Boron Catalysis in a Designer Enzyme

The creation of enzymes containing non-biological functionalities with activation modes outside of Nature’s canon paves the way towards fully programmable biocatalysis. Here, we present a fully genetically encoded boronic acid containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts. This boron enzyme catalyzes the kinetic resolution of hydroxyketones by oxime formation where crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign lead to a variant with natural enzyme like enantioselectivities for a number of different substrates. The unique activation mode of the boron enzyme was studied via X-ray crystallography, high resolution mass spectrometry and 11B NMR spectroscopy and opens up the possibility for a new class of boron dependent biocatalysts

Role of lymphocyte activation gene-3 (Lag-3) in conventional and regulatory T cell function in allogeneic transplantation.

Lag-3 has emerged as an important molecule in T cell biology. We investigated the role of Lag-3 in conventional T cell (Tcon) and regulatory T cell (Treg) function in murine GVHD with the hypothesis that Lag-3 engagement diminishes alloreactive T cell responses after bone marrow transplantation. We demonstrate that Lag-3 deficient Tcon (Lag-3(-/-) Tcon) induce significantly more severe GVHD than wild type (WT) Tcon and that the absence of Lag-3 on CD4 but not CD8 T cells is responsible for exacerbating GVHD. Lag-3(-/-) Tcon exhibited increased activation and proliferation as indicated by CFSE and bioluminescence imaging analyses and higher levels of activation markers such as CD69, CD107a, granzyme B, and Ki-67 as well as production of IL-10 and IFN-g early after transplantation. Lag-3(-/-) Tcon were less responsive to suppression by WT Treg as compared to WT Tcon. The absence of Lag-3, however, did not impair Treg function as both Lag-3(-/-) and WT Treg equally suppress the proliferation of Tcon in vitro and in vivo and protect against GVHD. Further, we demonstrate that allogeneic Treg acquire recipient MHC class II molecules through a process termed trogocytosis. As MHC class II is a ligand for Lag-3, we propose a novel suppression mechanism employed by Treg involving the acquisition of host MHC-II followed by the engagement of Lag-3 on T cells. These studies demonstrate for the first time the biologic function of Lag-3 expression on conventional and regulatory T cells in GVHD and identify Lag-3 as an important regulatory molecule involved in alloreactive T cell proliferation and activation after bone marrow transplantation

Lag-3^−/− Tcon proliferate faster than WT Tcon.

<p>(A) Quantitative analysis of photon emission <i>in vitro</i> by an equal number of <i>luc⁺</i> WT and <i>luc⁺</i> Lag-3^−/− Tcon. (B) Lethally irradiated Balb/c recipients were transplanted with 5×10⁶ TCD-BM and 5×10⁵<i>luc⁺</i> WT or Lag-3^−/− Tcon. <i>In vivo</i> BLI images at different times after transplantation show more expansion of Lag-3^−/− Tcon. The graph on the right represents the quantitative analysis of the BLI signal. (C) <i>Ex vivo</i> images of the intestinal tract on day 4 displayed increased BLI signal in MLN of recipients receiving <i>luc⁺</i> Lag-3^−/− Tcon. The ex vivo images are representative of 5 mice/group. The bar graph on the right represents the quantitative analysis of the <i>ex vivo</i> BLI signal from spleen, pLN, and MLN. Bar depicts mean plus or minus SD, n = 5 mice/group. (*P<0.05, **P<0.01, ***P<0.001).</p

Surface expression of Lag-3 increases upon T cell activation <i>in vitro</i> and <i>in vivo</i>.

<p>(A) T cells were incubated with anti-CD3/anti-CD28 activation beads in the presence of 1000 U/mL hrIL-2. Lag-3 expression was assessed over a period of 7 days. The grey solid histograms represent cells stained with isotype control Ab. Cells were gated on CD4⁺ and the numbers in the right side corner represent the percentage of Lag-3 positive cells. Data is representative of 3 independent experiments. (B) Balb/c (H-2K^d) recipients were co-transplanted with 5×10⁶ TCD-BM and 1×10⁶ Tcon from C57Bl/6 (H-2K^b) mice. At indicated time points after transplantation, donor T cells were re-isolated from spleen and LN and analyzed for Lag-3 expression. Grey histograms represent cells stained with isotype controls. Cells were gated on donor CD4⁺ cells and the numbers in the right side corner represent the percentage of Lag-3 positive cells. Data is representative of two independent experiments.</p

WT Treg and Lag-3^−/− Treg show similar protection against GVHD.

<p>(A) Foxp3 staining (upper panel) and Lag-3 staining (lower panel) of CD4⁺CD25⁺ T cells isolated from WT mice (black histogram) and Lag-3^−/− mice (dashed histogram). For Lag-3 staining, lower panel, regulatory T cells were activated for 2 days with anti-CD3/anti-CD28 beads. Cells were gated on CD4⁺Foxp3⁺. The shaded histogram represents the isotype staining control. The histograms are representative of 2 independent experiments. (B) ³H-thymidine incorporation of WT C57BL/6J responders to Balb/c stimulators in the presence of either WT Treg or Lag-3^−/− Treg at different Treg to responder ratios, R: responders, S: stimulators. (C–D) Lethally irradiated Balb/c recipients were co-transplanted with 5×10⁶ TCD-BM and 5×10⁵ WT or Lag-3^−/− Treg on day 0 followed by infusion of 1×10⁶<i>luc⁺</i> WT Tcon on day 2. (C) Percent survival of mice after transplantation. Graph contains data pooled from 3 independent experiments (n = 15). P = 0.034 for WT Treg+Tcon vs. Tcon and P = 0.033 for Lag-3^−/− Treg+Tcon vs. Tcon. (D) <i>In vivo</i> BLI images of mice receiving WT Tcon alone, WT Tcon+WT Treg and WT Tcon+Lag-3^−/− Treg. Upper panels: BLI images taken at different time points after transplantation; lower panels: the corresponding quantitative analysis of the BLI signal. The BLI images are representative of 3 independent experiments.</p

Lag-3^−/− Tcon accelerates GVHD.

