Oligoclonal dominance of immunoglobulin VH3 rearrangements following allogeneic bone marrow transplantation.

Normal numbers of circulating B lymphocytes are reached during the first 6 months following allogeneic BMT, but humoral immunity remains poor. The molecular basis for this lack of function in the first appearing B lymphocytes has not been clarified. Accordingly, we have studied the reconstitution of the VH3 containing Ig repertoire in two CML patients transplanted with allogeneic BM and one healthy control. PBMCs were isolated at several time-points after BMT and mRNA was prepared. VH3 containing Ig rearrangements were amplified with RT-PCR and then cloned and analyzed with colony hybridization using complementary determining region 3 (CDR3)-specific oligonucleotide probes. Four weeks after BMT, two individual clones together represented 52% of the analyzed CDR3 regions. At 6, 8 and 12 weeks after BMT the corresponding probes hybridized with 2-6% of the colonies. A similar pattern was obtained for the other patient. In samples from the healthy control no clones were detected using CDR3-specific oligonucleotide probes from the control. We conclude that the VH3 containing Ig repertoire after BMT is oligoclonal and that specific rearrangements dominate at different time-points. This restriction of the B cell repertoire may contribute to the impaired humoral immunity observed in BMT recipients.

Altered expression of receptors for thyroid hormone and insulin-like growth factor-I during reconstitution after allogeneic hematopoietic stem cell transplantation.

Treatment with neuroendocrine hormones has been suggested to promote reconstitution of the immune system after hematopoietic stem cell transplantation (HSCT). We investigated the expression of genes encoding receptors for growth hormone (GH), insulin-like growth factor-I (IGF-I) and triiodothyronine (T3), at various time points after HSCT in 16 patients and 15 healthy controls. Peripheral blood mononuclear cells were isolated and RNA for GH receptor (GHR), IGF-I receptor (IGF-IR) and thyroid hormone receptor (TRalpha1) was amplified by RT-PCR. The expression of the genes was compared with the expression of beta-actin. We demonstrate increased expression of TRalpha1 RNA in patients at 1.5 months post HSCT, compared to a group of healthy controls, and decreased expression of IGF-IR RNA at 2 and 3 months post HSCT, compared to the controls. Serum from three of the patients was also analyzed for levels of T3, T4, TSH and IGF-I at several time points after HSCT. Serum levels for T3, thyroxine (T4), thyroid stimulating hormone (TSH) and IGF-I were within the normal range in all samples. Our results on the molecular level indicate a role for thyroid hormones and IGF-I in immune reconstitution after HSCT, even though the serum levels of T3, T4, TSH and IGF-I are normal.

Preoperative feeding does not reverse postoperative insulin resistance in skeletal muscle in the rat.

Metabolic studies on injured and postoperative patients have shown impaired glucose disposal in peripheral tissues after trauma. Using small-bowel resection as a model of surgical trauma, we investigated whether substrate availability could ameliorate the changes in muscle glucose uptake induced by trauma. We also studied the effect of preoperative feeding on postoperative insulin-stimulated insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol (PI) 3-kinase activity in both Wistar rats and genetically non-insulin-dependent diabetic Goto-Kakazaki rats (GK rats). Serum glucose, insulin, plasma epinephrine, lactate, and plasma nonesterified free fatty acids (NEFAs) were measured as indicators of the metabolic state and surgical stress. Insulin-stimulated glucose transport was significantly reduced in fed traumatized Wistar rats compared with fed nontraumatized rats (P < .05). Significant increases in in vivo insulin-stimulated IRS-1-associated PI 3-kinase activity were found in fed traumatized Wistar rats compared with fed nontraumatized Wistar rats and fasted traumatized Wistar rats, as well as fed traumatized GK rats compared with fed nontraumatized GK animals (all P < .017). Serum insulin concentrations were significantly reduced in fed traumatized Wistar and GK rats compared with the respective fed nontraumatized groups (both P < .01). Serum glucose levels were significantly elevated in fed traumatized GK rats compared with fed nontraumatized animals (P < .01). In the present study, preoperative feeding did not prevent a postoperative reduction in insulin-stimulated glucose transport in skeletal muscle. The finding that insulin-stimulated PI 3-kinase activity increased after trauma in both Wistar and GK rats indicates that postoperative insulin resistance is not caused by an impairment in the early steps of the insulin signaling pathway. The postoperative decreases in serum insulin despite high blood glucose suggest that trauma impairs the insulin response to hyperglycemia.

