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Human T-lymphocyte differentiation in immunodeficiency diseases and after reconstitution by bone marrow or fetal thymus transplantation
Human T-Lymphocyte Differentiation in lmmunodeficiency Diseases and after Reconstitution by Bone Marrow or Fetal Thymus Transplantation JEAN-LOUIS Transplanrarion
and Itnmunohiology Uni/. Pnv. P.. HGpitcrl E. Herriot. Received
TOTRAINE. Clitric of Nrphtdogy 69374 Lyon Cede.x
tttd IN.SEH,M 2. Frcrncr
Uttit 80,
June 28. 1978
Characteristics of T lymphocytes. including differentiation antigens, capacity to form E rosettes. and proliferative responses to some phytomitogens and to allogeneic stimuli were looked for in peripheral blood lymphocytes from patients with certain immunodeficiency diseases. In virro induction of some T-cell characteristics on a fraction of bone marrow cells was possible in all normal donors, in patients with Di George syndrome. and in patients with common variable immunodeficiencies, but it was severely altered in all infants with severe combined immunodeficiency diseases (KID). Although SCID appear to be a very heterogeneous syndrome. some evidence is presented indicating that most cases of SCID may result from a block in early events of T-cell differentiation. After bone marrow transplantation from a compatible donor, T lymphocytes developed from the donor’s cells and the sequential stages of maturation confirmed our previously described model based on in \+o experiments: appearance of the HTLA’ phenotype, then of the capacity to form E rosettes and later of the responses to allogeneic cells or to phytomitogens. In Di George syndrome treated with fetal thymus transplantation. the recipient’s T-cell precursors differentiated and proliferated under the influence of the grafted thymus. following a comparable sequence of events.
INTRODUCTION
Recent knowledge of T-cell differentiation and of T-cell subsets has been derived from in vitro experiments using thymic factors as inducers ( 1, 3) and from analysis of functions associated with T cells of various phenotypes (3, 4). Based on such in vitro results we have proposed a model of sequential stages of T-lymphocyte differentiation in man (5, 6) : acquisition of human T-lymphocyte differentiation antigens (HTLA), then of the capacity to form E rosettes. and thereafter appearance of the responsiveness to allogeneic cells or to phytomitogens. These results are in agreement with the findings from ontogenetic studies. The purpose of this paper is to explore human T-cell differentiation after immunological reconstitution in vivo, to compare it with data obtained in induction experiments in vitro. and to see what information on immunodeficiencies can be obtained from the results of such assays. MATERIALS
AND METHODS
Two surface characteristics of T lymphocytes were studied: the HTLA’ phenotype and the capacity to form E rosettes. The presence of HTLA at the cell surface was revealed using a heterologous antiserum specifically cytotoxic for 228 0090-1229/79/020228Copyright All
rights
0 of
1979 reproductton
by
lO$Ol.OO/O Academic m anv
Press.
Inc
form
reserved
T-LYMPHOCYTE
DIFFERENTIATION
229
human T lymphocytes (7). E rosettes were formed after incubation of the lymphocytes with untreated sheep erythrocytes (SRBC) in the presence of SRBCabsorbed fetal calf serum (8). Bone marrow (BM) cells were separated in live layers by centrifugation on a discontinuous bovine serum albumin (BSA) density gradient (9). Thymocytes were prepared from thymuses of infants undergoing cardiac surgery. The induction assay consisted of the incubation, for various periods of time, of cells with thymic extracts (2) followed by the determination of T-cell characteristics (9). Lymphocyte cultures, in the presence of mitogens or of mitomycin-treated allogeneic lymphocytes, were performed as previously described ( 10). RESULTS
AND DISCUSSION
T-Lymphocyte Differentiation in Immunodejkiency Diseases Severe combined immunodeficiencies (SCID) are heterogeneous diseases. All patients reported here had a relative lymphopenia, a significant reduction in the number of T cells (low percentages of HTLA+ cells and of E rosette-forming cells) in peripheral blood and a variable proportion of immunoglobulin (Ig)-bearing B cells. No in vitro proliferative response to phytohemagglutinin (PHA) or concanavalin A (Con A) could be demonstrated and the allogeneic response was weak or absent. There was virtually no identifiable antibody synthesis in any of the patients but serum IgM levels were comparatively high in some of them. Genetic analysis allowed discrimination between X-linked forms of SCID and autosomal recessive forms. Two patients had an identified genetic deficiency in the enzyme adenosine deaminase (ADA), the others did not. Several of the patients had been studied repeatedly from birth until treatment (BM transplantation) was begun: in almost every instance, even before infection could be detected, the immunological abnormalities, including the defect in number and functions of peripheral blood T lymphocytes, tended to increase with time. In the induction assay (2)) bone marrow T-cell precursors susceptible to in vitro acquisition of the HTLA+ phenotype were absent or minimal in all patients with SCID (Table 1). No result comparable to that observed in normal marrow donors was ever found in SCID. However, this assay permitted the classification of patients into two groups: those with no inducible T-cell precursors (ITCP) and those with only a significantly reduced number of ITCP. In the latter group, there may be some patients without E-rosetting capacity in the induction assay (11) and this abnormality, according to our scheme (5), suggests a further block in early events of T-cell differentiation. As yet, no correlation has been found between the classification based on the degree of deficiency in ITCP and any particular form of SCID (X linkage, association with deficiency in ADA activity, presence of IgMbearing B lymphocytes, etc.). Only age was identified as one of the possible factors: in two infants with SCID, sequential studies showed that if the numbers of ITCP were indeed decreased at birth, they were much more so 2 to 3 months later. This finding, as well as the frequent existence of a few T lymphocytes and B lymphocytes at birth, argues against a complete absence of lymphoid stem cells in SCID. SCID therefore appear to result more often from a blockage of T-cell precursors during very early stages of T-lymphocyte differentiation, rather than from a genuine stem cell defect. This blockage seems to be generally related to
