A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer

Medullary thymic epithelial cells (mTECs) which produce and present self-antigens are essential for the establishment of central tolerance. Since mTEC numbers are limited, their function is complemented by thymic dendritic cells (DCs), which transfer mTEC-produced self-antigens via cooperative antigen transfer (CAT). While CAT is required for effective T cell selection, many aspects remain enigmatic. Given the recently described heterogeneity of mTECs and DCs, it is unclear whether the antigen acquisition from a particular TEC subset is mediated by preferential pairing with specific subset of DCs. Using several relevant Cre-based mouse models controlling the expression of fluorescent proteins, we found that in regards to CAT, each subset of thymic DCs preferentially targets distinct mTEC subset(s) and importantly, XCR1+ activated DCs represented the most potent subset in CAT. Interestingly, one thymic DC can acquire antigen repetitively and of these, monocyte-derived DCs (moDC) were determined to be the most efficient in repetitive CAT. moDCs also represented the most potent DC subset in the acquisition of antigen from other DCs. These findings suggest a preferential pairing model for the distribution of mTEC-derived antigens among distinct populations of thymic DCs.

Unraveling the Hexaploid Sweetpotato Inheritance Using Ultra-Dense Multilocus Mapping

The hexaploid sweetpotato (Ipomoea batatas (L.) Lam., 2n = 6x = 90) is an important staple food crop worldwide and plays a vital role in alleviating famine in developing countries. Due to its high ploidy level, genetic studies in sweetpotato lag behind major diploid crops significantly. We built an ultra-dense multilocus integrated genetic map and characterized the inheritance system in a sweetpotato full-sib family using our newly developed software, MAPpoly. The resulting genetic map revealed 96.5% collinearity between I. batatas and its diploid relative I. trifida. We computed the genotypic probabilities across the whole genome for all individuals in the mapping population and inferred their complete hexaploid haplotypes. We provide evidence that most of the meiotic configurations (73.3%) were resolved in bivalents, although a small portion of multivalent signatures (15.7%), among other inconclusive configurations (11.0%), were also observed. Except for low levels of preferential pairing in linkage group 2, we observed a hexasomic inheritance mechanism in all linkage groups. We propose that the hexasomic-bivalent inheritance promotes stability to the allelic transmission in sweetpotato.

Unraveling the hexaploid sweetpotato inheritance using ultra-dense multilocus mapping

The hexaploid sweetpotato (Ipomoea batatas (L.) Lam., 2n = 6x = 90) is an important staple food crop worldwide and has a vital role in alleviating famine in developing countries. Due to its high ploidy level, genetic studies in sweetpotato lag behind major diploid crops significantly. We built an ultra-dense multilocus integrated genetic map and characterized the inheritance system in a sweetpotato full-sib family using our newly implemented software, MAPpoly. The resulting genetic map revealed 96.5% collinearity between I. batatas and its diploid relative I. trifida. We computed the genotypic probabilities across the whole genome for all individuals in the mapping population and inferred their complete hexaploid haplotypes. We provide evidence that most of the meiotic configurations (73.3%) were resolved in bivalents, although a small portion of multivalent signatures (15.7%), among other inconclusive configurations (11.0%) were also observed. Except for low levels of preferential pairing in linkage group 2, we observed a hexasomic inheritance mechanism in all linkage groups. We propose that the hexasomic-bivalent inheritance promotes stability to the allelic transmission in sweetpotato.

A mechanistic model of linkage analysis in allohexaploids

Despite their pivotal role in agriculture and biological research, polyploids, a group of organisms with more than two sets of chromosomes, are very difficult to study. Increasing studies have used high-density genetic linkage maps to investigate the genome structure and function of polyploids and to identify genes underlying polyploid traits. However, although models for linkage analysis have been well established for diploids, with some essential modifications for tetraploids, no models have been available thus far for polyploids at higher ploidy levels. The linkage analysis of polyploids typically requires knowledge about their meiotic mechanisms, depending on the origin of polyplody. Here we describe a computational modeling framework for linkage analysis in allohexaploids by integrating their preferential chromosomal-pairing meiotic feature into a mixture model setting. The framework, implemented with the EM algorithm, allows the simultaneous estimates of preferential pairing factors and the recombination fraction. We investigated statistical properties of the framework through extensive computer simulation and validated its usefulness and utility by analyzing a real data from a full-sib family of allohexaploid persimmon. Our attempt in linkage analysis of allohexaploids by incorporating their meiotic mechanism lays a foundation for allohexaploid genetic mapping and also provides a new horizon to explore allohexaploid parental kinship.

Optimization of the HA-1-Specific T Cell Receptor for Gene Therapy of Hematological Malignancies.

