- Email: [email protected]
Variability in immunologic reconstitution following bone marrow transplantation
CLINICAL
IMMUNOLOGY
AND
Variability Following
2, t%l-%!-)
IMMUNOPATHOLOCY
in Immunologic Bone Marrow
Reconstitution Transplantation’
W. D. BIGGAR~ AND B. H. Pathology and Pediatrics,
University
(1974)
of Minnesota,
PARK Minneapolis,
Minnesota
Minneapolis,
Minnesota
B. DUPONT Department
of Pathology,
University of Minnesota,
AND R. A. GOOD Memorial
Sloan-Kettering
Cancer Center, New York
Received June 21,1973 Following the first successful bone marrow transplant which corrected both a genetically based immunodeficiency disease and a second marrow transplant that corrected an immunologically based marrow aplasia, 25 additional successful marrow transplants have been reported. In this report three patients are described to illustrate both the variability in the clinical and laboratory manifestations of combined immunodeficiency disease and the variability in the degree and pattern of immunologic reconstitution following marrow transplantation. These three patients emphasize that many factors that may influence the successful outcome of marrow grafting are not well understood and each patient must be considered as a unique undertaking.
Bone marrow transplantation (BMT) to achieve immunologic reconstitution of severe combined immunodeficiency disease (SCID), although still in its infancy, has recently undergone an explosive phase of growth and development. Prior to 1968, bone marrow transplantation had been used in an attempt to treat a variety of diseases. Success, however, had been limited to occasional patients with radiation induced bone marrow aplasia (l), aplastic anemia (2) and lymphoreticular malignancy (3,4). In 1967 it was postulated that children with severe deficiencies of both humoral and cellular immunity lacked a lymphoid precursor cell necessary for the development of normal immunity, but had normal precursor cells for the other cellular elements of the bone marrow and peripheral blood (5). In support of this postulate was the evidence of an existing pluripotential stem cell capable of differentiating into the formed elements of bone marrow, peripheral blood and lymphoid tissues. In addition, apparent stem cells could 1 Supported by The National Foundation-March of Dimes, The John A. Hartford Foundation, Inc., U. 8. Public Health Service (AI-88877 and HE-O8814), General Clinical Research Centers (RR400) Program of the Division of Research Resources NIH, the Levee Fund for Cellular Engineering, and the Danish State Research Foundation (512-1520). WDB is a Queen Elizabeth II Canadian Fellow. 2 Correspondence should be addressed to: Dr. W. D. Biggar, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto M5G 1X8, Ontario, Canada. 501 Copyright All rights
@ 1974 by Academic Press, Inc. of reproduction in any form reserved.
502
BIGGAR
ET
AL.
prevent the high mortality following lethal irradiation when grafted into syngeneic mice. With these clinical and experimental observations, it was predicted that suitably matched bone marrow stem cells could correct the deficiency of lymphoid precursor cells and the resultant immunodeficiency in those children with severe combined immunodeficiency disease (5,6). A suitably matched donor is one identical with the recipient at the major histocompatibility loci. This implies identical HL-A genotypes and mutual unresponsiveness in mixed lymphocyte testing (MLC) between the donor and the recipient. With only rare exceptions, this has proved to be true in HL-A identical siblings (7). These exceptions have occurred as a consequence of a recombination within the HL-A chromosomal region that gives rise to HL-A nonidentical sibling pairs which are mutually unresponsive in mixed lymphocyte culture (8). Extensive studies of these families clearly show that the genetic control of the HL-A antigens and the genetic control of the ability to stimulate and/or respond in mixed lymphocyte culture are not the same. Such was the case in our first successful correction of combined immunodeficiency disease by bone marrow transplantation (9,lO). Following the first successful BMT which corrected both a genetically based combined immunodeficiency disease and a second BMT that corrected an iatrogenic immunologically based aregenerative pancytopenia (lo,1 1) 25 additional successful BMT have been reported and 21 long-term survivors can be counted. It is apparent from studies in these children that the clinical and laboratory manifestations of this disease are variable. It is the purpose of this communication to report three patients who further emphasize the clinical variability of the disease and illustrate the variability of immunologic reconstitution following marrow transplantation. PATIENTS
AND
METHODS
The three patients were treated at the University of Minnesota Hospitals. The diagnosis in each case was severe combined immunodeficiency disease @CID) characterized by marked deficiencies of both humoral and cellular immunity. None of the children had family histories of immunodeficiency. Immunologic reconstitution was attempted in each patient by BMT from a matched sibling donor. The method of bone marrow transplantation has been published (12). Case 1 An 11-mo old female (K.S.) was referred to the University of Minnesota Hospitals because of recurrent sinopulmonary infection, persistent pneumonia and failure to thrive since 2 mo of age. On admission, the child was severely malnourished and dyspneic. Pneumocystis carinii pneumonia was diagnosed by open lung biopsy 12 hr after admission and a 19-day course of pentamidine therapy (4 mg/kg/day intramuscularly) was initiated. Roentgenograms of the chest following this therapy showed little improvement and a marked consolidation of the right upper lobe of the lung persisted. The patient was given 20 x lo6 bone marrow cells aspirated in a small
VARIABILITY
IMMUNOLOGIC
IN
IMMUNOLOGIC TABLE FOLLOWING
RESTORATION
1 BONE
Humoral
-~~__ Weeks post BMT
Serum
503
RECONSTITUTION
MARROW
TRANSPLANTATION
immunity
Immunoglobulins (mg So)
B-cell
Cellular receptors (%I
Control
Control
PHA
G
M
A
G
M
A
300 290 890 1410 1140 800 1000 661 I+219
4.0 29.0 120.0 75.0 18.0 3.0 31.5. 54 ?z 23
5 17 34 25 8 4 30 37 -t 18
1.1
6.1
0
10.0
5.8
6.4
13.8 22.0
13.8 17.0
12.8 10.2
14-19
4.4-9.5
3.6-9.4
CPM ~.--~-
-___ 0 3 4 6 8 13 17
immunity
20 7231 25,770 20,564 32,657 20,650 16,618
~.71 221 543 374 699 352 670
aliquot (12) per kg intraperitoneally (ip) from her matched sister and an identical graft was repeated 3 wk later. A mild and transient graft versus host (GVH) reaction began 14 days after the first BMT. A second lo-day course of pentamidine was given at this time. Three weeks after the first BMT clear evidence of immunologic reconstitution was seen. Five weeks after the first BMT full reconstitution of both humoral and cellular immunity was achieved (Table 1). The various parameters ‘of cellular immunity as tested by the in vitro lymphocyte responses to nonspecific mitogens, to specific antigens and to allogeneic cells were also restored in parallel (Fig. 1). Roentgenograms of ANALYSIS
I
PRE-BMT
OF CELLULAR
BMT + 4 WKS.
