- Email: [email protected]
David’s Story
C H A P T E R
25
David’s Story William T. Shearer1,2, Carol Ann Demaret1,2 1
The David Center, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas, USA 2 The Department of Pediatrics, Immunology, Allergy, and Rheumatology, Baylor College of Medicine, Houston, Texas, USA O U T L I N E
Introduction
313
Post-Mortem Discoveries
319
Birth and Early Years
313
Research Studies on David
314
David’s Immortalized B-cell Line: Contributions to Science
321
Immunological Studies
314
Personal Reflections
321
A Mother’s Recollection: Carol Ann Demaret
323
Haploidentical T-Cell Depleted Bone Marrow Transplant
318
INTRODUCTION David Vetter was born when the world of immunology was exploding with new discoveries of T-cells, B-cells, antibody replacement therapy, and restoration of severe immune deficiencies with bone marrow containing hematopoietic stem cells. David’s life within the sterile bubble taught the world about the power of the immune system by demonstrating what precautions a child with severe combined immunodeficiency (SCID) had to endure to avoid life-threatening infections. Investigators of primary immune deficiency disease and secondary
immune deficiency (e.g. HIV infection) benefited from the study of David’s immune system and his unfortunate demise subsequent to reactivation of Epstein–Barr (EBV)-containing donor lymphocytes that released infectious virus. Had David survived, he would be 43 years of age. Here is his story.
BIRTH AND EARLY YEARS David was the second son with SCID born in 1971 to young parents in suburban Houston. His brother had passed away within a few months
Primary Immunodeficiency Disorders: A Historic and Scientific Perspective Copyright © 2014 Elsevier Inc. All rights reserved
313
314
25. David’s Story
of his birth, due to uncertainty of the cause of his infections and delayed diagnosis. Soon after the brother’s diagnosis of SCID was made, he expired from Pnueumocystis jirovecii pneumonia. With the subsequent pregnancy, prenatal testing of David’s mother revealed that her fetus was a male, and preparations were made to deliver the baby by Caesarean section and to place him immediately in a sterile plastic isolator (originally designed by NASA engineers for the isolation of moon rocks and subsequently modified for David). Just after David entered that permanent isolation, he was baptized with sterile holy water by one of his doctors, Raphael Wilson, who was also a catholic brother of the Holy Cross Order. To the dismay of all, David’s lymphocyte testing showed a lack of E-rosetting cells (i.e. T-cells; see Chapter 5) and absence of stimulation of these cells with the universal lymphocyte stimulant, phytohemagglutinin (PHA). Also disappointing was the non-matching of David’s and his only sibling’s (Katherine) HLA antigens, thus preventing a regular bone marrow transplant. David’s doctors at that time, Mary Ann South, MD, Jack R. Montgomery, MD, MBA, and Raphael Wilson, PhD, were frustrated at the inability to restore David’s immune system because HLA-mismatched hematopoietic stem cell transplants were not available at that time. A decision was made by the parents and physicians to keep David in his sterile environment until further developments might provide immune reconstitution: either David’s immune system would develop in a germ-free environment, or technology would be designed to cross the HLA barrier. With that decision, the hospital provided an expanding suite of rooms to accommodate a growing child, (larger and larger bubbles). All items of life into and out of the sterile system had to be passed through a sterilizing chamber. An impressive list of consultants gave advice on every aspect of David’s life, including neurodevelopment and neurocognition, so that David would have as close to a normal life as possible, given the unique nature of his existence.
RESEARCH STUDIES ON DAVID Scientific publications on David were reviewed for preparation of this chapter that chronicles his life from birth in 1971 to his death in 1984. These publications include research on his family history (1–3), infection control (4, 5), immune system and responses (6–10), hematology (11), nutrition (12), mental and psychosocial development (13), speech and language development (14), psychiatric condition (15), bone marrow transplantation (16), development of cancer (17), detection of genetic mutation for SCID (17) and post-mortem B-cell studies (18,19).
IMMUNOLOGICAL STUDIES Many of these publications concerned David’s life up to four years of age and are contained in a monograph authored by the medical professionals who were responsible for his medical care, such as his nutritional status, neurological and language development, and psychiatric condition (1). These experts continued to follow David after his early years as the need arose. Aside from his immunological deficits, other studies revealed a normally developing boy without major problems, due to the intense efforts of parents and medical personnel to provide him with a normal young life. A longitudinal assessment of David’s few lymphocytes demonstrates that his serum immunoglobulin values and his T- and B-cell numbers changed little over his 12 year life span (Tables 25.1–25.4). At birth, his absolute lymphocyte count was 300–440 cells/mL (normal 3400–7600) and his lymphocytes showed no response to phytohemagglutinin (PHA) and only a weak response to allogeneic antigens in mixed lymphocyte culture. The serum IgM was present, but low; IgA was absent and the serum IgG value showed a decline of maternal IgG over the ensuing months eventually becoming undetectable (7) (Table 25.1). There was no antibody response to keyhole limpet hemocyanin;
315
Immunological Studies
TABLE 25.1 David’s serum immunoglobulin values over the first 4 years Immunoglobulins (mg/100 mL)
TABLE 25.2 Mitogenic response of David’s lymphocytes in tests using isolated leukocyte technique (birth to 27 months) Simulation index*: patient (control)
Age
IgM
IgG
IgA
Birth
16
560
0
Age, months (+ days)
1 wk
22
560
0
3 wk
30
ND*
ND
7 wk
10
305
0
3 mo
32
130
0
4 mo
29
135
0
6 mo
22
120
0
7 mo
17
105
0
10 mo
11
37
0
11 mo
8
27
0
12 mo
11
28
ND
13 mo
7
0
0
28 mo
6
0
0
39 mo
15
0
1.2
42 mo
17
0
3.7
43 mo
12
0
6.7
46 mo
10
0
<5.8
*ND: not done. Taken with permission from South MA, Montgomery JR, Richie E, et al. Immunologic studies. Pediatr Res 1977;11:71-78.
PHA
PWM
(Birth)
<1.0 (8.0)
1.5 (12.0)
0 (1)
0.6 (8.6)
1.5 (1.5)
1
1.0 (5.0)
1.3 (15.0)
4 (5)
1.4 (30.0)
1.3 (9.0)
5 (3)
1.2 (5.0)
0.9 (3.5)
2 (22)
MLC†
<1.0 (25.9) (5.9)
3 (16)
5 (20)
<1.0 (10.0)
1.5 (15.8) 1.2 (18.5)
6 (30) <1.0 (83.0) 11 (23)
<1.0 (453.0)
1.0 (77.0)
13 (20)
0.7 (483.0)
1.1 (188.0)
15 (20)
0.9 (127.0)
15 (23)
0.9 (101.0)
16 (12)
2.6 (31.0)
0.4 (114.0)
0.8 (250.0)
1.2 (122.0)
0.7 (24.0
1.0 (27.0)
0.7 (15.0)
2.0 (15.0)
2.4 (22.7) 18 (8)
David’s chest X-ray showed no thymic shadow; phagocytic function was normal by the Rebuck skin window technique; and serum complement values were normal. T-cell function was absent as measured by the blastogenic response to PHA and there was no change in the results over several years (7,10) (Tables 25.2, 25.4). Also, in assessing his T-cell responses, the child failed to reject an allogeneic skin patch (from Dr South) placed on his arm (7). Injections of thymosin did not change his T-cell function (7). Lymphocyte markers revealed an average of 50–100% for Bcells (surface Ig+) and 3–12 percent for T-cells (E-rosette forming lymphocytes). There was no
0.6 (130.0)
18 (23) 27 (26)
1.8 (11.0)
*The stimulation index is the ratio of counts per min in stimulated lymphocytes to counts per min in unstimulated lymphocytes in radioactive assay. †MLC: mixed lymphocyte culture; PHA: phytohemagglutinin; PWM: pokeweed mitogen. Values for control subject are within parentheses. Taken with permission from South MA, Montgomery JR, Richie E, et al. Immunologic studies. Pediatr Res 1977;11:71-78
evidence of a delayed ontogeny of the T-cell subsets taking place over the years. As David grew older, additional immunological studies confirmed all of the above findings with upgraded laboratory technology (10).
