Immunoglobulin idiotopes expressed by T cells. I. Expression of distinct idiotopes detected by monoclonal antibodies on antigen- specific suppressor T cells

The idiotopic repertoire expressed by antigen-specific suppressor T cells (Ts) generated by Streptococcus pneumoniae strain R36a (Pn) in BALB/c strain mice was investigated using a panel of five monoclonal anti-idiotopic antibodies against TEPC-15/HOPC-8 myeloma proteins. Previous studies suggested that the anti-idiotopic antibodies recognize distinct idiotopic determinants within the T15 idiotype, and that Pn-reactive B cells express all of those idiotopes as shown by a specific inhibitory effect of the anti-idiotopic antibodies on induction of anti-Pn response in vitro as well as on the mature antibody plaque-forming cells. In this study we asked the question of whether anti-idiotopic (Id) can block the inductive and/or effector phases of generation of Ts which act on the Pn-reactive B cells. The presence of anti-Id during the activation of T cells with Pn did not prevent the generation of Ts. However, suppression mediated by Ts on responder lymphocytes (cultures of spleen cells or B cels) was inhibited (reversed) by four out of five anti-Id. Some of the antibodies recognize hapten (phosphorylcholine)-inhibitable Id in the paratope of Ig whereas others are directed against nonparatopic Id. These data indicate that the antigen receptor on Ts includes VH sequences both within and without the immunoglobulin in paratope, and that the Id repertoir of Ts overlaps with that of B cells.     

Induction of cytotoxic T cell precursors in vivo. Role of T helper cells

Strain AS rats respond with two populations of cytotoxic T lymphocytes to stimulation in vitro by the major histocompatibility complex (MHC)-incompatible strain HL rat tumor (HL-A2T2). One is specific for MHC alloantigens present on both HL-A2T2 and normal HL targets, the other is tumor specific. The activation of these killer cells requires helper T lymphocytes. The tumor-specific helper cells depend on syngeneic radioresistant accessory cells to present the tumor antigens in an immunogenic form. The appropriate helper-accessory cell interaction results in the production of soluble factors which then induce the maturation of precursor cells into effective killer cells. Studies with a procedure for inducing negative selection of T cells in vivo showed that short-term exposure to HL-A2T2 tumor induced selection only for TH but not cytotoxic T lymphocyte precursors (CTLp). Simultaneous injection of supernatants from concanavalin A-activated spleen cell cultures, however, did produce selection of CTLp. These and other findings suggest that under normal circumstances in vivo, both signals (recognition of antigen and acceptance of maturation factors) are provided in the vicinity of an antigen presenting macrophage-like accessory cell.     

Antigen-binding T cells as helper cells. Separation of helper cells by immune rosette formation

The spleen T cells from mice immunized 6 days earlier with either chicken gamma globulin (CGG) or with donkey erythrocytes (DRC) were rosetted with CGG-coated sheep erythrocytes or with DRC. The immune rosettes (RFC) (antigen-binding cells) were separated from the bulk of nonrosette-forming cells (non-RFC) by 1-g velocity sedimentation and the RFC and non-RFC tested for helper activity in cooperative antihapten responses in vitro. RFC or non-RFC were mixed with normal or hapten-primed spleen cells, challenged with the appropriate hapten-carrier conjugate and cultured for 4 days in Marbrook tissue cultures. The helping activity was quantitated from the numbers of antihapten antibody-producing cells generated per culture. The results show that specific helper cell activity could be selectively recovered in the immune rosette-forming cell population whereas the non-RFC population was depleted of help. These findings indicate that the helper T cells express specific antigen binding receptors.     

Regulatory idiotypes. T helper cells recognize a shared VH idiotope on phosphorylcholine-specific antibodies

Priming of BALB/c mice with phosphorylcholine-hemocyanin (PC-Hy) induces T helper cells that are detected in splenic fragment cultures responding to immunization with trinitrophenylated PC-binding myeloma proteins, TEPC 15 (TNP-T15) and MOPC 167 (TNP-M167). Trinitrophenylation did not alter the binding site, idiotype, or isotype of the antibodies as demonstrated by binding studies. To assay idiotype-recognizing helper cells, Ly-2.2-depleted T cells from PC-Hy- primed donor mice were transferred to syngeneic athymic mice. Splenic anti-trinitrophenol fragment cultures were prepared from the nude recipients, and the response to TNP-T15 and TNP-M167 was measured by enzyme-linked immunosorbent assay. The number of responding fragments is dependent on the number of transferred primed T cells. The homing efficiency of 51Cr-labeled helper cells into the spleen of nude recipients was determined. The frequencies of T helper cells taken from PC-Hy-primed donors required for a B cell response to TNP-T15 or TNP- M167 were indistinguishable. The fine specificity of the anti-PC idiotype-recognizing T helper cells was studied by adding hapten (PC) or unconjugated myeloma proteins to fragment cultures as inhibitors at the time of immunization. PC and PC-bovine serum albumin, as well as T15 and M167, inhibited the helper function in vitro. Furthermore, free heavy chains of T15 and M167 partially inhibited T help, but free light chains of both idiotypes had no effect. These findings collectively show that T helper cells, induced by priming with antigen, recognize a shared idiotypic determination on T15 and M167 that is part of the PC binding site. The heavy chains of T15 and M167 appears to be the major structural component of this determinant. Evidently, T helper cells can recognize a shared determinant that is present on idiotypically different myeloma proteins. This determinant appears to be conserved throughout evolutionary and somatic mutations. The role of this shared, binding site-related idiotypic determinant as a regulatory idiotype in T-B cell interaction is discussed.     