T-cell receptor Vbeta repertoire after myeloablative and reduced intensity conditioning allogeneic haematopoietic stem cell transplantation.

T cells play an important role in the adaptive immune system. After haematopoietic stem cell transplantation (HSCT), T-cell function is impaired. This is reflected by the emergence of opportunistic infections, infections that are often difficult to treat because of the patient's insufficient immune function. T-cell receptor reconstitution was studied using CDR3 spectratyping to analyze the diversity of the T-cell repertoire at 3, 6 and 12 months after myeloablative and reduced intensity conditioning (RIC) HSCT in 23 patients. Immune function in vitro was tested by lymphocyte stimulation at 3, 6 and 12 months after HSCT. Lower diversity in the CDR3 repertoire was demonstrated in CD4+ cells after RIC HSCT at 3 and 6 months and in CD8+ cells at 3 months compared with healthy donors. After myeloablative HSCT, lower diversity was seen at 3, 6 and 12 months in CD4+ cells and at 6 and 12 months in CD8+ cells after HSCT. Acute and chronic graft-versus-host-disease (GVHD) did not affect diversity. Responses to phytohaemagglutinin (PHA), Concanavalin A (Con A) and Staphylococcus aureus protein A were significantly lower compared with healthy donors during the first 6 months after RIC HSCT. After myeloablative HSCT, lymphocyte response to Con A was significantly lower at 3 months compared with healthy donors. Decreased responses to cytomegalovirus and varicella zoster virus antigens were seen in patients suffering from acute GVHD grade II or chronic GVHD. The T-cell repertoire is skewed under the first year after HSCT, and immune reconstitution after HSCT with myeloablative and RIC conditioning seems to be comparable. GVHD, infections and age are more important for immune reconstitution than type of conditioning.

Reconstitution of the Ig heavy chain CDR3 repertoire after allogeneic haematopoietic stem cell transplantation with myeloablative or reduced-intensity conditioning regimens.

The objective of this study was to investigate B-lymphocyte reconstitution in patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) after myeloablative conditioning (MAC) or reduced-intensity conditioning (RIC) regimens. B-lymphocyte reconstitution was studied by monitoring the CDR3 repertoire with spectratyping. We demonstrate a delay in the recovery of the B-lymphocyte repertoire, measured by variation in size distribution of the immunoglobulin H CDR3 in patients conditioned with RIC compared to MAC. We found no general explanation for this finding, but when clinical data for each patient were studied in detail, we could identify a cause for the oligoclonality of the B-lymphocyte repertoire after HSCT with RIC for each of the patients. Older patients and donors, low cell dose at transplantation, relapse, graft-versus-host disease (GVHD) and its treatment as well as cytomegalovirus infection and its treatment are all possible causes for the restriction of the B-lymphocyte repertoire observed in this study. Taken together, reconstitution of the B-lymphocyte repertoire after HSCT is a process dependent on multiple factors and differs between patients. The conditioning regimen may be of importance, but data from this study suggest that individual factors and the various complications occurring after HSCT are more likely to determine the development of the B-lymphocyte repertoire.

Evidence for oligoclonal diversification of the VH6-containing immunoglobulin repertoire during reconstitution after bone marrow transplantation.

