Reduction and repopulation of recipient T4+ and T8+ T-lymphocytes in allogeneic bone marrow transplantation.

In eight recipients of allogeneic bone marrow grafts who had sex-mismatched donors, the reduction and subsequent repopulation of T4+ and T8+ T-lymphocytes of recipient origin were studied. The origin of the donor-recipient T4+ and T8+ T cells was studied using quinacrine staining of Y chromatin combined with T-cell typing for T4 and T8. Following chemoradiotherapy and bone marrow transplantation (BMT), T cells reached their nadir at a median of five (range 1-8) days after BMT. T8+ T cells decreased at a faster rate from the peripheral blood than T4+ T cells. The first T cells that appeared in the circulation at day 12 were predominantly T4+, and a large number of them were of recipient origin. Thereafter, they gradually decreased, and the numbers of T cells of donor origin increased. In the patients who had no or only minor complications, T4+ and T8+ T cells of donor origin repopulated the blood at similar rates. This pattern, however, was modified by severe graft-versus-host disease or by cytomegalovirus infection.

Regeneration of TdT+, pre-B, and B cells in bone marrow after allogeneic bone marrow transplantation.

In 15 children transplanted with allogeneic bone marrow for acute leukemia and in complete remission, regeneration of the early stages of the B cell system was studied. Bone marrow aspirates taken before and longitudinally after BMT were investigated for pre-B and B cells by immunofluorescence techniques; in some cases, TdT+ cells were also determined. Normal values were derived from bone marrow samples taken from 23 healthy individuals who served as bone marrow donors. In normal bone marrow, B cells outnumber pre-B cells and the latter are more numerous than TdT+ cells. Before BMT, the numbers of BM pre-B were outside the normal range in all cases; B cell numbers were abnormal in most of the 11 patients studied, probably due to the antileukemic remission induction/consolidation therapy. After BMT, two distinct patterns of regeneration of the B cell system were observed. In 9 patients, TdT+ cells were considerably increased early after BMT. This was followed by a rise in pre-B cells, with values well above the normal range, and resulting in ratios of TdT+:pre-B cells and of pre-B cells:B cells that were transiently greater than 1. In the other 6 patients, the regeneration of TdT+ cells varied and the reconstitution of the pre-B cells was more gradual than in the first group, with pre-B-to-B cell ratios less than 1 during the whole observation period. The only consistent difference between the patients of the two groups, possibly relevant to the regeneration of the B cell lineage, was the duration of corticosteroid therapy, which was much longer in the 6 patients with slow-pace reconstitution. The pace of regeneration of the B cell system in the bone marrow was correlated with the recovery of the humoral immunity, as indicated by a significant increase in specific antibody titers after the second vaccination with diphtheria-tetanus-poliomyelitis vaccine in 7 of 9 patients in the rapid-pace group, versus 2 of 6 patients in the slow-pace group.

Lymphoid chimerism after allogeneic bone marrow transplantation. Y-chromatin staining of peripheral T and B lymphocytes and allotyping of serum immunoglobulins.

Lymphoid cell engraftment was monitored for several years after bone marrow transplantation by Y-chromatin staining of T and B lymphocytes in the peripheral blood and/or by immunoglobulin allotyping in the serum of 20 of 52 pediatric patients grafted successively between October 1973 and October 1983. Data on 2 patients with severe combined immunodeficiency, grafted earlier in December 1968 and April 1971, are also included. These children received an allogeneic bone marrow graft for leukemia (n = 7), severe aplastic anemia (n = 11), or severe combined immunodeficiency (n = 4) and were informative for this study, because they differed from their donor by sex (n = 16) and/or by immunoglobulin phenotype (n = 13). Of 16 pairs in which the donor was of the opposite sex, 11 patients ultimately showed circulating T and B lymphocytes of donor origin after bone marrow transplantation; in the remaining 5, there was an incomplete chimerism of the circulating lymphoid cells. Of 13 pairs with a difference in immunoglobulin phenotype between donor and recipient, 8 patients exhibited donor allotypes 3 months or later after transplantation, in 3 of them together with recipient allotypes. In the remaining 5 patients, recipient allotypes were detected after transplantation, but the simultaneous presence of donor-type immunoglobulin production could not be excluded in 4. The persistence of either a split (T lineage of donor origin and B lineage of recipient origin) or mixed (T and/or B lineage of donor and recipient origin) chimerism was related to the type of disease. In 3 children circulating B cells of donor-origin did not fit with the recipient origin of the sessile immunoglobulin-secreting plasma cells. This implies that different immune compartments--e.g., bone marrow and peripheral lymphoid tissues--should be investigated following allogeneic bone marrow transplantation. A prolonged presence of recipient-type lymphoid cells increased the risk of leukemic relapse in the patients investigated.