Abstract 4093 Poster Board III-1028 Relapsed hematological malignancies after HLA-matched allogeneic stem cell transplantation (allo-SCT) are treated by donor lymphocyte infusion (DLI), inducing long-lasting complete remissions. However, treatment of relapsed hematological malignancies after allo-SCT with DLI is associated with induction of graft versus host disease (GvHD). It has been demonstrated that T cells recognizing minor histocompatibility antigens (mHags) selectively expressed on hematopoietic cells mediate anti-leukemic reactivity after allo-SCT without causing GvHD. mHags are derived from genetically polymorphic proteins that can be differentially expressed between donor and recipient. The mHag HA-1 is presented in the context of HLA-A2. The HA-1 tissue distribution is restricted to hematopoietic cells and carcinomas, making it an attractive target antigen to treat hematological malignancies relapsing after allo-SCT when the patient is HA-1+ and the donor is HA-1-. Therefore, adoptive transfer of HA-1-TCR gene modified T cells might be an attractive strategy to separate GvHD from graft versus leukemia effect (GvL). For optimal anti-leukemic reactivity, high expression of introduced TCRs and persistence of the gene modified T cells is important. Based on the previously reported low HA-1-TCR expression on HA-1-TCR modified T cells, optimization of the strategy is required. Several strategies to improve expression of the introduced TCR have been described. Protein expression of the TCR chains can be enhanced by codon optimization. In addition, preferential pairing facilitated by introduction of an extra disulfide bond in the constant regions of the TCR chains can increase the cell surface expression of the transferred TCR. Another strategy based on the fact that TCRs differ in their capacity to compete for cell surface expression, is to select recipient T cells with weak competitor phenotypes. In this study, we investigated the cause of low HA-1-TCR expression after gene transfer, and used the different strategies to increase HA-1-TCR expresssion. To study whether low HA-1-TCR expression was due to inefficiency of the TCRα and β chains to pair, TCR-deficient jurkat cells were transduced with the individual TCRα and TCRβ chains in combination with 17 different TCRα chains and TCRβ chains. Results indicated that low HA-1-TCR expression was not due to inefficient pairing of the HA-1-TCR chains, but caused by low HA-1-TCRβ chain expression on the cell surface. To investigate whether low cell surface expression of the HA-1-BV6S4 chain was due to intrinsic properties, the CDR1 and CDR3 region of the HA-1-TCR BV6S4 chain were exchanged with the CDR1 and CDR3 region of the HA-2-TCRβ (BV6S2) chain. We demonstrated that exchange of the HA-1-TCRβ CDR1 region with the HA-2-TCRβ CDR1 region resulted in improved TCR-expression, however, the HA-1-specificity was completely abolished, indicating that the HA-1-TCRβ CDR1 region is crucial for HA-1-specificity. Furthermore, since there is exclusive TCRBV6S4 chain usage of HA-1-specific T cells, we were unable to select for another HA-1-TCR for clinical use, and were pressed to optimize the HA-1-TCRβ chain. Both codon optimization of the HA-1-TCR chains aiming at improving protein expression and inclusion of cysteine residues in the HA-1-TCR chains aiming at inducing preferential pairing resulted in a significant increase in HA-1-TCR expression. Combining the two strategies increased the HA-1-TCR expression even more, resulting in 70% and 35% of tetramer positive HA-1-TCR transferred weak competitor and strong competitor T cells, respectively. In addition, the HA-1-TCR engineered T cells were able to efficiently recognize target cells that endogenously process and present HA-1, independent of whether the recipient T cells were strong or weak competitor T cells. These results illustrate that engineering of the HA-1-TCR by codon optimization and introduction of an extra cysteine bond resulted in high numbers of high-avidity HA-1-TCR in any T cell of choice irrespective of the properties of the endogenous TCR. Disclosures: No relevant conflicts of interest to declare.

An impact of environment on segregation ratio of qualitative traits in maize

The significant influence of environment was found on the segregation ratio in a dyhibrid inheritance in maize. Two possible causes are proposed for this segregation distortion: 1) environmental influence (selection) prior forming the gametes or/and 2) different preferential pairing in different environments. Further studies, however, on other self or cross-pollinated plant species, and with different traits are needed to better understand this phenomena.

Facilitating matched pairing and expression of TCR chains introduced into human T cells

Adoptive transfer of T lymphocytes is a promising treatment for a variety of malignancies but often not feasible due to difficulties generating T cells that are reactive with the targeted antigen from patients. To facilitate rapid generation of cells for therapy, T cells can be programmed with genes encoding the α and β chains of an antigen-specific T-cell receptor (TCR). However, such exogenous α and β chains can potentially assemble as pairs not only with each other but also with endogenous TCR α and β chains, thereby generating αβTCR pairs of unknown specificity as well as reducing the number of exogenous matched αβTCR pairs at the cell surface. We demonstrate that introducing cysteines into the constant region of the α and β chains can promote preferential pairing with each other, increase total surface expression of the introduced TCR chains, and reduce mismatching with endogenous TCR chains. This approach should improve both the efficacy and safety of ongoing efforts to use TCR transfer as a strategy to generate tumor-reactive T cells.