Patients who have undergone bone marrow transplantation (BMT) remain immunodeficient for months to years posttransplantation. To evaluate the basic molecular events underlying reconstitution of the humoral immune response, we have performed a detailed nucleotide sequence analysis of VH6 containing rearrangements in circulating B cells from two BM donor/recipient pairs. Our results show that the third complementarity determining region (CDR3) diversity is much lower early after transplantation, compared with that of the donors, and that the clonal variability remains low for 3 months. Repertoire diversification follows an oligoclonal pattern where B lymphopoiesis appears to occur in waves up to 6 months posttransplantation. The repertoire among donated marrow cells is not reflected in peripheral blood lymphocytes from the transplanted patients. There is differential D gene utilization among both donor and patient samples, whereas JH gene usage is biased toward JH4, 5, and 6. One month after transplantation the vast majority of the sequenced clones are functional and contain a high frequency of replacement mutations in the CDRs of the VH6 gene. We conclude that Ig gene expression is very restricted early after BMT and that development of the B-cell repertoire appears to follow a wavelike pattern.

On the validity of using lipopolysaccharide-driven limiting dilution systems for clonable B-cells to analyse functional antibody repertoires.

Analyses of B-cell repertoires by mitogen-driven limiting dilution systems (LDA) followed by specific antibody detection on immobilized antigens (ELISA), while constituting the only method available to determine absolute frequencies of any given specificity, provide no indications as to the functional ability of clonal precursors scored as positive to actually respond to antigen in vivo. We have now addressed this question by quantitating dextran alpha 1----6-specific clonal precursors among small, resting, and large activated B cells in the spleens of mice, before and after priming with optimal doses of antigen. The results demonstrate that virtually all B cells scored as antigen-specific in LDA/ELISA systems do respond to antigen and undergo blast transformation after priming in vivo.

Inverse correlation between the utilization of an idiotype in specific immune responses and its representation in pre-immune "natural" antibodies.

Previously we have characterized an idiotype (Id) that accounts for half of all specific anti-dextran B512 (Dex) antibodies in C57BL/6 mice. BALB/c mice produce the same Id in normal, pre-immune sera but fail to use it in antibody responses to Dex, although Id+ anti-Dex antibodies can be induced in this strain by anti-Id immunization. By limiting dilution analysis of B cell clonal precursors, we show here that the frequencies of Id+ B cells are comparable in both strains, but their state of activity is sharply distinct: while all Id+ B cells are small, resting lymphocytes in C57BL/6 mice, they are all large, naturally activated cells in BALB/c mice. The suggestion that naturally activated cells are poorly engaged in specific responses was supported by the delayed and lower Id+ responses obtained in BALB/c mice when they are immunized, in parallel with C57BL/6 animals, with a conjugate of anti-Id antibodies and lipopolysaccharide. Finally, C57BL/6 responder mice were found to closely reproduce the normal BALB/c situation, if analyzed 3 months after anti-Id priming: they produce low levels of serum Id and all Id+ B cells are in the large lymphocyte compartment. Upon immunization these animals develop serum Id+ responses that are undistinguishable from low-responder BALB/c mice. The relevance of these observations for the questions of physiologic self-reactivity is discussed.

The immune response to bacterial dextrans. VI. No correlation between the frequency of cells expressing a major anti-dextran idiotype and the idiotype profiles of specific antibody responses.

C57BL/6 mice respond to Dextran B512 (Dex) with a predominant idiotype (Id) (17-9) while no such Id-positive antibodies are identified in the specific antibody response of BALB/c mice. We used limiting dilution systems to determine the absolute frequencies of clonal B-cell precursors producing the 17-9 Id in these two mouse strains and analysed the correlation between Id-expression and antibody activity at the clonal level. The results show very similar frequencies of anti-Dex and Id-positive B cells in both strains, but C57BL/6 mice contained fourfold higher frequencies of Dex-specific clonal precursors which are Id-positive. This kind of clone, although not used in the specific response of BALB/c mice, constitute roughly 20% of their anti-Dex repertoire and they are readily induced in this strain. Thus, immunization of both strains with anti-idiotypic antibodies results in the production of Id-positive anti-Dex antibodies with serum titres that directly correlate with precursor frequencies. The results show, therefore, that the section of clonal repertoires utilized in a primary immune response varies with the immunogen, even if thymus-independent. These observations are discussed in the context of the genetic controls of anti-Dex antibody responses and on the general question of the utilization of available antibody repertoires in immune responses.

