Catastrophic relapse of Evans syndrome five years after allogeneic BMT notwithstanding full donor chimerism. Terminal hemolytic-uremic syndrome

A patient with severe Evans syndrome received an allo-BMT from his HLA-identical sister on November, 2000. Full marrow and blood donor chimerism were achieved only after 5 donor lymphocyte infusions (DLI), and coincided with complete clinical remission and disappearence of auto-antibodies. Five years later, hemolytic anemia recurred with rapid increase of serum bilirubin to over 50 mg%: he responded to combined therapy, but died on day +17 from admission of an acute hemolytic uremic syndrome (HUS). All circulating blood cells, including erythrocytes, were 100% donor. Ex vivo cultured and expanded T and B cells from the peripheral blood were also 100% donor. The supernatants from B cell cultures, containing either IgM or IgG, did not react with a panel of erythrocytes. Thus in this typical autoimmune disease with a predominant B cell pathogenesis the donor immune system resulted “innocent of autoimmunity”. The persistence of long-lived recipient autoreactive plasma-cell lines in survival niches, still producing autoantibodies, may be hypothesized for this and similar cases. The postulated graft-versus-autoimmunity (GVA) effect was apparently not sufficient to eradicate autoimmunity in this patient.     

Catastrophic relapse of Evans syndrome five years after allogeneic BMT notwithstanding full donor chimerism. Terminal hemolytic-uremic syndrome

A patient with severe Evans syndrome received an allo-BMT from his HLA-identical sister on November, 2000. Full marrow and blood donor chimerism were achieved only after 5 donor lymphocyte infusions (DLI), and coincided with complete clinical remission and disappearence of auto-antibodies. Five years later, hemolytic anemia recurred with rapid increase of serum bilirubin to over 50 mg%: he responded to combined therapy, but died on day +17 from admission of an acute hemolytic uremic syndrome (HUS). All circulating blood cells, including erythrocytes, were 100% donor. Ex vivo cultured and expanded T and B cells from the peripheral blood were also 100% donor. The supernatants from B cell cultures, containing either IgM or IgG, did not react with a panel of erythrocytes. Thus in this typical autoimmune disease with a predominant B cell pathogenesis the donor immune system resulted "innocent of autoimmunity". The persistence of long-lived recipient autoreactive plasma-cell lines in survival niches, still producing autoantibodies, may be hypothesized for this and similar cases. The postulated graft-versus-autoimmunity (GVA) effect was apparently not sufficient to eradicate autoimmunity in this patient.     

Diseases caused by reactions of T lymphocytes to incompatible structures of the major histocompatibility complex. IV. Autoantibodies to nuclear antigens.

We studied the requirements for induction of ANA formation in non-irradiated F1 hybrid mice undergoing a chronic graft-versus-host reaction (GVHR) after the injection of parental-strain lymphocytes. T lymphocytes in the donor cell inoculum were both needed and sufficient for the induction of ANA formation. For optimal ANA formation, the F1 recipient mice had to differ at H-2 from the parental donor strain. ANA belonged to the IgG1, IgG2, IgM and IgA (sub)classes of immunoglobulin. IgG ANA occurred in maximal serum titres of 1 in 5,120. ANA were not donor anti-host alloantibodies. At least some ANA were true autoantibodies, i.e. of F1 origin, because they carried the Ig allotypic markers characteristic of the F1 hybrid recipients. These findings are consistent with the concept that the pathogenic mechanism underlying autoantibody formation during the GVHR is an abnormal T-B-cell co-operation. In this process, donor T cells react against foreign histocompatibility antigens of the F1 recipient and generate non-specific help for B cells, including the autoreactive B cells.     

