An adenosine deaminase (ADA) allele contains two newly identified deleterious mutations (Y97C and L106V) that interact to abolish enzyme activity.

Genetic deficiency of the purine salvage enzyme adenosine deaminase (ADA) results in varying degrees of immunodeficiency, ranging from neonatal onset Severe Combined Immunodeficiency (SCID) to an adult onset immunodeficiency disorder. Multiple different mutations have now been identified in these immunodeficient patients. Additional mutations, initially identified in healthy individuals, abolish ADA in erythrocytes but retain 10-80% of activity in non-erythroid cells ('partial deficiency mutations'). In general, severity of disease correlates inversely with the amount of residual ADA expressed by the mutant enzymes and directly with the accumulation of the toxic metabolites deoxyATP and deoxyadenosine. We report two newly identified mutations (Y97C and L106V), both carried on the same allele of an immunodeficient patient who was diagnosed prenatally and successfully transplanted with haploidentical bone marrow. Based on the ability of mutant cDNAs to express ADA in vitro , the L106V mutation resulted in activity similar to 'partial' mutations (30% of normal) while the Y97C mutation resulted in detectable but markedly reduced activity (1.5% of normal). However, the presence of both mutations on the same allele virtually abolished detectable enzyme activity. Analysis of the crystallographic structure of ADA to understand the marked deleterious effect of the Y97C mutation suggested a previously unappreciated role of salt bridges in the catalytic mechanism of ADA. The patient was also heteroallelic for a previously described deletion of the promoter and exon 1. Testing of additional patients in whom we had not identified a mutation on the second allele revealed presence of this deletion in three of four patients tested. This deletion is therefore relatively common, accounting for 10% of almost 100 chromosomes studied by this and other laboratories, but is easily missed by currently used methods of mutation detection. Lastly, the finding of two mutations on the same allele that interact to reduce residual enzyme function emphasizes hazards in evaluating potential genotype-phenotype correlations in individuals analyzed only for the presence of single specific mutations.

Monoclonal antibody T-cell-depleted HLA-haploidentical bone marrow transplantation for Wiskott-Aldrich syndrome.

Four patients with Wiskott-Aldrich syndrome received bone marrow transplants (BMT) using monoclonal antibody T cell-depleted HLA-haploidentical marrow from a family member donor. The patients did not receive a significantly larger inoculum of mature T cells than other recipients of T cell-depleted marrow transplants. All four patients achieved quick engraftment, and three of the four patients are alive and well today. The three living patients have all had a complete return of normal T-cell and B-cell function. Infectious complications in the surviving patients were minimal; however, all three experienced some degree of graft-versus-host disease (GVHD). Two of these three patients received GVHD prophylaxis. The patient not receiving GVHD prophylaxis experienced severe GVHD and had a difficult posttransplant course. The patient who did not survive was chronically ill before BMT, whereas the other patients were in relatively good health at the time of BMT. Since the majority of individuals with this disease lack a matched bone marrow donor, our results using partially matched donors suggest that a greater number of patients can be successfully treated for Wiskott-Aldrich syndrome and that outcome is related to control of GVHD and state of health before BMT. Marrow transplantation should be offered earlier in the disease course before the onset of major infectious problems.

Immunologic reconstitution after haploidentical bone marrow transplantation for immune deficiency disorders: treatment of bone marrow cells with monoclonal antibody CT-2 and complement.

Patients with congenital T lymphocyte deficiency disorders received transplants with parental bone marrow depleted of mature T cells by the use of an anti-T cell monoclonal antibody (CT-2) and complement. Our results with 16 consecutive patients (20 transplants) showed rapid engraftment of donor cells; cytoreduction (busulfan, cytosine arabinoside [ara-C], cyclophosphamide) was used in six transplants, and marrow ablation was used in six (ara-C, cyclophosphamide, 1,365-cGy total body irradiation). No patient received prophylactic anti-graft-v-host disease (GVHD) therapy posttransplant, and only one patient developed significant GVHD, which involved the skin, liver, and gastrointestinal tract. Seven others showed some manifestations of GVHD, but these were of minimal clinical significance and required only occasional steroid therapy. Overall, eight patients are alive and well; eight did not survive. Polyclonal immunoglobulin synthesis by donor memory B cells was seen shortly after transplantation, with peak donor-derived serum levels seen approximately 2 months after transplantation. After this initial immunoglobulin synthesis waned, another wave of B cell responses developed. This immunoglobulin response appears to be permanent. T cell functions appeared as soon as 3 weeks after transplantation. This experience in a variety of patients with combined immunodeficiency who received transplants with monoclonal antibody T cell-depleted marrow shows gratifying results with a consistent T and B cell benefit.

Reconstitution of T- and B-cell function after T-lymphocyte-depleted haploidentical bone marrow transplantation in severe combined immunodeficiency due to adenosine deaminase deficiency.

A 4-month-old male received a T-lymphocyte-depleted haploidentical bone marrow transplant (BMT) for correction of severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency. Although previous haploidentical bone marrow transplants have been attempted in ADA-deficient SCID, complete reconstitution of both B-lymphocyte and T-lymphocyte function has not been obtained after a single transplant. In this patient, however, rapid, complete, and persistent engraftment occurred. Potential reasons for this successful reconstitution include the use of ablation by chemotherapy (busulfan, cyclophosphamide, and cytosine arabinoside), the in vitro technique of using monoclonal antibody (CT-2) and complement to deplete the donor cells of T lymphocytes, and the relative good health of the patient prior to the transplant. Further trials using this method of haploidentical BMT may prove it to be a successful method of immunologic reconstitution in ADA-deficient SCID patients for whom an HLA-identical marrow is not available.

Clinical trial depleting T lymphocytes from donor marrow for matched and mismatched allogeneic bone marrow transplants.