230
,JEAN-LOUIS
TOURAINE
intrinsic cellular defects or abnormalities (such as enzymatic deficiencies), but may occasionally be associated with extrinsic defects [e.g., abnormal function of the epithelial thymus? ( 12, 13)]. Different diseases with different causes are thus included in the KID syndrome but, in all cases, T-lymphocyte differentiation is prevented or severely altered at an early stage. The spectrum of abnormalities found in the differentiation of the B-cell system probably results either from an associated alteration of B-cell precursors or from the lack of normal T lymphocytes [Ref. ( 14) and unpublished personal results]. Three patients with partial Di George syndrome were studied. Peripheral blood T lymphocytes were relatively few and the percentages of B lymphocytes appeared to be high. In these children, as in every normal individual so far tested. substantial numbers of marrow cells from either layer II or layer III were converted from the HTLA- to the HTLA’ phenotype after incubation with thymic factors (Table 1). This result confirms that T-cell precursors from these patients can behave normally but do not differentiate spontaneously in viva due to lack of differentiation induction of thymic origin. Patients with other forms of immunodeficiency diseases were found to have normal percentages of ITCP in their bone marrow. Based on these results in the “i,z vitro differentiation assay” it is therefore possible that in common variable immunodeficiency patients having relatively few peripheral T lymphocytes (the two cases studied), the blockage of T-cell differentiation occurs at a later stage than in SCID patients. T-Lymphocyte Differentiation Transplantation
after Bone Marrow
or Fetal Thymus
The above-mentioned SCID patients have been studied and treated in various countries. We shall only describe the events in the four patients that we treated TABLE PERCFNI.AGES
OF INDUCIBLE
BONE
I MARROW
IN I.VMUNODEFICIEN~Y
Group
of patients
No. studied
Normal
25
Severe combined immunodeticiencies
10
Partial
Di George
syndrome
T-CELL
PRECURSORS
DISEASES”
Absence of conversion (G2%) -
Minimal conversion (3-10%)
Normal conversion (> 10%)
-
25
4
-
6
3
-
-
3
Common variable immunodeficiency
2
-
-
2
X-Linked
I
-
agammaglobulinemia
1
(I The patients’ bone marrow cells were separated in five layers by centrifugation on a discontinuous BSA density gradient. Cells from each layer were incubated for 2 hr with thymic factors then the number of HTLA+ cells was determined. The percentage of marrow cells which was induced to acquire the HTLA+ phenotype was above 10% among either the layer II cells or the layer III cells, with the exception of patients with SCID, only.
T-LYMPHOCYTE
DIFFERENTIATION
231
with bone marrow transplantation in Lyon and Saint-Etienne, in whom we have performed sequential investigations of T lymphocytes during the months and years following transplantation. An additional infant has recently received a fetal liver transplant with apparently good results but it is too early to evaluate the long-term efficiency of the treatment. For each of the four bone marrow transplants, the donor was an HLA A, B, and D identical sibling. Marrow aspirates were of small volume (less than 2 ml for each aspirate). Intravenous infusion of 1.5 x lOa marrow nucleated cells/kg was performed immediately, without any fractionation maneuver. Case reports are as follows. Patient C. T. (Prof. Jeune’s department) was a 3-month-old boy when the SCID was diagnosed. He had a severe pulmonary infection, a mucocutaneous candidiasis, and some signs of graft versus host reaction (GvHR) following a blood transfusion. Results of laboratory assays were as follows: 1% of HTLA+ lymphocytes in peripheral blood (normal, 49-78%), 0.5% of E rosette-forming cells (normal, 40-75%), 2% of Ig-bearing lymphocytes (normal, IO-32%), absence of proliferative response to phytomitogens and to allogeneic cells, absence of demonstrable antibody synthesis, and profound ADA deficiency in red blood cells (0.6% of normal), in leukocytes (5% of normal), and in fibroblasts (11% of normal). The father, the mother, and the only sister of the patient were ADA heterozygous and immunologically normal. BM transplantation was performed from the 4-year-old, ABO- and HLA-identical sister when the patient was 4-months old and repeated 3 months later. During these months, immunological functions developed and clinical condition improved remarkably. The infant recovered from previous infections and gained weight. A mild and transient GvHR (fugacious rash, hepatosplenomegaly, slight increase in serum transaminase activities) occurred 2 weeks after the first transplant but had a spontaneously favorable outcome. Peripheral blood lymphocytes increased and progressively differentiated: the percentage of HTLA+ cells rose, the capability to form E rosettes followed very shortly, and Ig-bearing B lymphocytes became more numerous (Fig. 1). Serum Ig levels increased with a transient peak of homogeneous IgG. Various antibodies were found. Lymphocyte proliferative responses to allogeneic stimuli and to Con A, then to PHA and pokeweed mitogen were observed in vitro many weeks after the appearance of HTLA+, E rosette-forming cells. All peripheral T lymphocytes were of the donor type (XX) and exhibited a relatively increased ADA activity. Myeloid marrow cells remained of the recipient type (XY) and erythrocyte ADA activity was not significantly modified. T-lymphocyte precursors able to undergo in vitro differentiation could be identified in the bone marrow 2 months after the first transplant. The patient is at present in perfect clinical condition, after a follow-up of 3.5 years. He responds well to vaccinations and to usual childhood infections, he has a positive skin test 1:o Cundidu antigens, he does not receive any treatment and he lives a normal life. Patient G. C. (Dr. Freycon’s department) was a girl with a family history suggestive of SCID with histiocytosis and the diagnosis was established soon after birth: significant lymphopenia and eosinophilia, relatively few T and B lymphocytes exhibiting surface markers, no in vitro proliferative response to mitogens.
