M. Louise Markert, MD, PhD
Departments of Immunology and Pediatrics

The 2 thrusts of research in our laboratory are to understand the molecular and immunological basis of inherited immunodeficiency diseases and to develop new therapies for these disorders. Through this work we hope to gain insights into basic human immunology. We currently are focusing on DiGeorge syndrome. Infants with DiGeorge syndrome are born with no thymus and have no circulating T cells. We are developing thymic transplantation for this disorder and are using these clinical experiments to learn about the role of the thymus in postnatal T-cell differentiation. We hypothesize that transplantation of human postnatal thymus will allow reconstitution of T-cell immunity in these patients. Other specific hypotheses include

the transplanted tissue (which can be maintained for 3 to 4 weeks in culture) will expand in the recipient and develop into areas resembling cortex and medulla that will express antigens characteristic for these regions;

prothymocytes from the host bone marrow will "home" to the transplanted donor thymic tissue and develop there into phenotypically normal thymocytes;

HLA restriction will develop to HLA antigens of both the donor and host; and

tolerance will develop to both host and donor MHC antigens.

We hope to apply the clinical work with thymic transplantation in DiGeorge syndrome to HIV infection. In HIV infection the thymus is severely damaged. In addition, in some patients the T-cell repertoire has been profoundly depleted. We are beginning a clinical trial in 16 HIV-seropositive patients who have CD4 T-cell counts between 200 and 500/mm3. All the patients will be treated with combination antiretroviral therapy. Half of the patients will receive thymic transplants. We will test the hypothesis that thymic function will be necessary for patients to achieve immunoreconstitution.

Preliminary Data

We have done 2 thymic transplants in patients with complete DiGeorge syndrome. Both patients initially had no T cells, no T-cell function, and no lymph nodes. Donor thymuses were obtained (after informed consent) from infants undergoing heart surgery. For the 1st patient, within 3 weeks, normal to elevated numbers of T cells appeared in the periphery; however, they were not functional (did not respond to the mitogens PHA, ConA or PWM). During this time the peripheral T-cell repertoire was oligoclonal. Also at this time the patient had numerous serious infections and massive lymphadenopathy developed. At 3 months after transplantation, a biopsy of the transplanted thymic tissue showed normal host T-cell differentiation in donor cortical and medullary thymic areas. All the thymocytes were host (as determined by HLA typing). T-cell function then began to develop and normal responses to mitogens, T-cell responses to tetanus and tolerance to the thymic donor by MLC developed. The TCR repertoire and the CD4:CD8 ratio normalized. As T-cell function developed, the lymphadenopathy resolved and the patient had no more infections. She is developing normally. HLA restriction studies are currently being done on this patient.

The 2nd patient had multiple birth defects in addition to DiGeorge syndrome. This patient eventually died of these problems. However, T-cell function developed 7 weeks after thymic transplantation. The functioning T cells were predominantly donor. The patient never developed signs of graft versus host disease.

Additional work in our laboratory has focused on elucidation of the molecular defects in patients with specific genetic defects leading to immune deficiency (such as adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency).

Selected Publications

Buckley RH, Schiff RI, Schiff SE, Markert ML, Williams LW, Harville TO, Roberts JL, Puck JM. Human severe combined immunodeficiency (SCID): genetic, phenotypic and functional diversity in 100 infants. J Pediatr 1997;130:378-87.

Davis CM, McLaughlin TM, Watson TJ, Buckley RH, Schiff SE, Hale LP, Haynes BF, Markert ML. Normalization of peripheral blood T-cell receptor V repertoire after postnatal thymic transplantation in DiGeorge syndrome. J Clin Immunol 1997;17:167-75.

Markert ML, Finkel BD, McLaughlin TM, Watson TJ, Collard HR, McMahon CP, Andrews LG, Barrett MJ, Ward FE. Mutations in purine nucleoside phosphorylase deficiency. Hum Mutat 1997;9:118-21.

Markert ML, Kostyu DD, Ward FE, McLaughlin TM, Watson TJ, Buckley RH, Schiff SE, Ungerleider RM, Gaynor JW, Oldham KT, and others. Successful formation of a chimeric human thymus allograft following transplantation of partially HLA-matched postnatal thymus. J Immunol 1997;158:998-1005.

Markert ML, Watson TJ, Kaplan I, Hale LP, Haynes BF. The human thymic microenvironment during organ culture. Clin Immunol Immunopathol 1997;82:26-36.

Garry RF, Fermin CD, Kohler PF, Markert ML, Luo H. Antibodies against retroviral proteins and nuclear antigens in a subset of idiopathic CD4+ T-lymphocytopenia patients. AIDS Res Hum Retroviruses 1996;12:931-40.

Nelson DM, Butters KA, Markert ML, Reinsmoen NL, McIvor RS. Correction of proliferative responses in purine nucleoside phosphorylase (PNP) deficient T lymphocytes by retroviral-mediated PNP gene transfer and expression. J Immunol 1995;154:3006-14.

Russell SM, Tayebi N, Nakajima H, Riedy MC, Roberts JL, Aman MJ, Migone TS, Noguchi M, Markert ML, Buckley RH, O'Shea JJ, Leonard WJ. Mutation of Jak3 in a patient with SCID: essential role of Jak3 in lymphoid development. Science 1995;270(5237):797-800.

Atasoy U, Norby-Slycord CJ, Markert ML. A missense mutation in Exon 4 of the human adenosine deaminase gene causes severe combined immunodeficiency. Hum Mol Genet 1993;2:1307-8.

Gossage DL, Norby-Slycord CJ, Hershfield MS, Markert ML. A homozygous five nucleotide deletion in the adenosine deaminase (AFA) mRNA in a child with ADA deficiency and very low levels of ADA mRNA and protein. Hum Mol Genet 1993;2:1493-4.

Aust MR, Andrews LG, Barrett MJ, Norby-Slycord CJ, Markert ML. Molecular analysis of mutations in a patient with purine nucleoside phosphorylase deficiency. Am J Hum Gen 1992;51:763-72.