Two separate effects contribute to regulatory T cell defect in systemic lupus erythematosus patients and their unaffected relatives.

Forkhead box P3 (FoxP3)+ regulatory T cells (Tregs ) are functionally deficient in systemic lupus erythematosus (SLE), characterized by reduced surface CD25 [the interleukin (IL)-2 receptor alpha chain]. Low-dose IL-2 therapy is a promising current approach to correct this defect. To elucidate the origins of the SLE Treg phenotype, we studied its role through developmentally defined regulatory T cell (Treg ) subsets in 45 SLE patients, 103 SLE-unaffected first-degree relatives and 61 unrelated healthy control subjects, and genetic association with the CD25-encoding IL2RA locus. We identified two separate, uncorrelated effects contributing to Treg CD25. (1) SLE patients and unaffected relatives remarkably shared CD25 reduction versus controls, particularly in the developmentally earliest CD4+ FoxP3+ CD45RO- CD31+ recent thymic emigrant Tregs . This first component effect influenced the proportions of circulating CD4+ FoxP3high CD45RO+ activated Tregs . (2) In contrast, patients and unaffected relatives differed sharply in their activated Treg CD25 state: while relatives as control subjects up-regulated CD25 strongly in these cells during differentiation from naive Tregs , SLE patients specifically failed to do so. This CD25 up-regulation depended upon IL2RA genetic variation and was related functionally to the proliferation of activated Tregs , but not to their circulating numbers. Both effects were found related to T cell IL-2 production. Our results point to (1) a heritable, intrathymic mechanism responsible for reduced CD25 on early Tregs and decreased activation capacity in an extended risk population, which can be compensated by (2) functionally independent CD25 up-regulation upon peripheral Treg activation that is selectively deficient in patients. We expect that Treg -directed therapies can be monitored more effectively when taking this distinction into account.

Genetic association of CD247 (CD3ζ) with SLE in a large-scale multiethnic study.

A classic T-cell phenotype in systemic lupus erythematosus (SLE) is the downregulation and replacement of the CD3ζ chain that alters T-cell receptor signaling. However, genetic associations with SLE in the human CD247 locus that encodes CD3ζ are not well established and require replication in independent cohorts. Our aim was therefore to examine, localize and validate CD247-SLE association in a large multiethnic population. We typed 44 contiguous CD247 single-nucleotide polymorphisms (SNPs) in 8922 SLE patients and 8077 controls from four ethnically distinct populations. The strongest associations were found in the Asian population (11 SNPs in intron 1, 4.99 × 10(-4) < P < 4.15 × 10(-2)), where we further identified a five-marker haplotype (rs12141731-rs2949655-rs16859085-rs12144621-rs858554; G-G-A-G-A; P(hap) = 2.12 × 10(-5)) that exceeded the most associated single SNP rs858554 (minor allele frequency in controls = 13%; P = 4.99 × 10(-4), odds ratio = 1.32) in significance. Imputation and subsequent association analysis showed evidence of association (P < 0.05) at 27 additional SNPs within intron 1. Cross-ethnic meta-analysis, assuming an additive genetic model adjusted for population proportions, showed five SNPs with significant P-values (1.40 × 10(-3) < P< 3.97 × 10(-2)), with one (rs704848) remaining significant after Bonferroni correction (P(meta) = 2.66 × 10(-2)). Our study independently confirms and extends the association of SLE with CD247, which is shared by various autoimmune disorders and supports a common T-cell-mediated mechanism.

Variants of the serotonin transporter gene (SLC6A4) significantly contribute to hyperserotonemia in autism.

The role of the serotonin system in the etiology and pathogenesis of autism spectrum disorders (ASD) is not clearly defined. High levels of platelet serotonin (5-HT) have been consistently found in a proportion of patients, and it is known that specific 5-HT transporter gene (SLC6A4) variants modulate transporter reuptake function, therefore possibly influencing the occurrence of hyperserotonemia in a subset of autistic patients. We have examined the association of platelet serotonin levels with two SLC6A4 polymorphisms, 5-HTT gene-linked polymorphic region (HTTLPR) in the promoter and intron 2 variable number of tandem repeats (VNTR), in a sample of 105 ASD patients, their parents, and 52 control children. Quantitative transmission disequilibrium test (QTDT) results showed a significant effect on 5-HT levels of each SLC6A4 marker (P=0.017 for HTTLPR; P=0.047 for intron 2 VNTR) and of haplotypes of the two markers (P=0.017), with a major contribution of the L.Stin2.10 haplotype (P=0.0013). A 5-HT mean value in the range of hyperserotonemia was associated with the homozygous L.Stin2.10 haplotype (H (1,N=97)=7.76, P=0.0054), which occurred in 33% of hyperserotonemic patients against 6% of patients with normal 5-HT levels (Fisher's exact test: P=0.013, OR=8). Allele interaction at the HTTLPR locus was found, with a significant dominance variance effect on 5-HT levels. We found no transmission disequilibrium of any of the SLC6A4 variants in ASD. Our results show that the SLC6A4 gene is a significant factor in the determination of 5-HT levels, and that specific SLC6A4 variants are associated with an increased risk for hyperserotonemia in our sample of autistic patients. The biological mechanism, however, is unlikely to involve the SLC6A4 gene solely. The associated SLC6A4 alleles likely interact with other genes or environmental factors to produce the abnormally high 5-HT levels observed in this subset of autistic patients, who possibly represent a separate etiological group.

Significant association between the skewed natural antibody repertoire of Xid mice and resistance to Trypanosoma cruzi infection.

