Public and private human T-cell clones respond differentially to HCMV antigen when boosted by CD3 copotentiation.

Human cytomegalovirus (HCMV) induces long-lasting T-cell immune responses that control but do not clear infection. Typical responses involve private T-cell clones, expressing T-cell antigen receptors (TCRs) unique to a person, and public T-cell clones with identical TCRs active in different people. Here, we report the development of a pretherapeutic immunostimulation modality against HCMV for human T cells, CD3 copotentiation, and the clonal analysis of its effects in recall assays at single-cell resolution. CD3 copotentiation of human T cells required identification of an intrinsically inert anti-CD3 Fab fragment that conditionally augmented signaling only when TCR was coengaged with antigen. When applied in recall assays, CD3 copotentiation enhanced the expansion of both public and private T-cell clones responding to autologous HLA-A2(+) antigen-presenting cells and immunodominant NLVPMVATV (NLV) peptide from HCMV pp65 protein. Interestingly, public vs private TCR expression was associated with distinct clonal expansion signatures in response to recall stimulus. This implied that besides possible differences in their generation and selection in an immune response, public and private T cells may respond differently to pharmacoimmunomodulation. Furthermore, a third clonal expansion profile was observed upon CD3 copotentiation of T-cell clones from HLA-A2(-) donors and 1 HLA-A2(+) presumed-uninfected donor, where NLV was of low intrinsic potency. We conclude that human T-cell copotentiation can increase the expansion of different classes of T-cell clones responding to recall antigens of different strengths, and this may be exploitable for therapeutic development against chronic, persistent infections such as HCMV.

Freeze-drying affects the starch digestibility of cooked potato tubers.

Freeze-drying (FD) has utility for phytonutrient screening but its reliability for starch measurements is unclear. The impact of FD was tested on total (TS), digestible (DS) and resistant starch (RS) for four potato varieties (PC Red, GG Red, GG Yellow, and Dolbec Yellow). The treatments included: (a) tubers boiled and then cooled for 1h at room temperature (RT) (control; Treatment 1) and 24h at 4°C; (b) tubers boiled and then cooled for 1h at RT with subsequent FD (Treatment 2); and (c) raw tubers that underwent FD, then were rehydrated, boiled, and cooled for 1h at RT (Treatment 3). TS and DS content did not differ between the control samples cooled for 1h or 24h with Treatment 1 but RS content at 24h was higher, which indicated starch retrogradation. Cultivar variations were observed in the percent increase in RS between 24h vs. 1h with the greatest increase in Dolbec Yellow (114.5±7.6%). Relative to controls, FD treatments modified measured TS content in three of four varieties including overestimation by 94.2±6.5% and 156.0±5.2% for GG Yellow with Treatments 2 and 3, respectively. FD caused overestimation of DS and underestimation of RS in the same three varieties relative to controls including overestimation of DS in GG Yellow by 122.9±4.7% (Treatment 2) and 205.7±13.8% (Treatment 3). PC Red showed the greatest underestimation in RS content compared to controls of 42.5±9.6% and 61.7±5.4% in Treatment 2 and 3, respectively. Modifications to cooking and rehydration procedures following FD of raw tuber samples did not improve reliability of TS, DS, and RS measurements. Microscopy showed that cells remained intact following cooking whereas cell wall integrity was reduced when FD followed cooking and that cooking followed by FD led to destruction of cellular structure. We conclude that FD leads to unreliable starch measurements, which was supported by morphological microscopic evidence. For accuracy of starch profile measurements, the use of freshly cooked potato samples is essential.

Coimmunoprecipitation of reversibly glycosylated polypeptide with sucrose synthase from developing castor oilseeds.

The sucrose synthase (SUS) interactome of developing castor oilseeds (COS; Ricinus communis) was assessed using coimmunoprecipitation (co-IP) with anti-(COS RcSUS1)-IgG followed by proteomic analysis. A 41-kDa polypeptide (p41) that coimmunoprecipitated with RcSUS1 from COS extracts was identified as reversibly glycosylated polypeptide-1 (RcRGP1) by LC-MS/MS and anti-RcRGP1 immunoblotting. Reciprocal Far-western immunodot blotting corroborated the specific interaction between RcSUS1 and RcRGP1. Co-IP using anti-(COS RcSUS1)-IgG and clarified extracts from other developing seeds as well as cluster (proteoid) roots of white lupin and Harsh Hakea consistently recovered 90 kDa SUS polypeptides along with p41/RGP as a SUS interactor. The results suggest that SUS interacts with RGP in diverse sink tissues to channel UDP-glucose derived from imported sucrose into hemicellulose and/or glycoprotein/glycolipid biosynthesis.