IMMUNITY
BMT + 6 WKS.
ALLOGENIC
DONOR
CELLS
0
0 t
Candid0
FIG. 1. In vitro Case 1.
testing
of cellular
immunity
before
and
after
bone
marrow
Albimnr
transplantation
in
504
BIGGAR
ET
AL.
the chest have returned to normal. The child is clinically munologically normal more than 1 yr following the BMT.
and
im-
Case 2 A 10 mo old female (T.T.) was referred to the University of Minnesota Hospitals with a history of recurrent pneumonia and failure to thrive since 10 wk of age. On admission, she was severely malnourished. Roentgenograms of the chest showed a collapsed right upper lobe. Quantitative immunoglobulins (in mg/lOO ml) were IgG, 150-200; IgA, 5-13; and IgM, 21-58. The patient was blood group 0, but had no isohemagglutinins. Blood lymphopenia was persistent. No delayed hypersensitivity skin reactions were obtained to a battery of antigens. Repeated analysis of the in vitro lymphocyte responses to phytohemagglutinin (PHA) showed absent or very minimal responses. There was no apparent lymphocyte response to allogeneic cells. A diagnosis of combined immunodeficiency was established. The patient was given a total of seven BMT over an 8-mo period. Each BMT was done at 4-6 wk intervals with gradual increments of cell doses (nucleated cells X 106/kg body weight 10,20,40, 113,220,220,220). The last three BMT were given intravenously. After the fifth BMT, (24 wk after the first BMT) a mild GVH reaction occurred and early evidence of lymphocyte responsiveness to PHA was observed for the first time. Lymphocyte responses in vitro to nonspecific mitogens, specific antigens and to allogeneic cells became normal soon thereafter but the serum immunoglobulins (in mg/lOO ml IgG, 250; IgA, 4; IgM, 22) and the lymphocytes bearing membrane bound immunoglobulin (B-cells) remained low. The child did not gain weight but remained clinically well and free of infection. Roentgenograms of the chest gradually returned to normal. Two more BMT were given and the child was discharged from the hospital clinically well. Twelve months after the seventh BMT, she had normal levels of serum IgM and IgA but the serum IgG remained somewhat low at 300 mg%. The patient had a vigorous antibody response to diphtheria and tetanus antigens and isohemagglutinins were present. In vitro lymphocyte responses to nonspecific mitogens, specific antigens and to allogeneic cells remained normal. The patient developed delayed hypersensitivity skin reactivity. She had gained 3 kg in the last 8 wk and is clinically well. Case 3 A 12-mo old male (C.Ma.) was referred to the University of Minnesota Hospitals with a history of recurrent sinopulmonary infections since 3 mo of age and a chronic, scaling, hyperkeratotic, eczematoid skin rash which had appeared 1 mo earlier. On admission, the patient was small and cachetic and had generalized, diffuse, chronic, irregular, raised erythematous and scaling skin lesions. Mild hepatomegaly was present and percutaneous biopsy of the liver showed mild fatty metamorphosis. Quantitative immunoglobulins were (in mg/lOO ml) IgG, 100-150; IgA, 10-20; IgM, 17-35; and no detectable IgE. Repeated in vitro lymphocyte responses to PHA and to allogeneic cells
VARIABILITY
IN
IMMUNOLOGIC
RECONSTITUTION
505
showed absent to minimal responses. Delayed hypersensitivity skin reactivity was absent. No leukoagglutinins, cytotoxic antibodies or serum inhibitors to in u&o lymphocyte testing could be demonstrated. Despite three BMTs (nucleated cells X lo6 kgm body weight, ip, 1, 5, 30) from an HL-A identical male sibling, no evidence of engraftment was seen and the patient died of E. coli sepsis 70 days after the first BMT. During his hospitalization, the patient remained critically ill. The skin eruption varied considerably and at times would involve 80% of the body surface as an acutely inflamed, confluent, and desquamative rash; a picture similar to severe GVH disease. Repeated skin biopsies were compatible with GVH disease. In summary, the patient had the immunodeficiencies and autopsy findings compatible with combined immunodeficiency disease but died after three BMTs from a matched sibling donor had failed to restore immunologic function. DISCUSSION Since 1968, a primary focus in treating patients with severe combined immunodeficiency has been to improve the technique of marrow grafting and to study, with improved methods, the pattern of immunologic reconstitution following BMT. The present three cases illustrate several important observations. Case 1 provided an opportunity to analyze a pattern of immunologic reconstitution in detail. Serial determinations of cellular and humoral immunity were obtained at weekly intervals before and after BMT. These are summarized in Table 1. These studies clearly demonstrate that rapid and full immunologic reconstitution of both cellular and humoral immunity is possible by grafting small numbers of carefully aspirated bone marrow cells intraperitoneally. Furthermore, the various parameters of cellular immunity [in vitro lymphocyte responses to PHA, allogeneic cells, pokeweed mitogen (PWM), and purified protein derivative (PPD), Candida albicans, staphylococcal and E. coli antigens] returned in parallel with the exception of the lymphocyte response to staphylococcal antigen which was delayed 1 wk (Fig. 1). The ideal bone marrow graft in these patients is one which will restore immunologic vigor with a minimum of GVH disease. Following BMT the severity of the GVH reaction seems to vary, in part, with the number and the immunocompetence of the cells engrafted. Since peripheral blood is rich in irnmunocompetent lymphoid cells capable of inducing a GVH reaction while the bone marrow is relatively devoid of such cells (13), precautionary measures are taken to minimize the contamination of the marrow aspirate by peripheral blood. Small aliquots (0.1-0.3 cc) of marrow yield a pure, readily standardized specimen (14) which yields a much lower response to PHA stimulation in vitro than do aliquots of marrow that have been aspirated in larger volumes (13). Thus, the nucleated cells contained in small volumes of carefully aspirated marrow would contain a higher concentration of precursor cells than would marrow that is heavily contaminated by
506
BIGGAR
ET
AL.
