Joseph L. Roberts

Hematopoietic Stem-Cell Transplantation for the Treatment of Severe Combined Immunodeficiency

BACKGROUND Since 1968 it has been known that bone marrow transplantation can ameliorate severe combined immunodeficiency, but data on the long-term efficacy of this treatment are limited. We prospectively studied immunologic function in 89 consecutive infants with severe combined immunodeficiency who received hematopoietic stem-cell transplants at Duke University Medical Center between May 1982 and September 1998. METHODS Serum immunoglobulin levels and lymphocyte phenotypes and function were assessed and genetic analyses performed according to standard methods. Bone marrow was depleted of T cells by agglutination with soybean lectin and by sheep-erythrocyte rosetting before transplantation. RESULTS Seventy-seven of the infants received T-cell-depleted, HLA-haploidentical parental marrow, and 12 received HLA-identical marrow from a related donor; 3 of the recipients of haploidentical marrow also received placental-blood transplants from unrelated donors. Except for two patients who received placental blood, none of the recipients received chemotherapy before transplantation or prophylaxis against graft-versus-host disease. Of the 89 infants, 72 (81 percent) were still alive 3 months to 16.5 years after transplantation, including all of the 12 who received HLA-identical marrow, 60 of the 77 (78 percent) who were given haploidentical marrow, and 2 of the 3 (67 percent) who received both haploidentical marrow and placental blood. T-cell function became normal within two weeks after transplantation in the patients who received unfractionated HLA-identical marrow but usually not until three to four months after transplantation in those who received T-cell-depleted marrow. At the time of the most recent evaluation, all but 4 of the 72 survivors had normal T-cell function, and all the T cells in their blood were of donor origin. B-cell function remained abnormal in many of the recipients of haploidentical marrow. In 26 children (5 recipients of HLA-identical marrow and 21 recipients of haploidentical marrow) between 2 percent and 100 percent of B cells were of donor origin. Forty-five of the 72 children were receiving intravenous immune globulin. CONCLUSIONS Transplantation of marrow from a related donor is a life-saving and life-sustaining treatment for patients with any type of severe combined immunodeficiency, even when there is no HLA-identical donor.

Clinical efficacy and immune regulation with peanut oral immunotherapy

BACKGROUND Oral immunotherapy (OIT) has been thought to induce clinical desensitization to allergenic foods, but trials coupling the clinical response and immunologic effects of peanut OIT have not been reported. OBJECTIVE The study objective was to investigate the clinical efficacy and immunologic changes associated with OIT. METHODS Children with peanut allergy underwent an OIT protocol including initial day escalation, buildup, and maintenance phases, and then oral food challenge. Clinical response and immunologic changes were evaluated. RESULTS Of 29 subjects who completed the protocol, 27 ingested 3.9 g peanut protein during food challenge. Most symptoms noted during OIT resolved spontaneously or with antihistamines. By 6 months, titrated skin prick tests and activation of basophils significantly declined. Peanut-specific IgE decreased by 12 to 18 months, whereas IgG(4) increased significantly. Serum factors inhibited IgE-peanut complex formation in an IgE-facilitated allergen binding assay. Secretion of IL-10, IL-5, IFN-gamma, and TNF-alpha from PBMCs increased over a period of 6 to 12 months. Peanut-specific forkhead box protein 3 T cells increased until 12 months and decreased thereafter. In addition, T-cell microarrays showed downregulation of genes in apoptotic pathways. CONCLUSION Oral immunotherapy induces clinical desensitization to peanut, with significant longer-term humoral and cellular changes. Microarray data suggest a novel role for apoptosis in OIT.