Reconstitution of lymphocyte subpopulations after paediatric bone marrow transplantation.

We prospectively studied the reconstitution of lymphocyte subpopulations in a group of 22 children, who survived disease-free at least 6 months after allogeneic BMT for a haematological malignancy. Absolute counts of total lymphocytes, B lymphocytes, T lymphocytes, and CD4+ helper T lymphocytes reached the 5th percentile (p5) of age-matched reference values within 6 months after BMT in 15, 17, 7 and 2 patients, respectively. In particular, CD4+ helper T lymphocyte reconstitution was very slow. Unexpectedly, CMV reactivation had a profound positive influence upon the number of CD4+ helper T lymphocytes in the children. In five patients, absolute B lymphocyte counts above the 95th percentile were reached from 6 months after BMT onwards, mimicking normal ontogeny. Unlike normal ontogeny, the percentages of helper T lymphocytes expressing the 'naive' CD45RA isoform were low and those expressing the 'memory' CD45RO isoform were high in the first 3 months after BMT, as described before. Thereafter, the CD45RA:CD45RO ratio slowly normalised. Also, CD7 expression was absent on up to 90% of T lymphocytes in the first months after BMT, and on a steadily decreasing percentage thereafter, as recently described in adults. However, the absolute counts of CD45RO+/CD4+ and CD7-/CD4+ helper T lymphocytes did not change significantly. So, we found no evidence of peripheral expansion of previously primed donor-derived 'memory' T lymphocytes during the follow-up period which spanned 1-18 months after BMT. The absolute counts of 'naive' CD45RA+ helper T lymphocytes did not show a faster increase after BMT than in adults, despite the presumed presence of a non-involuted thymus in children. Bone Marrow Transplantation (2000) 25, 267-275.

Risk factors for developing EBV-related B cell lymphoproliferative disorders (BLPD) after non-HLA-identical BMT in children.

B cell lymphoproliferative disorders (BLPD) are relatively frequent after genotypically non-HLA-identical BMT. We performed univariate analysis to study which BMT-related variables were associated with an increased risk of developing BLPD. Sixty-five recipients of other than genotypically HLA-identical BM grafts were included in the study. Seventy-seven recipients of genotypically HLA-identical BM grafts served as a comparison group. BLPD occurred in nine of 65 children after non-HLA-identical BMT (14%) and in none of the 77 children after HLA-identical BMT (0%). In all cases, BLPD was proven to be EBV-related. Our data suggest that the combined use of Campath 1G and anti-LFA1 was associated with an increased risk of developing BLPD, particularly children who had received a T cell-depleted BM graft, using albumen density gradient sedimentation followed by E-rosetting, and who were conditioned with Ara-C, CY and TBL. In addition, T cell numbers below 50/microliters at 1 month and below 100/microliters at 2 months after BMT, respectively, were associated with an increased risk of developing BLPD. Longitudinal determination of T cell numbers after non-HLA-identical BMT is a simple method for identifying patients at risk of developing BLPD. In addition to monitoring levels of circulating EBV-infected lymphocytes, monitoring T cell numbers may allow early intervention to prevent progression of BLPD.

[Combined immunologic deficiency]. / Gecombineerde immunologische deficiëntie.