Prostaglandin (PG) release in the mixed lymphocyte culture; effect of presensitization by a skin allograft; nature of the PG-producing cell

A release of prostaglandin (PG) has been demonstrated in lymphocyte culture supernatants. The amount of PG produced is strikingly increased in mixed cultures between allograft donor and recipient. This phenomenon is not yet detectable after 6 h of culture, appears at 24 h and reaches its maximum at 48 h. Little or no increase in PG production is found in the supernatant of primary mixed lymphocyte cultures (i.e. without previous allograft) at least in the first two days of culture. The difference between allografted and control animals begins at day 6 after grafting, reaches a maximum at day 12, remains high until day 20 and decreases thereafter. This increased amount of PG in mixed cultures between donor and recipient spleen cells is probably, at least to a large extent, produced by macrophages. Evidence for this interpretation includes the following observations: (a) cells responsible for the increase are adherent cells and (b) the phenomenon is no longer found following a treatment of spleen cells with silica. When mouse peritoneal macrophages (or adherent spleen cells) are incubated in vitro with supernatants of mixed cultures between allograft donor and recipient, their PG production is considerably increased. Hence, it is suggested that the above-described phenomenon could result from the stimulation of macrophages by lymphokines released in mixed cultures following allografts. If such a release of PG takes place within the graft, it may play a regulatory role in the control of the magnitude of the anti-allograft immune response.     

Strong Donor Mononuclear Cell Reactivity for Herpes Simplex Virus (HSV) Antigen in HSV Immune Donors Combined with Recipient Seropositivity for HSV is Associated with Acute Graft-versus-Host Disease

Prior to bone marrow transplantation (BMT) titres of IgG antibodies for cytomegalovirus (CMV), herpes simplex virus (HSV) and varicella zoster virus (VZV) were analysed in 51 donors and recipients of allogeneic bone marrow. Donor mononuclear cells from peripheral blood and bone marrow cells were stimulated with antigen prepared from CMV, HSV and VZV. High IgG titres for HSV in the recipient were associated with grade II-III acute graft-versus-host disease (GVHD) (P = 0.05). Furthermore, the combination of positive IgG titre for HSV antibodies in the recipient, and strong donor blood mononuclear cell reactivity to HSV antigen in HSV immune donors, significantly increased the incidence of grade II-III acute GVHD (P = 0.04). The data suggest that HSV immune donor mononuclear cells may initiate a GVH reaction.     

Immunosuppressive activity of serum from liver-grafted rats: in vitro specific inhibition of mixed lymphocyte reactivity by antibodies against class II RT1 alloantigens

The immunosuppressive activity of serum from PVG rats following orthotopic transplantation of DA liver has been examined in vitro. Liver grafts in this combination are never rejected, but induce a state of specific transplantation tolerance in the recipient. Serum from such tolerant animals was able to inhibit proliferation of normal PVG lymph node cells in response to DA stimulators in the mixed lymphocyte reaction (MLR); inhibition was specific for donor (DA) antigens. Interleukin-2 (IL-2) production during the MLR was also reduced. The production of anti-DA cytotoxic T cells developing in the MLR was not affected, but the total yield of such cells was reduced. Evidence was obtained that part of the inhibitory serum activity was due to IgG antibody against class II RT1a alloantigens. Thus, a purified IgG fraction retained much of the inhibitory activity which could be removed by an anti-IgG absorbent. Studies of MLR inhibition in different rat strains indicated the anti-class II specificity of the inhibitory IgG. Lymph node cells from DA-liver-grafted PVG rats responded normally against DA stimulators in vitro, and this MLR was also blocked by the inhibitory IgG. Our results suggest that anti-class II allo-antibody may play a role in immunosuppression and long-term graft survival following liver transplantation in this combination.     

Absence of hemolysis after a kidney transplant in an E+ recipient from a donor with anti-E

A 12-year-old Caucasian male with cystinosis received a kidney from his mother, whose red blood cells typed as group O, D+, E-. Her serum contained an anti-E with an IgG1 titer of 16 (score 31). The recipient's type was group O, D+, E+, with a negative antibody screen in the pretransplant period. The recipient and donor Rh phenotypes were most likely DCcEe and Dccee, respectively. Because the recipient's mother had no transfusion history, she was probably immunized by the fetal red blood cells of her one pregnancy (the recipient). The kidney had been immediately perfused with saline after removal from the donor. No acute or delayed hemolysis was observed clinically or in laboratory tests performed immediately after the transplant and at 7, 15, and 30 days after the transplant. Antibody screens were still negative at 6 months. In this case, anti-E was not present in the transplanted kidney in sufficient concentration to cause hemolysis of the recipient's red blood cells and transplanted lymphocytes did not synthesize sufficient anti-E to be detectable or to cause hemolysis.     