immunocompetent peripheral blood lymphocytes but poor in precursor cells. These factors, in addition to the small number of grafted cells, may have been important for the rapid and full immunologic reconstitution of this patient. The second BMT may not have been required in this instance but was given because difficulties in establishing full immunologic reconstitution in some patients with SCID had been encountered previously. The incidence of pneumocystis carinii infection in these patients is very high (15,16). Furthermore, responses to infection by microorganisms in these immunodeficient patients relates in a very interesting way to host-parasite interactions. Because these patients lack the capacity to mount an immunologically based inflammatory response against microorganisms, they fail to demonstrate many of the usual manifestations of disease. Thus, their infection remains relatively unchallenged and occult. When immunologic reconstitution is beginning, these patients, already heavily infected, have the immunologic capacity to express disease processes. Hence, a previously occult infection now becomes the target of an immunologically based inflammatory assault. What was initially an infection with minimal inflammation may become a disease process that is abrupt in onset and fatal in outcome. Two additional patients that were given BMTs for SCID have died of acute pneumocystis carinii penumonia at the time of immunologic reconstitution and others have observed this complication of immunologic reconstitution. In those cases, the organism was most certainly present prior to BMT but for the reasons mentioned, remained occult. Effective eradication of this microorganism seems to require participation of host immunologic mechanisms in addition to chemotherapy. Thus, chemotherapy alone in these patients, at best, can be expected to suppress the infection but not always completely eradicate the organism. For these reasons, we give a second course of therapy (as in Case 1) in proven cases of pneumocystis carinii pneumonia at the time immunologic reconstitution begins. If drugs less toxic than pentamidine, such as pyrimethamine and sulfadiazine (17) prove to be effective, one might consider using a drug(s) both as a therapeutic and a prophylactic agent in these patients in whom the incidence of both infection and mortality is so great (15,16). Case 2 had all the clinical and laboratory manifestations of severe combined immunodeficiency disease. Why immunologic reconstitution was so difficult to achieve in this patient by BMT is not clear. It may have related to the presence, in this case, of sufficient residual host immunity to reject the initial marrow grafts when immunologic reconstitution was first attempted. It is clear from variability in both the clinical and laboratory manifestations of this disease that, although immunologic functions are severely compromised, some have greater quantities of serum immunoglobulins and B-cells than do others. Furthermore, some patients have in vitro responses to nonspecific mitogens and allogeneic cells prior to BMT while these responses in other patients are undetectable. Indeed, our Case 2 had such minimal responses to PHA on two occasions prior to BMT. This minimal response may reflect a residual immunity, not adequate for survival but perhaps able to reject a BMT and thus interfere with the immunologic reconstitution.
VARIABILITY
IN IMMUNOLOGIC
RECONSTITUTION
507
This child emphasizes the difficulty in estimating, for any given patient, the optimum number of marrow cells required for full immunologic reconstitution. It may be that host and/or donor factors (age, sex, presence or absence of infection, nutrition, etc.) are also important in influencing the outcome of a transplant of any given number of marrow cells. In addition, as methodologies for the in vitro and in viva evaluation of immunity are improved, considerable variability in the amount of residual host immunity will be found. If a patient does have some residual immunity, it may take a greater number of marrow cells and/or several BMTs to achieve reconstitution than if he had no detectable immunity. It is also likely that those patients who have had a transient conversion from negative to positive of delayed hypersensitivity skin tests following transfer factor injections, will be found to have minimal cellular immunity if evaluations are carried out by sensitive in vitro quantitation of cellular immunity. Case 3, by our present-day methods of immunologic evaluation, had SCID. His clinical presentation was somewhat different from many of the other patients. He had a chronic generalized skin eruption that had many of the clinical and microscopic characteristics of GVH disease. Furthermore, the chronic malnutrition, diarrhea, hepatosplenomegaly and eosinophilia were compatible with this diagnosis. We postulated that the foreign graft initiating the GVH disease was of maternal origin and had occurred prior to, or at the time of delivery. The failure to demonstrate maternal cells in either the bone marrow, peripheral blood or the skin of this male patient does not exclude this diagnosis. In mice, for instance, donor derived cells can be found in the early post grafting period, after which time they are very difficult or impossible to demonstrate (18). Further, in important studies with chick embryos, Lafferty has created just such a model (19). The patient further illustrates the difficulty in predicting the optimum number of marrow cells and raises the possibility that an ongoing GVH reaction may jeopardize the success of immunologic reconstitution. The successful correction of combined immunodeficiency disease by BMT does not exclude the possibility that some patients,,in addition to lacking a lymphoid precursor cell, will also lack the normal microchemical environment(s) necessary for the maturation and differentiation of these precursor cells (e.g., thymus, bursa, etc.). Thus, in addition to transplanting a lymphoid precursor cell, these patients would also need a thymus and/or bursa equivalent for the complete differentiation of their central and peripheral lymphoid tissues. An alternative hypothesis might be that large numbers of marrow cells might establish a peripheral lymphoid population that is immunocompetent and expandable in the absence of a normal thymus and/or bursa equivalent as has been observed in mice (20). Such a peripheral reconstitution might be of somewhat shorter life and thus require retransplantation at a later date to maintain full immunologic vigor. In addition, some patients may, for reasons not clearly defined, but perhaps involving histocompatibility determinants, be unable to.accept a marrow graft- a phenomenon that has been observed in certain strains of mice (21,22). It is known that differences in histocompatibility antigens affect significantly cell traffic (22) and it is possible that the
508
BIGGAR
ET AL.