In the last 20 years 32 patients with severe combined immunodeficiency (SCID) were treated at our department. The clinical and immunological findings in these patients are presented. In connection with these patients, the recent WHO classification on combined immunodeficiency diseases and the pathogenesis in several forms of SCID, as far as is known, are discussed. SCID appears to represent a very heterogeneous group of disorders, which is affirmed by the findings in several of our patients. At this moment bone marrow transplantation (BMT) is the only way to cure patients with SCID. Without this treatment the prognosis of these patients is very poor. After BMT complete recovery is achieved for the majority of patients, even in the absence of a genotypically HLA identical donor. Besides the therapeutic aspects of SCID the recent developments with regard to carrier-detection and antenatal diagnosis are briefly discussed.

Characterization of early lymphoid precursor cells in the human fetus using monoclonal antibodies and anti-terminal deoxynucleotidyl transferase.

Monoclonal antibodies (MoAbs) directed primarily against immature lymphoid cells (VIL-A1, BA-2, OKT10) or recognizing antigens associated with the B cell lineage (VIB-C5, OKI1) were used for the identification of lymphoid cells in liver, bone marrow, spleen and thymus of human fetuses between 8 and 20 weeks of gestational age. Many lymphocytes in liver, bone marrow and spleen reacted with the MoAbs used. In the fetal thymus, however, cells did not bind to the VIL-A1 and VIB-C5 MoAbs and only a few cells were BA-2+ or OKI1+. In the liver and bone marrow the VIL-A1, VIB-C5 and BA-2 MoAbs reacted almost exclusively with terminal deoxynucleotidyl transferase (TdT) containing cells, pre-B and B cells. TdT+ cells were present in liver, bone marrow and thymus, but not in the spleen. In liver and bone marrow the relative numbers of TdT+ cells decreased during gestation, in the thymus they increased. The antigenic make-up of the TdT+ cells in liver and bone marrow was comparable to that of pre-B and B cells in these organs: most of them reacted with VIL-A1, VIB-C5 and OKT10 MoAbs and many were BA-2+ and OKI1+. TdT+ cells in liver and bone marrow did not bind to T-cell-markers, i.e. OKT6 and WT-1. A few lymphoid cells in these organs contained TdT and mu heavy chains. TdT+ cells in the thymus had a completely different phenotype: most of them were OKT6+ and they did not react with the VIL-A1 and VIB-C5 MoAbs. These findings suggest that TdT+ cells in fetal liver and bone marrow are precursors of the B cell lineage, whereas those in the thymus probably belong to the T cell lineage. In the fetal spleen almost all B cells displayed the VIB-C5 and OKI1 antigens. At 12 weeks of gestation greater than 80% of splenic B cells were also VIL-A1+ and BA-2+; with ongoing gestation far less B cells in spleen expressed these antigens, however, indicating that these B cells are more mature than those in fetal liver and bone marrow, but still less mature than the B cells in postnatal blood and bone marrow, which do not display the VIL-A1 and BA-2 markers. These findings suggest that some further maturation of B cell stages takes place in the spleen during human fetal life.

Use of monoclonal antibodies in a study of the development of T lymphocytes in the human fetus.

A panel of monoclonal antibodies (OKT3, 4, 6, 8, 10, 11) was used for the identification of T lymphocyte subpopulations in cell suspensions of human fetal liver, thymus, bone marrow and spleen. In liver suspensions of 8-16 week old fetuses and in bone marrow suspensions (12-20 weeks) less than 5% of lymphocytes reacted with either OKT3, 11, 4, 8 or 6, whereas the OKT10 antibody bound to, respectively, 35 and 86% of lymphocytes in these tissues. In liver suspensions of 17-20 week old fetuses, about 20% of lymphocytes carried either the T3, 11, 4 or 8 antigen and more than 60% of lymphocytes were OKT10+. The maturation stages in fetal thymus (11-20 weeks) are comparable to those in the post-natal thymus, with the exception that a substantial proportion of fetal thymocytes expresses the T3 and T6 antigen simultaneously. In the fetal spleen (12-20 weeks), 40% of lymphocytes reacts with OKT3. These OKT3+ spleen cells may be divided into two subsets expressing either the T4 antigen or the T8 antigen. These OKT3+/OKT4+ and OKT3+/OKT8+ lymphoid cells of the fetal spleen can be further subdivided into a T10+ and T10- subpopulation. These data suggest that T lymphoid precursor cells, reacting with either none of the monoclonal antibodies or only with OKT10, are generated in fetal liver (up till 16 weeks gestational age) and bone marrow. Further maturation takes place in the fetal thymus, but also to a certain extent in peripheral lymphoid organs such as the fetal spleen, as evidenced by the coexistence of a T3+/T10+ and T3+/T10- subpopulation in this organ.