Date Age Serum Ig levels (mg/dL) Unstimulated immunoglobulin-secreting cells/106 Mononuclear cells (mg/dL) IgG
IgA
IgM
10/24/79
8 yr 1 mo
5
20
40
12/9/80
9 yr 3 mo
8
16
49
6/7/82
10 yr 9 mo
8
13
37
6/1/83
11 yr 9 mo
8
12
42
Normal range*
631–1298
70–312
56–258
61–1384
IgG
IgA
2
93
102–2337
3–358
IgM
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TABLE 25.3 David’s immunoglobulin data 8–12 years of age Fluorescent anti-human Ig staining (%) IgM
IgD
IgG
IgA
13
18
30
42
1
6
1.6–14.4
0.0–8.0
0.0–4.7
6.1–17.5
Polyvalent 15
44
2.3–16.3
41
*95% confidence limits (normal values for serum Ig levels were 9-11-year-old control subjects). Taken with permission from Paschall VL, Brown LA, Lawrence EC, et al. Immunoregulation in an isolated 12-year-old boy with congenital severe combined immunodeficiency. Pediatr Res 1984;18: 723-728.
Date Age
Unstimulated response (cpm × 103)
PHA (10 mg/mL) net response (cpm × 103)
Con A (10 mg/mL) net response (cpm × 103)
PWM (1:640) net response (cpm × 103)
1.2 (109)†
23.2 (8)†
15.4 (7)†
17.2 (19)†
5/24/79
7 yr 8 mo
7/18/79
7 yr 10 mo
8/13/79
7 yr 11 mo
3/12/80
8 yr 6 mo
3/27/81
9 yr 6 mo
0.2 (28)
6.6 (4)
0.3 (.6)
6/7/82
10 yr 9 mo
0.8 (26)
13.5 (9)
6/7/83
11 yr 9 mo
0.2 (18)
Normal range*
0–3
21–235
0.02 (10)
Unstimulated response (cpm × 103)
Candida (1:100) net response (cpm × 103)
SK-SD (1:100) net response (cpm × 103)
Tetanus (1:100) net response (cpm × 103)
2.2 (0.8)† 0.2 (1)†
0.1 (.4)†
0.2 (.4)†
0.02 (2)
0.1 92)
0.02 (.2)
0.5 (7)†
5.4 (9)
0.4 (45)
0.1 (4)
0.2 (7)
0.1 (5)
8.4 (6)
9.4 (7)
0.2 (3)
0.2 (2)
0
0.2 (5)
9.8 (5)
7.3 (4)
5.0 (3)
0.3 (1)
0
0
0
6–125
34–226
0–6
0–13
0–17
0–9
>2.0
2.4 (2)
0.9 (1)
MLC response (cpm × 103)
4.5 (4)
3.0
Stimulation index †Percentage of control of the same day ‡Monocyte depleted §95% confidence limits. Only the concentration of mitogens or antigens showing the largest responses of the patient are presented. Taken with permission from Paschall VL, Brown LA, Lawrence EC, et al. Immunoregulation in an isolated 12-year-old boy with congenital severe combined immunodeficiency. Pediatr Res 1984;18: 723-728.
25. David’s Story
TABLE 25.4 David’s mitogen specific antigen, and mixed leukocyte culture responses 8–12 years of age
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Immunological Studies
Table 25.3 gives results of testing serum Ig levels and secreting B-cells in culture from age eight to 12 years of age. David’s B-cells secreted some IgM, IgD and IgG, but virtually no IgG. His in vitro T-cell responses to several mitogens and antigens were essentially zero, confirming his complete inability to activate his T-cells; however, his lymphocytes did possess weak responses to allogeneic antigens (see Tables 25.2, 25.4) (7, 10). With the arrival of the flow cytometer, it became possible to detect surface markers on blood mononuclear cells. These markers for T-cells and B-cells failed to demonstrate a movement to normal values with time. Table 25.5 compares the older E-rosetting technique with the new technique of staining surface markers (e.g. T3 equivalent to CD3) and the results generally confirm each other. Also shown is the high percentage of B-cells (B1 equivalent to CD20, the mature B-cell marker). Based on in vitro experiments of David’s B-cells cultured in the presence of normal monocytes,
his immunoglobulin production was only 12% of normal values. In addition to these T- and B-cell defects, David’s natural killer (NK) cell function was grossly abnormal. The results showed that his NK cells could bind to target tumor cells but could not kill them (10). Aside from the abnormal immunological system that David inherited, his life was as normal as could be expected, growing from infancy to early adolescence in a “sterile” world. Despite his weak T- and B-cell immune response, David possessed some measure of immune protection, presumably due to his normal neutrophil function, since after years of isolation, it was determined that his bubble system actually contained a biofilm of low virulence bacteria (5). David’s immune system from the beginning to the end was characterized as lymphopenia with low percentages and absolute counts of CD4+ T and CD8+ T-cells, and his T-cells did not respond to the usual extracellular signals, such as mitogens and antigens.
TABLE 25.5 Characteristics of David’s mononuclear cell subpopulations, 9–12 years of age A. Date
Age
E rosettes (%)
7/18/79
7 yr 10 mo
5.0
8/13/79
7 yr 11 mo
6.9
3/27/81
9 yr 6 mo
15.8
6/7/82
10 yr 9 mo
4.0
6/1/83
11 yr 9 mo
15.1
Normal range
43.4–81.0
B. Date
Age
5/20/81
%T3
%T4
%T8
9 yr 8 mo
30.9
19.3
7/21/81
9 yr 10 mo
40.4
10/26/81
10 yr 1 mo
12/16/81
10 yr 3 mo
6/7/83
11 yr 9 mo
Normal* range
%T11
T4:T8
%Ia1
15.3
1.3
47.7
21.1
17.7
1.2
24.9
17.7
13.4
25.0
14.3
3.7
3.9
49.3
42.2
6.2
12.9
5.5
1.0
1.1
5.5
60.1
32.6
25.0
11.6
11.0
12.7
3.8
0
13.1
3.3
68.1
45.4
29.7
54–97
60–85
45–60
15–25
0–2
3–5
1.5–3.5
5–20
5–10
10–25
22.7
%T6
%T10
%B1
%M1 0
Equivalence of surface markers: T11 (total T-cells); T3, (CD3), T8 (CD8), T6 (immature thymocytes), T10 (Immature activated T-Cells), Ia1 (DR), B1 (CD20), and M1 (CD14). *95% confidence limits. Taken with permission from Paschall VL, Brown LA, Lawrence EC, et al. Immunoregulation in an isolated 12-year-old boy with congenital severe combined immunodeficiency. Pediatr Res 1984;18(8):723-728.
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25. David’s Story
HAPLOIDENTICAL T-CELL DEPLETED BONE MARROW TRANSPLANT At 12 years of age, David began verbally to express his concern that he might never get out of his bubble. He was able to do that to a limited degree in the sterile transport isolator that he used for going to and from his suburban home, where another large isolator system maintained him. He had daily contact with grade school friends who came by with his homework assignments, and David was connected to his classroom through a speaker phone system. Despite this participation in home and school activities, David longed to be free from his isolator system. At this time, developments in transplantation were taking place in the research laboratories and hospitals whereby it would be possible to cross the HLA barrier and avoid fatal graftversus-host disease (20, 21). This new approach greatly reduced the number of mature T-cells contained in bone marrow given to patients. These mature T-cells were removed from donor bone marrow by lysis using, for instance, antiCD6 monoclonal antibody and complement (20); thus eliminating most attacking T-cells and allowing the stem cells in the donor bone marrow to repopulate the patient with donor stem cells that develop into normal T-cells. With approval of the Baylor College of Medicine Institutional Review Board and with David’s assent and his parent’s written informed consent, the transplantation of David’s sister’s bone marrow (7.87 × 107 cells/kg of T-cell depleted) was performed on October 21, 1983 without conditioning and without prophylaxis for graft-versushost disease (16). Resident T-cells in the donor bone marrow were reduced from 21 to 1% after the third lytic process. David had been brought from his home isolator via the transplant isolator to his hospital isolator at Texas Children’s Hospital for the procedure. He was monitored for chimerism, immune reconstitution and graft-versus-host disease.
David went home for the Christmas holiday, but when he returned to the hospital, he developed high-spiking fever, thrombocytopenia, erratic lymphocytosis and hyper IgM globulinemia (Fig. 25.1). At day 109 of the transplant, David had severe abdominal pain and bloody stools. David was removed from his isolator system with his parents’ consent and was noted
FIGURE 25.1 Clinical course of David after bone marrow transplantation. WBC denotes white blood cell count. Taken with permission from Shearer WT, Ritz J, Finegold MJ, et al. Epstein–Barr virus-associated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985;312:1151-1159.
Post-Mortem Discoveries
to be dehydrated with an enlarged liver. Intravenous IgG (400 mg/kg), and packed, washed, and irradiated blood and platelets were infused. Solumedrol (3 mg/kg) was given on the presumption of graft-versus-host disease on the basis of focal crypt epithelial necrosis seen at intestinal and rectal biopsy. Radiolabeled erythrocytes and nuclear scanning revealed multiple small bleeding sites in the terminal ileum, cecum, and colon. He expired on transplant day 124.