232
,pxN-I.ouIs
TouK.AISt
Response to allogeneic stimuli was low but present. Serum level of IgM was high (44 mg/dl). ADA activity was normal. Two sequential marrow transplants from the Y-year-old. ABO- and HLA-identical sister were performed. A very mild skin rash and a transient, partial alopecia suggested a possible and very minimal GvHR which disappeared without any specific treatment. One month following the marrow transplants, the number of eosinophils became normal and the lymphocyte count increased to over 1500/mm”. HTLA-+ cells augmented rapidly and after a few weeks were capable of E rosette formation (Fig. 1). Marrow T-cell precusors were detected in near normal numbers after 6 weeks. In vitro responses developed late and slowly but, at Z years of age, the stimulation index reached 71 for allogeneic stimuli, 17 for Con A, and 7 for PHA. Skin test to Ccindida antigens became positive and a transfer of the donor’s sensitivity to DNFB was demonstrable (the recipient had purposefully not been sensitized while the donor was). Serum Ig levels and antibody responses became normal. Due to the lack of an easily detectable genetic marker, the engraftment was difficult to ascertain. Some differences existed between the immunoglobulin Gm groups of the recipient and of the donor (as well as the mother) before transplantation and, following each of the two transplants, a significant decrease of the recipient’s Gm 1 group was observed. The present clinical condition of the patient is very satisfactory, more than 3 years after transplantation. She lives a normal life without any treatment and responds favorably to vaccinations and infections. Patient A. M. (Dr. Salle’s department), a boy. is the third child of North-African parents and was born after a girl who died of GvHR following blood transfusion. Immunobiological studies performed in A. M. at birth established that he similarly had a SCID: severe lymphopenia, virtual absence of T or B lymphocytes with surface markers in peripheral blood, absence of response to Con A and PHA, absence of plasma cells in bone marrow, and low levels of serum IgM. Marrow transplantation was performed at 6 weeks of age from an ABO- and HLA-identical sister, without any subsequent sign of GvHR. Immunological reconstitution was rapid: HTLA’ cells developed first, later acquiring the E rosette characteristic (Fig. I). Ig-bearing B lymphocytes appeared shortly thereafter. The lymphocyte proliferative responses and the secretion of serum immunoglobulins developed months later. Marrow ITCP became detectable in normal numbers. Chromosomal studies showed 46 XX karyotype in peripheral blood (T lymphocytes of the donor type) and 46 XY in bone marrow (myeloid cells of the recipient type). Three years after transplantation, the patient is in perfect clinical and immunological condition. Patient N. H. (Prof. Monnet’s department) was the ninth child of North-African parents. The family history of SCID was unfortunately not mentioned at birth and she was given a bacillus Calmette-Guerin (BCG) vaccination. When referred to us. the patient was in a very poor clinical condition including severe pneumonia, suppurative otitis, and cutaneous candidiasis. Examination showed no BCG scar. no lymph adenopathy. a moderate enlargement of liver and spleen. no skin reactions to purified protein derivative (PPD) or Cundidct antigens, and sensitization to DNCB was impossible. Laboratory investigations showed a moderate lymphopenia, very few T cells. present B cells, no plasma cells, low levels of Ig, no
T-LYMPHOCYTE
DIFFERENTIATION
233
response to mitogens or allogeneic cells, and a normal ADA activity. Two marrow transplants from an ABO-different, HLA-identical brother were performed at 5.5 and 6.5 months of age. The clinical condition continued to deteriorate: persistent fever, morbilliform rash, diarrhea, progressive appearance of pancytopenia without increase of serum transaminase activities or eosinophils, aggravation of pneumopathy resulting in death from respiratory failure, and extremely poor general condition. The last investigations on peripheral blood lymphocytes showed a beginning of reconstitution: 45.5% HTLA’ cells, 35% E rosette-forming cells, absence of PHA response. The cause of death was the BCG infection and post mortem examination of bone marrow and liver revealed extensive lesions of tuberculosis-like infection with epithelioid cells and acid-alcohol-resistant bacilli stained by Ziehl dye. On the whole, these four patients have demonstrated at least some degree of immunological reconstitution. Two patients received marrow transplants very early because of investigations at birth (family history suggestive of SCID) and the clinical course was uneventful. The two others were severely infected at the time of transplantation: one died of his BCG infection, the second patient was cured but clinical treatment was obviously less simple than in the uninfected infants. In all cases, T lymphocytes developed in viva following a sequence of differentiation events similar to that observed in in vitro experiments. Relatively long periods of time were, however, required for full development of mature T lymphocytes perhaps because, in a full in vivo development, proliferation and differentiation are closely linked processes. Several weeks were needed for the appearance of a large population of peripheral blood lymphocytes with an HTLA+ phenotype and the capacity to form E rosettes. In vitro response to allogeneic cells became significant only a few months later in the three long-term survivors. The response to Con A, then to PHA [characteristic of a T-cell subset different from that responding to allogeneic stimuli (lo)] appeared concomitantly to allogeneic responsiveness or was slightly delayed. These T lymphocytes developed from the donor’s marrow T-cell precursors. The latter cells were also observed in significant numbers in the recipient’s bone marrow a few weeks after transplantation. T-lymphocyte differentiation in viva has also been studied after fetal thymus transplantation in a patient with partial Di George syndrome. S. P. (Prof. Larbre’s department) had convulsions from the first days of life, followed by infections: purulent rhinitis and pneumonia. Hypoparathyroidism was confirmed: hypocalcemia, 50 mg/liter, hyperphosphoremia, 75 mg/liter, and undetectable level of serum parathormone. Facial abnormalities were noticed, no cardiovascular abnormality was detected. No thymic shadow was seen. Peripheral blood T lymphocytes were few: 28% of HTLA’ cells and 15% of E rosette-forming cells. The percentage of Ig-bearing B cells was high, 53%. Fetal thymus transplantation was carried out at 5 months of age. The thymus from a 12-week-old female fetus, kindly provided by the London “Tissue Bank,” was minced and implanted in a pouch created in the rectus abdominis muscle. Cell viability (trypan blue exclusion method) was 95% at the time of implantation. Clinical and immunological modifications were rapid after the thymus transplant. Pneumonia was resolved and rhinorrhea disappeared. No sign of GvHR was detecfed. T lymphocytes
