Jaak M. Vossen

Failure of lymphocyte-membrane HLA-A and -B expression in two siblings with combined immunodeficiency.

A diagnosis of partial combined immunodeficiency was made in two Turkish siblings with a history of multiple pyogenic infections and persistent candidiasis. They demonstrated severe hypo-γ-globulinemia, with B-lymphocytes, but deficient plasma cell differentiation. T-Lymphocytes were decreased in number and did not respond to antigens, but did proliferate in cultures with lectins and allogeneic cells. HLA-A and -B determinants were not detected on blood lymphocytes, but they were expressed by cultured lymphoblasts, cultured fibroblasts, and were present in serum. MLR-Stimulatory capacity was intermediate and only two of six anti-HLA-DRw7 antisera demonstrated B-cell reactivity. β-2-Microglobulin (B2M) was not detected on the surface of T-lymphocytes, but was found in cross-sectioned T-cell membranes. B-lymphocytes carried B2M normally. The absence of HLA-A and -B determinants on lymphocytes of patients with similar immunodeficiency syndromes suggests a role for HLA determinants in lymphocyte differentiation.

Measurement of primary in vivo IgM- and IgG-antibody response to KLH in humans: Implications of pre-immune IgM binding in antigen-specific ELISA

The antigen Keyhole Limpet Hemocyanin (KLH) is often used to test the primary in vivo antibody response capacity in humans. However, measurement of IgM anti-KLH antibodies in ELISA is complicated by the presence of natural antibodies in human serum. This problem occurs particularly at low antibody levels, i.e. after immunization with low doses of antigen and, under these conditions, it was found to be impossible to assess a dose-response curve by immunizing a series of individuals with different suboptimal doses of KLH. This problem was circumvented by choosing conditions for minimal binding of pre-immune IgM and to correct for such binding. Although signal-to-background ratios were markedly improved by modifying the ELISA conditions, pre-immune IgM still showed binding to KLH due to interaction with polysaccharide determinants. This non-specific binding was correlated with the total IgM content of the samples. When anti-KLH activities before and after immunization were expressed relative to total serum IgM, a significant correction was achieved, resulting in a diminished inter-individual variability with respect to both pre-immune and post-immunization values. As with IgG-class antibodies to KLH, virtually no binding was observed in pre-immune sera. After expression of the anti-KLH response as a ratio between the post-immunization and pre-immunization titres, a dose of 50 micrograms was found to be sufficient to evoke a detectable IgG-antibody response in the 10 subjects tested. To elicit a positive IgM response, a minimal dose of 250 micrograms was required.

Serological and molecular studies of Epstein-Barr virus infection in allogeneic marrow graft recipients.

We have shown in two allogeneic bone marrow transplant recipients that Epstein-Barr virus can be eradicated by the BMT procedure or its complications, and that these patients are susceptible to infection with a new EBV strain (1). This conclusion was based on a combination of EBV serology and virus strain identification (“Ebnotyping,” using the size variations of 5 EBV nuclear antigens). In the present study, we conducted a serological survey of EBV infection in 153 marrow graft recipients and their donors. Ten patients who were positive for IgG antibodies against EBV viral capsid antigens prior to BMT became completely seronegative at a median of 197 days post-BMT (range 106–320 days). Four of these patients, who had received seronegative marrow, remained seronegative during prolonged periods (222 to 2105 days). Six patients had received seropositive marrow. Two of them remained seronegative during their subsequent periods of follow-up (895 and 1437 days). An additional 10 patients showed a 100-fold or greater decrease in VCA IgG antibody titers. Their titers reached a nadir of 10 (the lower limit of positive) at a median of 134 days post BMT (range 83–386 days). The serological patterns of the above 20 patients were particularly frequent among patients with chronic graft-versus-host disease; 12 of 20 patients with decreasing VCA titers (60%) developed chronic GVHD versus only 22 of 73 patients with stable or increasing VCA titers (30%). These results suggest that GVHD may contribute to the elimination of residual EBV-carrying recipient cells. Establishment of EBV-carrying lymphoblastoid cell lines (LCL) was attempted in 60 donor-recipient pairs whose cryopreserved peripheral blood mononuclear cells were available. LCL were established from 18 of 51 EBV-seropositive marrow donors and 10 of 57 seropositive recipients prior to BMT. The same EBV strain was detected in 4 of the 6 cases in which LCL could be established from both the donor and the recipient prior to BMT. The persistence of the original EBV strain was demonstrated in a recipient of a T cell–depleted graft who showed only transient hematological recovery and no GVHD, and was associated with the persistence of B cells of recipient origin.

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.

Transient Elevation of Serum IgE after Allogeneic Bone-Marrow Transplantation

Serum IgE levels were followed longitudinally twice a week for up to 100 days in 60 children treated for leukemia, severe aplastic anemia or severe combined immunodeficiency: 52 underwent allogeneic bone-marrow transplantation and 8 immunosuppressive treatment. In 55 of 58 treatment periods which could be analyzed (95%), a transient sharp increase of serum IgE was detected, irrespective of the initial diagnosis and mode of treatment. A second IgE peak was recorded in 16% of evaluable treatment periods. In the transplanted leukemia and aplastic anemia patients, the rise of serum IgE levels occurred at the same time, i.e. at a mean of 14 days after transplantation; it occurred significantly later in children grafted for severe combined immunodeficiency. In children who received immunosuppression for the treatment of severe aplastic anemia, IgE elevations were always seen within 2 weeks after institution of therapy. No relation was found between either the occurrence of clinically acute graft-versus-host disease or infections after treatment, and the time of onset of IgE elevations. It is suggested that the phenomenon of IgE peaks in the population of patients investigated was due to disturbance of T-cell regulation, i.e. a temporary impairment of T-suppressor cell activity.