POST-MORTEM DISCOVERIES The principal post-mortem finding was the presence of numerous small tumor nodules scattered throughout the patient’s intestines and infiltrating his liver, gall bladder, spleen, thymus, parathyroids and stomach. Light microscopy
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revealed highly pleomorphic infiltrates of plasma cells and large and small lymphoid cells. Many mitotic figures were observed in these infiltrating cells. In addition, immunoblasts with vesicular nuclei were prominently seen in the jejunum. A large retroperitoneal mass of lymphoproliferative cells was seen. Hassall’s corpuscles were absent in the apparent thymus, but mostly infiltrating cells of a malignant nature were found. None of the tumor tissue contained the sister’s HLA-B35 haplotype (not shared by David). Several tests of David’s pre-mortem mononuclear blood cells did not contain this donor antigen, suggesting that no engraftment of donor cells had taken place. Instead Epstein–Barr virus was found in transformed B-cells in tumor masses and in peripheral blood B-cells from day 117 of the transplant (Fig. 25.2). The source of the virus was likely the donor B-cells contained in
FIGURE 25.2 Autoradiograms of slot-blot DNA hybridizations for EBV genomes in multiple tumor specimens. DNA specimens were hybridized with a radiolabeled EBV-specific DNA probe after application of tissue DNA to nylon membranes. Tissue DNA was applied to the membrane with a slot-blot apparatus that deposits the DNA in a uniform area on the membrane. Specimen A was derived from a thymic lesion, B from a lung lesion, C from a liver lesion, D from a spleen lesion, E from a jejunal lesion, F from an appendiceal lesion, G from a retroperitoneal lesion, H from a grossly and microscopically normal kidney, and I from a peripheral-blood leukocyte fraction from the allograft donor. J was derived from grossly and microscopically normal heart tissue removed at autopsy. For each tissue sample the intensity of the dark hybridization signal correlates with the number of EBV genomes in the tissue. For use as standards, varying amounts of EBV probe DNA were added to EBV-negative tonsil DNA in mixtures totaling 5 mg of DNA, before application to the membrane. The numbers of genome equivalents per cellular DNA equivalent in these standards are indicated at the right. Taken with permission from Shearer WT, Ritz J, Finegold MJ, et al. Epstein–Barr virus-associated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985;312:1151-1159.
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25. David’s Story
the transplant. The EBV serology of the donor was positive for past infection (antibody to viral capsid antigens): IgM titer 1:5 and IgG titer 1:320; antibody to EBV nuclear antigen 1:8, and antibody to early antigen (R or D) <1:10. Studies of immunoglobulin gene rearrangement in
multiple tumor specimens indicated that the tumors arose from monoclonal and oligoclonal B-cell proliferation (14). In retrospect, the events of David’ attempted bone marrow transplant indicated that the donor lymphocytes never engrafted (Table 25.6).
TABLE 25.6 Results of the pre- and post haploidentical T-cell-depleted transplant analysis of peripheral-blood lymphocytes After Transplant
Before Transplant
10 days
30 days
60 days
90 days
120 days
Normal Range (±2 S.D.)
5.1
5.5
4.7
3.4
3.3
3.0
4.5–14.5
Per cent
4
7
7
6
8
27
28–48
Per microliter
204
385
329
204
264
810
1260–6900
E rosettes (T-cells)
15
6
1
2
ND
ND
43–74
SIg cells (B-cells)
41
38
47
63
61
55
3–19
T3
11
7
8
3
2
1
38–86
T4
13
5
5
2
2
2
23–58
T8
4
2
7
2
4
2
13–33
T6
1
3
0
0
1
0
0–4
T10
13
5
15
12
11
58
0–26
T11
12
11
12
13
21
7
51–100
NKH1
ND
4
15
15
20
40
5–33
B1
45
17
15
13
25
49
2–14
Mo2
ND
12
27
19
14
14
1–30
68
18
38
25
21
79
11–40
Phytohemagglutinin
10
7
4
1
0
0
64–297
Concanavalin A
7
2
1
0
0
0
69–283
Pokeweed mitogen
5
5
2
1
0
0
34–226
Leukocytes (per microliter; ×10−3) Lymphocytes
Surface markers (%)
Antigens on cells (%)
Ia –3
Mitogen reactivity (×10 cpm)
*SIg denotes suface immunoglobulin, and ND not done. See Table 25.5 for identification of surface markers. NK H1: natural killer cells, Mo2: monocytes. Taken with permission Shearer WT, Ritz J, Finegold MJ, et al. Epstein-Barr virus-associated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985;312:1151-1159.
Personal Reflections
Suggestive evidence is that the weak 2-to-3-fold response to alloantigen in mixed lymphocyte cultures prevented engraftment (10). Also, there must have been some degree of immune resistance in David since he lived 12 years in a “germfree” environment that contained small amounts of several bacteria. Malignant B-cells, all containing the EBV, caused David’s death. None of these tumors appeared on the surface of David’s body (as they have in other cases of post-transplant Bcell lymphoproliferative disease) rendering definitive diagnosis only at autopsy.
DAVID’S IMMORTALIZED B-CELL LINE: CONTRIBUTIONS TO SCIENCE In the early 1990s, intense efforts were initiated to establish the genetic causes of primary immunodeficiencies, particularly the genetic defect that caused David’s disease, namely X-linked SCID (13). Noguchi et al. (17) were the first to report that X-linked SCID was caused by mutations in the gamma chain of the interleukin-2 receptor. This landmark discovery was made in the lymphocytes of three male unrelated children who carried the phenotypic diagnosis of X-linked SCID. One of these patient specimens was that of David, his EBV-transformed B-cell line (18,19) (Fig. 25.3). Each of the children’s lymphocytes carried a different mutation in the Xg13 region of the human X chromosome. The concordance of the genetic defect observed in David’s B-cell line with that of two other nonrelated children firmly established the nature and chromosome locations of the gene responsible for X-linked SCID.
PERSONAL REFLECTIONS Memories of David the Bubble Boy On my first day as a new recruit to Baylor College of Medicine, Department of Pediatrics, and
321
Texas Children’s Hospital, I knew from David’s firm handshake through plastic gloves, his direct eye contact and strong personality that he was a special child, far advanced in mental age and very much aware of his extraordinary life. On that first day at Texas Children’s Hospital on September 1, 1978 (Fig. 25.4), I had no idea what this young boy would teach the world about human courage, immune resistance to infection and cancer, and about the gene discovery of the cause of X-linked SCID. David taught the world about T-cells, graft-versus-host disease, inheritance patterns of immunodeficiency, escape of virus from carrier B-cells and infection of target cells, rapid growth of virus-transformed tumors and how protective isolation prevents infection. I became part of David’s extended family, and grew to know his mother, Carol Ann Demaret, his father, David Vetter and his sister, Katherine Vetter, then just entering her teenage years. My team of faculty and fellows saw David almost every day and we were privileged to witness his bravery and his brilliance in understanding who he was to the world – a child who lived his entire life in a highly technological world never before and never since attempted. His questions to me were always sharp and well thought out, and many times caught me off-guard. David would have been 40 years of age on September 21, 2011, but this celebration in his honor with his parents and family, friends, doctors, nurses and hospital staff is testimony to his immortal spirit, alive with perpetual youth and undiminished hope. Far more than any professor, book, or lecture, David’s life was a lesson that will forever continue to teach us about the intricate yet mysterious mechanisms that permit life in a hostile microbial world. Today, I speak to David’s family and to all the families who have entrusted the care of their children to us. Thank you. Your children have taught us so much, not only about human immunity and its power to preserve life, but also about their will to succeed against great obstacles and emerge victorious. We are grateful for
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25. David’s Story
FIGURE 25.3 XSCID patients have mutations in the IL-2Rg gene. (A) Pedigrees of the XSCID patients’ studies. Circles, female; squares, male; closed squares, males with SCID; squares with slashes, deceased males. Small squares and diamond in the pedigree of patient 2 indicate miscarriages (male and of undetermined sex, respectively). David is patient 3. (B) Sequencing of the IL-2Rg gene. Shown is the sequence of DNA from a normal donor (left panel in each pair) and of DNAs from patients 1, 2, and 3 (right panel in each pair). Patient 1 has an AAA (Lys) to TAA (stop codon) transversion in exon 3, resulting in a truncation of the carboxy-terminal 251 amino acids; patient 2 has a CGA (Arg) to TGA (stop codon) transition in exon 7, resulting in the truncation of 81 amino acids; and patient 3 has a TCG (Ser) to TAG (stop codon) transversion in exon 7, resulting in the truncation of 62 amino acids. The location of each mutation is indicated in the sequence to the right of each set of panels. The sequence shown is complementary to the coding strand. The boxed nucleotides for patients 1, 2, and 3 are complementary to TAA, TGA, and TAG stop codons, respectively. (C) Schematic showing locations of the artificial stop codons (diagonal arrows) present in the XSCID patients. Patient 1 has a premature stop codon in exon 3, and patients 2 and 3 have premature stop codons in exon 7. UTR: Untranslated region. Taken with permission from Noguchi M, Yi H, Rosenblatt HM, et al. Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 1993;73:147-157.