234
JEAN-LOUIS
TOURAINE
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T-LYMPHOCYTE
235
DIFFERENTIATION
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236
JEAN-LOCTIS
‘I-UURAINE
developed readily acquiring the HTLA’ phenotype, then the E-rosetting capacity (Fig. 1). The rapid T-cell reconstitution following transplantation can be regarded as an evidence against a spontaneous cure of this patient’s condition. The responses to mitogens progressively increased: The Con A response was less than 30% of normal at 3 months, 80% at 1 year, and 100% at I.5 year. All peripheral T lymphocytes were of the recipient type (XY) confirming that they derived from the patient’s own T-cell precursors differentiating under the influence of the transplanted thymus. This good clinical and immunological condition has not altered since transplantation and the patient lives normally at the present time, 3.5 years later. The response to vaccinations and infections has been normal. The only treatment presently given to the patient is vitamin D. A surprising finding has been the relatively long duration of the thymic function, as evaluated by secretion of a serum factor with an inhibitory activity on azathioprine-sensitive rosettes (1.5): the thymic factor activity measured in serum by Drs. M. Dardenne and J. F. Bach was unusually low ( I :2) before transplantation, confirming the thymic defect, rose rapidly after it, and was still found at a perfectly normal level ( 1:32) in two determinations more than 2 years later. In this patient with Di George syndrome, as in SCID patients treated with bone marrow transplantation, but somewhat more rapidly than in the latter cases. T lymphocytes matured sequentially and the differentiation stages were very comparable to those observed in induction experiments performed in t*itro. Analysis of some other markers and functions of human T lymphocytes will permit the elaboration of a more complete scheme of T-lymphocyte differentiation and such a model will hopefully provide a basis for a more rational classification of all T-cell abnormalities. ADDENDUM
More recently we have treated a SCID patient with a fetal liver and thymus transplant. After a very long period, cells from the donor (with a completely different HLA haplotype than recipient’s cells) were found in significant number in the peripheral blood. The child is now fully reconstituted and has been taken off isolation. Since then he developed two minor viral infections and appeared to be normally capable of limiting the virus spread. He is presently in perfect condition without any further therapy. When T lymphocytes proliferated and differentiated. their development followed the same stages as in the previous cases but required more than 6 months. These results demonstratively confirmed the in vi\~ sequence of T-lymphocyte differentiation, following transplantation of even more immature cells than bone marrow cells (which contain a few postthymic cells in addition to prothymocytes). ACKNOWLEDGMENTS We are grateful to Drs. F. Touraine. R. A. Good. and G. S. Incefy for their help and advice in these studies. Clinical treatment was provided in collaboration with Drs. B. B&end, H. Betuel. B. Chataing, L. Cret, R. Francois. F. Freycon, C. Genin, R. Gilly, M. Jeune. F. Larbre. B. Lam-as. S. D. Lawler. P. Monnet. N. Philippe. H. Plauchu. P. Richard, B. Salle. and G. Souillet. The excellent technical assistance of 0. de Bouteiller is acknowledged. This work was supported by Grants ATP 11. 74. 32 and 76.59 from the “lnstitut National de la Sante et de la Recherche Medicale” and ATP 21. 27 and 36.17 from the “Centre National de la Recherche Scientifique.” A part of this work was presented at the
T-LYMPHOCYTE
DIFFERENTIATION
Third Workshop of the International Cooperative Group for Bone Marrow Transplantation Tarrytown, New York, August 19-22, 1976.
237 in Man,
REFERENCES 1. Komuro, K., and Boyse, E. A., Lancer 1, 740, 1973. 2. Touraine, J. L., Incefy, G. S., Touraine, F., Rho, Y. M., and Good, R. A., Clin. Exp. Immunol. 17, 151, 1974. 3. Cantor, H., and Boyse, E. A., J. Exp. Med. 141, 1376, 1975. 4. Kisielow, P., Hirst, J. A., Shiku, H., Beverley, P. C. L., Hoffman, M. K., Boyse, E. A., and Oettgen, H. F., Nature (London) 253, 219, 1975. 5. Touraine, J. L., In “Leukocyte Membrane Determinants Regulating Immune Reactivity” (V. P. Eijsvoogel, D. Roos, W. P. Zeijkemaker, Eds.), pp. 71 l-717, Academic Press, New York, 1976. 6. Touraine. J. L., Hadden, J. W.. and Good, R. A., Proc. Nat. Acad. Sci. USA 74, 3414, 1977. 7. Touraine, J. L., Touraine, F., Kiszkiss, D. F., Choi, Y. S., and Good, R. A., C/in. Exp. Zmmunol. 16, 503, 1974. 8. Bach, J. F., Transplant. Rev. 16, 196, 1973. 9. Touraine, J. L., Touraine, F., Incefy, G. S., and Good, R. A., Ann. N. Y. Acad. Sci. 249, 335, 1975. 10. Touraine. J. L.. Touraine, F., Hadden, J. W., Hadden, E. M., and Good, R. A., Int. Arch. Aliergy Appl. Immunol. 52, 105, 1976. 11. Incefy, G. S., Grimes, W. A., Kagan, G., Goldstein, G., Smithwick, E., O’Reilly, R., and Good, R. A., Clin. Exp. Immunol. 25, 462, 1976. 12. Pyke, K. W., Dosch, H. M., Ipp, M. M., and Gelfand, E. W., N. Engl. J. Med. 293, 424, 1975. 13. Hong, R.. Santosham, M.. Schulte-Wissermann. H., Horowitz, S., Hsu, S., and Winkelstein. J. A.. Lancer 2, 1270, 1976. 14. Seeger, R. C., Robins, A. R., Stevens, R. H., Klein, R. B., Waldman, D. J., Zeltzer, P. M., and Kessler, S. W., C/in. Exp. Immunol. 26, 1, 1976. 15. Bach, J. F., Dardenne, M., Papiernik, M., Barois, A., Levasseur, P., and Le Brigand, H., Lancer 2, 1056, 1972.