failure to achieve marrow engraftment and immunologic reconstitution in Patient 3 could reflect such subtle and as yet poorly defined influences. In support of this view are recent observations that the autosomal chromosome region controlling histocompatibility is very complex. In addition to controlling histocompatibility characteristics, it controls capacity to stimulate antigenically to respond to antigens (8,23) and vulnerability to develop certain diseases. The recent observation that some patients with SCID have no detectable adenosine-deaminase (A.D.A.) in their red blood cells (24) and the reported close association between the genetic locus for A.D.A. and the HL-A locus (25) further adds to the complexity of this disease. Thus, this general chromosomal region is extraordinarily complicated. In addition to the known immunologic functions genetically controlled by this chromosomal region, new parameters of cellular function may indeed await discovery. From analyses of these three patients, it is clear that as yet each patient must be considered as a unique undertaking dealing with many unresolved questions. In spite of great progress in recent years yielding important insights and real improvement in marrow transplantation, many of the principles essential to making this form of cellular engineering a relatively simple form of therapy remain to be elucidated. REFERENCES 1. MATHE, G., T~BIANA, MERY, A. 2. PILLOW, R.
AMIEL, J. L., SCHWARZENBERG, L., CATTAN, A., SCHNEIDER, M., DE VRIES, M. J., M., LALANNE, C., BINET, J. J., PAPEIRNIK, M., SEMEN, G., MATSUKIJRA, M., M., SCHARZMANN, V., AND FLAISLER, A., Blood 25,179, 1965. P., EPSTEIN, R. B., BUCKNER, C. D., GIBLETT, E. R., AND THOMAS, E. D., N. En&
J. Med. 275,94,1966. 3. BEILBY,
J. 0. W., CADE,
I. S., JELLIFFEE,
A. M., PARKIN,
D. M.,
AND STEWART,
J. W.,
Brit.
Med. J. 1,96,1966. 4. STEWART, J. W., Brit. Med. J. 1,304,1964. 5. GOOD, R. A., PETERSON, R. D. A., PEREY, munologic Deficiency Diseases in Man,”
D. Y., FINSTAD, J., AND COOPER, M. D., In “Impp. 17-34, National Foundation Press, New York,
1964. 6. HONG, R., KAY, H. E. M., COOPER, M. D., MEUWISSEN,.H., ALLEN, R. A., Lancet I, 503, 1968. 7. AMOS, D. B., AND BACH, F. J., J. Erp. Med. 128,623, 1968. 8. YUMS, E. J., AND AMOS, B., hoc. Nat. Acad. Sci. 68,3031, 1971. 9. GATTI, R: A., MEUWISSEN, 1971. 10. GATTI, R. A., MEU~ISSEN,
H. J., TERASAM, H. J., ALLEN,
P. I., AND GOOD,
M.
J. G., AND
GOOD,
Antigens
1, 239,
R. A., Tissue
H. D., HONG,
R., AND GOOD,
P. I., HONG,
R., AND GOOD,
R. A.,
Lancet 2, 1366,
1968. 11. MEUWISSEN,
281,691,
H. J., GA~I,
R. A., TERASAKI,
R. A., N.
Engl. J. Med.
1969.
12. BIGCAR, W. D., GOOD, R. A., AND PARK, B. H., J. Pedtit. 81,301, 1972. 13. PARK, B. H., BIGGAR, W. D., AND GOOD, R. A., Transplantation 14,384, 1972. 14. GOOD, R. A., AND CAMPBELL, B., Amer. J. Med. 9,330, 1950. 15. BURKE, B. A., AND GOOD, R. A., Medicine, 52,23, 1973. 16. WALZER, P. D., SCHULTZ, M. G., WESTERN, K. A., AND ROBBINS, J. B., J. Pediat. 82,416,
Ii’. 18.
1973. KIRBY, H. B., KENAMORE, B., AND GUCKLAN, FOX, M., Immunology 5,489, 1962.
S. C.,
Ann. Int. Med. 75,505, 1971.
VARIABILITY
IN IMMUNOLOGIC
FWCONSTITUTION
509
19. WALKER, K. Z., SCHOEFL, G. T., ANJJ LAFFERTY, K. J.,Aust. J. Erp. Biol. Med. Sci. 59,675, 1972. 29. YUNIS, E. J., HILGARD, H. R., MARTINEZ, C., AND GOOD, R. A., J. Erp. Med. 121,607, 1965. 21. GOODMAN, J. W., MARTIN, F. B., AND CONGDON, C. C., Arch. Pathol. 89,226, 1970. 22. STUTMAN, O., YUNIS, E. J., AND GOOD, R. A.,]. Immunol. 103,92, 1969. 23. PARK, B. H., AND GOOD, R. A., Proc. Nat. Acad. Sci 69, 1490, 1972. 24. GIBLETT, E. R., ANDERSON, J. E., COHEN, F., POLLARA, B., AND MEUWISSEN, H. J.,Lancet 172, 1667, 1971. 25. EDWARDS, J. H., ALLEN, F. H., GLENN, K. P., LAMM, L. V., AND ROBSON, E. B., In “Histocompatibility Testing” (J. Dausset and J. Colombani, Eds.) 1972 ed., pp. 745-751, Munksgaard, Copenhagen, 1973.