A Mother’s Recollection: Carol Ann Demaret
323
FIGURE 25.4 The first time I met David, the bubble boy. Dr William T. Shearer meets David on September 1, 1978 on his first day at Texas Children’s Hospital and shakes hands with his new patient. Dr Buford Nicols who directed David’s nutritional program looks on. David is almost seven years of age in this picture.
the legacy David and all the special children after him have given to the world. Delivered in David’s memory upon the 40th anniversary of his birth, September 21, 2011, The David Center Texas Children’s Hospital Houston, Texas.
A MOTHER’S RECOLLECTION: CAROL ANN DEMARET Now and then I dream of David and we laugh and I feel touched and good when I awaken. In many ways, David’s life at home was “normal”. I could care for him, change his diapers as a baby and clothed him and fed him the foods that were packaged in sterile, air-locked space age “supple cylinders” that were replenished weekly. I
couldn’t touch him, but I could feel his warmth, as I embraced and held him close through the plastic and I believed he could feel mine, both as an infant and a boy. I cut his hair, and bathed him and changed his clothes when he was small and did most of the child care that all mothers do. As he grew, his bubbles grew in size and numbers – at home and at the hospital. He was happy to be at home with his family and he was happy to be at the hospital with his doctors and nurses and all the attention he received. However, he did not like having his blood drawn which was not an easy task. As he grew, he had sterilized toys and games, was educated by way of at-home teachers, (sometimes accompanied by classmates). He received Holy Communion, after the host had been sterilized and consecrated. He had
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25. David’s Story
FIGURE 25.5 The wall of plastic that separated David and his mother, Carol Ann Demaret. David is on the inside of his sterile isolator while his mother, Carol Ann, is on the outside. Among her many memories of David, she recalls with pleasure cutting his hair with sterile scissors. David is 11 years of age in this picture.
religious training, and family and friends who visited often. His sister slept on the floor next to him and they would talk and plot and giggle. He watched television and enjoyed favorite programs. He talked on the telephone, even had pets (outside the bubble, of course). He had a parakeet that he tried to teach to talk. There was a large window in his room, both at home and at the hospital, where he could view his surroundings. David Phillip grew with fine health, dark and handsome, highly intelligent and astonishingly perceptive, with black locks and penetrating eyes that some said seemed to see into a far beyond (Fig. 25.5). When a possible new medical technique that showed great promise finally became available, David himself decided that he wanted to participate. It was a complex treatment that involved a cellular transplant. At first it appeared to be working. But then it failed. All attempts to reverse the process were unsuccessful, and my gallant son died on February 22, 1984, age 12. We were devastated. We wept. And we felt a nation wept with us.
David not only lives on in my soul, but in other ways, too. Within a few days of David’s passing, his family was approached by Texas Children’s Hospital with an idea in tribute to David’s memory to create “The David Center” where SCID babies, and all children born with compromised or faulty immune systems whether genetic or acquired, can be successfully treated, and with continued research, immune systems could be stirred into more vigorous action, and where important advances have been made in discovering the genetic defect for SCID. A school was named after him, “The David Elementary” in the Woodlands, Texas, at one time said to be the only school in the country named after a child. One of David’s unrealized dreams was to be able “to walk barefoot in the grass”. With this in mind and spirit, each year the school sponsors a “David Dream Run” and raises contributions to “The David Center”. There is a David Memorial Drive in the neighborhood where he lived. In December of 2012, it was time to celebrate David’s legacy as it marked the beginning of
A Mother’s Recollection: Carol Ann Demaret
SCID New born Screening in Texas. Texas joins 12 other states that are currently screening all newborns for SCID. I now believe my prayer has been fully answered, and “that the bubble has burst” for all time. For David’s family and those who shared in his life, other lessons gleaned from David are important, too. He taught us that handicap of body is not necessary handicap of mind and spirit, that handicap cannot just be endured, but nobly endured. He has reminded the world of humble marvels, such as grass under our feet. All children leave behind memories and a sense of yearning and loss, but David left behind some things for humankind, too, and it helps. “He never touched the world, but the world was touched by him”, are the words inscribed on his gravestone. I believe the promise for the very near future is not just to preserve some portion of precious life, but to enhance it at the core. I carry in my heart sweet reminders of the past and great expectations for the future for all generations that follow. Presented at the 3rd Pediatric Immunodeficiency Treatment Consortium Third Annual Workshop, May 2, 2013 The David Center Texas Children’s Hospital Houston, Texas.
Acknowledgment We thank Janice Hopkins and Janelle Allen for assistance in manuscript preparation, and for reviewing the memorabilia of David’s life saved at the David Center, Texas Children’s Hospital.
References 1. Williamson AP, Montgomery JR, South MA, et al. A special report: four-year study of a boy with combined immune deficiency maintained in strict reverse isolation from birth. Pediatr Res 1977;11:63–4. 2. Blattner RJ. Introduction. Pediatr Res 1977;11:65. 3. Montgomery JR, South MA, Wilson R, et al. Family background, early history, and diagnosis. Pediatr Res 1977;11:65–6.
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4. Wilson R, Taylor GR, Kropp K, et al. Gnotobiotic care and infectious disease prevention. Pediatr Res 1977;11: 67–71. 5. Guerra IC, Shearer WT. Environmental control in management of immunodeficient patients: experience with “David”. Clin Immunol Immunopathol 1986;40:128–35. 6. Mukhopadhyay N, Richie E, Montgomery J, et al. Peripheral blood T and B-cell characteristics in a patient with severe combined immune deficiency (SCID) maintained in a gnotobiotic environment. Exp Hematol 1976;4: 1–9. 7. South MA, Montgomery JR, Richie E, et al. Immunologic studies. Pediatr Res 1977;11:71–8. 8. Mukhopadhyay N, Richie E, Mackler BF, et al. A longitudinal study of T and B lymphocytes from a three-yearold patient with severe combined immunodeficiency (SCID) in ‘gnotobiotic protection’. Exp Hematol 1978;6: 129–34. 9. Mackler BF, O’Neill PA. T-lymphocyte induction of nonT-cell-mediated nonspecific cytotoxicity. II. A clinical study of a severe combined immunodeficiency patient maintained in a gnotobiotic environment. Clin Immunol Immunopathol 1979;12:358–66. 10. Paschall VL, Brown LA, Lawrence EC, et al. Immunore gulation in an isolated 12-year-old boy with congenital severe combined immunodeficiency. Pediatr Res 1984;18: 723–8. 11. Fernbach DJ, Starling KA, Falletta JM. Hematology. Pediatr Res 1977;11:78–9. 12. Potts E, Huang CTL, Nichols BL. Nutritional care and related studies. Pediatr Res 1977;11:80–2. 13. Molish B, Murphy M, Desmond M. Mental, psychomotor, and psychosocial development. Pediatr Res 1977;11:82–3. 14. Musher KK. Speech and language development. Pediatr Res 1977;11:84–5. 15. Freedman DA. Psychiatric evaluation. Pediatr Res 1977;11:85–6. 16. Shearer WT, Ritz J, Finegold MJ, et al. Epstein-Barr virusassociated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985;312:1151–9. 17. Noguchi M, Yi H, Rosenblatt HM, et al. Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 1993;73: 147–57. 18. Rosenblatt HM, Green CG, McClure JE, et al. Antibody to human lymphocyte actin regulates immunoglobulin secretion by an EBV-transformed human B-cell line. Biochem Biophys Res Commun 1986;140:399–405. 19. Cleary ML, Nalesnik MA, Shearer WT, et al. Clonal analy sis of transplant-associated lymphoproliferations based on the structure of the genomic termini of the Epstein-Barr virus. Blood 1988;72:349–52.
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20. Reinherz EL, Geha R, Rappeport JM, et al. Reconstitution after transplantation with T-lymphocyte-depleted HLA haplotype-mismatched bone marrow for severe combined immunodeficiency. Proc Natl Acad Sci USA 1982;79:6047–51.
21. Reisner Y, Kapoor N, Kirkpatrick D, et al. Transplantation for severe combined immunodeficiency with HLA-A, B, D, DR incompatible parental marrow cells fractionated by soybean agglutinin and sheep red blood cells. Blood 1983;61:341–8.
