Self-reactive B Cells Revealed In Normal Repertoire: Distribution, V Gene Usage And Their Role In Maintenance Of Immune Tolerance

In the past decades or so, several lines of studies have shed new light on the principle and functions of autoimmunity. It has been widely accepted that autoreactive cells are present in healthy individuals other than patients with autoimmune diseases. Selective amplifications of the intrinsic autoimmunity via self-Ag immunization have been proved to be effective in some autoimmune diseases and tissue repair. These new observations present a compelling new picture of beneficial autoimmunity that is presumably designed to counteract the unfavorable damage.Recent data also showed that self-reactive B cells are seeded to the peripheral immune compartment and form the physiological autoimmune repertoire. By secreting natural autoantibodies (NAA), these B cells have been implicated in self-maintenance and immunological tolerance. However, the mechanisms responsible for beneficial effects of self-reactive B cells or NAA remain to be unclear. To address these issues, we chose keratin-reactive B cells and NAA and investigated their mechanisms of tolerance in normal repertoires.Objectives: The anatomical distribution, relevant ratio and V gene usage of keratin-reactive B cells in normal repertoire were characterized in advance to clarify the existence of physiological autoimmunity. To explore the underlying mechanism mediated by keratin-reactive B cell, their antigen presentation and the involvement in the clearance of apoptotic cell were investigated respectively.Methods: All works are divided into five sections. The experimentalprocedures are described as follows:1.Evaluation of anatomical distribution and relevant ratio of keratin-reactive B cells in normal repertoireFive antigens were initially conjugated with flurescein. FCM was used to identify the anatomical distribution and relevant ratio of keratin-reactive B cells derived from different species (i.e. human or mice), different lymphoid organs (i.e. spleen, peritoneal cavity, lymph node, thymus and bone marrow) and different grow phases (i.e. peripheral blood from adult or umbilical cord from newborn).2. Determination of V gene usage from keratin-reactive NAAThe splenocytes of non-immunized mice were fused with SP2/0 myeloma cells using 45% polyethylene glycol and culture supernatants were tested to screen the hybridomas secreting keratin-reactive NAA. Western-blot and immuno-histochemical techniques were adopted for their characterization. Total RNA from hybridoma cell line was isolated and cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit. Vh or Vk-specific ds-cDNA was generated by polymerase chain reaction (PCR) using antibody universal primer. DNA fragments isolated from agrose gel were ligated to pMD18-T vector and transfected into Escherichia Coli JM-109. The nucleotide sequences of amplified Vh genes were determined by the dideoxynucleotide chain termination reaction using the Sequenase kit. Sequence alignment analysis was carried out by searching the Entrez databases of the National Center for Biotechnology Information (NCBI) using the BLAST program. Vector NTI software was also used for comparing the disparity of the cloned sequences.3.Subpopulation and developmental phase of keratin-reactive B cells Biotin-conjugated keratin was prepared in advance. Fresh isolated cells from mice spleen or human PBMC were labeled with biotin-conjugated keratin and incubated with avidin-beads subsequently. Keratin-reactive B cells were then immunobead sorted by positive selections using BD? IMagnet. The purity of sorted B cells was characterized using anti-mouse B220-PE or anti-human CD19-PerCP. FITC-conjugated keratin was used to identify thekeratin-binding specificity. Subpopulation and developmental phase of sorted B cells were characterized by FCM using antibodies against CD5 and mAb493.4. Antigen presentation of keratin-reactive B cellsBiotin-conjugated keratin in Freund's adjuvants was injected subcutaneous in the footpad and the base of the tail. After 7 days, inguinal lymph nodes were isolated and T cells were enriched by nylon separation column. Enriched lymph node T cells from Ag-immunized mice were incubated with keratin-reactive B cells a ratio of 5:1 in the presence of keratin at 37°C for 96hr. Splenic cells as Ag-presentators were used as positive control. The proliferation indexes were compared by MTT colorimetric determination.The sorted B cells were incubated with fluorescent antibodies against MHC- II, CD80, CD86, CD40. The expression of these molecules was analyzed by FCM to investigate the underlying mechanisms of antigen-presentation.5. Involvements of NAA in clearance of apoptotic cellsThe assay for polyreactivity was performed by ELISA using the following antigens: keratin, actin, myosin, transferrin, pepsin. Keratin-reactive NAA was isolated using MBP-binding chromatography