IMMUNOLOGY
AND
Variability Following
2, t%l-%!-)
IMMUNOPATHOLOCY
in Immunologic Bone Marrow
Reconstitution Transplantation’
W. D. BIGGAR~ AND B. H. Pathology and Pediatrics,
University
(1974)
of Minnesota,
PARK Minneapolis,
Minnesota
Minneapolis,
Minnesota
B. DUPONT Department
of Pathology,
University of Minnesota,
AND R. A. GOOD Memorial
Sloan-Kettering
Cancer Center, New York
Received June 21,1973 Following the first successful bone marrow transplant which corrected both a genetically based immunodeficiency disease and a second marrow transplant that corrected an immunologically based marrow aplasia, 25 additional successful marrow transplants have been reported. In this report three patients are described to illustrate both the variability in the clinical and laboratory manifestations of combined immunodeficiency disease and the variability in the degree and pattern of immunologic reconstitution following marrow transplantation. These three patients emphasize that many factors that may influence the successful outcome of marrow grafting are not well understood and each patient must be considered as a unique undertaking.
Bone marrow transplantation (BMT) to achieve immunologic reconstitution of severe combined immunodeficiency disease (SCID), although still in its infancy, has recently undergone an explosive phase of growth and development. Prior to 1968, bone marrow transplantation had been used in an attempt to treat a variety of diseases. Success, however, had been limited to occasional patients with radiation induced bone marrow aplasia (l), aplastic anemia (2) and lymphoreticular malignancy (3,4). In 1967 it was postulated that children with severe deficiencies of both humoral and cellular immunity lacked a lymphoid precursor cell necessary for the development of normal immunity, but had normal precursor cells for the other cellular elements of the bone marrow and peripheral blood (5). In support of this postulate was the evidence of an existing pluripotential stem cell capable of differentiating into the formed elements of bone marrow, peripheral blood and lymphoid tissues. In addition, apparent stem cells could 1 Supported by The National Foundation-March of Dimes, The John A. Hartford Foundation, Inc., U. 8. Public Health Service (AI-88877 and HE-O8814), General Clinical Research Centers (RR400) Program of the Division of Research Resources NIH, the Levee Fund for Cellular Engineering, and the Danish State Research Foundation (512-1520). WDB is a Queen Elizabeth II Canadian Fellow. 2 Correspondence should be addressed to: Dr. W. D. Biggar, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto M5G 1X8, Ontario, Canada. 501 Copyright All rights
@ 1974 by Academic Press, Inc. of reproduction in any form reserved.
502
BIGGAR
ET
AL.
prevent the high mortality following lethal irradiation when grafted into syngeneic mice. With these clinical and experimental observations, it was predicted that suitably matched bone marrow stem cells could correct the deficiency of lymphoid precursor cells and the resultant immunodeficiency in those children with severe combined immunodeficiency disease (5,6). A suitably matched donor is one identical with the recipient at the major histocompatibility loci. This implies identical HL-A genotypes and mutual unresponsiveness in mixed lymphocyte testing (MLC) between the donor and the recipient. With only rare exceptions, this has proved to be true in HL-A identical siblings (7). These exceptions have occurred as a consequence of a recombination within the HL-A chromosomal region that gives rise to HL-A nonidentical sibling pairs which are mutually unresponsive in mixed lymphocyte culture (8). Extensive studies of these families clearly show that the genetic control of the HL-A antigens and the genetic control of the ability to stimulate and/or respond in mixed lymphocyte culture are not the same. Such was the case in our first successful correction of combined immunodeficiency disease by bone marrow transplantation (9,lO). Following the first successful BMT which corrected both a genetically based combined immunodeficiency disease and a second BMT that corrected an iatrogenic immunologically based aregenerative pancytopenia (lo,1 1) 25 additional successful BMT have been reported and 21 long-term survivors can be counted. It is apparent from studies in these children that the clinical and laboratory manifestations of this disease are variable. It is the purpose of this communication to report three patients who further emphasize the clinical variability of the disease and illustrate the variability of immunologic reconstitution following marrow transplantation. PATIENTS
AND
METHODS
The three patients were treated at the University of Minnesota Hospitals. The diagnosis in each case was severe combined immunodeficiency disease @CID) characterized by marked deficiencies of both humoral and cellular immunity. None of the children had family histories of immunodeficiency. Immunologic reconstitution was attempted in each patient by BMT from a matched sibling donor. The method of bone marrow transplantation has been published (12). Case 1 An 11-mo old female (K.S.) was referred to the University of Minnesota Hospitals because of recurrent sinopulmonary infection, persistent pneumonia and failure to thrive since 2 mo of age. On admission, the child was severely malnourished and dyspneic. Pneumocystis carinii pneumonia was diagnosed by open lung biopsy 12 hr after admission and a 19-day course of pentamidine therapy (4 mg/kg/day intramuscularly) was initiated. Roentgenograms of the chest following this therapy showed little improvement and a marked consolidation of the right upper lobe of the lung persisted. The patient was given 20 x lo6 bone marrow cells aspirated in a small
VARIABILITY
IMMUNOLOGIC
IN
IMMUNOLOGIC TABLE FOLLOWING
RESTORATION
1 BONE
Humoral
-~~__ Weeks post BMT
Serum
503
RECONSTITUTION
MARROW
TRANSPLANTATION
immunity
Immunoglobulins (mg So)
B-cell
Cellular receptors (%I
Control
Control
PHA
G
M
A
G
M
A
300 290 890 1410 1140 800 1000 661 I+219
4.0 29.0 120.0 75.0 18.0 3.0 31.5. 54 ?z 23
5 17 34 25 8 4 30 37 -t 18
1.1
6.1
0
10.0
5.8
6.4
13.8 22.0
13.8 17.0
12.8 10.2
14-19
4.4-9.5
3.6-9.4
CPM ~.--~-
-___ 0 3 4 6 8 13 17
immunity
20 7231 25,770 20,564 32,657 20,650 16,618
~.71 221 543 374 699 352 670
aliquot (12) per kg intraperitoneally (ip) from her matched sister and an identical graft was repeated 3 wk later. A mild and transient graft versus host (GVH) reaction began 14 days after the first BMT. A second lo-day course of pentamidine was given at this time. Three weeks after the first BMT clear evidence of immunologic reconstitution was seen. Five weeks after the first BMT full reconstitution of both humoral and cellular immunity was achieved (Table 1). The various parameters ‘of cellular immunity as tested by the in vitro lymphocyte responses to nonspecific mitogens, to specific antigens and to allogeneic cells were also restored in parallel (Fig. 1). Roentgenograms of ANALYSIS
I
PRE-BMT
OF CELLULAR
BMT + 4 WKS.
