struggled to achieve consistently positive results for the treatment of infectious disease in humans. We hypothesized that a strategy of building mAbs based on human immune memory B cells (MBCs) could be advantageous. This thesis describes efforts to develop mAbs with plausible therapeutic potential against two microbes that drive severe morbidity in vulnerable patients: Pseudomonas aeruginosa and SARS-CoV-2. Diverging from historical, animal-based methods for mAb discovery, we used the B cell receptor sequences of human, antigen-specific B cells, with special focus on MBCs, as templates for novel mAbs. Focusing first on P. aeruginosa, we generated anti-bacterial mAbs using source B cells obtained from patients seen at a cystic fibrosis (CF) clinic. CF is a genetic condition associated with unusually frequent P. aeruginosa infections, but not defects in adaptive immunity. In an in vivo pneumonia model with a highly virulent strain, 2 of 2 human-derived mAbs exhibited prominent protective activity. Notably, our new mAbs were noninferior to an extensively engineered mAb that comprises half of the bi-specific clinical candidate, gremubamab. A second panel of MBC-derived mAbs that showed promise in vitro remain to be tested in future work, highlighting the efficiency of our mAb discovery strategy. Further, we believe our study contributes to understanding of immunity in CF, being the first to confirm the presence of P. aeruginosa-specific MBCs in CF patients. In contrast to P. aeruginosa, mAbs targeting SARS-CoV-2 were developed from human B cells contemporaneously by multiple groups including our team. However, circulating escape variants have significantly limited clinical use. Importantly, consistent with nearly all mAbs in clinical use, these vulnerable mAbs were of the monomeric isotype, IgG. Building on our prior studies of MBCs and evidence that an alternative isotype, IgM, was an important component in virus-neutralizing human serum, we cloned new mAbs from SARS-CoV-2-specific IgM MBCs. We also found that diverse MBC-derived mAbs gained greater neutralizing potency when expressed as the naturally pentameric IgM isotype. Importantly, we showed that IgM mAbs retained neutralizing activity against viral variants that evaded otherwise identical mAbs made in the IgG isotype. These results suggest a biological role for IgM MBCs in protection against rapidly mutating pathogens, and illuminate the potential for IgM mAbs in the search for new treatments for infectious diseases

Obesity Triggers Enhanced MDSC Accumulation in Murine Renal Tumors via Elevated Local Production of CCL2

<div><p>Obesity is one of the leading risk factors for developing renal cell carcinoma, an immunogenic tumor that is treated clinically with immunostimulatory therapies. Currently, however, the mechanisms linking obesity with renal cancer incidence are unclear. Using a model of diet-induced obesity, we found that obese BALB/c mice with orthotopic renal tumors had increased total frequencies of myeloid-derived suppressor cells (MDSC) in renal tumors and spleens by d14 post-tumor challenge, relative to lean counterparts. Renal tumors from obese mice had elevated concentrations of the known myeloid cell chemoattractant CCL2, which was produced locally by increased percentages of dendritic cells, macrophages, B cells, and CD45^- cells in tumors. MDSC expression of the CCL2 receptor, CCR2, was unaltered by obesity but greater percentages of CCR2⁺ MDSCs were present in renal tumors from obese mice. Of note, the intracellular arginase levels and per-cell suppressive capacities of tumor-infiltrating and splenic MDSCs were unchanged in obese mice relative to lean controls. Thus, our findings suggest that obesity promotes renal tumor progression via development of a robust immunosuppressive environment that is characterized by heightened local and systemic MDSC prevalence. Targeted intervention of the CCL2/CCR2 pathway may facilitate immune-mediated renal tumor clearance in the obese.</p></div

β1-adrenoceptor Arg389Gly polymorphism confers differential β-arrestin-binding tropism in cardiac myocytes

AIM: The β METHODS: We tested the β RESULTS: βarr1 binds both variants upon isoproterenol, carvedilol or metoprolol treatment in neonatal rat ventricular myocytes. Conversely, the potentially beneficial in the heart βarr2 only interacts with the Arg389 receptor in response to isoproterenol or carvedilol. CONCLUSION: Arg389 confers unique βarr2-interacting tropism to the

Obese mice have elevated concentrations of the MDSC chemoattractant CCL2 in renal tumors.