Recipient expression of ligands for donor inhibitory KIRs enhances NK‐cell function to control leukemic relapse after haploidentical transplantation

Natural killer (NK) cells that express self‐HLA‐specific receptors (where HLA is human leukocyte antigen) are “licensed” and more readily activated than unlicensed cells; therefore, NK‐cell licensing could influence the antileukemia effects of NK cells following haploidentical stem cell transplantation (haplo‐SCT). In this study, we compared the functionality of reconstituting NK cells, based on CD107α expression and interferon‐γsecretion, in a cohort of 29 patients that expressed (n = 8) or lacked (n = 21) class I human leukocyte antigens for donor inhibitory killer cell immunoglobulin‐like receptors (KIRs) following T‐cell‐replete haplo‐SCT. We also addressed whether recipient expression of class I ligands for donor inhibitory KIRs could predict relapse occurrence in another cohort of 188 patients. A longitudinal analysis indicated that patients presenting class I for all donor inhibitory KIRs showed more capable functional NK effector cells when tested against class I negative K562 cells and primary leukemic cells within 3 months of transplantation. The lowest 7‐year relapse incidence was observed when donor KIRs were ligated by recipient class I (n = 60) compared with donor–host partnerships where donor KIR⁺ cells were ligated by donor, but not recipient class I (n = 86, p = 0.026) or KIRs that were ligated by neither donor nor recipient class I (n = 42, p = 0.043). This study suggests that haplo‐SCT recipients presenting class I for donor inhibitory KIRs promote NK‐cell licensing, leading to decreased relapse rates.     

Human immunoglobulin production in immunodeficient mice: enhancement by immunosuppression of host and in vitro activation of human mononuclear cells.

The affect of host and donor related factors on successful engraftment of human cells into mice was examined to minimize the variability that has been observed in successful development of human-mouse chimera for the study of human disease and immune physiology and regulation. Human immunoglobulin production in severe combined immunodeficiency (SCID) mice engrafted with human peripheral blood mononuclear cells (PBMC) was augmented by immunosuppressing recipient mice and activating donor PBMC. Immunosuppression of recipient mice with 3 Gy of gamma-irradiation induced a 10-fold increase in human IgG in the sera of engrafted SCID mice. Variation in production of human IgG in recipient mice correlated with preinjection phenotype and activation status of injected PBMC. Mice injected with PBMC with a low CD4/CD8 ratio (less than 0.5) produced no detectable circulating human immunoglobulin. When the CD4/CD8 ratio was greater than 1.5, human IgG was detected in sera of PBMC-recipient SCID mice. Serum IgG increased 10-fold following in vitro activation of donor PBMC with anti-CD3, IL-2 and Staphylococcus aureus. Successful engraftment and serum IgG production was evidenced by an increase in the recovery of activated human IgG+ cells in the spleens of mice with maximal IgG production. Optimization of functional engraftment required modification of both the host (SCID mice) and the donor cells.     

Evidence for the production of a histamine-releasing factor (HRF) during mixed lymphocyte culture in an allograft model

An increased production of histamine has been demonstrated during mixed cultures between allograft donor and recipient lymphocytes. This phenomenon is not yet detectable after 6 h of culture, appears at 24 h, reaches its maximum at 48 h and remains at the same level at 72 h. Little or no increase in histamine production is found during primary mixed lymphocyte culture (without previous allograft).This increase in histamine production during secondary MLC results from the action of a non-dialyzable factor (histamine-releasing factor or HRF) released by recipient cells in the presence of donor cells. This factor may be obtained from lymph nodes as well as spleen cells. Conversely, it acts on a cell population which is present in spleen, peritoneal or bone marrow cells, but absent or present in smaller amounts in lymph node and thymus cells. The effect of HRF-containing supernatant on histamine production from normal spleen cells appears after 24 h of incubation and reaches its maximum at 48 h. It is not yet detectable after 5 h. The activity is usually lost by 20–40-fold dilution of the supernatant.     