IgM purified column.Thymocytes from Balb/c mice were maintained in 10% FCS supplemented DMEM medium in the presence of dexamethasone(l U M). The nuclei were visualized by staining Hoechst33342. The PS exposure and apoptotic ratio were estimated by Annexin-V/PI staining using FCM. After incubation with purified 3B4 or pooled mice serum, the apoptotic cells was stained with anti- u -FITC and subjected to FCM analysis. The binding of IgM against apoptotic cells was determinated and the IgM binding positive response was also compared with apoptotic cells ratio. The apoptotic phase(early or late) of IgM binding was further exminated by 3-color fluorescence staining(Anti-IgM-PE> Annexin-V, 7-AAD).Thymocytes labeled with PKH67 were incubated with dexamethasone as described above. Macrophages labeled with PKH26 were then incubated withapoptotic thymocytes in the presence or absence of 3B4. The double or single positive ratio of macrophage was detected by FCM to learn the uptake of apoptotic cells.Results: All results were divided into five sections and described as follows:1. Existence of keratin-reactive B cells in normal repertoireFCM analysis showed the presence of self-reactive B cells in normal repertoires. The ratios of keratin , actin, myosin-reactive B cells in bone marrow(BM) were 33%, 20%, 16%. In spleen(Sp), the presence of self-reactive B cells against keratin actin, myosin was 20%, 22%, 14%. The percentage of self-reactive B cells was highest in peritoneal cavity(Pc), with the ratio of 65%, 62%, 40%. The corresponding ratio in lymph node(LN) was 18%, 16%, 18%. The ratio of self-reactive B cells in thymus was extremely low. With regard to B cells from lymphoid organs, OVA or BSA bindings may be neglected for considerably low ratio.In peripheral blood from adult, self-reactive B cells against keratin, actin, myosin were 19%, 16%, 11%. The corresponding ratios in umbilical cord blood were 48%, 49%, 43%. OVA or BSA bindings are similar within the different grow phase, with the ratio of 4%, 4% (peripheral blood from adult) , 6%, 4%( umbilical cord blood).2. Molecules structure of keratin-reactive NAAFour hybridoma strains secreting keratin-reactive NAA were isolated and sequenced. They are of novel gene segments and were deposited in DDBJ/EMBL/GenBank data bank. Sequence alignment analysis revealed that the isolated Vh and Vk genes had high homologies with their germ-line counterpart. The identities of aligned Vh genes amounted to 97%-98%. The obtained Vk genes showed similar homologies to their germ line match, with identities of 96%-97%.3. Subpopulation and develpmetal phase of keratin-reactive B cellsThe sorted B cells showed high purity and keratin binding specificity. FCM analysis of CD5 expression indicated that self-reactive B cells containCD5+and CD5' subpopulations. It was also revealed that self-reactive BCR is expressed on the surface of mature B cells.4. Antigen presentation of keratin-reactive B cellsAfter incubation of the sorted B cells with enriched T cells, they failed to induce proliferation of T cells in the presence of keratin-Ag. Although high expression of MHC- II molecules were found on surface of keratin-reactive B cells, it also showed that they had considerably low expressions of CD80 and CD86 as well as moderate expression of CD40. With regard to the expression of antigen presentation related molecules, similar patterns were confirmed in human and mice periphery.5. Clearance of apoptotic cells mediated by 3B4The binding of 3B4 to different unrelated antigens was confirmed by ELISA, which implied that 3B4 is polyreactive.Hoechst33342 staining showed that chromatin of thymocytes demonstrated a condensed, converged appearances after induction with dexamethasone(l u M). Annexin-V/PI double staining revealed that apoptotic thymocytes after 4hr dexamethasone induction was mainly at early phase. Followed by incubation dexamethasone for lOhr, mice thymocytes consisted of early and late apoptotic phases. After induction for 20hr, thymocytes were predominantly at late apoptotic phase. The kinetic of 3B4 binding to apoptotic cells was more closely approximated to entry of PI into the cells. Three-color fluorescence staining further demonstrated that 3B4 bound to antigens exposed on late phase apoptotic cells. In the presence of 3B4, the uptake ofapoptotic cells was significantly more efficient in macrophages. Conclusions:1. Self-reactive B cells are widely distributed in Pc, Sp, LN and peripheral blood of normal repertories, implies that physiological autoimmunity is constitutely existed in healthy individuals. The presence of self-reactive B cells varied accordingly from different lymphoid organs. Additionally, in view of high percentage of self-reactive B cells in umbilical cord blood, it is concluded that they are the predominant cell type in the newborn B cell repertoire.2. Based on high homologies of V gene usage from keratin-reactive NAA...