Biology subjects

Dillard, P.

Publications and source records attributed to Dillard, P..

3 recordsLinked to original sources

bioRxiv2020

Combinatorial CAR design improves target restriction

CAR T cells targeting the B-lymphocyte antigen CD19 have led to remarkable clinical results in B-cell leukemia and lymphoma, but eliminate all B-lineage cells, leading to increased susceptibility to severe infections. As malignant B cells will express either immunoglobulin (Ig) light chain {kappa} or {lambda}, we designed a second-generation CAR targeting Ig{kappa}, IGK CAR. This construct demonstrated high target specificity, but displayed reduced efficacy in the presence of serum IgG. Since CD19 CAR is insensitive to serum IgG, we designed various combinatorial CAR constructs in order to maintain the CD19 CAR T cell efficacy, but with IGK CAR target selectivity. The Kz-19BB design, combining CD19 CAR containing a 4-1BB co-stimulatory domain with an IGK CAR containing a CD3zeta stimulatory domain, maintained the target specificity of IgK CAR and was resistant to the presence of soluble IgG. Our results demonstrate that a combinatorial CAR approach can improve target selectivity and efficacy.

immunology↗

bioRxiv2019

Mutational Analysis of an Intrinsically Disordered Region in the E. coli Phosphatase CheZ, a Regulator of Chemotaxis

As a model for Intrinsically Disordered Region (IDR) structure-function, we investigated the IDR present in the E. coli chemotaxis regulatory phosphatase CheZ. The CheZ IDR (amino acids 169-200) is attractive for study because CheZ is part of an exceptionally well characterized and easily tractable system and has an available high-resolution crystal structure. The CheZ IDR contains striking evolutionarily conserved regions that are functionally critical as shown by the fact that changes in single specific amino acids in the conserved IDR regions are able to abolish normal chemotaxis. We have focused on identifying suppressor mutations in the coding region of CheZ G188E, a variant with a >80% reduction in chemotactic swarm activity. Our screen identified 6 suppressor mutations that restored swarm activity to wild-type levels. Interestingly, the suppressor mutations were found not in the CheZ coding region, but in CheY, the phosphoprotein substrate of the CheZ phosphatase. The 6 suppressor mutations were restricted to two amino acid codons. Three suppressor mutations were found at position CheY A42 and three at position CheY M60. A model for how the changes in CheY restore CheZ activity is presented. These studies in dissection of the CheZ IDR will be useful in extending our understanding of IDR structure-function. The accumulating knowledge of IDR principles will be a significant component of a broadening understanding of protein biophysics with direct implications in basic protein structure and protein engineering research.

biochemistry↗

bioRxiv2017

Biphasic mechanosensitivity of TCR mediated adhesion of T lymphocytes

Force sensitivity of the T cell receptor (TCR) is now believed to be essential for immune recognition, but cellular mechanosensitivity of T cells is still poorly understood. Here we show that T cells adhering via the TCR-complex respond to environmental stiffness in an unusual biphasic fashion. As the stiffness increases, adhesion and spreading first increase, then decrease, attaining their maximal values on an optimally stiff surface, with stiffness comparable to certain antigen presenting cells. Remarkably, in presence of additional ligands for the integrin LFA-1, spreading increases monotonously with stiffness up to a saturation value. Using a mesoscopic semi-analytical model linking spreading to molecular characteristics of bonds, we identify force sensitivity of the off-rate and the effective bond stiffness as the crucial parameters that determine monotonic or biphasic mechanosensitive behavior.

biophysics↗