and Itnmunohiology Uni/. Pnv. P.. HGpitcrl E. Herriot. Received
TOTRAINE. Clitric of Nrphtdogy 69374 Lyon Cede.x
tttd IN.SEH,M 2. Frcrncr
Uttit 80,
June 28. 1978
Characteristics of T lymphocytes. including differentiation antigens, capacity to form E rosettes. and proliferative responses to some phytomitogens and to allogeneic stimuli were looked for in peripheral blood lymphocytes from patients with certain immunodeficiency diseases. In virro induction of some T-cell characteristics on a fraction of bone marrow cells was possible in all normal donors, in patients with Di George syndrome. and in patients with common variable immunodeficiencies, but it was severely altered in all infants with severe combined immunodeficiency diseases (KID). Although SCID appear to be a very heterogeneous syndrome. some evidence is presented indicating that most cases of SCID may result from a block in early events of T-cell differentiation. After bone marrow transplantation from a compatible donor, T lymphocytes developed from the donor’s cells and the sequential stages of maturation confirmed our previously described model based on in \+o experiments: appearance of the HTLA’ phenotype, then of the capacity to form E rosettes and later of the responses to allogeneic cells or to phytomitogens. In Di George syndrome treated with fetal thymus transplantation. the recipient’s T-cell precursors differentiated and proliferated under the influence of the grafted thymus. following a comparable sequence of events.
INTRODUCTION
Recent knowledge of T-cell differentiation and of T-cell subsets has been derived from in vitro experiments using thymic factors as inducers ( 1, 3) and from analysis of functions associated with T cells of various phenotypes (3, 4). Based on such in vitro results we have proposed a model of sequential stages of T-lymphocyte differentiation in man (5, 6) : acquisition of human T-lymphocyte differentiation antigens (HTLA), then of the capacity to form E rosettes. and thereafter appearance of the responsiveness to allogeneic cells or to phytomitogens. These results are in agreement with the findings from ontogenetic studies. The purpose of this paper is to explore human T-cell differentiation after immunological reconstitution in vivo, to compare it with data obtained in induction experiments in vitro. and to see what information on immunodeficiencies can be obtained from the results of such assays. MATERIALS
AND METHODS
Two surface characteristics of T lymphocytes were studied: the HTLA’ phenotype and the capacity to form E rosettes. The presence of HTLA at the cell surface was revealed using a heterologous antiserum specifically cytotoxic for 228 0090-1229/79/020228Copyright All
rights
0 of
1979 reproductton
by
lO$Ol.OO/O Academic m anv
Press.
Inc
form
reserved
T-LYMPHOCYTE
DIFFERENTIATION
229
human T lymphocytes (7). E rosettes were formed after incubation of the lymphocytes with untreated sheep erythrocytes (SRBC) in the presence of SRBCabsorbed fetal calf serum (8). Bone marrow (BM) cells were separated in live layers by centrifugation on a discontinuous bovine serum albumin (BSA) density gradient (9). Thymocytes were prepared from thymuses of infants undergoing cardiac surgery. The induction assay consisted of the incubation, for various periods of time, of cells with thymic extracts (2) followed by the determination of T-cell characteristics (9). Lymphocyte cultures, in the presence of mitogens or of mitomycin-treated allogeneic lymphocytes, were performed as previously described ( 10). RESULTS
AND DISCUSSION
T-Lymphocyte Differentiation in Immunodejkiency Diseases Severe combined immunodeficiencies (SCID) are heterogeneous diseases. All patients reported here had a relative lymphopenia, a significant reduction in the number of T cells (low percentages of HTLA+ cells and of E rosette-forming cells) in peripheral blood and a variable proportion of immunoglobulin (Ig)-bearing B cells. No in vitro proliferative response to phytohemagglutinin (PHA) or concanavalin A (Con A) could be demonstrated and the allogeneic response was weak or absent. There was virtually no identifiable antibody synthesis in any of the patients but serum IgM levels were comparatively high in some of them. Genetic analysis allowed discrimination between X-linked forms of SCID and autosomal recessive forms. Two patients had an identified genetic deficiency in the enzyme adenosine deaminase (ADA), the others did not. Several of the patients had been studied repeatedly from birth until treatment (BM transplantation) was begun: in almost every instance, even before infection could be detected, the immunological abnormalities, including the defect in number and functions of peripheral blood T lymphocytes, tended to increase with time. In the induction assay (2)) bone marrow T-cell precursors susceptible to in vitro acquisition of the HTLA+ phenotype were absent or minimal in all patients with SCID (Table 1). No result comparable to that observed in normal marrow donors was ever found in SCID. However, this assay permitted the classification of patients into two groups: those with no inducible T-cell precursors (ITCP) and those with only a significantly reduced number of ITCP. In the latter group, there may be some patients without E-rosetting capacity in the induction assay (11) and this abnormality, according to our scheme (5), suggests a further block in early events of T-cell differentiation. As yet, no correlation has been found between the classification based on the degree of deficiency in ITCP and any particular form of SCID (X linkage, association with deficiency in ADA activity, presence of IgMbearing B lymphocytes, etc.). Only age was identified as one of the possible factors: in two infants with SCID, sequential studies showed that if the numbers of ITCP were indeed decreased at birth, they were much more so 2 to 3 months later. This finding, as well as the frequent existence of a few T lymphocytes and B lymphocytes at birth, argues against a complete absence of lymphoid stem cells in SCID. SCID therefore appear to result more often from a blockage of T-cell precursors during very early stages of T-lymphocyte differentiation, rather than from a genuine stem cell defect. This blockage seems to be generally related to
