CHAPTER Thymus Transplantation 30 M. Louise Markert, Blythe H. Devlin, Elizabeth A. McCarthy, Ivan K. Chinn, Laura P. Hale monoclonal antibody reagents that identified com- History ponents of the human thymus [10-12] and the earliest stages of human thymocyte development Thymus transplantation was first attempted in the [13, 14]. In the 1990s, the ability to stain the cul- 1960s and 1970s using fetal thymus tissue [1, 2]. The tured tissue for cytokeratin and other thymic ele- results overall were disappointing [3-6]. In part the ments allowed Markert and Haynes to develop cul- poor outcomes related to the lack of reagents need- ture conditions that maximized the viability of the ed to characterize and identify the patients into those who were truly athymic (complete DiGeorge anom- cultured thymus slices [15]. Other monoclonal an- aly) and those who had bone marrow stem cell prob- tibodies were developed to identify naïve T-cells lems (severe combined immunodeficiency). It is al- [16]. This progress allowed accurate determination so possible that the fetal thymus tissue was too small of the presence of thymically derived T-cells. At the to reconstitute a human infant [7]. The use of fetal same time the underlying immunodeficiencies were thymus carried the risk of fatal graft versus host dis- better defined. In particular, the difference between ease since mature T-cells can be found in the human partial and complete DiGeorge anomaly was clari- thymus by the end of the first trimester [3]. By 1986, fied, with the partial DiGeorge anomaly patients in a review of 26 infants treated with fetal thymus having a small thymus versus the complete transplantation, 22 had died; the other 4 patients had DiGeorge anomaly patients having no thymus at all achieved a 3-year survival [6]. [17-19]. The former had some thymic-derived Important research was conducted in animals in T-cells that could reject transplants. The latter did the 1970s and 1980s which would provide the back- not have thymically derived T-cells; thus engraft- ground for improved outcomes. Hong and colleagues ment was facilitated. showed that thymus transplantation from complete- ly mismatched mouse strains could reconstitute T-cells in nude mice [8]. In the 1980s and 1990s Patient Population Haynes and colleagues performed animal experi- ments in which postnatal human tissue was trans- The target population for thymus transplantation is planted into mice [9]. Dr. Haynes had reported in the the group of athymic infants with complete DiGeorge early 1990s [9] that fragments of postnatal human anomaly. DiGeorge anomaly is characterized by de- thymus (readily available as discarded tissue from fects in organs derived from the 3rd and 4th pharyn- exposure of the heart in congenital cardiac surgery) geal pouches and the intervening 4th pharyngeal arch could be transplanted in the SCID/human mouse [20]. The parathyroid, thymus and heart are variably model. If the mice were pretreated with an antibody affected [19, 21-25]. Most infants have some para - against murine NK cells and macrophages, the human thyroid deficiency and require calcium replacement thymus tissue remained viable and murine T-cells [26]. Typical heart defects include interrupted aortic colonized the thymus within 1-3 months. Human arch type B and truncus arteriosus, although some thymopoiesis did not develop as there was not a patients have no cardiac defect at all [23, 26]. In source of human stem cells. complete DiGeorge anomaly, the thymus is absent. In addition to the animal experiments that were Other common problems in infants with complete essential for the development of the current thymus DiGeorge anomaly include speech delay, aspiration, transplantation trials, other advances in the 1980s gastroesophageal reflux, rib or vertebral anomalies, and 1990s allowed for critical advancement of the renal abnormalities, atypical facies, developmental field. Dr. Haynes and other investigators developed delay, hearing or visual deficits, 7th nerve palsies, 256 M.L. Markert et al. and cleft palate. Approximately half of children with procedure, all transplantation is conducted under In- complete DiGeorge anomaly have 22q11 hemizy- stitutional Review Board (Ethics Committee)- gosity [26-28]; approximately 20% have CHARGE approved and FDA-reviewed protocols. Informed association (coloboma, heart defect, choanal atresia, consent is obtained from parents of the donors and growth or developmental retardation, genital hy- recipients prior to thymus transplantation. poplasia, and ear anomaly or deafness) [26, 29, 30] The recipients are screened prior to transplanta- often with CHD7 mutations [31]; approximately tion to confirm the diagnosis of athymia and to bet- 15% are infants of diabetic mothers [26, 32, 33]; and ter characterize the subject. For the diagnosis of the remaining infants have no genetic or syndromic athymia, the subject must have fewer than 50/mm3 associations [26]. All athymic infants have a fatal naïve T-cells in the peripheral blood on flow cytom- condition and succumb to infection within the first 2 etry. In atypical complete