25
David’s Story William T. Shearer1,2, Carol Ann Demaret1,2 1
The David Center, Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas, USA 2 The Department of Pediatrics, Immunology, Allergy, and Rheumatology, Baylor College of Medicine, Houston, Texas, USA O U T L I N E
Introduction
313
Post-Mortem Discoveries
319
Birth and Early Years
313
Research Studies on David
314
David’s Immortalized B-cell Line: Contributions to Science
321
Immunological Studies
314
Personal Reflections
321
A Mother’s Recollection: Carol Ann Demaret
323
Haploidentical T-Cell Depleted Bone Marrow Transplant
318
INTRODUCTION David Vetter was born when the world of immunology was exploding with new discoveries of T-cells, B-cells, antibody replacement therapy, and restoration of severe immune deficiencies with bone marrow containing hematopoietic stem cells. David’s life within the sterile bubble taught the world about the power of the immune system by demonstrating what precautions a child with severe combined immunodeficiency (SCID) had to endure to avoid life-threatening infections. Investigators of primary immune deficiency disease and secondary
immune deficiency (e.g. HIV infection) benefited from the study of David’s immune system and his unfortunate demise subsequent to reactivation of Epstein–Barr (EBV)-containing donor lymphocytes that released infectious virus. Had David survived, he would be 43 years of age. Here is his story.
BIRTH AND EARLY YEARS David was the second son with SCID born in 1971 to young parents in suburban Houston. His brother had passed away within a few months
Primary Immunodeficiency Disorders: A Historic and Scientific Perspective Copyright © 2014 Elsevier Inc. All rights reserved
313
314
25. David’s Story
of his birth, due to uncertainty of the cause of his infections and delayed diagnosis. Soon after the brother’s diagnosis of SCID was made, he expired from Pnueumocystis jirovecii pneumonia. With the subsequent pregnancy, prenatal testing of David’s mother revealed that her fetus was a male, and preparations were made to deliver the baby by Caesarean section and to place him immediately in a sterile plastic isolator (originally designed by NASA engineers for the isolation of moon rocks and subsequently modified for David). Just after David entered that permanent isolation, he was baptized with sterile holy water by one of his doctors, Raphael Wilson, who was also a catholic brother of the Holy Cross Order. To the dismay of all, David’s lymphocyte testing showed a lack of E-rosetting cells (i.e. T-cells; see Chapter 5) and absence of stimulation of these cells with the universal lymphocyte stimulant, phytohemagglutinin (PHA). Also disappointing was the non-matching of David’s and his only sibling’s (Katherine) HLA antigens, thus preventing a regular bone marrow transplant. David’s doctors at that time, Mary Ann South, MD, Jack R. Montgomery, MD, MBA, and Raphael Wilson, PhD, were frustrated at the inability to restore David’s immune system because HLA-mismatched hematopoietic stem cell transplants were not available at that time. A decision was made by the parents and physicians to keep David in his sterile environment until further developments might provide immune reconstitution: either David’s immune system would develop in a germ-free environment, or technology would be designed to cross the HLA barrier. With that decision, the hospital provided an expanding suite of rooms to accommodate a growing child, (larger and larger bubbles). All items of life into and out of the sterile system had to be passed through a sterilizing chamber. An impressive list of consultants gave advice on every aspect of David’s life, including neurodevelopment and neurocognition, so that David would have as close to a normal life as possible, given the unique nature of his existence.
RESEARCH STUDIES ON DAVID Scientific publications on David were reviewed for preparation of this chapter that chronicles his life from birth in 1971 to his death in 1984. These publications include research on his family history (1–3), infection control (4, 5), immune system and responses (6–10), hematology (11), nutrition (12), mental and psychosocial development (13), speech and language development (14), psychiatric condition (15), bone marrow transplantation (16), development of cancer (17), detection of genetic mutation for SCID (17) and post-mortem B-cell studies (18,19).
IMMUNOLOGICAL STUDIES Many of these publications concerned David’s life up to four years of age and are contained in a monograph authored by the medical professionals who were responsible for his medical care, such as his nutritional status, neurological and language development, and psychiatric condition (1). These experts continued to follow David after his early years as the need arose. Aside from his immunological deficits, other studies revealed a normally developing boy without major problems, due to the intense efforts of parents and medical personnel to provide him with a normal young life. A longitudinal assessment of David’s few lymphocytes demonstrates that his serum immunoglobulin values and his T- and B-cell numbers changed little over his 12 year life span (Tables 25.1–25.4). At birth, his absolute lymphocyte count was 300–440 cells/mL (normal 3400–7600) and his lymphocytes showed no response to phytohemagglutinin (PHA) and only a weak response to allogeneic antigens in mixed lymphocyte culture. The serum IgM was present, but low; IgA was absent and the serum IgG value showed a decline of maternal IgG over the ensuing months eventually becoming undetectable (7) (Table 25.1). There was no antibody response to keyhole limpet hemocyanin;
315
Immunological Studies
TABLE 25.1 David’s serum immunoglobulin values over the first 4 years Immunoglobulins (mg/100 mL)
TABLE 25.2 Mitogenic response of David’s lymphocytes in tests using isolated leukocyte technique (birth to 27 months) Simulation index*: patient (control)
Age
IgM
IgG
IgA
Birth
16
560
0
Age, months (+ days)
1 wk
22
560
0
3 wk
30
ND*
ND
7 wk
10
305
0
3 mo
32
130
0
4 mo
29
135
0
6 mo
22
120
0
7 mo
17
105
0
10 mo
11
37
0
11 mo
8
27
0
12 mo
11
28
ND
13 mo
7
0
0
28 mo
6
0
0
39 mo
15
0
1.2
42 mo
17
0
3.7
43 mo
12
0
6.7
46 mo
10
0
<5.8
*ND: not done. Taken with permission from South MA, Montgomery JR, Richie E, et al. Immunologic studies. Pediatr Res 1977;11:71-78.
PHA
PWM
(Birth)
<1.0 (8.0)
1.5 (12.0)
0 (1)
0.6 (8.6)
1.5 (1.5)
1
1.0 (5.0)
1.3 (15.0)
4 (5)
1.4 (30.0)
1.3 (9.0)
5 (3)
1.2 (5.0)
0.9 (3.5)
2 (22)
MLC†
<1.0 (25.9) (5.9)
3 (16)
5 (20)
<1.0 (10.0)
1.5 (15.8) 1.2 (18.5)
6 (30) <1.0 (83.0) 11 (23)
<1.0 (453.0)
1.0 (77.0)
13 (20)
0.7 (483.0)
1.1 (188.0)
15 (20)
0.9 (127.0)
15 (23)
0.9 (101.0)
16 (12)
2.6 (31.0)
0.4 (114.0)
0.8 (250.0)
1.2 (122.0)
0.7 (24.0
1.0 (27.0)
0.7 (15.0)
2.0 (15.0)
2.4 (22.7) 18 (8)
David’s chest X-ray showed no thymic shadow; phagocytic function was normal by the Rebuck skin window technique; and serum complement values were normal. T-cell function was absent as measured by the blastogenic response to PHA and there was no change in the results over several years (7,10) (Tables 25.2, 25.4). Also, in assessing his T-cell responses, the child failed to reject an allogeneic skin patch (from Dr South) placed on his arm (7). Injections of thymosin did not change his T-cell function (7). Lymphocyte markers revealed an average of 50–100% for Bcells (surface Ig+) and 3–12 percent for T-cells (E-rosette forming lymphocytes). There was no
0.6 (130.0)
18 (23) 27 (26)
1.8 (11.0)
*The stimulation index is the ratio of counts per min in stimulated lymphocytes to counts per min in unstimulated lymphocytes in radioactive assay. †MLC: mixed lymphocyte culture; PHA: phytohemagglutinin; PWM: pokeweed mitogen. Values for control subject are within parentheses. Taken with permission from South MA, Montgomery JR, Richie E, et al. Immunologic studies. Pediatr Res 1977;11:71-78
evidence of a delayed ontogeny of the T-cell subsets taking place over the years. As David grew older, additional immunological studies confirmed all of the above findings with upgraded laboratory technology (10).
Date Age Serum Ig levels (mg/dL) Unstimulated immunoglobulin-secreting cells/106 Mononuclear cells (mg/dL) IgG
IgA
IgM
10/24/79
8 yr 1 mo
5
20
40
12/9/80
9 yr 3 mo
8
16
49
6/7/82
10 yr 9 mo
8
13
37
6/1/83
11 yr 9 mo
8
12
42
Normal range*
631–1298
70–312
56–258
61–1384
IgG
IgA
2
93
102–2337
3–358
IgM
316
TABLE 25.3 David’s immunoglobulin data 8–12 years of age Fluorescent anti-human Ig staining (%) IgM
IgD
IgG
IgA
13
18
30
42
1
6
1.6–14.4
0.0–8.0
0.0–4.7
6.1–17.5
Polyvalent 15
44
2.3–16.3
41
*95% confidence limits (normal values for serum Ig levels were 9-11-year-old control subjects). Taken with permission from Paschall VL, Brown LA, Lawrence EC, et al. Immunoregulation in an isolated 12-year-old boy with congenital severe combined immunodeficiency. Pediatr Res 1984;18: 723-728.