IMMUNITY
BMT + 6 WKS.
ALLOGENIC
DONOR
CELLS
0
0 t
Candid0
FIG. 1. In vitro Case 1.
testing
of cellular
immunity
before
and
after
bone
marrow
Albimnr
transplantation
in
504
BIGGAR
ET
AL.
the chest have returned to normal. The child is clinically munologically normal more than 1 yr following the BMT.
and
im-
Case 2 A 10 mo old female (T.T.) was referred to the University of Minnesota Hospitals with a history of recurrent pneumonia and failure to thrive since 10 wk of age. On admission, she was severely malnourished. Roentgenograms of the chest showed a collapsed right upper lobe. Quantitative immunoglobulins (in mg/lOO ml) were IgG, 150-200; IgA, 5-13; and IgM, 21-58. The patient was blood group 0, but had no isohemagglutinins. Blood lymphopenia was persistent. No delayed hypersensitivity skin reactions were obtained to a battery of antigens. Repeated analysis of the in vitro lymphocyte responses to phytohemagglutinin (PHA) showed absent or very minimal responses. There was no apparent lymphocyte response to allogeneic cells. A diagnosis of combined immunodeficiency was established. The patient was given a total of seven BMT over an 8-mo period. Each BMT was done at 4-6 wk intervals with gradual increments of cell doses (nucleated cells X 106/kg body weight 10,20,40, 113,220,220,220). The last three BMT were given intravenously. After the fifth BMT, (24 wk after the first BMT) a mild GVH reaction occurred and early evidence of lymphocyte responsiveness to PHA was observed for the first time. Lymphocyte responses in vitro to nonspecific mitogens, specific antigens and to allogeneic cells became normal soon thereafter but the serum immunoglobulins (in mg/lOO ml IgG, 250; IgA, 4; IgM, 22) and the lymphocytes bearing membrane bound immunoglobulin (B-cells) remained low. The child did not gain weight but remained clinically well and free of infection. Roentgenograms of the chest gradually returned to normal. Two more BMT were given and the child was discharged from the hospital clinically well. Twelve months after the seventh BMT, she had normal levels of serum IgM and IgA but the serum IgG remained somewhat low at 300 mg%. The patient had a vigorous antibody response to diphtheria and tetanus antigens and isohemagglutinins were present. In vitro lymphocyte responses to nonspecific mitogens, specific antigens and to allogeneic cells remained normal. The patient developed delayed hypersensitivity skin reactivity. She had gained 3 kg in the last 8 wk and is clinically well. Case 3 A 12-mo old male (C.Ma.) was referred to the University of Minnesota Hospitals with a history of recurrent sinopulmonary infections since 3 mo of age and a chronic, scaling, hyperkeratotic, eczematoid skin rash which had appeared 1 mo earlier. On admission, the patient was small and cachetic and had generalized, diffuse, chronic, irregular, raised erythematous and scaling skin lesions. Mild hepatomegaly was present and percutaneous biopsy of the liver showed mild fatty metamorphosis. Quantitative immunoglobulins were (in mg/lOO ml) IgG, 100-150; IgA, 10-20; IgM, 17-35; and no detectable IgE. Repeated in vitro lymphocyte responses to PHA and to allogeneic cells
VARIABILITY
IN
IMMUNOLOGIC
RECONSTITUTION
505
showed absent to minimal responses. Delayed hypersensitivity skin reactivity was absent. No leukoagglutinins, cytotoxic antibodies or serum inhibitors to in u&o lymphocyte testing could be demonstrated. Despite three BMTs (nucleated cells X lo6 kgm body weight, ip, 1, 5, 30) from an HL-A identical male sibling, no evidence of engraftment was seen and the patient died of E. coli sepsis 70 days after the first BMT. During his hospitalization, the patient remained critically ill. The skin eruption varied considerably and at times would involve 80% of the body surface as an acutely inflamed, confluent, and desquamative rash; a picture similar to severe GVH disease. Repeated skin biopsies were compatible with GVH disease. In summary, the patient had the immunodeficiencies and autopsy findings compatible with combined immunodeficiency disease but died after three BMTs from a matched sibling donor had failed to restore immunologic function. DISCUSSION Since 1968, a primary focus in treating patients with severe combined immunodeficiency has been to improve the technique of marrow grafting and to study, with improved methods, the pattern of immunologic reconstitution following BMT. The present three cases illustrate several important observations. Case 1 provided an opportunity to analyze a pattern of immunologic reconstitution in detail. Serial determinations of cellular and humoral immunity were obtained at weekly intervals before and after BMT. These are summarized in Table 1. These studies clearly demonstrate that rapid and full immunologic reconstitution of both cellular and humoral immunity is possible by grafting small numbers of carefully aspirated bone marrow cells intraperitoneally. Furthermore, the various parameters of cellular immunity [in vitro lymphocyte responses to PHA, allogeneic cells, pokeweed mitogen (PWM), and purified protein derivative (PPD), Candida albicans, staphylococcal and E. coli antigens] returned in parallel with the exception of the lymphocyte response to staphylococcal antigen which was delayed 1 wk (Fig. 1). The ideal bone marrow graft in these patients is one which will restore immunologic vigor with a minimum of GVH disease. Following BMT the severity of the GVH reaction seems to vary, in part, with the number and the immunocompetence of the cells engrafted. Since peripheral blood is rich in irnmunocompetent lymphoid cells capable of inducing a GVH reaction while the bone marrow is relatively devoid of such cells (13), precautionary measures are taken to minimize the contamination of the marrow aspirate by peripheral blood. Small aliquots (0.1-0.3 cc) of marrow yield a pure, readily standardized specimen (14) which yields a much lower response to PHA stimulation in vitro than do aliquots of marrow that have been aspirated in larger volumes (13). Thus, the nucleated cells contained in small volumes of carefully aspirated marrow would contain a higher concentration of precursor cells than would marrow that is heavily contaminated by
506
BIGGAR
ET
AL.
