Blythe H. Devlin

First use of thymus transplantation therapy for FOXN1 deficiency (nude/SCID): a report of 2 cases.

FOXN1 deficiency is a primary immunodeficiency characterized by athymia, alopecia totalis, and nail dystrophy. Two infants with FOXN1 deficiency were transplanted with cultured postnatal thymus tissue. Subject 1 presented with disseminated Bacillus Calmette-Guérin infection and oligoclonal T cells with no naive markers. Subject 2 had respiratory failure, human herpes virus 6 infection, cytopenias, and no circulating T cells. The subjects were given thymus transplants at 14 and 9 months of life, respectively. Subject 1 received immunosuppression before and for 10 months after transplantation. With follow up of 4.9 and 2.9 years, subjects 1 and 2 are well without infectious complications. The pretransplantation mycobacterial disease in subject 1 and cytopenias in subject 2 resolved. Subject 2 developed autoimmune thyroid disease 1.6 years after transplantation. Both subjects developed functional immunity. Subjects 1 and 2 have 1053/mm(3) and 1232/mm(3) CD3(+) cells, 647/mm(3) and 868/mm(3) CD4(+) T cells, 213/mm(3) and 425/mm(3) naive CD4(+) T cells, and 10 200 and 5700 T-cell receptor rearrangement excision circles per 100 000 CD3(+) cells, respectively. They have normal CD4 T-cell receptor β variable repertoires. Both subjects developed antigen-specific proliferative responses and have discontinued immunoglobulin replacement. In summary, thymus transplantation led to T-cell reconstitution and function in these FOXN1 deficient infants.

Statistical analysis of antigen receptor spectratype data

MOTIVATION The effectiveness of vertebrate adaptive immunity depends crucially on the establishment and maintenance of extreme diversity in the antigen receptor repertoire. Spectratype analysis is a method used in clinical and basic immunological settings in which antigen receptor length diversity is assessed as a surrogate for functional diversity. The purpose of this paper is to describe the systematic derivation and application of statistical methods for the analysis of spectratype data. RESULTS The basic probability model used for spectratype analysis is the multinomial model with n, the total number of counts, indeterminate. We derive the appropriate statistics and statistical procedures for testing hypotheses regarding differences in antigen receptor distributions and variable repertoire diversity in different treatment groups. We then apply these methods to spectratype data obtained from several healthy donors to examine the differences between normal CD4+ and CD8+ T cell repertoires, and to data from a thymus transplant patient to examine the development of repertoire diversity following the transplant.

Human FOXN1-deficiency is associated with αβ double-negative and FoxP3+ T-cell expansions that are distinctly modulated upon thymic transplantation.

Forkhead box N1 (FOXN1) is a transcription factor crucial for thymic epithelium development and prevention of its involution. Investigation of a patient with a rare homozygous FOXN1 mutation (R255X), leading to alopecia universalis and thymus aplasia, unexpectedly revealed non-maternal circulating T-cells, and, strikingly, large numbers of aberrant double-negative αβ T-cells (CD4negCD8neg, DN) and regulatory-like T-cells. These data raise the possibility that a thymic rudiment persisted, allowing T-cell development, albeit with disturbances in positive/negative selection, as suggested by DN and FoxP3+ cell expansions. Although regulatory-like T-cell numbers normalized following HLA-mismatched thymic transplantation, the αβDN subset persisted 5 years post-transplantation. Involution of thymus allograft likely occurred 3 years post-transplantation based on sj/βTREC ratio, which estimates intrathymic precursor T-cell divisions and, consequently, thymic explant output. Nevertheless, functional immune-competence was sustained, providing new insights for the design of immunological reconstitution strategies based on thymic transplantation, with potential applications in other clinical settings.

Factors Affecting Success of Thymus Transplantation for Complete DiGeorge Anomaly

Thymus transplantation shows promise for the treatment of athymia in complete DiGeorge anomaly. This report reviews the effects of dose of thymus tissue, ABO compatibility, HLA matching, culture conditions, age of donor and immunosuppression of recipient on immune outcomes at 1 year after transplantation. Forty‐nine athymic subjects have been treated with cultured postnatal allogeneic thymus tissue; 36 (73%) survive with only one subject on immunosuppression at 1.5 years. Of 31 surviving subjects more than 1 year after transplantation, 30 (97%) developed naive T cells, T‐cell proliferative responses to mitogens and a diverse T‐cell receptor beta variable (TCRBV) repertoire. The dose of thymus tissue, HLA matching and use of immunosuppression had nonsignificant effects on these outcome variables. Removal of deoxyguanosine from culture medium and length of culture did not adversely affect outcomes. Use of thymus tissue from donors over 1 month of age, versus under 1 month, resulted in higher total T‐cell numbers (p = 0.03). However, this finding must be confirmed in a prospective trial. Although subtle immune effects may yet be associated with some of the factors tested, it is remarkable that consistently good immune outcomes result despite variation in dose, HLA matching and use of immunosuppression.