Idiotype-specific regulation might contribute to specific unresponsiveness in dextran-primed mice.

Priming of adult responder mice with optimal immunogenic doses of dextran alpha, 1-6 results in reduced antibody responses to secondary antigenic challenge. We have now quantitated dextran-specific clonal precursors in the large and small B-lymphocyte compartments within several days or several months after priming with either dextran or antiidiotypic antibodies directed to a dominant idiotype of C57BL/6 mice which accounts for more than 50% of the antibody response. The results show that "secondary unresponsiveness" correlates with idiotype-directed depletion of the appropriate specificities from the immunocompetent resting B-cell pool.

The immune response to bacterial dextrans. III. Ontogenic development and strain distribution of specific clonal precursors.

The frequencies of B512 dextran (Dex)-specific B cell precursors were determined by limiting dilution analysis in a number of mouse strains originally described as "high responder", "low responder" and "nonresponder" to this antigen. No significant difference in the frequencies of Dex-specific precursors was found in C57BL/6, B10.BR, C3H/Tif, BALB/c and A/Sn adult mice. Together with the large intra-strain variability in the magnitude of anti-Dex PFC responses in vivo, these results established that differential reactivity in vivo cannot be ascribed to genetically controlled absence or wide variation in the frequency of Dex-specific immunocompetent precursors. A similar analysis of the Dex-specific precursor frequency was carried out in C57BL/6 mice between 1 week and 3 months of age. While no Dex-specific antibody response was detected in vivo before the age of 3 weeks, clonal precursor analysis revealed that the appearance of these specificities parallels the development of competent (IgM-producing) B lymphocyte clonal precursors, such that no significant difference in absolute frequencies of Dex-specific precursors could be observed among these age groups. This is interpreted to suggest that the late development of the Dex-specific antibody responses is regulatory rather than due to late rearrangement and activation of the appropriate V genes and a sequential expression of antibody specificities in ontogenic development.

The immune response to bacterial dextrans. V. A "dominant" idiotype in IgCHb mice.

A battery of syngeneic monoclonal anti-idiotypic antibodies was prepared against a monoclonal C57BL/6 anti-Dextran B512 antibody (17-9). Two such anti-idiotypes were shown to bind to sites on the 17-9 molecule which are related to the Dex-binding site and were used to characterize the anti-Dex antibody response in a number of inbred mouse strains. The results show that the 17-9 idiotype is recurrently expressed by all mice carrying the IgCHb haplotype, regardless of H-2 or background genes, and that this idiotype accounts for roughly one-half of the primary, specific response to Dex B512 in C57BL/6 mice. Backcross analysis confirmed the allotype-linkage of idiotype expression in the antibody response. Mice carrying other allotypes, however, had detectable levels of the 17-9 idiotype in normal sera, which was not associated with anti-Dex antibody activity and was not raised by specific immunization. Together with previous observations, these results characterize a second "recurrent" idiotype in the anti-alpha,1-6 response of IgCHb mice, both of which are expressed in the normal serum of all mouse strains tested.

Selective usage of VH genes in adult human B lymphocyte repertoires.

The authors have compared the VH gene utilization patterns among small resting immunocompetent B cells and large naturally activated B lymphocytes of healthy human adults. They employed a non-radioactive RNA in situ hybridization technique that allows detection of VH gene family expression at the single cell level. Pokeweed mitogen stimulated and unmanipulated mononuclear cells from peripheral blood and spleen of unrelated individuals were hybridized to digoxigenin-labelled antisense RNA probes specific for human VH families 1-6 and for the constant region genes C mu and C gamma. The observed VH gene family utilization patterns did not correlate with the genomic complexity of human VH genes. The VH3 gene family was most frequently used among resting B cells in both peripheral blood and spleen. Among naturally activated lymphocytes the VH6 gene was markedly over-represented, while expression of the VH1 and VH3 gene families was decreased. The data show that V-region mediated selection participates in shaping the peripheral antibody repertoire in healthy adults.

Enhanced scavenger receptor expression in monocyte-macrophages in dialysis patients.