Anti-idiotypic antibodies in a patient with a well-functioning renal graft without azathioprine. II. Anti-idiotypic antibodies directed to a clonotype of a certain cytotoxic T lymphocyte

A recipient who received a renal graft from a living related donor showed good renal function for as long as 15 years after the withdrawal of azathioprine. The patient's creatinine clearance was maintained at 60 ml/min in spite of the administration of only 10 mg of predonisolon per day. Anti-idiotypic antibodies in the patient's serum were found by testing for specific inhibition of the mixed lymphocyte reaction (MLR) and cytotoxic T lymphocyte (CTL) response. The treatment of responder or effector cells with the IgG fraction from the patient's serum resulted in the specific inhibition of the MLR and CTL killing reaction against target cells of the graft donor. These findings indicate that the patient's serum contains anti-idiotypic antibodies against antigen recognition structures of a certain CTL as well as a certain MLR to donor alloantigens.     

Analyses of Ia restriction specificity of helper T cells in H-2 subregion compatible bone marrow chimera in mice

Using irradiation bone marrow chimeras which had partial compatibility in H-2 subregions between donor and recipient mice, we found that H-2I matching was sufficient for the chimeras to generate anti-sheep erythrocyte plaque-forming cell (PFC) responses. In such chimeras, T cells appeared to encounter appropriate partner cells bearing the same Ia antigens as those which they had learned to recognize as self in the recipient micro-environment. Furthermore, the PFC number seen in I-A compatible chimeras was only about half of that seen in I-A, I-E compatible chimeras, suggesting the existence of two independent subpopulations of helper T cells. When incompatibility of donor and recipient mice existed on the left side of the H-2I region, the responses were very weak. However, even in such chimeras, marked responses were observed for both IgM and IgG type PFC following a sufficient period after immunization. This observation appears to indicate the existence of a minor subpopulation of helper T cells which can expand and interact effectively with antigen presenting cells of donor type.     

The IgG antibody reactivity of sera from patients with active chronic hepatitis to a crude liver antigen and liver specific protein (LSP): analysis by ELISA and immunoblotting.

This report extends our previous study on experimental autoimmune hepatitis in C57BL/6(B6) mice. Cellular immunity involved in the induction of liver injury in this model was studied by transfer of primed spleen cells from hepatitis donor mice to syngeneic normal recipient mice. The most prominent liver damage in recipient B6 mice was induced by transfer of nylon wool adherent spleen cells from hepatitis donor mice, and T cells in this fraction were the essential requirement for the liver damage in the recipient mice. Nylon wool adherent spleen cells from hepatitis donor mice after depletion of the suppressor T-cell function by low-dose (300 rad) irradiation induced more severe liver injury compared to the same cells without irradiation. When the recipient mice were depleted of lymphocytes by low or high dose (700 rad) whole body irradiation, transfer of primed spleen cells from hepatitis donor mice did not induce liver lesion in the lymphocyte-depleted mice. This low susceptibility of lymphocyte-depleted recipient mice to primed spleen cells of hepatitis mice was no longer demonstrated after reconstitution with normal spleen cells. In a cell-migration study using 51Cr-labelled spleen cells, it was shown that a considerable number of infiltrating cells in the liver of recipient mice were derived from recipient mice themselves. These results seem to indicate that cell-to-cell interaction between radiosensitive precursor cells of recipient mice and liver-antigen-primed T cells from hepatitis donor mice play an essential role in the induction of liver injury in the recipient mice.     

