Elizabeth Dunn

Establishing diagnostic criteria for severe combined immunodeficiency disease (SCID), leaky SCID, and Omenn syndrome: The Primary Immune Deficiency Treatment Consortium experience

BACKGROUND The approach to the diagnosis of severe combined immunodeficiency disease (SCID) and related disorders varies among institutions and countries. OBJECTIVES The Primary Immune Deficiency Treatment Consortium attempted to develop a uniform set of criteria for diagnosing SCID and related disorders and has evaluated the results as part of a retrospective study of SCID in North America. METHODS Clinical records from 2000 through 2009 at 27 centers in North America were collected on 332 children treated with hematopoietic stem cell transplantation (HCT), enzyme replacement therapy, or gene therapy for SCID and related disorders. Eligibility for inclusion in the study and classification into disease groups were established by using set criteria and applied by an expert review group. RESULTS Two hundred eighty-five (86%) of the patients were determined to be eligible, and 47 (14%) were not eligible. Of the 285 eligible patients, 84% were classified as having typical SCID; 13% were classified as having leaky SCID, Omenn syndrome, or reticular dysgenesis; and 3% had a history of enzyme replacement or gene therapy. Detection of a genotype predicting an SCID phenotype was accepted for eligibility. Reasons for noneligibility were failure to demonstrate either impaired lymphocyte proliferation or maternal T-cell engraftment. Overall (n = 332) rates of testing were as follows: proliferation to PHA, 77%; maternal engraftment, 35%; and genotype, 79% (mutation identified in 62%). CONCLUSION Lack of complete laboratory evaluation of patients before HCT presents a significant barrier to definitive diagnosis of SCID and related disorders and prevented inclusion of subjects in our observational HCT study. This lesson is critical for patient care, as well as the design of future prospective treatment studies for such children because a well-defined and consistent study population is important for precision in outcomes analysis.

Megadose CD34+ Cell Grafts Improve Recovery of T Cell Engraftment but not B Cell Immunity in Patients with Severe Combined Immunodeficiency Disease Undergoing Haplocompatible Nonmyeloablative Transplantation

To determine whether T cell engraftment and recovery of B cell immunity could be improved, we prospectively treated 15 children with severe combined immunodeficiency disease (SCID) with megadoses of haplocompatible CD34(+) cells and a fixed number of CD3(+) cells without previous myeloablative chemotherapy. Evidence of T cell engraftment was seen in 73% of patients (95% confidence interval [CI] = 48%-90%). Engraftment was more likely in patients with X-linked SCID and in those with evidence of maternal engraftment at the time of diagnosis. In patients with T cell engraftment, the median time to development of a CD4 count > 200 cells/mm(3) and a phytohemagglutinin response > 50% of control was 1.2 and 4.9 months, respectively. Clearance of preexisting infections occurred after a median of 2.8 months. B cell function developed in 33% of engrafted patients (95% CI = 14%-61%). The 1-year event-free survival (EFS) rate was 60% (95% CI = 36%-80%), and the overall survival (OS) rate was 87% (95% CI = 61%-98%), with a median follow-up of 39 months. The use of megadoses of CD34(+) cells with a fixed number of CD3(+) cells in nonmyeloablative hematopoietic stem cell transplantation (HSCT) in patients with SCID is associated with excellent engraftment, T cell recovery, and OS; however, B cell function does not recover in most patients.

Targeted disruption of the Artemis murine counterpart results in SCID and defective V(D)J recombination that is partially corrected with bone marrow transplantation

Artemis is a mammalian protein, the absence of which results in SCID in Athabascan-speaking Native Americans (SCIDA). This novel protein has been implicated in DNA double-strand break repair and V(D)J recombination. We have cloned the Artemis murine counterpart, mArt, and generated a mouse with a targeted disruption of mArt. Artemis-deficient mice show a similar T−B− NK+ immunodeficiency phenotype, and carry a profound impairment in coding joint rearrangement, while retaining intact signal ends and close to normal signal joint formation. mArt−/− embryonic fibroblasts show increased sensitivity to ionizing radiation. Hemopoietic stem cell (HSC) transplantation using 500-5000 enriched congenic, but not allogeneic mismatched HSC corrected the T cell and partially corrected the B cell defect. Large numbers (40,000) of allogeneic mismatched HSC or pretreatment with 300 cGy of radiation overcame graft resistance, resulting in limited B cell engraftment. Our results suggest that the V(D)J and DNA repair defects seen in this mArt−/− mouse model are comparable to those in humans with Artemis deficiency, and that the recovery of immunity following HSC transplantation favors T rather than B cell reconstitution, consistent with what is seen in children with this form of SCID.