Human severe combined immunodeficiency: Genetic, phenotypic, and functional diversity in one hundred eight infants

OBJECTIVE To determine the relative frequencies of the different genetic forms of severe combined immunodeficiency (SCID) and whether there are distinctive characteristics of the particular genotypes. STUDY DESIGN The demographic, genetic, and immunologic features of 108 infants with SCID who were treated consecutively at Duke University Medical Center were analyzed. RESULTS Eighty-nine subjects were boys and 19 were girls; there were 84 white infants, 16 black infants, and 8 Hispanic infants. Forty-nine had X-linked SCID with mutations of common cytokine receptor gamma chain (gamma c), 16 had adenosine deaminase (ADA) deficiency, 8 had Janus kinase 3 (Jak3) deficiency, 21 had unknown autosomal recessive mutations, 1 had reticular dysgenesis, 1 had cartilage hair hypoplasia, and 12 (all boys) had SCID of undetermined type. Deficiency of ADA caused the most profound lymphopenia; gamma c or Jak3 deficiency resulted in the most B cells and fewest natural killer (NK) cells; NK cells and function were highest in autosomal recessive and unknown types of SCID. CONCLUSIONS Different SCID genotypes are associated with distinctive lymphocyte characteristics. The presence of NK function in ADA-deficient, autosomal recessive, and unknown type SCIDs, and low NK function in a majority of gamma c and Jak3 SCIDs indicates that some molecular lesions affect T, B, and NK cells (gamma c and Jak3), others primarily T cells (ADA deficiency), and others just T and B cells.

Unexpected Effects of FERM Domain Mutations on Catalytic Activity of Jak3: Structural Implication for Janus Kinases

Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.

Thymic output, T-cell diversity, and T-cell function in long-term human SCID chimeras

Severe combined immunodeficiency (SCID) is a syndrome of diverse genetic cause characterized by profound deficiencies of T, B, and sometimes NK-cell function. Nonablative human leukocyte antigen-identical or rigorously T cell-depleted haploidentical parental bone marrow transplantation (BMT) results in thymus-dependent genetically donor T-cell development in the recipients, leading to long-term survival. We reported previously that normal T-cell numbers, function, and repertoire developed by 3 to 4 months after transplantation in SCID patients, and the repertoire remained highly diverse for the first 10 years after BMT. The T-cell receptor diversity positively correlated with T-cell receptor excision circle levels, a reflection of thymic output. However, the fate of thymic function in SCID patients beyond 10 to 12 years after BMT remained to be determined. In this greater than 25-year follow-up study of 128 patients with 11 different molecular types of SCID after nonconditioned BMT, we provide evidence that T-cell function, thymic output, and T-cell clonal diversity are maintained long-term.

Cooperation among Multiple Transcription Factors Is Required for Access to Minimal T-Cell Receptor α-Enhancer Chromatin In Vivo

ABSTRACT To understand the molecular basis for the dramatic functional synergy between transcription factors that bind to the minimal T-cell receptor α enhancer (Eα), we analyzed enhancer occupancy in thymocytes of transgenic mice in vivo by genomic footprinting. We found that the formation of a multiprotein complex on this enhancer in vivo results from the occupancy of previously identified sites for CREB/ATF, TCF/LEF, CBF/PEBP2, and Ets factors as well as from the occupancy of two new sites 5′ of the CRE site, GC-I (which binds Sp1 in vitro) and GC-II. Significantly, although all sites are occupied on a wild-type Eα, all sites are unoccupied on versions of Eα with mutations in the TCF/LEF or Ets sites. Previous in vitro experiments demonstrated hierarchical enhancer occupancy with independent binding of LEF-1 and CREB. Our data indicate that the formation of a multiprotein complex on the enhancer in vivo is highly cooperative and that no single Eα binding factor can access chromatin in vivo to play a unique initiating role in its assembly. Rather, the simultaneous availability of multiple enhancer binding proteins is required for chromatin disruption and stable binding site occupancy as well as the activation of transcription and V(D)J recombination.

