Not All SCID Pigs Are Created Equally: Two Independent Mutations in the Artemis Gene Cause SCID in Pigs.

Mutations in >30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as SCID. SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T(-)B(-)NK(+) SCID, representing, to our knowledge, the first example of naturally occurring SCID in pigs. In this study, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed that two mutations were present in the Artemis gene, which in the homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented in the present study reveals two mutations in the Artemis gene that cause T(-)B(-)NK(+) SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues.

PD-1 is not required for natural or peripherally induced regulatory T cells: Severe autoimmunity despite normal production of regulatory T cells.

The expression of the coinhibitor PD-1 on T cells is important for the establishment of immune homeostasis. We previously found that PD-1 is particularly critical for the control of self-tolerance during lymphopenia-induced proliferation of recent thymic emigrants (RTEs). Previous studies suggested that PD-1 modulates the generation of Treg cells, particularly peripherally induced Treg (pTreg) cells, and controls Th17 cells. However, these conclusions were derived indirectly from studies on the ligand PD-L1, and not PD-1 itself. Herein we directly tested whether T-cell PD-1 expression was needed for Treg cell generation and examined if a paucity of Treg cells or enhanced Th17 cells could explain the severe lymphopenia-potentiated autoimmunity caused by PD-1 KO RTEs. Employing the murine FoxP3(EGFP) reporter system to simultaneously monitor conversion of WT and PD-1 KO T cells to pTreg cells in the same animal, we found that PD-1 deficiency did not inhibit pTreg cell generation or lead to Th17-cell-mediated autoimmunity. Surprisingly, pTreg cell numbers were increased in PD-1 KO versus WT cell populations. Furthermore, we noted an increased conversion to pTreg cells by RTEs. Our data suggest that the primary role for PD-1 is to restrain T-cell activation/proliferation to self-Ags rather than promote generation of Treg cells.

Evaluating antigen-specific cytotoxic T lymphocyte responses by a novel mouse granzyme B ELISPOT assay.

We have established novel ELISA- and ELISPOT-based assays specific for the detection of a potent cytotoxic mediator, granzyme B (GrB), for the assessment of antigen-specific cytotoxic T lymphocyte responses in mice. The sensitivity and specificity of our assays was demonstrated by ELISA using purified mouse GrB and supernatants and cell lysates of cytotoxic lymphocytes derived from GrB-deficient mice. No reactivity was observed by the GrB ELISA in GrB-deficient cells. The mouse GrB ELISPOT was successfully employed to detect antigen-specific effector cell responses of two CTL clones, producing GrB ELISPOT results that correlated strongly with target cell lysis, as assessed by 51Cr-release. Furthermore, we were able to demonstrate direct correlations between GrB ELISPOT and killing by LCMV gp33-specific effector and memory T cells generated in vivo. Thus, the mouse GrB ELISPOT may be used to detect cytotoxic responses, at the single-cell level, for the functional assessment of antigen-specific CD8+ T cell responses in mouse models of infection.

Porcine reproductive and respiratory syndrome virus replication and quasispecies evolution in pigs that lack adaptive immunity.

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) was studied in a line of pigs possessing a severe combined immunodeficiency (SCID). Real-time RT-PCR revealed a unique course of infection for the SCID group. During the course of infection, viremia was initially significantly lower than normal littermates, but by 21 days was significantly elevated. Deep sequencing of the viral structural genes at days 11 and 21 identified seven amino acid substitutions in both normal and SCID pigs. The most significant change was a W99R substitution in GP2, which was present in the inoculum at a frequency of 35%, but eventually disappeared from all pigs regardless of immune status. Therefore, amino acid substitutions that appear during acute infection are likely the result of the adaptation of the virus to replication in pigs and not immune selection.

Granzyme H induces cell death primarily via a Bcl-2-sensitive mitochondrial cell death pathway that does not require direct Bid activation.

Natural killer and T cell-mediated cytotoxicity is important for the elimination of viruses and transformed cells. The granule lytic pathway utilizes perforin and granzymes to induce cell death, while receptor-mediated lytic pathways rely on molecules such as FasL. Pro-apoptotic activities of Granzyme B (GrB) and Fas are well-established, and many of their cellular targets have been identified. However, humans express additional related granzymes - GrA, GrM, GrK, and GrH. Neither the cytotoxic potential of GrH, nor the mechanism by which GrH may induce target cell death is currently understood. We proposed that GrH would have pro-apoptotic activity that would be distinct from that of GrB and FasL, which could be relevant when Fas/FasL or GrB activity or death pathways were impaired. Our results, using a purified recombinant form of GrH, revealed that GrH induced cell death via a Bcl-2-sensitive mitochondrial pathway without direct processing of Bid. Additionally, neither the apoptosome nor caspase-3 was essential to the induction of GrH-mediated cell death. However, GrH did directly process DFF45, potentially leading to DNA damage. Our findings support the idea that multiple, non-redundant death pathways may be initiated by cytotoxic cells to counteract various immune evasion strategies.