The endogenous X-linked phosphatidyl inositol glycan class A gene (Pig-a) can be used as a reporter of in vivo somatic cell mutation in rats and mice. Pig-a mutant cells are deficient in specific protein surface markers and can be identified and quantified by immunofluorescent staining followed by high-throughput flow cytometry. Pig-a mutation detection is commonly performed with red blood cells (RBCs) because: (1) the low volumes of blood required for determining mutant frequencies in RBCs allow multiple samplings on small laboratory animals over extended periods of time; (2) the execution of the RBC assay is easy and the interpretation of the results is straightforward; and (3) RBC Pig-a mutant frequencies are known within hours of sample collection. Two endpoints are determined in the assay: the frequency of mutant total RBCs and the frequency of mutant reticulocytes. When Pig-a mutation is used to assess the in vivo mutagenic potential of suspect hazards, the frequency of mutant reticulocytes is an early indicator of mutagenic potential, while the mutant frequency in total RBCs can be measured more rapidly and with greater precision.

Mycobacterium tuberculosis (M.tb)-derived antigens capable of inducing strong cellular and/or humoral responses are potential targets for both immunodiagnosis and vaccine development against tuberculosis (TB). In the present study, we identified adenylate kinase (ADK, Rv0733) as an antigen that induces high cellular and antibody responses in active TB patients. We consequently tested the use of ADK-specific T cells and antibodies as biomarkers for TB diagnosis. The ADK-specific IFN-γ-producing cells detected by ELISPOT assay showed a sensitivity of 85.0 % and specificity of 94.15 % for TB diagnosis while ADK-specific IgG antibody showed a sensitivity of 40.35 % and specificity of 96.43 %. Combining ADK-specific cellular and antibody responses increased the sensitivity to 91.59 % and the specificity to 96.15 %. Immunogenicity and protection against M.tb infection were further tested in a murine model. Immunization with ADK protein elicited strong specific T- and B-cell responses, and provided protection against the virulent H37Rv stain of M.tb resulting in lower bacilli load in the spleens and lungs. More ADK-specific polyfunctional Th1 cells were observed in the lungs when compared to adjuvant-immunized mice. ADK thus may serve as a novel M.tb antigen for TB immunodiagnosis and development of subunit vaccines.
ADK induces strong immune responses both in humans and mice. ADK-specific IFN-γ production and B-cell responses have high potential for TB diagnosis. ADK immunization provides protection against M.tb infection.

The endogenous X-linked phosphatidyl inositol glycan class A gene (Pig-a) can be used as a reporter of in vivo somatic cell mutation in rats and mice. Pig-a mutant cells are deficient in specific protein surface markers and can be identified and quantified by immunofluorescent staining followed by high-throughput flow cytometry. Pig-a mutation detection is commonly performed with red blood cells (RBCs) because (1) the low volumes of blood required for determining mutant frequencies in RBCs allow multiple samplings on small laboratory animals over extended periods of time; (2) the execution of the RBC assay is easy and the interpretation of the results is straightforward; and (3) RBC Pig-a mutant frequencies are known within hours of sample collection. Two endpoints are determined in the assay: the frequency of mutant total RBCs and the frequency of mutant reticulocytes. When Pig-a mutation is used to assess the in vivo mutagenic potential of suspect hazards, the frequency of mutant reticulocytes is an early indicator of mutagenic potential, while the mutant frequency in total RBCs can be measured more rapidly and with greater precision.

Genetic modification for enhancing cellular function has been continuously pursued for fighting diseases. Messenger RNA (mRNA) transfection is found to be a promising solution in modifying hematopoietic and immune cells for therapeutic purpose. We have developed a flow electroporation-based system for large volume electroporation of cells with various molecules, including mRNA. This allows robust and scalable mRNA transfection of primary cells of different origin. Here we describe transfection of chimeric antigen receptor (CAR) mRNA into NK cells to modulate the ability of NK cells to target tumor cells. High levels of CAR expression in NK cells can be maintained for 3-7 days post transfection. CD19-specific CAR mRNA transfected NK cells demonstrate targeted lysis of CD19-expressing tumor cells OP-1, primary B-CLL tumor cells, and autologous CD19+ B cells in in vitro assays with enhanced potency: >80% lysis at effector-target ratio of 1:1. This allows current good manufacturing practices (cGMP) and regulatory compliant manufacture of CAR mRNA transfected NK cells for clinical delivery.

Natural killer (NK) cells are emerging as a new tool for cell therapy of cancer. However, some cancer subtypes are relatively resistant to NK cell cytotoxicity. Expression of anti-CD19 chimeric signaling receptors can enhance NK-cell reactivity against CD19+ leukemia and lymphoma cells. Here we describe a method to enforce expression of such receptors in human NK cells relying on electroporation of mRNA and compare it to retroviral transduction of cDNA. These methods are applicable to the reprogramming of NK cells with chimeric receptors specific for other antigens expressed on cancer cells as well as with molecules that can modulate NK cell function.