Epstein-Barr virus infection in allogeneic marrow grafting: lessons for transplant physicians and virologists*

SummaryThe relationship between Epstein-Barr virus (EBV) and the host is profoundly disturbed by allogeneic bone marrow transplantation (BMT) because EBV resides in the recipients hematopoietic system, which has to be destroyed in the majority of cases, and in the donors hematopoietic system, i.e., the marrow graft. We have shown that EBV may be eradicated from some BMT recipients and that the virus may be transferred with the marrow graft. During the immediate post-transplant period oropharyngeal EBV excretion may occur which, by infecting passing B lymphocytes, may act as co-factor for acute graft-versus-host disease and help the virus to survive, despite the temporary depletion of its reservoir. The coexistence of totally different EBV strains in BMT recipients but not in healthy, untransfused controls, suggests that superinfection may by possible in case of immunodeficiency; alternatively, transfer of the virus by the reservoir itself (the B lymphocytes) might be the only effective route for superinfection. The generation of ‘variant’ strains during viral replication may form the basis of the vast polymorphism between wild-type EBV isolates in the population.

Bone marrow transplantation in children with severe aplastic anemia: reconstitution of cellular immunity.

Cellular immune functions were evaluated longitudinally in seven children with severe aplastic anemia, who were successfully transplanted with bone marrow cells from an HLA-identical, mixed lymphocyte culture (MLC)-negative sibling. Several parameters were followed: the number of lymphocytes and E rosette-forming cells in the peripheral blood and the lymphocyte reactivity toward various mitogens, antigens, and allogeneic lymphocytes. Some patients already displayed decreased in vitro lymphocyte reactivity before transplantation, especially with regard to the response to pokeweed mitogen (PWM). After transplantation, a severe cellular immunodeficiency developed in all patients, with low numbers of T cells and markedly impaired responsiveness to mitogens, antigens, and allogeneic lymphocytes. Variations between patients were substantial, both with regard to the severity and duration of the immunodeficiency and to the pattern of the recovery of lymphocyte responses to mitogens and antigens. This variability might be attributable to an imbalanced proliferation of different lymphocyte subsets and/or the sequence of appearance of receptors for mitogens on the cell surface.

Hypogommoglobul inmio and HLA gene poducts

Two siblings of unlike sex from a Turkish family suffered from a severe hypogammaglobuline mia but with B-cells present in normal numbers. The serum immunoglobulin was mainly IgM, but contained no measurable antibody activity. The numbers of T-cells were severe reduced and did not proliferate when stimulated by antigens, while mitogenic stimulation gave normal responses. Peripheral blood lymphocytes appeared able to differentiate to IgM containing blasts only, when activated by PWM. HLA-antigens on lymphocytes were undetectable. HLA-A antigens were found in serum and on cultured fibroblasts. B2M was present only on B-cells using viable lymphocyte suspensions. In tissues and after fixation of cells B2M seemed also present on T-cells. The coincidence of absence of HLA-gene products with failing differentiation of B-cells to plasmacells suggests a role of these products in T-cell dependent immune responses.

GRADUAL ESTABLISHMENT OF SPLIT (AUTOLOGOUS B, HOMOLOGOUS T) IMMUNOLOGICAL RECONSTITUTION FOLLOWING TRANSPLANTATIONS OF STEM CELL ENRICHED ONE NARROW FRACTIONS IN A PATIENT WITH C.I.D.

The diagnosis of C.I.D. with inmunoglobulins was made at the age of 15 months in a girl with a positive family history, who suffered from recurrent pulmonary infections and moderate failure to thrive. The number of blood T cells (E. rosettee) waa below noroal (x 1100 per μl). The in vitro response of the peripheral blood lymphocytes waa absent to mitogena and decreased x 21% of controls) to alloganeic cells. The delayed hyperaensitivity skin tests were negative and a skin graft was not rejected. The number of B cells (sIg pos.) was above normal x 1600 per μl). Serum Ig levels were either normal (IgM, IgG) or increased (IgA, IgD) and of reatricted heterogeneity. Isohemagglutinin titrea were normal (anti A 1/8, anti B 1/64, bloodgroup O), but antibody formation to protein antigens was lacking. The human serum thymic factor (SF) was normal. Neither excess of suppressor nor lack of helper T cells could be found by in ivitro stimulation tests of mixtures of the patients lymphocytes with lymphocytes of her SD and LD Identical brother. Treatment with thymic humoral factor at 22 months of age whan the patients SF level waa very low, was unsuccessful. A first transplantation at 27 months of age with a stem cell enriched PHA-negative bone marrow fraction 3 (alb.gradient) of her SD-LD) identical brother (bloodgroup A) restored only the cellular inmuns capacity; also SF levels became normal. A second transplantation at 33 months of age with stem cell enriched PHA-positive bone marrow fraction 4 (alb.fradient; kept at -196° C) restored also the humoral immune capacity, B cells, however, remained of receptor type, as well as the sarum Ig allotype, whereas T cells were of donor type (quinacrine Y chromoe.staining); anti A isohamagglutinins were no longer >raaent in the serum.

T-Cell Profile and Reactivity in vitro During Immune Reconstitution Following Bone Marrow Transplantation

Abstract Shortly after bone marrow transplantation for aplastic anemia the T-cell profile in peripheral blood, identified by monoclonal antibodies 0KT, is compatible with the presence of immature T cells, i.e. increased binding of 0KT 10 (thymocytes) and reversed 0KT 4 (helper/inducer T) to 0KT8 (cytotoxic/suppressor T) ratio; the lymphocyte responses in vitro are decreased. On recovery of reactivity in vitro, no correlation with the profile of 0KT binding of PBL is seen, except for a crossing of a threshold of 20% 0KT4+cells before responsiveness to mitogens increases.