immunocompetent peripheral blood lymphocytes but poor in precursor cells. These factors, in addition to the small number of grafted cells, may have been important for the rapid and full immunologic reconstitution of this patient. The second BMT may not have been required in this instance but was given because difficulties in establishing full immunologic reconstitution in some patients with SCID had been encountered previously. The incidence of pneumocystis carinii infection in these patients is very high (15,16). Furthermore, responses to infection by microorganisms in these immunodeficient patients relates in a very interesting way to host-parasite interactions. Because these patients lack the capacity to mount an immunologically based inflammatory response against microorganisms, they fail to demonstrate many of the usual manifestations of disease. Thus, their infection remains relatively unchallenged and occult. When immunologic reconstitution is beginning, these patients, already heavily infected, have the immunologic capacity to express disease processes. Hence, a previously occult infection now becomes the target of an immunologically based inflammatory assault. What was initially an infection with minimal inflammation may become a disease process that is abrupt in onset and fatal in outcome. Two additional patients that were given BMTs for SCID have died of acute pneumocystis carinii penumonia at the time of immunologic reconstitution and others have observed this complication of immunologic reconstitution. In those cases, the organism was most certainly present prior to BMT but for the reasons mentioned, remained occult. Effective eradication of this microorganism seems to require participation of host immunologic mechanisms in addition to chemotherapy. Thus, chemotherapy alone in these patients, at best, can be expected to suppress the infection but not always completely eradicate the organism. For these reasons, we give a second course of therapy (as in Case 1) in proven cases of pneumocystis carinii pneumonia at the time immunologic reconstitution begins. If drugs less toxic than pentamidine, such as pyrimethamine and sulfadiazine (17) prove to be effective, one might consider using a drug(s) both as a therapeutic and a prophylactic agent in these patients in whom the incidence of both infection and mortality is so great (15,16). Case 2 had all the clinical and laboratory manifestations of severe combined immunodeficiency disease. Why immunologic reconstitution was so difficult to achieve in this patient by BMT is not clear. It may have related to the presence, in this case, of sufficient residual host immunity to reject the initial marrow grafts when immunologic reconstitution was first attempted. It is clear from variability in both the clinical and laboratory manifestations of this disease that, although immunologic functions are severely compromised, some have greater quantities of serum immunoglobulins and B-cells than do others. Furthermore, some patients have in vitro responses to nonspecific mitogens and allogeneic cells prior to BMT while these responses in other patients are undetectable. Indeed, our Case 2 had such minimal responses to PHA on two occasions prior to BMT. This minimal response may reflect a residual immunity, not adequate for survival but perhaps able to reject a BMT and thus interfere with the immunologic reconstitution.
VARIABILITY
IN IMMUNOLOGIC
RECONSTITUTION
507
This child emphasizes the difficulty in estimating, for any given patient, the optimum number of marrow cells required for full immunologic reconstitution. It may be that host and/or donor factors (age, sex, presence or absence of infection, nutrition, etc.) are also important in influencing the outcome of a transplant of any given number of marrow cells. In addition, as methodologies for the in vitro and in viva evaluation of immunity are improved, considerable variability in the amount of residual host immunity will be found. If a patient does have some residual immunity, it may take a greater number of marrow cells and/or several BMTs to achieve reconstitution than if he had no detectable immunity. It is also likely that those patients who have had a transient conversion from negative to positive of delayed hypersensitivity skin tests following transfer factor injections, will be found to have minimal cellular immunity if evaluations are carried out by sensitive in vitro quantitation of cellular immunity. Case 3, by our present-day methods of immunologic evaluation, had SCID. His clinical presentation was somewhat different from many of the other patients. He had a chronic generalized skin eruption that had many of the clinical and microscopic characteristics of GVH disease. Furthermore, the chronic malnutrition, diarrhea, hepatosplenomegaly and eosinophilia were compatible with this diagnosis. We postulated that the foreign graft initiating the GVH disease was of maternal origin and had occurred prior to, or at the time of delivery. The failure to demonstrate maternal cells in either the bone marrow, peripheral blood or the skin of this male patient does not exclude this diagnosis. In mice, for instance, donor derived cells can be found in the early post grafting period, after which time they are very difficult or impossible to demonstrate (18). Further, in important studies with chick embryos, Lafferty has created just such a model (19). The patient further illustrates the difficulty in predicting the optimum number of marrow cells and raises the possibility that an ongoing GVH reaction may jeopardize the success of immunologic reconstitution. The successful correction of combined immunodeficiency disease by BMT does not exclude the possibility that some patients,,in addition to lacking a lymphoid precursor cell, will also lack the normal microchemical environment(s) necessary for the maturation and differentiation of these precursor cells (e.g., thymus, bursa, etc.). Thus, in addition to transplanting a lymphoid precursor cell, these patients would also need a thymus and/or bursa equivalent for the complete differentiation of their central and peripheral lymphoid tissues. An alternative hypothesis might be that large numbers of marrow cells might establish a peripheral lymphoid population that is immunocompetent and expandable in the absence of a normal thymus and/or bursa equivalent as has been observed in mice (20). Such a peripheral reconstitution might be of somewhat shorter life and thus require retransplantation at a later date to maintain full immunologic vigor. In addition, some patients may, for reasons not clearly defined, but perhaps involving histocompatibility determinants, be unable to.accept a marrow graft- a phenomenon that has been observed in certain strains of mice (21,22). It is known that differences in histocompatibility antigens affect significantly cell traffic (22) and it is possible that the
508
BIGGAR
ET AL.