The macrophage scavenger receptor (SR), which has two isoforms named type I and II, plays a leading role in the atherosclerotic process. To elucidate the mechanism of atherosclerosis in dialysis patients, SR expression was studied in monocyte-macrophages from thirteen dialysis patients and eight healthy controls. SR mRNA expression was examined in four hemodialysis patients and four controls matched for age and sex. Monocytes were allowed to differentiate into macrophages in in vitro cultures for seven days and SR type I and II mRNA expression were analyzed with the reverse transcription-polymerase chain reaction and quantitated using radiation densities of Southern blots. Only SR type I expression increased during differentiation and was accelerated by one or two days and enhanced after five days in patients, as compared to controls. To detect SR protein expression, uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) was evaluated by flow cytometry. The mean fluorescence intensity of labeled cells was significantly higher in hemodialysis and continuous ambulatory peritoneal dialysis patients than in controls, although the number of SR-positive cells remained constant. In conclusion, SR expression is enhanced in macrophages from dialysis patients, probably by up-regulation of type I, which may contribute to the development of atherosclerosis in these patients.

Prophylactic intravenous immunoglobulin treatment influences serum immunoglobulin M repertoire development after allogeneic bone marrow transplantation.

Patients treated with allogeneic bone marrow transplantation (BMT) suffer from a deficient humoral immunity during the post-transplant period. To prevent infections patients may receive prophylactic intravenous immunoglobulin (IVIG) therapy from 1 week before to 3 months after BMT. We have studied the effect of IVIG treatment on reconstitution of immunoglobulin repertoires in transplanted patients. Sera obtained from 13 IVIG-treated and 31 non-IVIG-treated patients before and at different time points after BMT, ranging from 3 days to 3 years, and from 18 healthy controls, were analyzed using a quantitative immunoblot system. The average immunoglobulin (Ig)M and IgG reactivity profiles against antigens derived from human liver, muscle and skin as well as Staphylococcus epidermidis protein extracts were similar in both patient groups and in controls. Both IgG and IgM reactivity profiles are, however, less heterogeneous among the individuals in the IVIG-treated patient group. Around 1 year after BMT the heterogeneity of the IgM reactivity profiles against allogeneic protein extracts is much lower in the IVIG-treated group compared to the non-IVIG-treated group and the healthy controls. This effect remains months to years after the IVIG treatment has been completed. Our results suggest that IVIG influences selection of the natural antibody repertoire mediated by the variable (V)-region during reconstitution after BMT.

Evidence for a functional idiotypic network among natural antibodies in normal mice.

We monitored in normal adult BALB/c mice the serum concentrations of four natural IgM antibodies, two of which show idiotypic complementarity in in vitro assays. In each individual, serum concentration of all four idiotypes were found to fluctuate in complex dynamical patterns with low correlation. The spectral power of some such patterns was found to be compatible with the existence of a chaotic regime. Groups of normal adult mice were injected intravenously with low (10 ng) or moderate (10 micrograms) doses of either of the two complementary idiotypes in saline. This treatment resulted in a pronounced inhibition of the fluctuation in the serum concentration of both complementary idiotypes for periods up to 3 months. Such compensations were not detected for the two unrelated natural idiotypes and were specifically induced, for they did not occur following the injection of unrelated antibodies. These results indicate the functional operation of an idiotypic network among natural antibodies.

Immune modulatory effects of immunoglobulins on cell-mediated immune responses in vitro.