The Dynamics of T-Cell Receptor Repertoire Diversity Following Thymus Transplantation for DiGeorge Anomaly

T cell populations are regulated both by signals specific to the T-cell receptor (TCR) and by signals and resources, such as cytokines and space, that act independently of TCR specificity. Although it has been demonstrated that disruption of either of these pathways has a profound effect on T-cell development, we do not yet have an understanding of the dynamical interactions of these pathways in their joint shaping of the T cell repertoire. Complete DiGeorge Anomaly is a developmental abnormality that results in the failure of the thymus to develop, absence of T cells, and profound immune deficiency. After receiving thymic tissue grafts, patients suffering from DiGeorge anomaly develop T cells derived from their own precursors but matured in the donor tissue. We followed three DiGeorge patients after thymus transplantation to utilize the remarkable opportunity these subjects provide to elucidate human T-cell developmental regulation. Our goal is the determination of the respective roles of TCR-specific vs. TCR-nonspecific regulatory signals in the growth of these emerging T-cell populations. During the course of the study, we measured peripheral blood T-cell concentrations, TCRβ V gene-segment usage and CDR3-length spectratypes over two years or more for each of the subjects. We find, through statistical analysis based on a novel stochastic population-dynamic T-cell model, that the carrying capacity corresponding to TCR-specific resources is approximately 1000-fold larger than that of TCR-nonspecific resources, implying that the size of the peripheral T-cell pool at steady state is determined almost entirely by TCR-nonspecific mechanisms. Nevertheless, the diversity of the TCR repertoire depends crucially on TCR-specific regulation. The estimated strength of this TCR-specific regulation is sufficient to ensure rapid establishment of TCR repertoire diversity in the early phase of T cell population growth, and to maintain TCR repertoire diversity in the face of substantial clonal expansion-induced perturbation from the steady state.

SpA: web-accessible spectratype analysis: data management, statistical analysis and visualization

SUMMARY SpA is a web-accessible system for the management, visualization and statistical analysis of T-cell receptor spectratype data. Users upload data from their spectratype analyzers to SpA, which saves the raw data and user-defined supplementary covariates to a secure database. The statistical engine performs several data analyses and statistical summaries. The visualization engine displays spectratype histograms in a Java applet and in an image file suitable for download. All of these results are also saved to the database and remain accessible to the user. Additional statistical tools specific to the analysis of multiple spectratypes are also available through the SpA interface. AVAILABILITY The service is freely accessible via the web at http://www.duke.edu/~kepler/spa.html. Additional technical support and specialized statistical analysis and consultation are available by arrangement with the authors and, depending on the service requested, may be subject to fee.

Use of Allograft Biopsies to Assess Thymopoiesis after Thymus Transplantation

Thymus allograft biopsies were performed in athymic infants with complete DiGeorge anomaly after thymus transplantation to assess whether the thymus allograft tissue was able to support thymopoiesis. Forty-four consecutive infants were treated with postnatal cultured thymus allografts. Thirty biopsies and six autopsies evaluating the allograft site were obtained in 33 infants, 23 of whom survive. The allograft was examined by immunohistochemistry for evidence of thymopoiesis. Grafted thymus tissue was found in 25 of 30 biopsies, 23 of which showed thymopoiesis. All 19 survivors with thymopoiesis on biopsy developed naive T cells and T cell function. Autopsies were done in six subjects, three of whom had biopsies. All autopsy samples contained thymus tissue including one for which the biopsy had not contained graft. Of the six autopsies, one had evidence of thymopoiesis. Epithelium without thymopoiesis was seen in two of 25 biopsies in which thymus tissue was detected and in five of six autopsies. Graft rejection was seen in one autopsy. Biopsies were important for showing the following: 1) the damaging effect of pulse steroids on thymopoiesis; 2) the need for adequate immunosuppression of atypical subjects; and 3) the presence of thymopoiesis in the presence of ongoing immunosuppression. In addition, the biopsy could rule out graft rejection in the atypical subjects who had oligoclonal T cells that could cause rejection. In summary, combining biopsy and autopsy data, allogeneic thymus tissues showed thymopoiesis in 24 of 29 (86%) evaluable transplants. The results of these biopsies led to improved care of these complex patients.

