Background The phosphorus (P) stocks of arable subsoils not only influence crop production but also fertilizer P sequestration. However, the extent of this influence is largely unknown. This study aimed to (i) determine the extent of P sequestration with soil depth, (ii) analyze P speciation after long-term P fertilization, and (iii) compare soil P tests in predicting crop yields. We analyzed four long-term fertilizer trials in Germany to a depth of 90 cm. Treatments received either mineral or organic P, or a combination of both, for 16 to 113 years. We determined inorganic and organic P pools using sequential extraction, and P speciation using 31P nuclear magnetic resonance (NMR) and X-ray absorption near edge structure (XANES) spectroscopy. In addition, we applied three P soil tests, double-lactate (DL), calcium acetate lactate (CAL), and diffusive gradients in thin films (DGT). Results The results suggested that plants are capable of mobilizing P from deeper soil layers when there is a negative P budget of the topsoil. However, fertilization mostly only showed insignificant effects on P pools, which were most pronounced in the topsoil, with a 1.6- to 4.4-fold increase in labile inorganic P (Pi; resin-P, NaHCO3–Pi) after mineral fertilization and a 0- to 1.9-fold increase of organic P (Po; NaHCO3–Po, NaOH–Po) after organic P fertilization. The differences in Po and Pi speciation were mainly controlled by site-specific factors, e.g., soil properties or soil management practice rather than by fertilization. When modeling crop yield response using the Mitscherlich equation, we obtained the highest R2 (R2 = 0.61, P < 0.001) among the soil P tests when using topsoil PDGT. However, the fit became less pronounced when incorporating the subsoil. Conclusion We conclude that if the soil has a good P supply, the majority of P taken up by plants originates from the topsoil and that the DGT method is a mechanistic surrogate of P plant uptake. Thus, DGT is a basis for optimization of P fertilizer recommendation to add as much P fertilizer as required to sustain crop yields but as low as necessary to prevent harmful P leaching of excess fertilizer P.

CD3-engaging bispecific antibodies (BsAbs) enable the formation of an immune synapse between T cells and tumor cells, resulting in robust target cell killing not dependent on a preexisting tumor specific T cell receptor. While recent studies have shed light on tumor cell-specific factors that modulate BsAb sensitivity, the T cell-intrinsic determinants of BsAb efficacy and response durability are poorly understood. To better clarify the genes that shape BsAb-induced T cell responses, we conducted targeted analyses and a large-scale unbiased in vitro CRISPR/Cas9-based screen to identify negative regulators of BsAb-induced T cell proliferation. These analyses revealed that CD8+ T cells are dependent on CD4+ T cell-derived signaling factors in order to achieve sustained killing in vitro. Moreover, the mammalian target of rapamycin (mTOR) pathway and several other candidate genes were identified as intrinsic regulators of BsAb-induced T cell proliferation and/or activation, highlighting promising approaches to enhancing the utility of these potent therapeutics.

Studies conducted in mice and humans have shown that IFN-γ+ CD8 T cell are the key effectors against liver-stage (LS) malaria; however, owing to complexities associated with the development of Plasmodia parasites, specificities of CD8 T cell effectors have been difficult to establish. Direct detection of Ag-specific CD8 T cells in high-throughput manner became possible with the development of caged MHC-tetramer technology, based on conditional photo-cleavable MHC class I ligands that can be displaced during UV induced exchange with a peptide of interest. On the basis of previously identified P. falciparum (Pf) genes expressed exclusively during LS infection, we identified P. berghei (Pb) orthologues of Pf genes and validated their expression by qRT-PCR in Pb infected C57Bl/6 mice. Several Pb LS antigens, administered as DNA vaccines, significantly reduced LS parasite burden in mice infected with Pb sporozoites. Using sequences of the 28 novel Pb LS genes we screened ~400 peptides by the caged MHC class I tetramer approach. Our preliminary results demonstrate that 2 H-2Kb- and 1 H-2Db-restricted Pb peptides that correspond to protective Pb LS Ags form MHC class I tetramers that specifically bound to CD8 T cells from mice immunized with radiation-attenuated Pb sporozoites. Identification of these epitopes is crucial towards resolving the role of antigen-specific CD8 T cell responses and establishing these responses as correlates of protection to Plasmodia infection.

A Plasmodium falciparum (Pf) genetically attenuated parasite (Pf GAP) vaccine, engineered by deletion of 2 pre-erythrocytic genes, was administered to 6 subjects by bites from Anopheles mosquitoes. Previous studies of immune responses elicited by recombinant malaria vaccines show that protection is linked to Ag-specific T cells producing IFN-γ, TNF, and/or IL-2. In this study we asked if Pf GAP vaccine induced Ag-specific T cell responses characterized by inflammatory cytokines. We analyzed pre- and post vaccination PBMC for Ag-specific T cell responses to well characterized pre-erythrocytic and erythrocytic P. falciparum protein and peptide Ags. Additionally, we examined responses to several novel Pf liver-stage Ag (Pf LSA) because Pf LSA-dependent immunity might contribute to GAP-induced protection. IFN-γ and TNF responses to multiple antigens were significantly enhanced post vaccination; IFN-γ responses were primarily attributed to CD8+ T cells while TNF was secreted primarily by CD4+ T cells. Notably, a subject with a breakthrough peripheral parasitemia did show positive IFN-γ responses to erythrocytic-stage Ags, but no responses were recalled with the novel Pf LSA. In summary, two exposures to a Pf GAP vaccine induced persisting T cell responses specific for Pf LSA in humans. Future trials that assess the efficacy of Pf GAP vaccines should examine the role of LSA-specific responses in protection.