Usage of TCRAV and TCRBV gene families in human fetal and adult TCR rearrangements.

We have investigated fetal and adult T-cell receptor (TCR) A and B V-gene repertoires both by fluorescence-activated cell sorter (FACS) analysis with the available TCR V region-specific mAbs and by the polymerase chain reaction (PCR) with TCR V gene family-specific oligonucleotides. Among the low number of CD3+ T cells, most of the TCR V regions tested for could be detected by FACS analysis in liver, bone marrow, and spleen derived from a 14-week-old fetus and two 15-week-old fetuses. Similarly, the PCR analysis showed that the majority of the TCRAV and TCRBV families were expressed in the peripheral organs of the 13-week-old fetus, although an apparent absence of particular TCR V families was found in liver and bone marrow. This was most probably the consequence of the low number of CD3+ T cells in these organs. In 17-week-old fetal thymi the level of expression of some TCRAV and TCRBV gene families, in particular those that contain a single member, was lower compared to post-partum thymi and adult peripheral blood mononuclear cells. The combined data of FACS and PCR analysis demonstrate that TCR V genes belonging to the majority of TCR V gene families can be used in TCR alpha and beta chain rearrangements during early human fetal life. Our data also suggest that the expression levels of some of the single member TCR V gene families may be influenced by the developmental stage.

Relationship between patterns of engraftment in peripheral blood and immune reconstitution after allogeneic bone marrow transplantation for (severe) combined immunodeficiency.

We report the outcome of allogeneic bone marrow transplantation (BMT) as treatment for severe combined immunodeficiency disease (SCID) in 31 patients grafted from 1968 until 1992. The patients received a graft from an HLA-identical related (n = 10), an HLA-haplo-identical related (n = 19), or a closely HLA-matched unrelated (n = 2) donor that resulted in the long-term survival of 6 of 10, 9 of 19, and 0 of 2 children, respectively. Major complications included failure of engraftment and early death caused by respiratory failure. The chimerism pattern and immunologic reconstitution were evaluated in 15 children who survived more than 1 year with sustained engraftment. The pattern of engraftment was investigated within flow-sorted peripheral blood (PB) T- and B-lymphoid, natural killer (NK), and myelomonocytic cell populations using the amplification of variable number of tandem repeats by the polymerase chain reaction. The immunologic reconstitution was assessed by various in vitro and in vivo parameters. Although the number of PB T cells and the in vitro T-cell proliferative response was in the lower region of normal in the majority of cases and even subnormal in some, in all cases donor T-cell engraftment and reconstitution of T-cell immunity was observed. Residual host-type T cells (1% to 5%) were detected in eight cases at multiple occasions. All children showed normal serum IgM and IgG subclass levels and produced specific IgG antibodies after vaccination, irrespective of donor B-cell engraftment. However, three HLA haplo-identical graft recipients with host-type B lymphoid and myeloid cells have a persistent selective IgA deficiency. NK cells were either of donor, host, or mixed origin. Donor NK cell engraftment restored defective in vitro NK cell function of the recipient. We conclude that determination of lineage-specific engraftment patterns provides valuable information for the understanding of the immunologic reconstitution after allogeneic BMT for SCID.