The Effect of Natural Killer Cell Killer Ig-Like Receptor Alloreactivity on the Outcome of Bone Marrow Stem Cell Transplantation for Severe Combined Immunodeficiency (SCID)

Natural killer (NK) cell alloreactions against recipient cells in the setting of bone marrow transplantation have been associated with decreased rates of graft-versus-host disease (GVHD) and improved survival in transplant recipients with myeloid leukemia. These alloreactions are predicted by the absence of recipient HLA class I ligands for donor inhibitory killer Ig-like receptors (KIR). We hypothesized that donor NK cell alloreactions against recipient cells may affect the development of T and B-cell functions and incidence of GVHD in infants with severe combined immunodeficiency (SCID). Of the 156 patients with SCID who had received related bone marrow transplants without pretransplant chemotherapy or posttransplant GVHD prophylaxis, 137 patient-donor pairs were evaluated for the absence of recipient HLA class I ligands for donor inhibitory KIR. Analysis showed that the absence of a KIR ligand had no effect on the incidence or severity of GVHD (RR [corrected] = 0.95, p = 0.84), development of T-cell function (RR [corrected] = 1.05, p = 0.69), production of IgA (p = 0.46) or IgM (p = 0.33), or on 5-year survival (RR [corrected] = 1.21, p = 0.10). Further, in patients possessing native NK cells, the absence of KIR ligands in donors for recipient-inhibitory KIR did not alter transplantation outcomes. This study suggests that inhibitory KIR/HLA interactions do not play a significant role in bone marrow transplantation for SCID.     

Natural transmission of Streptococcus sobrinus in rats: saliva and serum antibody responses to colonization.

One hundred and twenty weanling rats fed diet NIH 2000 that were free of Streptococcus sobrinus and other mutans streptococci were employed in this study. Sixty rats were inoculated orally with S. sobrinus 6715. Each infected rat (donor) was paired and housed with an uninfected recipient. Saliva and serum samples were collected from 24 (12 donor and 12 recipient) rats at the baseline (day 0) and from groups of 12 recipients sacrificed on days 10, 24, 38, and 52, and the level of infection with S. sobrinus was monitored. Salivary immunoglobulin A (IgA) and IgG and serum IgM and IgG antibodies reactive with whole cells (WC), glucosyltransferase (GTF), and the serotype carbohydrate (g) of S. sobrinus were measured by an indirect enzyme-linked immunosorbent assay. Although the rats were free of S. sobrinus and other mutans streptococci at baseline, they exhibited salivary IgA and serum IgM antibodies reactive with S. sobrinus WC, GTF, and g and serum IgG antibodies reactive with WC and GTF. Infection of recipients with S. sobrinus did not induce salivary antibodies reactive with WC, GTF, or g. In contrast, increases in serum IgM and IgG antibodies reactive with WC and serum IgM antibodies reactive with g were observed.     

Allotype Analysis to Distinguish the Origin of Varicella-Zoster Virus Immunoglobulin G after Allogeneic Stem Cell Transplantation

Varicella-zoster virus (VZV) reactivation is a frequent complication after allogeneic hematopoietic stem cell transplantation (HSCT). Although previous studies have revealed that cellular immunity is important for suppressing reactivation, the role of humoral immunity against VZV has been poorly evaluated. We analyzed inherited polymorphisms in the immunoglobulin G (IgG) heavy chain constant regions of 50 HSCT recipient-donor pairs to distinguish donor-derived and recipient-derived antibodies. Twelve pairs were informative regarding the origin of IgG, since either the donors (n = 3) or recipients (n = 9) were homozygous null for the IgG1m(f) allotype. In these 9 homozygous-null recipients, allotype-specific IgG against VZV were measured by enzyme-linked immunosorbent assay and compared with measles-IgG. All 9 homozygous-null recipients were monitored for more than 1 year after HSCT, with (n = 4, localized zoster) or without (n = 5) clinical VZV disease. In 3 patients with VZV disease, donor-derived IgG against VZV was elevated between 500 to 700 days after HSCT after the episode of VZV disease. In 1 patient who suffered from VZV disease just before HSCT, donor-derived VZV IgG was elevated within 3 months after HSCT. On the other hand, 2 patients who received reduced-intensity conditioning (RIC) transplantation from an IgG1m(f) null donor maintained recipient-derived IgG against VZV for more than 1 year, whereas it was decreased within 3 months in 1 recipient who received conventional conditioning. In conclusion, the production of anti-VZV IgG by recipient plasma cells persists long after RIC. In patients without symptomatic VZV reactivation, donor-derived anti-VZV IgG did not reach titers comparable to those measured in healthy virus carriers.     