<p>(A) DIO and NW mice were challenged as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118784#pone.0118784.g001" target="_blank">Fig. 1</a>. Tumor-bearing kidneys (TK) or tumor-free contralateral kidneys (CK) were harvested at the times indicated. Homogenates were tested via Multiplex array for CCL2. n = 4–8 mice per group, combined from three experiments. Bars indicate mean +/- sd. * = p < 0.05. (B) Histograms showing intracellular CCL2 for the indicated cell populations. (C) Intracellular CCL2 from pooled DIO versus NW mice for the indicated cell populations, with n = 3–5 mice per group. Bars indicate mean +/- sd. * = p < 0.05.</p

Obesity does not alter the suppressive capacity of MDSC.

<p>Tumor-bearing kidneys and spleens from the same mice were harvested between days 21–23. (A) Monocytic (Ly6C⁺) and granulocytic MDSC (Ly6G⁺) were sort-purified from each organ, then placed into culture with naive DUC18 T cells and stimulatory splenic DC from tumor-free NW mice. MDSC and stimulatory DC were pulsed with the DUC18 cognate peptide tERK. n = 6–8 mice per group, combined from three experiments. Bars indicate mean +/- sd. * = p < 0.05. TI = tumor-infiltrating: sp = splenic. (B) Bulk tumor and spleen homogenates were surface stained as described in Methods to distinguish monocytic versus granulocytic MDSC subpopulations, then stained intracellularly for arginase-1 expression. Dots indicate results from individual mice. No statistical differences were present in the percentages of arginase⁺ MDSC subpopulations from like organs in obese versus NW mice.</p

Equivalent surface expression of CCR2 on MDSCs from DIO and NW mice.

<p>(A) DIO and NW mice were challenged as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118784#pone.0118784.g001" target="_blank">Fig. 1</a>. Tumor-bearing kidneys (TK) or tumor-free contralateral kidneys (CK) were harvested at the times indicated. Histograms show representative surface expression of CCR2 on Ly6C⁺ or Ly6G⁺ MDSC from DIO or NW mice. Open histograms = isotype control; shaded histograms = CCR2 staining. (B) Pooled data showing the % of MDSCs that express CCR2 in tumor-bearing kidneys (TK) and contralateral kidneys (CK). Gating on Ly6C⁺ or Ly6G⁺ MDSC shows strong and equivalent expression of CCR2 on MDSC subpopulations from DIO and NW mice. (C) Increased overall frequencies of CCR2⁺ MDSC from both Ly6C⁺ and Ly6G⁺ subsets when calculated as a percentage of total live cells within tumor-bearing kidneys. For B and C, n = 4–6 mice per group from two experiments. Bars indicate mean +/- sd. * = p < 0.05.</p

Evaluating the Effect of Heteroatoms on the Photophysical Properties of Donor–Acceptor Conjugated Polymers Based on 2,6-Di(thiophen-2-yl)benzo[1,2-b:4,5-b′]difuran: Two-Photon Cross-Section and Ultrafast Time-Resolved Spectroscopy

We investigate the influence of the heteroatom on the electronic and photophysical properties of four conjugated polymers based on 3,7-didodecyl-2,6-di­(thiophen-2-yl)­benzo­[1,2-<i>b</i>:4,5-<i>b</i>′]­difuran (BDF) as the donor and 3,6-di­(thiophen-2-yl)-1,4-diketopyrrolo­[3,4-<i>c</i>]­pyrrole (TDPP) or 3,6-di­(2-furanyl)-1,4-diketopyrrolo­[3,4-<i>c</i>]­pyrrole (FDPP) as the acceptor. The polymers with a furan as the linker showed higher extinction coefficients than their thiophene counterparts. Ultrafast fluorescence decay showed that the exciton relaxation process is affected by the type of linker in these conjugated polymers. Theoretical calculations showed that the polymers with a furan as the linker are more planar than their thiophene analogues. Also, theoretical calculation showed that the polymers with a thiophene as the linker have larger transition dipole moments. The two-photon absorption cross sections (TPACS) of the polymers with a furan as the linker were larger than their thiophene polymer analogues. These results suggest that the polymers with a furan as the linker have higher charge transfer character than their thiophene polymers analogues. The photovoltaics performance for these polymers are correlated with their optical properties. These results suggest that furan-derivatives are good candidates for synthetic exploration for long-range energy transport materials in photovoltaic applications

Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

Gene editing by homology-directed recombination (HDR) can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR) T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC) locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA) CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics