Generation of Non-Alloreactive Antiviral Central Memory CD8 Human Veto T Cells for Cell Therapy.

Previous studies in mice demonstrated that CD8 T cells exhibit marked veto activity enhancing engraftment in several models for T cell-depleted bone marrow (TDBM) allografting. To reduce the risk of graft-versus-host disease (GVHD) associated with allogeneic CD8 veto T cells, these studies made use of naive CD8 T cells stimulated against third-party stimulators under cytokine deprivation and subsequent expansion in the presence of IL-15. More recently, it was shown that mouse CD8 veto T cells can be generated by stimulating CD8 memory T cells from ovalbumin immunized mice under cytokine deprivation, using ovalbumin as a third-party antigen. These cells also exhibited substantial enhancement of BM allografting without GVHD. In this study, we tested the hypothesis that stimulation and expansion of human CD8 memory T cells under IL-15 and IL-7 deprivation during the early phase of activation against recall viral antigens can lead to substantial loss of alloreactive T clones while retaining marked veto activity. Memory CD8 T cells were enriched by removal of CD45RA+, CD4+, and CD56+ cells from peripheral blood of cytomegalovirus (CMV)- and Epstein-Barr virus (EBV)-positive donors. In parallel, CD14+ monocytes were isolated; differentiated into mature dendritic cells (mDCs); pulsed with a library of CMV, EBV, adenovirus, and BK virus peptides; and irradiated. The CD8 T cell-enriched fraction was then cultured with the pulsed mDCs in the presence of IL-21 for 3 days, after which IL-15 and IL-7 were added. After 12 days of culture, the cells were tested by limiting dilution analysis for the frequency of alloreactive T cell clones and their veto activity. In preclinical runs using GMP reagents, we established that within 12 days of culture, a large number of highly homogenous CD8 T cells, predominantly expressing a central memory phenotype, could be harvested. These cells exhibited marked veto activity in vitro and >3-log depletion of alloreactivity. Based on these preclinical data, a phase 1-2 clinical trial was initiated to test the safety and efficacy of these antiviral CD8 central memory veto cells in the context of nonmyeloablative (NMA) T cell-depleted haploidentical hematopoietic stem cell transplantation (HSCT). In 2 validation runs and 11 clinical runs using GMP reagents, >1 × 1010 cells were generated from a single leukapheresis in 12 out of 13 experiments. At the end of 12 days of culture, there were 97 ± 2.5% CD3+CD8+ T cells, of which 84 ± 9.0% (range, 71.5% to 95.1%) exhibited the CD45RO+CD62L+ CM phenotype. Antiviral activity tested by intracellular expression of INF-γ and TNF-α and showed an average of 38.8 ± 19.6% positive cells on 6 hours of stimulation against the viral peptide mixture. Our results demonstrate a novel approach for depleting alloreactive T cell clones from preparations of antiviral CD8 veto cells. Based on these results, a phase 1-2 clinical trial is currently in progress to test the safety and efficacy of these veto cells in the context of NMA haploidentical T cell-depleted HSCT. Studies testing the hypothesis that these non-alloreactive CD8 T cells could potentially offer a platform for off-the-shelf veto chimeric antigen receptor T cell therapy in allogenic recipients, are warranted.

Effectiveness and safety of standard chemotherapy in older patients with ovarian cancer: a retrospective analysis by age group and treatment regimen.

Objective: To evaluate the effectiveness and safety of standard chemotherapy administered to patients >70 years with advanced ovarian cancer (OC). Methods: Medical records of 956 advanced-stage patients with OC treated between 2002-2020 with standard surgery and paclitaxel-carboplatin chemotherapy in a three-weekly (PC-3W) or weekly (PC-1W) regimen were reviewed. Treatment response and tolerability were compared between patients ≤70 years (N=723) and >70 years (N=233) with stratification to septuagenarians (>70-80 years) and octogenarians (>80 years). Results: Median overall survival (mOS) in patients >70 was 41.26 months (95% confidence interval [Cl], 37.22-45.14) and median progression-free survival (mPFS) was 11.04 months (95% Cl, 8.97-15.74). No statistically significant differences in mPFS and mOS were observed between septuagenarians and octogenarians. Patients >70 treated with PC-1W versus PC-3W had significantly longer mOS (57.17 versus 30.00 months) and mPFS (19.09 versus 8.15 months). Toxicity rates were mostly similar between younger and older patients. Among patients >70 treated with PC-1W, the rate of neutropenia (75.7% versus 51.8%, p=0.0005), thrombocytopenia (41.0% versus 22.2%, p=0.0042) and anemia (78.1% versus 51.9%, p<0.0001) were significantly higher and the rate of grade 2 alopecia was statistically significantly lower compared with those >70 treated with PC-3W. Significantly more patients treated with PC-1W completed ≥6 chemotherapy cycles, suggesting better tolerability of this regimen. Conclusions: Older patients with OC may benefit from improved OS with reasonable toxicity if treated with standard chemotherapy. Older patients treated with PC-1W are more likely to complete the full chemotherapy course and survive longer compared with those treated with conventional PC-3W.

Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem.

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community-either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.

Who's more generous than me? Children's self-evaluation of their prosociality in normative social comparisons.

We examined the development of children's self-evaluation of their prosociality in normative social comparisons (with an average peer). Results suggest that when comparing themselves with an average other in the abstract (i.e., without reference to actual behavior), elementary school children (aged 6-12 years) demonstrated the better than average (BTA) effect of perceiving themselves as more prosocial than their average peer (Study 1). However, when they evaluated other children's prosociality (sharing), after experiencing an actual opportunity to share their endowment with others (Studies 2 and 3), the younger children (at first-grade level) exhibited the worse than average (WTA) effect in that they assumed that their peers would act more generously than themselves. Task difficulty predicted relative self-evaluations across all examined ages, such that greater difficulty was related to a lower BTA effect (or a greater WTA effect). However, whereas the older children used abstract difficulty perceptions to evaluate themselves relative to others, the younger children's evaluations were affected only by the difficulty that they themselves experienced. In all age groups, the BTA effect was driven mostly by participants who were above the mean in the extent of their sharing, whereas the WTA effect was driven by those who shared below the mean of their age group.

Exploring the Prokaryotic Community Associated With the Rumen Ciliate Protozoa Population.

Ciliate protozoa are an integral part of the rumen microbiome and were found to exert a large effect on the rumen ecosystem itself as well as their host animal physiology. Part of these effects have been attributed to their ability to harbor a diverse ecto- and endo-symbiotic community of prokaryotic cells. Studies on the relationship between the protozoa population and their associated prokaryotic community in the rumen mainly focused on the methanogens, revealing that protozoa play a major role in enhancing methanogenesis potential. In contrast, little is known about the composition and function of the bacteria associated with rumen protozoa and the extent of this association. In this study, we characterize the prokaryotic communities associated with different protozoa populations and compare their structure to the free-living prokaryotic population residing in the cow rumen. We show that the overall protozoa associated prokaryotic community structure differs significantly compared to the free-living community in terms of richness and composition. The methanogens proportion was significantly higher in all protozoa populations compared to the free-living fraction, while the Lachnospiraceae was the most prevalent bacterial family in the protozoa associated bacterial communities. Several taxa not detected or detected in extremely low abundance in the free-living community were enriched in the protozoa associated bacterial community. These include members of the Endomicrobia class, previously identified as protozoa symbionts in the termite gut. Our results show that rumen protozoa harbor prokaryotic communities that are compositionally different from their surroundings, which may be the result of specific tropism between the prokaryotic community and protozoa.