230
,JEAN-LOUIS
TOURAINE
intrinsic cellular defects or abnormalities (such as enzymatic deficiencies), but may occasionally be associated with extrinsic defects [e.g., abnormal function of the epithelial thymus? ( 12, 13)]. Different diseases with different causes are thus included in the KID syndrome but, in all cases, T-lymphocyte differentiation is prevented or severely altered at an early stage. The spectrum of abnormalities found in the differentiation of the B-cell system probably results either from an associated alteration of B-cell precursors or from the lack of normal T lymphocytes [Ref. ( 14) and unpublished personal results]. Three patients with partial Di George syndrome were studied. Peripheral blood T lymphocytes were relatively few and the percentages of B lymphocytes appeared to be high. In these children, as in every normal individual so far tested. substantial numbers of marrow cells from either layer II or layer III were converted from the HTLA- to the HTLA’ phenotype after incubation with thymic factors (Table 1). This result confirms that T-cell precursors from these patients can behave normally but do not differentiate spontaneously in viva due to lack of differentiation induction of thymic origin. Patients with other forms of immunodeficiency diseases were found to have normal percentages of ITCP in their bone marrow. Based on these results in the “i,z vitro differentiation assay” it is therefore possible that in common variable immunodeficiency patients having relatively few peripheral T lymphocytes (the two cases studied), the blockage of T-cell differentiation occurs at a later stage than in SCID patients. T-Lymphocyte Differentiation Transplantation
after Bone Marrow
or Fetal Thymus
The above-mentioned SCID patients have been studied and treated in various countries. We shall only describe the events in the four patients that we treated TABLE PERCFNI.AGES
OF INDUCIBLE
BONE
I MARROW
IN I.VMUNODEFICIEN~Y
Group
of patients
No. studied
Normal
25
Severe combined immunodeticiencies
10
Partial
Di George
syndrome
T-CELL
PRECURSORS
DISEASES”
Absence of conversion (G2%) -
Minimal conversion (3-10%)
Normal conversion (> 10%)
-
25
4
-
6
3
-
-
3
Common variable immunodeficiency
2
-
-
2
X-Linked
I
-
agammaglobulinemia
1
(I The patients’ bone marrow cells were separated in five layers by centrifugation on a discontinuous BSA density gradient. Cells from each layer were incubated for 2 hr with thymic factors then the number of HTLA+ cells was determined. The percentage of marrow cells which was induced to acquire the HTLA+ phenotype was above 10% among either the layer II cells or the layer III cells, with the exception of patients with SCID, only.
T-LYMPHOCYTE
DIFFERENTIATION
231
with bone marrow transplantation in Lyon and Saint-Etienne, in whom we have performed sequential investigations of T lymphocytes during the months and years following transplantation. An additional infant has recently received a fetal liver transplant with apparently good results but it is too early to evaluate the long-term efficiency of the treatment. For each of the four bone marrow transplants, the donor was an HLA A, B, and D identical sibling. Marrow aspirates were of small volume (less than 2 ml for each aspirate). Intravenous infusion of 1.5 x lOa marrow nucleated cells/kg was performed immediately, without any fractionation maneuver. Case reports are as follows. Patient C. T. (Prof. Jeune’s department) was a 3-month-old boy when the SCID was diagnosed. He had a severe pulmonary infection, a mucocutaneous candidiasis, and some signs of graft versus host reaction (GvHR) following a blood transfusion. Results of laboratory assays were as follows: 1% of HTLA+ lymphocytes in peripheral blood (normal, 49-78%), 0.5% of E rosette-forming cells (normal, 40-75%), 2% of Ig-bearing lymphocytes (normal, IO-32%), absence of proliferative response to phytomitogens and to allogeneic cells, absence of demonstrable antibody synthesis, and profound ADA deficiency in red blood cells (0.6% of normal), in leukocytes (5% of normal), and in fibroblasts (11% of normal). The father, the mother, and the only sister of the patient were ADA heterozygous and immunologically normal. BM transplantation was performed from the 4-year-old, ABO- and HLA-identical sister when the patient was 4-months old and repeated 3 months later. During these months, immunological functions developed and clinical condition improved remarkably. The infant recovered from previous infections and gained weight. A mild and transient GvHR (fugacious rash, hepatosplenomegaly, slight increase in serum transaminase activities) occurred 2 weeks after the first transplant but had a spontaneously favorable outcome. Peripheral blood lymphocytes increased and progressively differentiated: the percentage of HTLA+ cells rose, the capability to form E rosettes followed very shortly, and Ig-bearing B lymphocytes became more numerous (Fig. 1). Serum Ig levels increased with a transient peak of homogeneous IgG. Various antibodies were found. Lymphocyte proliferative responses to allogeneic stimuli and to Con A, then to PHA and pokeweed mitogen were observed in vitro many weeks after the appearance of HTLA+, E rosette-forming cells. All peripheral T lymphocytes were of the donor type (XX) and exhibited a relatively increased ADA activity. Myeloid marrow cells remained of the recipient type (XY) and erythrocyte ADA activity was not significantly modified. T-lymphocyte precursors able to undergo in vitro differentiation could be identified in the bone marrow 2 months after the first transplant. The patient is at present in perfect clinical condition, after a follow-up of 3.5 years. He responds well to vaccinations and to usual childhood infections, he has a positive skin test 1:o Cundidu antigens, he does not receive any treatment and he lives a normal life. Patient G. C. (Dr. Freycon’s department) was a girl with a family history suggestive of SCID with histiocytosis and the diagnosis was established soon after birth: significant lymphopenia and eosinophilia, relatively few T and B lymphocytes exhibiting surface markers, no in vitro proliferative response to mitogens.