Intravenous Immunoglobulin (IVIG) at a concentration of 5 mg/ml, significantly inhibited mitogenic responses to phytohaemagglutinin (PHA), concanavalin A (conA) and pokeweed mitogen (PWM) by peripheral blood cells from healthy donors. No difference in inhibition by IVIG was seen when stimulating different T-lymphocyte cell subsets. Inhibition by IVIG was dose-dependent. An increased response was observed when IVIG was added more than 12 h after PHA compared to adding 1 h before [P = 0.05]. Intravenous immunoglobulin added to mixed lymphocyte cultures (MLC), reduced the median response by more than 60% (range 14-89%; P = 0.03) and almost completely abrogated the lymphocyte response to Staphylococcus aureus protein A (SPA), whose median inhibition was 94% (range 90-99%; P = 0.02). When comparing 12 different commercial IVIG preparations at a concentration of 2.5 mg/ml, the median inhibition of the PHA stimulation ranged from 4% to 35% and the MLC response from 0% to 66%. In the presence of IVIG the lymphocyte response to different herpes virus antigens was reduced by > 50%. No difference in inhibitory effect was seen when comparing IVIG and cytomegalovirus (CMV) hyper Ig, but CMV negative Ig resulted in lower inhibition [P = 0.05]. Three out of five IgG preparations (2.5 mg/ml) made from single donors inhibited PHA stimulation significantly more than commercial IVIG [P < 0.05]. Mean inhibition was 61% compared to 35%. Inhibition by pooled IgG from five donors was 56%. F(ab')2 fragments of IVIG inhibited the MLC response by more than 50% (range 34-75%), SPA stimulation by 97% (83-104%) and PHA stimulation by more than 30% (26-37%). One of two Fc preparations tested had an inhibitory effect, but the inhibition was less than that obtained with the F(ab')2 fragments [P = 0.04]. These results further strengthen the notion that IVIG exerts its immune modulatory effect by binding to leukocyte surface receptors. A clear inhibition was obtained with concentrations corresponding to the serum levels obtained when IVIG is given 250-500 mg/kg bodyweight. F(ab')2 fragments have the same inhibitory effect as intact IgG molecules but the role of Fc fragments still remains unclear. Differences in the immunosuppressive effect of various IVIG preparations may be associated with the method of preparation.

Altered VH6-D-JH repertoire in human insulin-dependent diabetes mellitus and autoimmune idiopathic thrombocytopenic purpura.

We have characterized the peripheral B cell repertoire in T cell-mediated insulin-dependent diabetes mellitus (IDMM) and in B cell-mediated autoimmune idiopathic thrombocytopenic purpura (AITP). The VH6-containing repertoire in adult patients with IDDM or AITP and healthy control subjects was investigated by PCR amplification using VH6- and JH-specific primers. Nucleotide sequence analysis of VH6-D-JH rearrangements showed an abnormally high frequency of somatic mutations in non-functional rearrangements from diabetic (3. 58 %) as well as AITP patients (3.18 %), compared to controls (0.4 % and 1.43 %, respectively; p < 0.05). In contrast, the mutation frequency among functional rearrangements was 2.4 - 3 times lower in patients compared to controls ( p < 0.05). Detailed analysis of the VH6 genes carrying mutations showed that the underlying mechanism for this observation is probably different for the two diseases. Analysis of D- and JH gene usage revealed additional deviations from the normal pattern. Taken together, these results suggest defects in the mechanisms controlling selection of the B cell repertoire in patients with IDDM or AITP.

Age-related impaired affinity maturation and differential D-JH gene usage in human VH6-expressing B lymphocytes from healthy individuals.

To elucidate the basic molecular events underlying humoral immunity during ontogeny and senescence, we analyzed a panel of 179 polymerase chain reaction-derived VH6-D-JH rearrangements from cord blood, peripheral blood, and spleen. Nucleotide sequence analysis of the CDR3 region shows that there is a difference in D and JH gene usage in functional rearrangements between lymphocytes from peripheral blood and spleen. Analysis of the VH6 gene shows that the mutational frequencies rise from 0.81% in cord blood to 1.96% in peripheral blood lymphocytes derived from young adults, and decrease to 0.80% in samples from individuals older than 50 years. The number of rearrangements carrying mutations follows a similar pattern: 22% in cord blood, 73% in the age group 20-49 years, and 57% in the age group over 50 years. The mutational frequencies among the mutated genes are, however, similar for cord blood and young adults, 2.76% and 2.51%, respectively, and 1.3% in older adults. These data show an age-related impaired affinity maturation which might relate to the decrease in immunological responsiveness among the elderly.