Individualized Treatment Approach for Rectal Adenocarcinoma in the Setting of Congenital Neutropenia.

Congenial neutropenia is a rare genetic disorder that puts individuals at risk of life-threatening bacterial infections early in life, and the current standard of care includes the use of colony-stimulating factors or curative intent bone marrow transplant. Cancer treatment strategies that include surgery, chemotherapy, radiation, and immunotherapy present significant challenges to an individual with a baseline immunodeficiency as seen in this condition. Evidence-based national guidelines aid physicians and patients in moving through complex cancer care regimens. However, these are altered when the intensity of the patient's comorbidities puts them at increased risk of developing a potentially life-threatening infection. Here, we present a patient treated for rectal carcinoma in the setting of severe congenital neutropenia.

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses.

The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1-/HLAG1+ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1+ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger's sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous ß2-microglobulin (ß2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4+ and CD8+ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1+ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1+ may extend survival of porcine pancreatic islet and organ xenografts.

Human-porcine MHC-I homology allows for antibody cross-reactivity.

Pigs are especially useful large animal models, however, limited availability of commercially available antibodies for immunoblotting presents a significant obstacle facing preclinical xenotransplantation research. Major histocompatibility complex class I (MHC-I) molecule expression enhancement by nucleotide-binding oligomerization domain (NOD)-like receptor family with a caspase recruitment domain (CARD) containing caspase 5 (NLRC5) is fundamental to understanding porcine xenoantigen presentation. Swine Leukocyte Antigens (SLAs) are the porcine MHC homologs for human leukocyte antigens. SLA-I is a known xenoantigen that causes T cell activation. NLRC5, SLA-I, and B2M are all targets of immune modulation in genetically engineered pigs in xenotransplantation research with the goal to reduce SLA-I expression. In the present study, the human anti-NLRC5 (ab105411), anti-NLRC5 (ab117624), anti-NLRC5 N-terminal (ab178767), anti-HLA E (ab203082), anti-HLA E (ab135826), anti-HLA E (ab2216) and anti-ß2 M (ab75853) antibodies were examined using immunoblots for porcine cross-reactivity. The anti-human antibodies ab117624, ab105411, ab178767, ab2216, and ab75853 cross reacted with cognate proteins in porcine cell lysates. Antibody reagents from this study will allow for validation of NLRC5, B2M, MHC-I expression in future research studies. In addition, following the methodology described in this study for other xenotransplantation targets may provide an alternative to custom antibody development.