A Non-Leaky Artemis-Deficient Mouse That Accurately Models the Human Severe Combined Immune Deficiency Phenotype, Including Resistance to Hematopoietic Stem Cell Transplantation

Two Artemis-deficient (mArt(-/-)) mouse models, generated independently on 129/SvJ backgrounds, have the expected T(-)B(-)NK(+) severe combined immune deficiency (SCID) phenotype but fail to mimic the human disease because of CD4(+) T cell leakiness. Moreover, immune reconstitution after hematopoietic stem cell transplantation is achieved more readily in these leaky mouse models than in Artemis-deficient humans. To develop a more clinically relevant animal model, we backcrossed the mArt(-/-) mutation onto the C57Bl/6 (B6) background (99.9%), which resulted in virtually no CD4(+) T cell leakiness compared with 129/SvJ mArt(+/-) mice (0.3% +/- 0.25% vs 19.5% +/- 15.1%, P < .001). The nonleaky mouse also was uniquely resistant to engraftment using allogeneic mismatched hematopoietic stem cells, comparable to what is seen in human Artemis deficiency. The genetic background also influenced Artemis-associated radiation sensitivity, with differing degrees of x-ray hypersensitivity evident in 129/SvJ and B6 backgrounds with both the mArt(-/-) and mArt(+/-) genotypes. Our results indicate that immunogenic and DNA repair phenotypes associated with Artemis deficiency are significantly altered by genetic background, which has important implications for the diagnosis and treatment of SCID. Moreover, the B6 mArt(-/-) mouse provides a more accurate model for the human disease and a more appropriate system for studying human Artemis deficiency and for developing improved transplantation and gene therapy regimens for the treatment of children with SCID.

A novel missense RAG-1 mutation results in T-B-NK+ SCID in Athabascan-speaking Dine Indians from the Canadian Northwest Territories.

DNA double-strand repair factors in the non-homologous end joining (NHEJ) pathway resolve DNA double-strand breaks introduced by the recombination-activating gene (RAG) proteins during V(D)J recombination of T and B lymphocyte receptor genes. Defective NHEJ and subsequent failure of V(D)J recombination leads to severe combined immunodeficiency disease (SCID). We originally linked T−B−NK+ SCID in Athabascan-speaking Native Americans in the Southwestern US and Northwest Territories of Canada to chromosome 10. However, despite a common ancestry, the null mutation in the Artemis gene that we found to be causal in the SCID among the Navajo and Apache Indians was not present in the Dine Indians in the Northwest Territories. We now report a novel homozygous missense mutation (R776W) in RAG-1 in three children with T−B−NK+ SCID from two related families of Athabascan-speaking Dine Indians in the Canadian Northwest Territories. As expected, we found no increased sensitivity to ionizing radiation in patient fibroblasts. The impaired activity of this RAG-1 mutant in V(D)J recombination was confirmed by the EGFP-based V(D)J recombination assays. Overexpression of wild type RAG-1 in patient fibroblasts complemented V(D)J recombination, with recovery of both coding and signal joint formation. Our results indicate that the novel R776W missense mutation in RAG-1 is causal in the T−B−NK+ SCID phenotype in Athabascan-speaking Dine Indians from the Canadian Northwest Territories.

Genotype, Phenotype and Outcomes of Nine Patients with T-B+NK+ SCID

Yu GP, Nadeau KC, Berk DR, de Saint Basile G, Lambert N, Knapnougel P, Roberts J, Kavanau K, Dunn E, Stiehm ER, Lewis DB, Umetsu DT, Puck JM, Cowan MJ. Genotype, phenotype, and outcomes of nine patients with T‐B+NK+ SCID.
Pediatr Transplantation 2011: 15: 733–741.

T Cell and B Cell Immunity can be Reconstituted with Mismatched Hematopoietic Stem Cell Transplantation Without Alkylator Therapy in Artemis-Deficient Mice Using Anti-Natural Killer Cell Antibody and Photochemically Treated Sensitized Donor T Cells

Children with Artemis-deficient T(-)B(-)NK(+) severe combined immunodeficiency are at high risk for graft rejection from natural killer (NK) cells and toxicity from increased sensitivity to the alkylating agents used in mismatched hematopoietic stem cell transplantation (HSCT). We evaluated the use of a nonalkylating agent regimen before HSCT in Artemis-deficient (mArt(-/-)) C57Bl/6 (B6) mice to open marrow niches and achieve long-term multilineage engraftment with full T cell and B cell immune reconstitution. We found that partial depletion of both recipient NK cells using anti-NK1.1 monoclonal antibody and donor T cells sensitized to recipient splenocytes was necessary. BALB/c-sensitized T cells (STCs) were photochemically treated (PCT) with psoralen and UVA light to inhibit proliferation, reduce the risk of graft-versus-host disease (GVHD), and target host hematopoietic stem cells (HSCs). A dose of 4 × 10(5) PCT STCs coinjected with 1 × 10(5) lineage-depleted c-kit(+) BALB/c HSCs resulted in 43.9% ± 3.3% CD4(+) and 10.9% ± 1.2% CD8(+) donor T cells in blood, 29% ± 7.8% and 21.7% ± 4.0 donor B220(+) IgM(+) in spleen and bone marrow, and 15.0% ± 3.6% donor Gran-1(+) cells in bone marrow at 6 months post-HSCT versus 0.02% ± 0.01%, 0.13% ± 0.10%, 0.53% ± 0.16%, 0.49% ± 0.09%, and 0.20% ± 0.06%, respectively, in controls who did not receive PCT STCs. We found that STCs target host HSCs and that PCT STCs are detectable only up to 24 hours after infusion, in contrast to non-photochemically treated STCs, which proliferate resulting in fatal GVHD. Increased mortality in the groups receiving 4-6 × 10(5) PCT STCs was associated with evidence of GVHD, particularly in the recipients of 6 × 10(5) cells. These results demonstrate that blocking NK cell-mediated resistance and making niches in bone marrow are both essential to achieving multilineage engraftment of mismatched donor cells and T cell and B cell reconstitution, even though GVHD is not completely eliminated.