Developmental regulation of V(D)J recombination at the TCR a/5 locus

Summary: The T‐cell receptor (TCR) α/σ locus includes a large number of V, D, J and C gene segments that are used lo produce functional TCR 8 and TCR a chains expressed by distinct subsets of T lymphocytes. V(D)J recombination events within the locus are regulated as a function of developmental stage and cell lineage during T‐lymphocyte differentiation in the thymus. The process of V(D)J recombination is regulated by cis‐acting elements that modulate the accessibility of chromosomal substrates to the recombinase. Here we evaluate how the assembly of transcription factor complexes onto enhancers, promoters and other regulatory elements within the TCR α/σ locus imparts developmental control to VDJσ and VJα rearrangement events. Furthermore, we develop the notion that within a complex locus such as the TCR α/σ locus, highly localized and region‐specific control is likely to require an interplay between positive regulatory elements and blocking or boundary elements that restrict the influence of the positive elements to defined regions of the locus.

Flexible Stereospecific Interactions and Composition within Nucleoprotein Complexes Assembled on the TCRα Gene Enhancer

During thymocyte maturation, enhancers of genes encoding for TCRδ (Tcrd) and TCRα (Tcra), Eδ8, and Eα, work as a developmental switch controlling transition from Tcrd to Tcra activity at the Tcrad locus. Previous experiments revealed that an Eα fragment, Tα1-Tα2, which constitutes a well-characterized compact nucleoprotein structure led to premature activation of V(D)J recombination compared with that observed for the entire Eα or Tα1-Tα4. These experiments indicated that Tα3-Tα4 collaborates with factors bound to Tα1-Tα2 for the strict developmental regulation of Tcra rearrangement. The compact enhanceosome created on Tα1-Tα2 explained the molecular basis for requirement of intact Tα2 TCF/LEF and ets sites for enhancer function. We have created a mutant version of Eα, EαMC, in which Eδ myb and runx sites have been substituted for Tα2 runx and ets sites, that argues against the notion of a requirement for strict Eα enhanceosome structure for function. EαMC resulted in a very potent enhancer indicating that stereospecific interactions among proteins that form an Eα enhanceosome are rather flexible. Activation of V(D)J recombination by EαMC during thymocyte development resulted, however, to be premature and indistinguishable from that of Tα1-Tα2. These results indicate that Tα3-Tα4 itself is not sufficient to impart a developmental delay to a chimeric “early” enhancer, and indicate the need for functional collaboration between Tα2 runx/ets sites binding proteins and proteins bound to Tα3-Tα4 for proper developmental activation. The possibility of assembly of distinct sets of proteins on Eα might represent a more flexible form of information processing during thymocyte development.

Partial splenectomy but not total splenectomy preserves immunoglobulin M memory B cells in mice

PURPOSE The mechanism by which partial splenectomy preserves splenic immune function is unknown. Immunoglobulin (Ig) M memory B cells are critical for the immune response against encapsulated bacteria and are reduced in asplenic patients, although it is unknown whether partial splenectomy can preserve memory B cells. We hypothesized that IgM memory B cells (murine B-1a cells) would be preserved after partial splenectomy but not after total splenectomy in mice. METHODS We performed total splenectomy (n = 17), partial splenectomy (n = 10), or sham laparotomy (n = 16) on C57BL/6J mice. Mice were killed on postoperative day 10 or 30, and peritoneal washings were analyzed by multiparameter flow cytometry for expression of murine B-1a cells (IgM(pos)IgD(dull)CD5(pos)B220(dull)). RESULTS We found that B-1a cells were significantly reduced after both total and partial splenectomies compared with sham laparotomy in the early postoperative period, although normal levels of B-1a cells returned by postoperative day 30 in mice undergoing partial splenectomy but not total splenectomy. CONCLUSION Partial splenectomy but not total splenectomy preserves the B-1a B-cell population in mice within 30 days after surgery. Maintenance of these critical B cells may contribute to the preservation of a splenic-dependent immune response after partial splenectomy.

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.