DiGeorge patients with years of life because of their profound immunodefi- oligoclonal T-cells, less than 5% of circulating T-cells ciency [17]. can be naïve in phenotype. Stimulation of peripher- Complete DiGeorge anomaly may present with al blood mononuclear cells with the mitogen phyto- two different phenotypes. The majority of infants hemagglutinin is done to characterize the patient’s have “typical” complete DiGeorge anomaly. These T-cell response and determine if immunosuppression infants usually have very few T-cells (<50/mm3) and will be required. Every subject is tested for 22q11 always have fewer than 50 naïve T-cells/mm3. Naïve hemizygosity. T-cells are recent thymic emigrants that co-express In infants with rash and circulating T-cells, ad- CD45RA and CD62L [16]. Almost all of these in- ditional studies are performed. The clonality of the fants will lack a proliferative response to the mito- T-cells is assessed by flow cytometry and spec- gen phytohemagglutinin (PHA) [17]. These infants tratyping [40]. T-cell receptor rearrangement exci- do not have a rash. At some point after birth many sion circles (TREC) are quantified [41]. TRECs are infants with complete DiGeorge anomaly will de- episomes of DNA that form when the V, D, and J velop circulating oligoclonal T-cells associated with segments of DNA come together to encode the vari- rash and lymphadenopathy [34-36]. This phenotype able portion of the T-cell receptor chains. Absence is called “atypical” complete DiGeorge anomaly of TRECs confirms the diagnosis of athymia made [34]. Patients with atypical complete DiGeorge by the flow cytometry showing a lack of naïve anomaly resemble those with Omenn’s syndrome T-cells. [37-39]. The skin on biopsy shows spongiotic der- In the atypical patients, maternal engraftment matitis with T-cell infiltration [34]. The T-cells ap- [42] and graft versus host disease (GVHD) from pear to have developed without having been “edu- unirradiated blood transfusions must be ruled out. cated”. The oligoclonal T-cells seem to attack the in- DNA is obtained from the infant’s buccal swab and fant and do not protect against opportunistic from the mother. The DNA samples are compared infections. These T-cells have infiltrated the liver, as- using molecular methods to DNA extracted from sociated with hepatomegaly and elevated liver T-cells isolated from the infant’s peripheral blood. transaminases (unpublished). Strikingly, the oligo- GVHD from a blood transfusion is a life-threatening clonal T-cells can expand to very high numbers such complication. The only infant who presented with as 40,000/mm3 (unpublished). Despite the high T-cell GVHD in our series died despite intensive therapy numbers, less than 5% are naïve in phenotype. The to try to suppress the third party T-cells. Maternal peripheral T-cells may or may not proliferate in re- cells are rarely seen in atypical complete DiGeorge sponse to PHA. The two phenotypes of complete anomaly. Their affect on transplant outcomes is not DiGeorge anomaly must be distinguished because known at this time. the atypical patients can reject thymus transplants. To prepare for transplantation, standard testing is Atypical patients require peritransplantation im- conducted to assess the medical condition of the in- munosuppression. fant. Testing includes electrolytes, liver transami- nases, renal function (creatinine, blood urea nitrogen, urinalysis, renal ultrasound), and HLA typing. A car- Screening of Recipients for Transplantation diac evaluation is performed to assess suitability for surgery. As for other transplant recipients, the sub- Currently in the USA, an Investigator New Drug jects are screened for HIV-1 and hepatitis B and C. (IND) application with the Food and Drug Adminis- Subjects are also screened for human herpes virus 6 tration (FDA) is required for thymus transplantation. (HHV6), Epstein Barr virus (EBV), and cyto - Because thymus transplantation is an experimental megalovirus (CMV). HHV6 may have a detrimental Chapter 30 • Thymus Transplantation 257 affect on thymus development [43]. EBV and CMV erythematosis, Crohn’s disease, ulcerative colitis, are worrisome infections for infants with profound and rheumatoid arthritis. immunodeficiency as they can cause severe disease The donor’s mother is tested for the same infec- and may also be associated with lymphoproliferative tions as the donor plus toxoplasmosis and Chagas disease [44-47]. Parents are counseled during the in- disease. If the mother is IgG positive for toxoplas- formed consent process that these infections may af- mosis, the infant is tested as well. The mother is test- fect outcomes. If EBV or CMV is present, anti-viral ed for antibodies to CMV and EBV. Results consis- therapy is instituted. tent with acute infection lead to exclusion of the Infants are screened for autoimmune disease donor. In addition, extensive questionnaires are re- with complete blood counts, thyroid studies, viewed with the donor’s parents to review risk fac- Coombs antibody test, a urinalysis, and anti-HLA tors for Creutzfeldt-Jacob disease, small pox expo- antibodies.