The Xid mutation predominantly affects the development of B cells and consequently the levels and composition of natural antibodies in sera. In contrast to the congenic and susceptible BALB/c strain, immunodeficient BALB.Xid mice display a resistant phenotype both to acute Trypanosoma cruzi infection and to the development of severe cardiopathy. Because natural antibodies are known to be basically self-antigen driven, IgM and IgG natural antibody repertoires (NAR) were compared before and during infection in these two strains. The analysis revealed fundamental alterations of IgM and IgG NAR in pre- and post-infected Xid mice. In particular, relatively increased natural (pre-existing) autoreactive IgG, dominated by the unique recognition of a single band in autologous heart extracts, was typical for uninfected Xid mice. This natural autoreactive IgG directed to heart antigens disappeared early after infection not only in Xid, but also in individual BALB/c mice that survived the acute infection. Conversely, the subgroup of BALB/c mice that died early after infection presented the most pronounced instances of the rapid, relative increase of IgM reactivities to self and non-self proteins. These results suggest that self-reactive NAR may play a role in an immunoregulatory mechanism relevant for the determination of susceptibility/resistance to infections. This may act either by influencing specific responses, or by modulating the self-aggressive components responsible for pathology.

Serum IgM repertoire reactions to MBP/CFA immunization reflect the individual status of EAE susceptibility.

Lewis rats develop experimental allergic encephalomyelitis (EAE) in response to immunization with myelin basic protein (MBP) in CFA, while Fischer rats are usually resistant. These strains, while comparably producing anti-MBP antibodies, also differ in their repertoire reactions to immunization, as measured by patterns of serum IgM reactivity with various autologous proteins. We have now scored IgM repertoire reactions to MBP/CFA immunization after treatments that alter EAE susceptibility in either strain. The results show that abrogation of EAE susceptibility in Lewis rats by a previous experience of T cell-induced passive EAE provoked a novel set of IgM reactivities that otherwise characterized the Fischer's repertoire reaction. Conversely, these reactivities were delayed in the response of Fischer rats that had been rendered EAE-susceptible by cyclophosphamide. Another IgM reactivity with a significant association to individual EAE severity in Lewis rats behaved reciprocally. Together with previous results, these observations suggest that putative regulatory mechanisms concordantly affect EAE resistance and IgM repertoire reactions, operating naturally in Fischer rats and abrogatable by cyclophosphamide treatment, whereas naturally suppressed, but restorable in Lewis rats. Other treatments altering EAE susceptibility, however, did not share these characteristics and may thus be mediated by other mechanisms.

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.

Natural antibodies in childhood: development, individual stability, and injury effect indicate a contribution to immune memory.

Natural, often autoreactive antibodies are present in normal sera in large quantity and show alterations in specificity in diverse pathological situations. They have, however, usually not been studied longitudinally. Here we investigated some representative serum reactivities of natural antibodies in 67 normal children and 10 with injury during childhood, followed up for 3 years. Normal children showed an individually characteristic and relatively stable level of most IgM, IgG, and IgA reactivities when measured with ELISA by reference to a standard. Injured children showed some very rapidly enhanced reactivities within 3 days after trauma, which thereafter slowly diminished over years before coming back to a normal level. This period exceeds by far the lifetime of antibodies and plasma cells. We conclude that natural antibodies contribute to the establishment and maintenance of immune memory in a manner that is distinct from classical immune reactions.

Structured reactions of serum IgM repertoires to immunization are dependent on major histocompatibility complex genes.

In normal animals, responses to immunization include alterations in the serum IgM antibody repertoire, as scored on autologous tissue antigens with no respect for the immunizing antigen. These immunogen-nonspecific antibody reactions were found previously to display specific structures dependent on strain and immunization protocols. Using major histocompatability complex (MHC)-congenic Lewis rats, we show that such IgM repertoire reactions are under the control of MHC-linked genes, including a class I locus. This strongly suggests the involvement of T cells restricted by both class I and class II MHC, in regulating serum IgM repertoires. Immunogen-nonspecific repertoire reactions to immunization may, therefore, represent degenerate, but prototypical, reactions or regulatory mechanisms embodying the natural repertoires of T- and B cells connected to autoantigens. Natural (auto)immunity could so serve to regulate the effector class of adaptive immune responses, particularly in order to avoid pathogenic autoreactivity following specific immunization with self-cross-reacting antigens. Appropriate analysis of nonspecific repertoire reactions could therefore contribute to the understanding of general structures of immune regulation and natural tolerance.

Dynamics of serum IgM autoreactive repertoires following immunization: strain specificity, inheritance and association with autoimmune disease susceptibility.

Immunization of Lewis rats with myelin basic protein (MBP) in complete Freud's adjuvant (CFA) provokes experimental allergic encephalomyelitis (EAE). Here we compare, irrespective of antigen specificity, the structure and dynamics of serum IgM autoreactive repertoires following immunization with MBP/CFA in EAE-susceptible Lewis and relatively resistant Fischer rats. Prior to the appearance of clinical symptoms, Lewis rats developed a specific modification of serum IgM autoreactivities that, scored on other determinants than MBP itself, showed a prognostic association with EAE symptoms. Although comparable in their production of MBP-specific serum IgM and IgG antibodies, Fischer rats did not share these MBP/CFA-induced IgM autoreactivities of Lewis rats when immunized in the same manner. Moreover, while the Lewis-type repertoire reaction was specific for MBP/CFA alone, the respective Fischer reaction was not qualitatively different from that observed in this strain upon non-pathogenic immunization with self-related or -unrelated antigens. In general, the repertoire reactions differed qualitatively between the strains, consisting of components with typical behavior and strain preferences. The EAE-associated, as well as the other components of both Lewis- and Fischer-type repertoire reactions were usually co-dominantly inherited in F1 animals. These results indicate that a global antibody repertoire analysis may serve as a tool to describe prototypical response structures, possibly involved in immune regulation and susceptibility to pathogenic autoimmunity.