232
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Response to allogeneic stimuli was low but present. Serum level of IgM was high (44 mg/dl). ADA activity was normal. Two sequential marrow transplants from the Y-year-old. ABO- and HLA-identical sister were performed. A very mild skin rash and a transient, partial alopecia suggested a possible and very minimal GvHR which disappeared without any specific treatment. One month following the marrow transplants, the number of eosinophils became normal and the lymphocyte count increased to over 1500/mm”. HTLA-+ cells augmented rapidly and after a few weeks were capable of E rosette formation (Fig. 1). Marrow T-cell precusors were detected in near normal numbers after 6 weeks. In vitro responses developed late and slowly but, at Z years of age, the stimulation index reached 71 for allogeneic stimuli, 17 for Con A, and 7 for PHA. Skin test to Ccindida antigens became positive and a transfer of the donor’s sensitivity to DNFB was demonstrable (the recipient had purposefully not been sensitized while the donor was). Serum Ig levels and antibody responses became normal. Due to the lack of an easily detectable genetic marker, the engraftment was difficult to ascertain. Some differences existed between the immunoglobulin Gm groups of the recipient and of the donor (as well as the mother) before transplantation and, following each of the two transplants, a significant decrease of the recipient’s Gm 1 group was observed. The present clinical condition of the patient is very satisfactory, more than 3 years after transplantation. She lives a normal life without any treatment and responds favorably to vaccinations and infections. Patient A. M. (Dr. Salle’s department), a boy. is the third child of North-African parents and was born after a girl who died of GvHR following blood transfusion. Immunobiological studies performed in A. M. at birth established that he similarly had a SCID: severe lymphopenia, virtual absence of T or B lymphocytes with surface markers in peripheral blood, absence of response to Con A and PHA, absence of plasma cells in bone marrow, and low levels of serum IgM. Marrow transplantation was performed at 6 weeks of age from an ABO- and HLA-identical sister, without any subsequent sign of GvHR. Immunological reconstitution was rapid: HTLA’ cells developed first, later acquiring the E rosette characteristic (Fig. I). Ig-bearing B lymphocytes appeared shortly thereafter. The lymphocyte proliferative responses and the secretion of serum immunoglobulins developed months later. Marrow ITCP became detectable in normal numbers. Chromosomal studies showed 46 XX karyotype in peripheral blood (T lymphocytes of the donor type) and 46 XY in bone marrow (myeloid cells of the recipient type). Three years after transplantation, the patient is in perfect clinical and immunological condition. Patient N. H. (Prof. Monnet’s department) was the ninth child of North-African parents. The family history of SCID was unfortunately not mentioned at birth and she was given a bacillus Calmette-Guerin (BCG) vaccination. When referred to us. the patient was in a very poor clinical condition including severe pneumonia, suppurative otitis, and cutaneous candidiasis. Examination showed no BCG scar. no lymph adenopathy. a moderate enlargement of liver and spleen. no skin reactions to purified protein derivative (PPD) or Cundidct antigens, and sensitization to DNCB was impossible. Laboratory investigations showed a moderate lymphopenia, very few T cells. present B cells, no plasma cells, low levels of Ig, no
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response to mitogens or allogeneic cells, and a normal ADA activity. Two marrow transplants from an ABO-different, HLA-identical brother were performed at 5.5 and 6.5 months of age. The clinical condition continued to deteriorate: persistent fever, morbilliform rash, diarrhea, progressive appearance of pancytopenia without increase of serum transaminase activities or eosinophils, aggravation of pneumopathy resulting in death from respiratory failure, and extremely poor general condition. The last investigations on peripheral blood lymphocytes showed a beginning of reconstitution: 45.5% HTLA’ cells, 35% E rosette-forming cells, absence of PHA response. The cause of death was the BCG infection and post mortem examination of bone marrow and liver revealed extensive lesions of tuberculosis-like infection with epithelioid cells and acid-alcohol-resistant bacilli stained by Ziehl dye. On the whole, these four patients have demonstrated at least some degree of immunological reconstitution. Two patients received marrow transplants very early because of investigations at birth (family history suggestive of SCID) and the clinical course was uneventful. The two others were severely infected at the time of transplantation: one died of his BCG infection, the second patient was cured but clinical treatment was obviously less simple than in the uninfected infants. In all cases, T lymphocytes developed in viva following a sequence of differentiation events similar to that observed in in vitro experiments. Relatively long periods of time were, however, required for full development of mature T lymphocytes perhaps because, in a full in vivo development, proliferation and differentiation are closely linked processes. Several weeks were needed for the appearance of a large population of peripheral blood lymphocytes with an HTLA+ phenotype and the capacity to form E rosettes. In vitro response to allogeneic cells became significant only a few months later in the three long-term survivors. The response to Con A, then to PHA [characteristic of a T-cell subset different from that responding to allogeneic stimuli (lo)] appeared concomitantly to allogeneic responsiveness or was slightly delayed. These T lymphocytes developed from the donor’s marrow T-cell precursors. The latter cells were also observed in significant numbers in the recipient’s bone marrow a few weeks after transplantation. T-lymphocyte differentiation in viva has also been studied after fetal thymus transplantation in a patient with partial Di George syndrome. S. P. (Prof. Larbre’s department) had convulsions from the first days of life, followed by infections: purulent rhinitis and pneumonia. Hypoparathyroidism was confirmed: hypocalcemia, 50 mg/liter, hyperphosphoremia, 75 mg/liter, and undetectable level of serum parathormone. Facial abnormalities were noticed, no cardiovascular abnormality was detected. No thymic shadow was seen. Peripheral blood T lymphocytes were few: 28% of HTLA’ cells and 15% of E rosette-forming cells. The percentage of Ig-bearing B cells was high, 53%. Fetal thymus transplantation was carried out at 5 months of age. The thymus from a 12-week-old female fetus, kindly provided by the London “Tissue Bank,” was minced and implanted in a pouch created in the rectus abdominis muscle. Cell viability (trypan blue exclusion method) was 95% at the time of implantation. Clinical and immunological modifications were rapid after the thymus transplant. Pneumonia was resolved and rhinorrhea disappeared. No sign of GvHR was detecfed. T lymphocytes