Date Age
Unstimulated response (cpm × 103)
PHA (10 mg/mL) net response (cpm × 103)
Con A (10 mg/mL) net response (cpm × 103)
PWM (1:640) net response (cpm × 103)
1.2 (109)†
23.2 (8)†
15.4 (7)†
17.2 (19)†
5/24/79
7 yr 8 mo
7/18/79
7 yr 10 mo
8/13/79
7 yr 11 mo
3/12/80
8 yr 6 mo
3/27/81
9 yr 6 mo
0.2 (28)
6.6 (4)
0.3 (.6)
6/7/82
10 yr 9 mo
0.8 (26)
13.5 (9)
6/7/83
11 yr 9 mo
0.2 (18)
Normal range*
0–3
21–235
0.02 (10)
Unstimulated response (cpm × 103)
Candida (1:100) net response (cpm × 103)
SK-SD (1:100) net response (cpm × 103)
Tetanus (1:100) net response (cpm × 103)
2.2 (0.8)† 0.2 (1)†
0.1 (.4)†
0.2 (.4)†
0.02 (2)
0.1 92)
0.02 (.2)
0.5 (7)†
5.4 (9)
0.4 (45)
0.1 (4)
0.2 (7)
0.1 (5)
8.4 (6)
9.4 (7)
0.2 (3)
0.2 (2)
0
0.2 (5)
9.8 (5)
7.3 (4)
5.0 (3)
0.3 (1)
0
0
0
6–125
34–226
0–6
0–13
0–17
0–9
>2.0
2.4 (2)
0.9 (1)
MLC response (cpm × 103)
4.5 (4)
3.0
Stimulation index †Percentage of control of the same day ‡Monocyte depleted §95% confidence limits. Only the concentration of mitogens or antigens showing the largest responses of the patient are presented. Taken with permission from Paschall VL, Brown LA, Lawrence EC, et al. Immunoregulation in an isolated 12-year-old boy with congenital severe combined immunodeficiency. Pediatr Res 1984;18: 723-728.
25. David’s Story
TABLE 25.4 David’s mitogen specific antigen, and mixed leukocyte culture responses 8–12 years of age
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Immunological Studies
Table 25.3 gives results of testing serum Ig levels and secreting B-cells in culture from age eight to 12 years of age. David’s B-cells secreted some IgM, IgD and IgG, but virtually no IgG. His in vitro T-cell responses to several mitogens and antigens were essentially zero, confirming his complete inability to activate his T-cells; however, his lymphocytes did possess weak responses to allogeneic antigens (see Tables 25.2, 25.4) (7, 10). With the arrival of the flow cytometer, it became possible to detect surface markers on blood mononuclear cells. These markers for T-cells and B-cells failed to demonstrate a movement to normal values with time. Table 25.5 compares the older E-rosetting technique with the new technique of staining surface markers (e.g. T3 equivalent to CD3) and the results generally confirm each other. Also shown is the high percentage of B-cells (B1 equivalent to CD20, the mature B-cell marker). Based on in vitro experiments of David’s B-cells cultured in the presence of normal monocytes,
his immunoglobulin production was only 12% of normal values. In addition to these T- and B-cell defects, David’s natural killer (NK) cell function was grossly abnormal. The results showed that his NK cells could bind to target tumor cells but could not kill them (10). Aside from the abnormal immunological system that David inherited, his life was as normal as could be expected, growing from infancy to early adolescence in a “sterile” world. Despite his weak T- and B-cell immune response, David possessed some measure of immune protection, presumably due to his normal neutrophil function, since after years of isolation, it was determined that his bubble system actually contained a biofilm of low virulence bacteria (5). David’s immune system from the beginning to the end was characterized as lymphopenia with low percentages and absolute counts of CD4+ T and CD8+ T-cells, and his T-cells did not respond to the usual extracellular signals, such as mitogens and antigens.
TABLE 25.5 Characteristics of David’s mononuclear cell subpopulations, 9–12 years of age A. Date
Age
E rosettes (%)
7/18/79
7 yr 10 mo
5.0
8/13/79
7 yr 11 mo
6.9
3/27/81
9 yr 6 mo
15.8
6/7/82
10 yr 9 mo
4.0
6/1/83
11 yr 9 mo
15.1
Normal range
43.4–81.0
B. Date
Age
5/20/81
%T3
%T4
%T8
9 yr 8 mo
30.9
19.3
7/21/81
9 yr 10 mo
40.4
10/26/81
10 yr 1 mo
12/16/81
10 yr 3 mo
6/7/83
11 yr 9 mo
Normal* range
%T11
T4:T8
%Ia1
15.3
1.3
47.7
21.1
17.7
1.2
24.9
17.7
13.4
25.0
14.3
3.7
3.9
49.3
42.2
6.2
12.9
5.5
1.0
1.1
5.5
60.1
32.6
25.0
11.6
11.0
12.7
3.8
0
13.1
3.3
68.1
45.4
29.7
54–97
60–85
45–60
15–25
0–2
3–5
1.5–3.5
5–20
5–10
10–25
22.7
%T6
%T10
%B1
%M1 0
Equivalence of surface markers: T11 (total T-cells); T3, (CD3), T8 (CD8), T6 (immature thymocytes), T10 (Immature activated T-Cells), Ia1 (DR), B1 (CD20), and M1 (CD14). *95% confidence limits. Taken with permission from Paschall VL, Brown LA, Lawrence EC, et al. Immunoregulation in an isolated 12-year-old boy with congenital severe combined immunodeficiency. Pediatr Res 1984;18(8):723-728.
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25. David’s Story
HAPLOIDENTICAL T-CELL DEPLETED BONE MARROW TRANSPLANT At 12 years of age, David began verbally to express his concern that he might never get out of his bubble. He was able to do that to a limited degree in the sterile transport isolator that he used for going to and from his suburban home, where another large isolator system maintained him. He had daily contact with grade school friends who came by with his homework assignments, and David was connected to his classroom through a speaker phone system. Despite this participation in home and school activities, David longed to be free from his isolator system. At this time, developments in transplantation were taking place in the research laboratories and hospitals whereby it would be possible to cross the HLA barrier and avoid fatal graftversus-host disease (20, 21). This new approach greatly reduced the number of mature T-cells contained in bone marrow given to patients. These mature T-cells were removed from donor bone marrow by lysis using, for instance, antiCD6 monoclonal antibody and complement (20); thus eliminating most attacking T-cells and allowing the stem cells in the donor bone marrow to repopulate the patient with donor stem cells that develop into normal T-cells. With approval of the Baylor College of Medicine Institutional Review Board and with David’s assent and his parent’s written informed consent, the transplantation of David’s sister’s bone marrow (7.87 × 107 cells/kg of T-cell depleted) was performed on October 21, 1983 without conditioning and without prophylaxis for graft-versushost disease (16). Resident T-cells in the donor bone marrow were reduced from 21 to 1% after the third lytic process. David had been brought from his home isolator via the transplant isolator to his hospital isolator at Texas Children’s Hospital for the procedure. He was monitored for chimerism, immune reconstitution and graft-versus-host disease.
David went home for the Christmas holiday, but when he returned to the hospital, he developed high-spiking fever, thrombocytopenia, erratic lymphocytosis and hyper IgM globulinemia (Fig. 25.1). At day 109 of the transplant, David had severe abdominal pain and bloody stools. David was removed from his isolator system with his parents’ consent and was noted
FIGURE 25.1 Clinical course of David after bone marrow transplantation. WBC denotes white blood cell count. Taken with permission from Shearer WT, Ritz J, Finegold MJ, et al. Epstein–Barr virus-associated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985;312:1151-1159.
Post-Mortem Discoveries
to be dehydrated with an enlarged liver. Intravenous IgG (400 mg/kg), and packed, washed, and irradiated blood and platelets were infused. Solumedrol (3 mg/kg) was given on the presumption of graft-versus-host disease on the basis of focal crypt epithelial necrosis seen at intestinal and rectal biopsy. Radiolabeled erythrocytes and nuclear scanning revealed multiple small bleeding sites in the terminal ileum, cecum, and colon. He expired on transplant day 124.