failure to achieve marrow engraftment and immunologic reconstitution in Patient 3 could reflect such subtle and as yet poorly defined influences. In support of this view are recent observations that the autosomal chromosome region controlling histocompatibility is very complex. In addition to controlling histocompatibility characteristics, it controls capacity to stimulate antigenically to respond to antigens (8,23) and vulnerability to develop certain diseases. The recent observation that some patients with SCID have no detectable adenosine-deaminase (A.D.A.) in their red blood cells (24) and the reported close association between the genetic locus for A.D.A. and the HL-A locus (25) further adds to the complexity of this disease. Thus, this general chromosomal region is extraordinarily complicated. In addition to the known immunologic functions genetically controlled by this chromosomal region, new parameters of cellular function may indeed await discovery. From analyses of these three patients, it is clear that as yet each patient must be considered as a unique undertaking dealing with many unresolved questions. In spite of great progress in recent years yielding important insights and real improvement in marrow transplantation, many of the principles essential to making this form of cellular engineering a relatively simple form of therapy remain to be elucidated. REFERENCES 1. MATHE, G., T~BIANA, MERY, A. 2. PILLOW, R.
AMIEL, J. L., SCHWARZENBERG, L., CATTAN, A., SCHNEIDER, M., DE VRIES, M. J., M., LALANNE, C., BINET, J. J., PAPEIRNIK, M., SEMEN, G., MATSUKIJRA, M., M., SCHARZMANN, V., AND FLAISLER, A., Blood 25,179, 1965. P., EPSTEIN, R. B., BUCKNER, C. D., GIBLETT, E. R., AND THOMAS, E. D., N. En&
J. Med. 275,94,1966. 3. BEILBY,
J. 0. W., CADE,
I. S., JELLIFFEE,
A. M., PARKIN,
D. M.,
AND STEWART,
J. W.,
Brit.
Med. J. 1,96,1966. 4. STEWART, J. W., Brit. Med. J. 1,304,1964. 5. GOOD, R. A., PETERSON, R. D. A., PEREY, munologic Deficiency Diseases in Man,”
D. Y., FINSTAD, J., AND COOPER, M. D., In “Impp. 17-34, National Foundation Press, New York,
1964. 6. HONG, R., KAY, H. E. M., COOPER, M. D., MEUWISSEN,.H., ALLEN, R. A., Lancet I, 503, 1968. 7. AMOS, D. B., AND BACH, F. J., J. Erp. Med. 128,623, 1968. 8. YUMS, E. J., AND AMOS, B., hoc. Nat. Acad. Sci. 68,3031, 1971. 9. GATTI, R: A., MEUWISSEN, 1971. 10. GATTI, R. A., MEU~ISSEN,
H. J., TERASAM, H. J., ALLEN,
P. I., AND GOOD,
M.
J. G., AND
GOOD,
Antigens
1, 239,
R. A., Tissue
H. D., HONG,
R., AND GOOD,
P. I., HONG,
R., AND GOOD,
R. A.,
Lancet 2, 1366,
1968. 11. MEUWISSEN,
281,691,
H. J., GA~I,
R. A., TERASAKI,
R. A., N.
Engl. J. Med.
1969.
12. BIGCAR, W. D., GOOD, R. A., AND PARK, B. H., J. Pedtit. 81,301, 1972. 13. PARK, B. H., BIGGAR, W. D., AND GOOD, R. A., Transplantation 14,384, 1972. 14. GOOD, R. A., AND CAMPBELL, B., Amer. J. Med. 9,330, 1950. 15. BURKE, B. A., AND GOOD, R. A., Medicine, 52,23, 1973. 16. WALZER, P. D., SCHULTZ, M. G., WESTERN, K. A., AND ROBBINS, J. B., J. Pediat. 82,416,
Ii’. 18.
1973. KIRBY, H. B., KENAMORE, B., AND GUCKLAN, FOX, M., Immunology 5,489, 1962.
S. C.,
Ann. Int. Med. 75,505, 1971.
VARIABILITY
IN IMMUNOLOGIC
FWCONSTITUTION
509
19. WALKER, K. Z., SCHOEFL, G. T., ANJJ LAFFERTY, K. J.,Aust. J. Erp. Biol. Med. Sci. 59,675, 1972. 29. YUNIS, E. J., HILGARD, H. R., MARTINEZ, C., AND GOOD, R. A., J. Erp. Med. 121,607, 1965. 21. GOODMAN, J. W., MARTIN, F. B., AND CONGDON, C. C., Arch. Pathol. 89,226, 1970. 22. STUTMAN, O., YUNIS, E. J., AND GOOD, R. A.,]. Immunol. 103,92, 1969. 23. PARK, B. H., AND GOOD, R. A., Proc. Nat. Acad. Sci 69, 1490, 1972. 24. GIBLETT, E. R., ANDERSON, J. E., COHEN, F., POLLARA, B., AND MEUWISSEN, H. J.,Lancet 172, 1667, 1971. 25. EDWARDS, J. H., ALLEN, F. H., GLENN, K. P., LAMM, L. V., AND ROBSON, E. B., In “Histocompatibility Testing” (J. Dausset and J. Colombani, Eds.) 1972 ed., pp. 745-751, Munksgaard, Copenhagen, 1973.