Nine patients received T-lymphocyte-depleted histocompatible bone marrow and 28 patients received T-lymphocyte-depleted histoincompatible bone marrow. Eight of nine patients receiving matched bone marrow quickly engrafted without severe graft-versus-host disease (GvHD). None of the eight patients received anti-GvHD prophylaxis medications. Two of these eight patients are currently alive. Nonengraftment and severe GvHD were problems seen in some of the patients given the histoincompatible bone marrow. Additional cytarabine pretransplant permitted engraftment in those patients undergoing histoincompatible transplants for treatment of malignancy, and prednisone and cyclosporine posttransplant reduced the incidence of acute GvHD in those given T-lymphocyte-depleted grafts. Seven of these 28 patients are currently alive. T-lymphocyte-depleted marrow can reduce the occurrence or prevent severe acute GvHD, especially when combined with additional prednisone and cyclosporine; however, the impact on relapse patterns and survival remains to be determined. The occurrence of nonengraftment and treatment-related lymphomas are formidable problems to overcome.

Autoimmune hemolytic anemia as a manifestation of T-suppressor-cell deficiency.

This report describes two children in whom autoimmune hemolytic anemia was the initial clinical manifestation of an underlying T-cell deficiency. Further investigation revealed a profound deficiency of T suppressor cells in both children as detected by monoclonal T-cell antibody (OKT8) and/or functional assays. In vitro incubation of their lymphocytes with cultured thymus epithelium or thymic factors induced T suppressor cells. In vivo treatment with cultured thymus epithelium or calf thymosin fraction 5 resulted in increased T-suppressor-cell numbers and/or function in both and possibly decreased hemolytic activity in one. These results suggest that the autoimmune process in some patients with autoimmune hemolytic anemia is associated with T-suppressor-cell deficiency and that in vivo therapy with agents that modulate T-cell function may be of therapeutic value.

Diminished reactivity to alloantigen following transplantation of cultured thymic fragments.

Transplantation of allogeneic thymus into thymic deficient individuals will restore T-cell function including ability to demonstrate alloreactivity. In allogeneically reconstituted athymic (nude) mice, alloreactivity as manifested by positive mixed leukocyte reactivity, cell mediated lympholysis and skin graft rejection is present for all alloantigens, save those of the thymus donor. Such reconstituted animals possess double tolerance, for self and for donor. Cultured thymic fragments have been used to correct the immunodeficiency of thymic deficient humans. In a patient with severe combined immunodeficiency who acquired T-killer activity after allogeneic cultured thymic fragment transplant, mixed leukocyte response to the thymus donor was nearly absent. Patients with cancer of the lung, although not profoundly T deficient, received cultured thymic fragments in a project designed to enhance their immunity. Diminished alloreactivity for donor cells was seen in 2 of 3 such patients.

The cerebro-hepato-renal syndrome of Zellweger: similarity to and differentiation from the DiGeorge syndrome.

A child with the cerebro-hepato-renal syndrome of Zellweger, who was originally diagnosed as having the DiGeorge syndrome, was studied and transplanted unsuccessfully with cultured thymus. The pertinent literature is reviewed and the importance of distinguishing the two disorders emphasized. Autopsy studies reveal that transplanted cultured thymic fragments can attract lymphoid aggregates as early as 2 wk after transplantation.

Fatal lymphoma after transplantation of cultured thymus in children with combined immunodeficiency disease.

A fatal, widespread, polyclonal, B-cell immunoblastic lymphoproliferative disorder developed in three children with combined immunodeficiency shortly after intra-abdominal transplantation of cultured thymus epithelium for immunoreconstitution. All three had surface immunoglobulin-bearing cells (15 to 20 per cent) in the peripheral blood before transplantation and polyclonally elevated immunoglobulins afterward. Abnormal immunoregulation was demonstrated by a lack of concanavalin A-induced suppressor-cell activity in mixed leukocyte culture in all three patients before transplantation and in two afterward. We suggest that the transplant acted as a promoter through immunostimulation or production of promoter factors, and that excessive polyclonal B-cell proliferation resulted because of inadequate immunoregulatory mechanisms. Although this complication occurred in only three of 30 patients with various forms of immunodeficiency treated with cultured thymus, these cases illustrate a potential problem in immunoreconstitution of combined immunodeficiency disorders.

Transplantation of cultured thymic fragments. II. Results in nude mice.

Nine nude mice were transplanted with cultured thymic fragments derived from syngeneic (three recipients) or allogeneic (six recipients) sources. All transplanted mice survived for periods of up to 8-10 mo thereafter, at which time they were sacrificed. Weight gain had been progressive and the animals were in excellent health. Four nontransplanted littermates housed in the same cages died at the age of 4 mo. In the nontransplanted mice, the usual deficits of T and B cells were observed. In transplanted mice, normalization of IgG1 and IgA levels as well as IgG antibodies to sheep erythrocytes and precipitating antibodies to rabbit serum occurred. Lymphocyte counts and Thy-1 bearing cells increased to approximately 50% of normal values. Proliferative responses to phytohemagglutinin and concanavalin A, mixed leukocyte reactivity, and cell-mediated lympholysis were variably restored from approximately 10-100% of normal. Attained responses were the same in recipients of syngeneic or allogeneic tissues and these, in turn, were equal or superior to responses measured in animals transplanted with whole noncultured thymuses. Skin grafts from third party donors were vigorously rejected, whereas those derived from second party (allogeneic thymus donor strain) may have been accepted or slowly rejected. Cultured thymic fragments, consisting primarily of epithelial elements, can effectively repair the thymic deficiency of nude mice. Experiments to date do not indicate that syngeneic tissues enjoy an advantage over allogeneic grafts in this restoration procedure.