POST-MORTEM DISCOVERIES The principal post-mortem finding was the presence of numerous small tumor nodules scattered throughout the patient’s intestines and infiltrating his liver, gall bladder, spleen, thymus, parathyroids and stomach. Light microscopy
319
revealed highly pleomorphic infiltrates of plasma cells and large and small lymphoid cells. Many mitotic figures were observed in these infiltrating cells. In addition, immunoblasts with vesicular nuclei were prominently seen in the jejunum. A large retroperitoneal mass of lymphoproliferative cells was seen. Hassall’s corpuscles were absent in the apparent thymus, but mostly infiltrating cells of a malignant nature were found. None of the tumor tissue contained the sister’s HLA-B35 haplotype (not shared by David). Several tests of David’s pre-mortem mononuclear blood cells did not contain this donor antigen, suggesting that no engraftment of donor cells had taken place. Instead Epstein–Barr virus was found in transformed B-cells in tumor masses and in peripheral blood B-cells from day 117 of the transplant (Fig. 25.2). The source of the virus was likely the donor B-cells contained in
FIGURE 25.2 Autoradiograms of slot-blot DNA hybridizations for EBV genomes in multiple tumor specimens. DNA specimens were hybridized with a radiolabeled EBV-specific DNA probe after application of tissue DNA to nylon membranes. Tissue DNA was applied to the membrane with a slot-blot apparatus that deposits the DNA in a uniform area on the membrane. Specimen A was derived from a thymic lesion, B from a lung lesion, C from a liver lesion, D from a spleen lesion, E from a jejunal lesion, F from an appendiceal lesion, G from a retroperitoneal lesion, H from a grossly and microscopically normal kidney, and I from a peripheral-blood leukocyte fraction from the allograft donor. J was derived from grossly and microscopically normal heart tissue removed at autopsy. For each tissue sample the intensity of the dark hybridization signal correlates with the number of EBV genomes in the tissue. For use as standards, varying amounts of EBV probe DNA were added to EBV-negative tonsil DNA in mixtures totaling 5 mg of DNA, before application to the membrane. The numbers of genome equivalents per cellular DNA equivalent in these standards are indicated at the right. Taken with permission from Shearer WT, Ritz J, Finegold MJ, et al. Epstein–Barr virus-associated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985;312:1151-1159.
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25. David’s Story
the transplant. The EBV serology of the donor was positive for past infection (antibody to viral capsid antigens): IgM titer 1:5 and IgG titer 1:320; antibody to EBV nuclear antigen 1:8, and antibody to early antigen (R or D) <1:10. Studies of immunoglobulin gene rearrangement in
multiple tumor specimens indicated that the tumors arose from monoclonal and oligoclonal B-cell proliferation (14). In retrospect, the events of David’ attempted bone marrow transplant indicated that the donor lymphocytes never engrafted (Table 25.6).
TABLE 25.6 Results of the pre- and post haploidentical T-cell-depleted transplant analysis of peripheral-blood lymphocytes After Transplant
Before Transplant
10 days
30 days
60 days
90 days
120 days
Normal Range (±2 S.D.)
5.1
5.5
4.7
3.4
3.3
3.0
4.5–14.5
Per cent
4
7
7
6
8
27
28–48
Per microliter
204
385
329
204
264
810
1260–6900
E rosettes (T-cells)
15
6
1
2
ND
ND
43–74
SIg cells (B-cells)
41
38
47
63
61
55
3–19
T3
11
7
8
3
2
1
38–86
T4
13
5
5
2
2
2
23–58
T8
4
2
7
2
4
2
13–33
T6
1
3
0
0
1
0
0–4
T10
13
5
15
12
11
58
0–26
T11
12
11
12
13
21
7
51–100
NKH1
ND
4
15
15
20
40
5–33
B1
45
17
15
13
25
49
2–14
Mo2
ND
12
27
19
14
14
1–30
68
18
38
25
21
79
11–40
Phytohemagglutinin
10
7
4
1
0
0
64–297
Concanavalin A
7
2
1
0
0
0
69–283
Pokeweed mitogen
5
5
2
1
0
0
34–226
Leukocytes (per microliter; ×10−3) Lymphocytes
Surface markers (%)
Antigens on cells (%)
Ia –3
Mitogen reactivity (×10 cpm)
*SIg denotes suface immunoglobulin, and ND not done. See Table 25.5 for identification of surface markers. NK H1: natural killer cells, Mo2: monocytes. Taken with permission Shearer WT, Ritz J, Finegold MJ, et al. Epstein-Barr virus-associated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985;312:1151-1159.
Personal Reflections
Suggestive evidence is that the weak 2-to-3-fold response to alloantigen in mixed lymphocyte cultures prevented engraftment (10). Also, there must have been some degree of immune resistance in David since he lived 12 years in a “germfree” environment that contained small amounts of several bacteria. Malignant B-cells, all containing the EBV, caused David’s death. None of these tumors appeared on the surface of David’s body (as they have in other cases of post-transplant Bcell lymphoproliferative disease) rendering definitive diagnosis only at autopsy.
DAVID’S IMMORTALIZED B-CELL LINE: CONTRIBUTIONS TO SCIENCE In the early 1990s, intense efforts were initiated to establish the genetic causes of primary immunodeficiencies, particularly the genetic defect that caused David’s disease, namely X-linked SCID (13). Noguchi et al. (17) were the first to report that X-linked SCID was caused by mutations in the gamma chain of the interleukin-2 receptor. This landmark discovery was made in the lymphocytes of three male unrelated children who carried the phenotypic diagnosis of X-linked SCID. One of these patient specimens was that of David, his EBV-transformed B-cell line (18,19) (Fig. 25.3). Each of the children’s lymphocytes carried a different mutation in the Xg13 region of the human X chromosome. The concordance of the genetic defect observed in David’s B-cell line with that of two other nonrelated children firmly established the nature and chromosome locations of the gene responsible for X-linked SCID.
PERSONAL REFLECTIONS Memories of David the Bubble Boy On my first day as a new recruit to Baylor College of Medicine, Department of Pediatrics, and
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Texas Children’s Hospital, I knew from David’s firm handshake through plastic gloves, his direct eye contact and strong personality that he was a special child, far advanced in mental age and very much aware of his extraordinary life. On that first day at Texas Children’s Hospital on September 1, 1978 (Fig. 25.4), I had no idea what this young boy would teach the world about human courage, immune resistance to infection and cancer, and about the gene discovery of the cause of X-linked SCID. David taught the world about T-cells, graft-versus-host disease, inheritance patterns of immunodeficiency, escape of virus from carrier B-cells and infection of target cells, rapid growth of virus-transformed tumors and how protective isolation prevents infection. I became part of David’s extended family, and grew to know his mother, Carol Ann Demaret, his father, David Vetter and his sister, Katherine Vetter, then just entering her teenage years. My team of faculty and fellows saw David almost every day and we were privileged to witness his bravery and his brilliance in understanding who he was to the world – a child who lived his entire life in a highly technological world never before and never since attempted. His questions to me were always sharp and well thought out, and many times caught me off-guard. David would have been 40 years of age on September 21, 2011, but this celebration in his honor with his parents and family, friends, doctors, nurses and hospital staff is testimony to his immortal spirit, alive with perpetual youth and undiminished hope. Far more than any professor, book, or lecture, David’s life was a lesson that will forever continue to teach us about the intricate yet mysterious mechanisms that permit life in a hostile microbial world. Today, I speak to David’s family and to all the families who have entrusted the care of their children to us. Thank you. Your children have taught us so much, not only about human immunity and its power to preserve life, but also about their will to succeed against great obstacles and emerge victorious. We are grateful for
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25. David’s Story
FIGURE 25.3 XSCID patients have mutations in the IL-2Rg gene. (A) Pedigrees of the XSCID patients’ studies. Circles, female; squares, male; closed squares, males with SCID; squares with slashes, deceased males. Small squares and diamond in the pedigree of patient 2 indicate miscarriages (male and of undetermined sex, respectively). David is patient 3. (B) Sequencing of the IL-2Rg gene. Shown is the sequence of DNA from a normal donor (left panel in each pair) and of DNAs from patients 1, 2, and 3 (right panel in each pair). Patient 1 has an AAA (Lys) to TAA (stop codon) transversion in exon 3, resulting in a truncation of the carboxy-terminal 251 amino acids; patient 2 has a CGA (Arg) to TGA (stop codon) transition in exon 7, resulting in the truncation of 81 amino acids; and patient 3 has a TCG (Ser) to TAG (stop codon) transversion in exon 7, resulting in the truncation of 62 amino acids. The location of each mutation is indicated in the sequence to the right of each set of panels. The sequence shown is complementary to the coding strand. The boxed nucleotides for patients 1, 2, and 3 are complementary to TAA, TGA, and TAG stop codons, respectively. (C) Schematic showing locations of the artificial stop codons (diagonal arrows) present in the XSCID patients. Patient 1 has a premature stop codon in exon 3, and patients 2 and 3 have premature stop codons in exon 7. UTR: Untranslated region. Taken with permission from Noguchi M, Yi H, Rosenblatt HM, et al. Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 1993;73:147-157.