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developed readily acquiring the HTLA’ phenotype, then the E-rosetting capacity (Fig. 1). The rapid T-cell reconstitution following transplantation can be regarded as an evidence against a spontaneous cure of this patient’s condition. The responses to mitogens progressively increased: The Con A response was less than 30% of normal at 3 months, 80% at 1 year, and 100% at I.5 year. All peripheral T lymphocytes were of the recipient type (XY) confirming that they derived from the patient’s own T-cell precursors differentiating under the influence of the transplanted thymus. This good clinical and immunological condition has not altered since transplantation and the patient lives normally at the present time, 3.5 years later. The response to vaccinations and infections has been normal. The only treatment presently given to the patient is vitamin D. A surprising finding has been the relatively long duration of the thymic function, as evaluated by secretion of a serum factor with an inhibitory activity on azathioprine-sensitive rosettes (1.5): the thymic factor activity measured in serum by Drs. M. Dardenne and J. F. Bach was unusually low ( I :2) before transplantation, confirming the thymic defect, rose rapidly after it, and was still found at a perfectly normal level ( 1:32) in two determinations more than 2 years later. In this patient with Di George syndrome, as in SCID patients treated with bone marrow transplantation, but somewhat more rapidly than in the latter cases. T lymphocytes matured sequentially and the differentiation stages were very comparable to those observed in induction experiments performed in t*itro. Analysis of some other markers and functions of human T lymphocytes will permit the elaboration of a more complete scheme of T-lymphocyte differentiation and such a model will hopefully provide a basis for a more rational classification of all T-cell abnormalities. ADDENDUM
More recently we have treated a SCID patient with a fetal liver and thymus transplant. After a very long period, cells from the donor (with a completely different HLA haplotype than recipient’s cells) were found in significant number in the peripheral blood. The child is now fully reconstituted and has been taken off isolation. Since then he developed two minor viral infections and appeared to be normally capable of limiting the virus spread. He is presently in perfect condition without any further therapy. When T lymphocytes proliferated and differentiated. their development followed the same stages as in the previous cases but required more than 6 months. These results demonstratively confirmed the in vi\~ sequence of T-lymphocyte differentiation, following transplantation of even more immature cells than bone marrow cells (which contain a few postthymic cells in addition to prothymocytes). ACKNOWLEDGMENTS We are grateful to Drs. F. Touraine. R. A. Good. and G. S. Incefy for their help and advice in these studies. Clinical treatment was provided in collaboration with Drs. B. B&end, H. Betuel. B. Chataing, L. Cret, R. Francois. F. Freycon, C. Genin, R. Gilly, M. Jeune. F. Larbre. B. Lam-as. S. D. Lawler. P. Monnet. N. Philippe. H. Plauchu. P. Richard, B. Salle. and G. Souillet. The excellent technical assistance of 0. de Bouteiller is acknowledged. This work was supported by Grants ATP 11. 74. 32 and 76.59 from the “lnstitut National de la Sante et de la Recherche Medicale” and ATP 21. 27 and 36.17 from the “Centre National de la Recherche Scientifique.” A part of this work was presented at the
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Third Workshop of the International Cooperative Group for Bone Marrow Transplantation Tarrytown, New York, August 19-22, 1976.
237 in Man,
REFERENCES 1. Komuro, K., and Boyse, E. A., Lancer 1, 740, 1973. 2. Touraine, J. L., Incefy, G. S., Touraine, F., Rho, Y. M., and Good, R. A., Clin. Exp. Immunol. 17, 151, 1974. 3. Cantor, H., and Boyse, E. A., J. Exp. Med. 141, 1376, 1975. 4. Kisielow, P., Hirst, J. A., Shiku, H., Beverley, P. C. L., Hoffman, M. K., Boyse, E. A., and Oettgen, H. F., Nature (London) 253, 219, 1975. 5. Touraine, J. L., In “Leukocyte Membrane Determinants Regulating Immune Reactivity” (V. P. Eijsvoogel, D. Roos, W. P. Zeijkemaker, Eds.), pp. 71 l-717, Academic Press, New York, 1976. 6. Touraine. J. L., Hadden, J. W.. and Good, R. A., Proc. Nat. Acad. Sci. USA 74, 3414, 1977. 7. Touraine, J. L., Touraine, F., Kiszkiss, D. F., Choi, Y. S., and Good, R. A., C/in. Exp. Zmmunol. 16, 503, 1974. 8. Bach, J. F., Transplant. Rev. 16, 196, 1973. 9. Touraine, J. L., Touraine, F., Incefy, G. S., and Good, R. A., Ann. N. Y. Acad. Sci. 249, 335, 1975. 10. Touraine. J. L.. Touraine, F., Hadden, J. W., Hadden, E. M., and Good, R. A., Int. Arch. Aliergy Appl. Immunol. 52, 105, 1976. 11. Incefy, G. S., Grimes, W. A., Kagan, G., Goldstein, G., Smithwick, E., O’Reilly, R., and Good, R. A., Clin. Exp. Immunol. 25, 462, 1976. 12. Pyke, K. W., Dosch, H. M., Ipp, M. M., and Gelfand, E. W., N. Engl. J. Med. 293, 424, 1975. 13. Hong, R.. Santosham, M.. Schulte-Wissermann. H., Horowitz, S., Hsu, S., and Winkelstein. J. A.. Lancer 2, 1270, 1976. 14. Seeger, R. C., Robins, A. R., Stevens, R. H., Klein, R. B., Waldman, D. J., Zeltzer, P. M., and Kessler, S. W., C/in. Exp. Immunol. 26, 1, 1976. 15. Bach, J. F., Dardenne, M., Papiernik, M., Barois, A., Levasseur, P., and Le Brigand, H., Lancer 2, 1056, 1972.