Interleukin-4-induced IgG subclass and IgE secretion by mononuclear cells from atopic donors

To investigate the effect of interleukin-4 (IL-4) on the secretion of IgG subclasses and IgE in atopic individuals, peripheral blood mononuclear cells from 10 atopic and 10 normal donors were incubated with IL-4, and the production of IgE, IgG1, IgG2, IgG3, and IgG4 was determined after a 14-day culture period. Like normal peripheral blood mononuclear cells, atopic mononuclear cells were stimulated by IL-4 to secrete IgE and IgG4, but not the other IgG subclasses. This response was in the same quantitative ranges in both donor groups. Addition of interferon gamma to IL-4-containing cultures efficiently antagonized the IL-4-induced IgE and IgG4 secretion. These results do not support the hypothesis that an atopic condition is due to a discordant effect of IL-4 on IgE compared to IgG4 production or to an altered response to the potent antagonist interferon gamma.     

Neointimal and tubulointerstitial infiltration by recipient mesenchymal cells in chronic renal-allograft rejection.

BACKGROUND: Tissue remodeling depends on mesenchymal cells (fibroblasts and myofibroblasts) and is a prominent feature of chronic renal-transplant rejection. It is not known whether the mesenchymal cells that participate in remodeling originate locally or from circulating precursor cells. METHODS: We obtained biopsy specimens of renal allografts from six male recipients of an allograft from a female donor, four female recipients of an allograft from a male donor, two male recipients of an allograft from a male donor, and two female recipients of an allograft from a female donor. All the allografts were undergoing chronic rejection. All but two specimens were obtained within six months after transplantation. We used immunohistochemical methods to identify mesenchymal cells with smooth-muscle alpha-actin and in situ hybridization to identify mesenchymal cells with Y-chromosome DNA. RESULTS: No Y-chromosome bodies were identified in the case of the two renal-allograft specimens in which both the donor and the recipient were female. In the case of the two renal-allograft specimens in which both the donor and the recipient were male, approximately 40 percent of mesenchymal cells contained a Y-chromosome body. In the case of the six specimens in which the donor was female and the recipient was male, a mean (+/-SD) of 34+/-16 percent of mesenchymal cells in the neointima, 38+/-12 percent of such cells in the adventitia, and 30+/-7 percent of such cells in the interstitium contained the Y-chromosomal marker, indicating that they originated from the recipient rather than the donor. In the case of the four renal-allograft specimens in which the donor was male and the recipient was female, the respective values were 24+/-15 percent, 33+/-9 percent, and 23+/-8 percent, indicating a persistent population of donor mesenchymal cells. CONCLUSIONS: The presence of mesenchymal cells of host origin in the vascular and interstitial compartments of renal allografts undergoing chronic rejection provides evidence that a circulating mesenchymal precursor cell has the potential to migrate to areas of inflammation.     

Cell culture of clonal ginbuna crucian carp hematopoietic cells: differentiation of cultured cells into erythrocytes in vivo

We cultivated kidney hematopoietic cells from clonal triploid ginbuna crucian carp, Carassius auratus langsdorfii. Proliferating cells from hematopoietic cell cultures were harvested and injected into tetraploid hybrids (clonal triploid ginbuna and goldfish hybrid) which possess three sets of chromosomes from a triploid clone and a haploid set of chromosomes from goldfish (Carassius auratus). After injection of cultured triploid cells (donor cells), blood samples were obtained from tetraploid hybrids (recipients) every other week. Blood cells stained with acridine orange were measured by flow cytometry to trace the injected donor cells by means of differences in DNA content. Although erythrocytes were not produced in donor cell cultures, such cultures maintained precursor cells capable of differentiation into erythrocytes in vivo. After 4-12 weeks of transplantation, mature erythrocytes derived from the donors were observed in the blood circulation of the recipient fish. These results indicated that the ginbuna hematopoietic cell culture system is an in vivo situation suitable for the study of hematopoietic control mechanisms.