A Mother’s Recollection: Carol Ann Demaret
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FIGURE 25.4 The first time I met David, the bubble boy. Dr William T. Shearer meets David on September 1, 1978 on his first day at Texas Children’s Hospital and shakes hands with his new patient. Dr Buford Nicols who directed David’s nutritional program looks on. David is almost seven years of age in this picture.
the legacy David and all the special children after him have given to the world. Delivered in David’s memory upon the 40th anniversary of his birth, September 21, 2011, The David Center Texas Children’s Hospital Houston, Texas.
A MOTHER’S RECOLLECTION: CAROL ANN DEMARET Now and then I dream of David and we laugh and I feel touched and good when I awaken. In many ways, David’s life at home was “normal”. I could care for him, change his diapers as a baby and clothed him and fed him the foods that were packaged in sterile, air-locked space age “supple cylinders” that were replenished weekly. I
couldn’t touch him, but I could feel his warmth, as I embraced and held him close through the plastic and I believed he could feel mine, both as an infant and a boy. I cut his hair, and bathed him and changed his clothes when he was small and did most of the child care that all mothers do. As he grew, his bubbles grew in size and numbers – at home and at the hospital. He was happy to be at home with his family and he was happy to be at the hospital with his doctors and nurses and all the attention he received. However, he did not like having his blood drawn which was not an easy task. As he grew, he had sterilized toys and games, was educated by way of at-home teachers, (sometimes accompanied by classmates). He received Holy Communion, after the host had been sterilized and consecrated. He had
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25. David’s Story
FIGURE 25.5 The wall of plastic that separated David and his mother, Carol Ann Demaret. David is on the inside of his sterile isolator while his mother, Carol Ann, is on the outside. Among her many memories of David, she recalls with pleasure cutting his hair with sterile scissors. David is 11 years of age in this picture.
religious training, and family and friends who visited often. His sister slept on the floor next to him and they would talk and plot and giggle. He watched television and enjoyed favorite programs. He talked on the telephone, even had pets (outside the bubble, of course). He had a parakeet that he tried to teach to talk. There was a large window in his room, both at home and at the hospital, where he could view his surroundings. David Phillip grew with fine health, dark and handsome, highly intelligent and astonishingly perceptive, with black locks and penetrating eyes that some said seemed to see into a far beyond (Fig. 25.5). When a possible new medical technique that showed great promise finally became available, David himself decided that he wanted to participate. It was a complex treatment that involved a cellular transplant. At first it appeared to be working. But then it failed. All attempts to reverse the process were unsuccessful, and my gallant son died on February 22, 1984, age 12. We were devastated. We wept. And we felt a nation wept with us.
David not only lives on in my soul, but in other ways, too. Within a few days of David’s passing, his family was approached by Texas Children’s Hospital with an idea in tribute to David’s memory to create “The David Center” where SCID babies, and all children born with compromised or faulty immune systems whether genetic or acquired, can be successfully treated, and with continued research, immune systems could be stirred into more vigorous action, and where important advances have been made in discovering the genetic defect for SCID. A school was named after him, “The David Elementary” in the Woodlands, Texas, at one time said to be the only school in the country named after a child. One of David’s unrealized dreams was to be able “to walk barefoot in the grass”. With this in mind and spirit, each year the school sponsors a “David Dream Run” and raises contributions to “The David Center”. There is a David Memorial Drive in the neighborhood where he lived. In December of 2012, it was time to celebrate David’s legacy as it marked the beginning of
A Mother’s Recollection: Carol Ann Demaret
SCID New born Screening in Texas. Texas joins 12 other states that are currently screening all newborns for SCID. I now believe my prayer has been fully answered, and “that the bubble has burst” for all time. For David’s family and those who shared in his life, other lessons gleaned from David are important, too. He taught us that handicap of body is not necessary handicap of mind and spirit, that handicap cannot just be endured, but nobly endured. He has reminded the world of humble marvels, such as grass under our feet. All children leave behind memories and a sense of yearning and loss, but David left behind some things for humankind, too, and it helps. “He never touched the world, but the world was touched by him”, are the words inscribed on his gravestone. I believe the promise for the very near future is not just to preserve some portion of precious life, but to enhance it at the core. I carry in my heart sweet reminders of the past and great expectations for the future for all generations that follow. Presented at the 3rd Pediatric Immunodeficiency Treatment Consortium Third Annual Workshop, May 2, 2013 The David Center Texas Children’s Hospital Houston, Texas.
Acknowledgment We thank Janice Hopkins and Janelle Allen for assistance in manuscript preparation, and for reviewing the memorabilia of David’s life saved at the David Center, Texas Children’s Hospital.
References 1. Williamson AP, Montgomery JR, South MA, et al. A special report: four-year study of a boy with combined immune deficiency maintained in strict reverse isolation from birth. Pediatr Res 1977;11:63–4. 2. Blattner RJ. Introduction. Pediatr Res 1977;11:65. 3. Montgomery JR, South MA, Wilson R, et al. Family background, early history, and diagnosis. Pediatr Res 1977;11:65–6.
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4. Wilson R, Taylor GR, Kropp K, et al. Gnotobiotic care and infectious disease prevention. Pediatr Res 1977;11: 67–71. 5. Guerra IC, Shearer WT. Environmental control in management of immunodeficient patients: experience with “David”. Clin Immunol Immunopathol 1986;40:128–35. 6. Mukhopadhyay N, Richie E, Montgomery J, et al. Peripheral blood T and B-cell characteristics in a patient with severe combined immune deficiency (SCID) maintained in a gnotobiotic environment. Exp Hematol 1976;4: 1–9. 7. South MA, Montgomery JR, Richie E, et al. Immunologic studies. Pediatr Res 1977;11:71–8. 8. Mukhopadhyay N, Richie E, Mackler BF, et al. A longitudinal study of T and B lymphocytes from a three-yearold patient with severe combined immunodeficiency (SCID) in ‘gnotobiotic protection’. Exp Hematol 1978;6: 129–34. 9. Mackler BF, O’Neill PA. T-lymphocyte induction of nonT-cell-mediated nonspecific cytotoxicity. II. A clinical study of a severe combined immunodeficiency patient maintained in a gnotobiotic environment. Clin Immunol Immunopathol 1979;12:358–66. 10. Paschall VL, Brown LA, Lawrence EC, et al. Immunore gulation in an isolated 12-year-old boy with congenital severe combined immunodeficiency. Pediatr Res 1984;18: 723–8. 11. Fernbach DJ, Starling KA, Falletta JM. Hematology. Pediatr Res 1977;11:78–9. 12. Potts E, Huang CTL, Nichols BL. Nutritional care and related studies. Pediatr Res 1977;11:80–2. 13. Molish B, Murphy M, Desmond M. Mental, psychomotor, and psychosocial development. Pediatr Res 1977;11:82–3. 14. Musher KK. Speech and language development. Pediatr Res 1977;11:84–5. 15. Freedman DA. Psychiatric evaluation. Pediatr Res 1977;11:85–6. 16. Shearer WT, Ritz J, Finegold MJ, et al. Epstein-Barr virusassociated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985;312:1151–9. 17. Noguchi M, Yi H, Rosenblatt HM, et al. Interleukin-2 receptor gamma chain mutation results in X-linked severe combined immunodeficiency in humans. Cell 1993;73: 147–57. 18. Rosenblatt HM, Green CG, McClure JE, et al. Antibody to human lymphocyte actin regulates immunoglobulin secretion by an EBV-transformed human B-cell line. Biochem Biophys Res Commun 1986;140:399–405. 19. Cleary ML, Nalesnik MA, Shearer WT, et al. Clonal analy sis of transplant-associated lymphoproliferations based on the structure of the genomic termini of the Epstein-Barr virus. Blood 1988;72:349–52.
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20. Reinherz EL, Geha R, Rappeport JM, et al. Reconstitution after transplantation with T-lymphocyte-depleted HLA haplotype-mismatched bone marrow for severe combined immunodeficiency. Proc Natl Acad Sci USA 1982;79:6047–51.
21. Reisner Y, Kapoor N, Kirkpatrick D, et al. Transplantation for severe combined immunodeficiency with HLA-A, B, D, DR incompatible parental marrow cells fractionated by soybean agglutinin and sheep red blood cells. Blood 1983;61:341–8.