<p>Lethally irradiated Balb/c recipients were transplanted with 5×10⁶ TCD-BM and various doses of Tcon from either Lag-3^−/− or WT C57Bl/6 mice and monitored for survival (A–C) and GVHD score (D–E). (A) Mice were infused with TCD-BM and 1×10⁶ Tcon (n = 15/group, P = 0.009), (B) TCD-BM and 7.5×10⁵ Tcon (n = 10/group, P<0.001), and (C) TCD-BM and 5×10⁵ Tcon (n = 10/group, P<0.001). P value is calculated for survival rate differences between mice receiving Lag-3^−/− Tcon and WT Tcon. Data in graphs are pooled from two independent experiments. (D) and (E) represent the GVHD score of mice 5 days (D) and 25 days (E) after transplantation. Mice receiving TCD-BM and Lag-3^−/− Tcon had a significantly higher GVHD score than mice receiving TCD-BM and WT Tcon (P<0.005). (F) Histopathologic analysis of GVHD target organs isolated at day 8 after transplant. High magnification photomicrographs of colon (upper panels) and small intestine (lower panels) reveal a more severe GVHD in mice transplanted with Lag-3^−/− Tcon as evidenced by increased inflammatory cell infiltrate in the lamina propria (black asterisk) and intestinal gland lumina (white arrowhead), loss of microarchitecture of intestinal glands that are lined by poorly-differentiated epithelial cells and a larger number of apoptotic bodies within the epithelial layer (black arrowhead). H&E stain, scale bar = 50 microns. (G) Mice received 5×10⁶ TCD-BM and 1×10⁶ different combinations of CD4 and CD8 T cells. One group of mice received Lag-3^−/− CD4+ WT-CD8 T cells (n = 10), one received WT-CD4+ Lag-3^−/− CD8 T cells (n = 10, P<0.0001), one group received Lag-3^−/−CD4+Lag-3^−/−CD8T cells (Lag-3^−/− Tcon, n = 15) and the last group received WT-CD4+WT-CD8 T cells (WT Tcon, n = 15). P values are calculated for survival rate differences between mice receiving Lag-3^−/− Tcon and WT-CD4+Lag-3^−/−CD8 T cells. Data are pooled from two independent experiments.</p

Regulatory T cells acquire MHC class II complexes from host APC.

<p>(A) Lethally irradiated Balb/c (H-2K^d) recipients were co-transplanted with 5×10⁶ TCD-BM, 1×10⁶ Tcon from CD45.1⁺ C57BL/6 (H-2K^b) mice and 1×10⁶ WT or Lag-3^−/− Treg. MHC class II expression on donor Treg was assessed 5 days after transplantation. Cells were gated on CD45.1 negative, H2Kb positive (panel i), then CD4 and Foxp3 positive events (panel ii). Panels iii and iv represent the MHC class II expression level on day 0 and day 5 respectively. (B) MHC class II staining on Treg of donor origin (WT or Lag-3^−/−). Grey histogram represents cells stained with isotype control. Cells were gated on donor CD4 positive, Foxp3 positive events. The histograms are representative of 2 independent experiments each having 4 mice/group.</p

Lag-3^−/− Tcon show increased activation and proliferation.

<p>Lethally irradiated Balb/c recipients were transplanted with 5×10⁶ TCD-BM and 7.5×10⁵ CFSE-labeled WT or Lag-3^−/− Tcon. Donor T cells were re-isolated 3 days after transplant. (A) CFSE histograms of donor CD4 T cells (left panels) and CD8 T cells (right panels) re-isolated from spleen (top), pLN (middle), and MLN (bottom). Histograms are representative of 5 mice/group. Numbers represent percentage of proliferated cells. (B) Bar graphs indicating the percentage of proliferated donor CD4 T cells (left panels) and CD8 T cells (right panels) re-isolated from spleen (top), pLN (middle), and MLN (bottom). Bar depicts mean plus or minus SD, n = 5 mice/group. (C) and (D) Mice were transplanted with 5×10⁶ TCD-BM and 7.5×10⁵ WT or Lag-3^−/− Tcon. Donor T cells were re-isolated 4 days after transplant. To facilitate retention of intracellular cytokines <i>in vivo</i>, mice were injected i.p. with 250 µg brefeldin A 6 h before spleen and LN harvest. (C) Frequency of indicated activation markers gated on donor CD4 T cells (upper panels) and donor CD8 T cells (lower panels) re-isolated from spleen. (D) Mean fluorescence intensity (MFI) of IFN-γ and IL-10 gated on donor CD4 and CD8 T cells re-isolated from spleen. Bar depicts mean plus or minus SD, each data point represents two pooled mice, n = 4/group. Statistical significance indicated by *(* P<0.05, **P<0.01, ***P<0.001).</p