Quantification of total T-cell receptor diversity by flow cytometry and spectratyping

BackgroundT-cell receptor diversity correlates with immune competency and is of particular interest in patients undergoing immune reconstitution. Spectratyping generates data about T-cell receptor CDR3 length distribution for each BV gene but is technically complex. Flow cytometry can also be used to generate data about T-cell receptor BV gene usage, but its utility has not been compared to or tested in combination with spectratyping.ResultsUsing flow cytometry and spectratype data, we have defined a divergence metric that quantifies the deviation from normal of T-cell receptor repertoire. We have shown that the sample size is a sensitive parameter in the predicted flow divergence values, but not in the spectratype divergence values. We have derived two ways to correct for the measurement bias using mathematical and statistical approaches and have predicted a lower bound in the number of lymphocytes needed when using the divergence as a substitute for diversity.ConclusionsUsing both flow cytometry and spectratyping of T-cells, we have defined the divergence measure as an indirect measure of T-cell receptor diversity. We have shown the dependence of the divergence measure on the sample size before it can be used to make predictions regarding the diversity of the T-cell receptor repertoire.

Mechanisms of Tolerance to Parental Parathyroid Tissue when Combined with Human Allogeneic Thymus Transplantation

BACKGROUND The induction of tolerance toward third-party solid organ grafts with allogeneic thymus tissue transplantation has not been previously demonstrated in human subjects. OBJECTIVE Infants with complete DiGeorge anomaly (having neither thymus nor parathyroid function) were studied for conditions and mechanisms required for the development of tolerance to third-party solid organ tissues. METHODS Four infants who met the criteria received parental parathyroid with allogeneic thymus transplantation and were studied. RESULTS Two of 3 survivors showed function of both grafts but subsequently lost parathyroid function. They demonstrated alloreactivity against the parathyroid donor in mixed lymphocyte cultures. For these 2 recipients, parathyroid donor HLA class II alleles were mismatched with the recipient and thymus. MHC class II tetramers confirmed the presence of recipient CD4(+) T cells with specificity toward a mismatched parathyroid donor class II allele. The third survivor has persistent graft function and lacks alloreactivity toward the parathyroid donor. All parathyroid donor class II alleles were shared with either the recipient or the thymus graft, with minor differences between the parathyroid (HLA-DRB1∗1104) and thymus (HLA-DRB1∗1101). Tetramer analyses detected recipient T cells specific for the parathyroid HLA-DRB1∗1104 allele. Alloreactivity toward the parathyroid donor was restored with low doses of IL-2. CONCLUSION Tolerance toward parathyroid grafts in combined parental parathyroid and allogeneic thymus transplantation requires matching of thymus tissue to parathyroid HLA class II alleles to promote negative selection and suppression of recipient T cells that have alloreactivity toward the parathyroid grafts. This matching strategy may be applied toward tolerance induction in future combined thymus and solid organ transplantation efforts.

Genotypic divergence precedes clinical dissemination in a case of synchronous bilateral B-cell malignant lymphoma of the testes

Malignant lymphoma of the testis occurs bilaterally more often than any other tumor type. We report the case of a 62-year-old man who presented with synchronous, bilateral, testicular malignant lymphomas without clinical or radiologic evidence of extratesticular disease. The patient received no therapy other than bilateral orchiectomy and subsequently developed widespread disease 6 months later. Southern blot DNA analysis was performed on the initial orchiectomy samples for immunoglobulin (Ig) gene rearrangements. These genotypic analyses showed different clonal rearrangements in the Ig heavy chain JH region but identical clonal rearrangements in the Ig light chain C Kappa region. To our knowledge this is the first genotypic demonstration of a common clonal origin in synchronous, bilateral, testicular malignant lymphomas. We interpret these findings as molecular evidence that the patients malignant lymphoma was already disseminated at initial presentation, although it was clinically undetectable at that time.