Induction of idiotype-bearing, nuclease-specific helper T cells by in vivo treatment with anti-idiotype

Treatment of BALB/c mice with purified pig anti-(BALB/c anti-nuclease) anti-idiotypic antibodies has been found to induce the appearance of idiotype-bearing immunoglobulins (Id') in the serum of these mice in the absence of detectable antigen binding activity. This phenomenon appeared to require T cells in the hosts because no Id' was detected in the serum of nude mice similarly treated. Furthermore, the spleens of BALB/c mice treated with anti-idiotype were found to contain helper T cells capable of providing help in an in vitro plaque-forming cell response to trinitrophenyl-nuclease equivalent to that provided by helper T cells from the spleens of nuclease-primed animals. Helper T cells from both anti-idiotype-treated and nuclease-treated animals were found to be antigen-specific and to be similarly susceptible to elimination by treatment with anti-idiotype plus complement. Therefore, treatment with both antigen and anti-idiotype appeared to prime similar populations of antigen-specific helper T cells, while having different effects on the induction of antibody. These findings are consistent with the network theory of receptor interactions in the immune response, and may provide a means for studying individual cell populations involved in such interactions.     

Induction of anti-allo-class I H-2 tolerance by inactivation of CD8+ helper T cells, and reversal of tolerance through introduction of third-party helper T cells

The intravenous sensitization of C57BL/6 (B6) mice with class I H-2-disparate B6-C-H-2bm1 (bm1) spleen cells resulted in the abrogation of CD8+ T cell-mediated anti-bm1 (proliferative and interleukin 2-producing) T helper (Th) cell activities. In vitro stimulation of lymphoid cells from these mice with bm1 cells, however, generated a reduced, but appreciable, anti-bm1 cytotoxic T lymphocyte (CTL) response. Moreover, the anti-bm1 CTL response, upon stimulation with [bm1 x B6-C-H-2bm12 (bm12)]F1 spleen cells, was enhanced when compared with the response induced upon stimulation with bm1 cells. These in vitro results were reflected on in vivo graft rejection responses; bm1 skin grafts engrafted in the bm1-presensitized B6 mice exhibited prolonged survival, whereas (bm1 x bm12)F1 grafts placed collateral to bm1 grafts (dual engrafted mice) inhibited the tolerance to bm1. In the B6 mice 1-2 d after rejecting the bm1 grafts, anti-bm1 Th activities remained marginal, whereas potent anti-bm1 CTL responses were found to be generated from their spleen cells. Administration in vivo of anti-CD4 antibody into bm1-presensitized, dual graft-engrafted mice prolonged bm1 graft survival and interfered with enhanced induction of anti-bm1 CTL activity. These results indicate that anti-class I alloantigen (bm1) tolerance as induced by intravenous presensitization with the relevant antigens is not ascribed to the elimination of CD8+ CTL precursors, but to the specific inactivation of CD8+ Th cells, whose function can be bypassed by activating third-party Th cells.     

T cell receptor-independent immunosuppression induced by dexamethasone in murine T helper cells.

The immune and inflammatory responses are largely inhibited by glucocorticosteroids. In thymocytes, for example, glucocorticosteroids cause apoptosis, whereas they suppress the activity of phospholipase A2 and the production of eicosanoids in tissues actively engaged in inflammation. The immunosuppressive action of dexamethasone (DEX) was studied in vitro by employing a model cell system, namely the murine Th2 clone D10.G4.1 (D10) and its clonotypic anti-T cell receptor (TCR) mAb 3D3. Although the proliferative response of D10 cells to 3D3 stimulation was not affected by DEX, the costimulation provided by IL-1 was dramatically inhibited. Substitution of 3D3 by exogenous IL-4 (as the IL-1 costimulant) failed to prevent the inhibition of proliferation caused by DEX. Yet, when 3D3-mediated stimulation of TCR was supplemented with IL-2, D10 cells were capable of proliferating, even in the presence of DEX. Thus, TCR stimulation on D10 cells remained intact and their resulting propagation was not compromised by DEX treatment. These results provide evidence that immunosuppression caused by DEX is TCR independent and involves an early cytokine-signalling event.     

T-independent and T-dependent B lymphoblasts: helper T cells prime for interleukin 2-induced growth and secretion of immunoglobulins that utilize downstream heavy chains

Resting B cells enlarge, enter the cell cycle, and change their surface phenotype when activated via the surface immunoglobulin (Ig) receptor, but subsequent cell growth and antibody production is relatively limited. To identify stimuli that might prime B cells for enhanced function in vitro, we have compared the effects of anti-Ig with helper T (Th) cells on the formation of B lymphoblasts and the subsequent ability of the blasts to grow and secrete Ig. The B blasts first were induced by either anti-Ig, anti-Ig plus T cell-derived lymphokines, or alloreactive T blasts. Each population of B blasts showed enhanced expression of cell surface adhesion molecules, interleukin 2 receptor (IL-2R) p55, and MHC products, as well as decreased expression of IgD. The allo-activated B blasts were distinctive in expressing low levels of Thy-1 and increased reactivity with peanut agglutinin, a marker of germinal center B blasts in situ. The function of the different populations of B blasts was also different. Whereas anti-Ig or anti-Ig plus lymphokines primed for enhanced responses to lipopolysaccharide (LPS), the B blasts induced by Th cells were insensitive to LPS. B lymphoblasts that had been activated in the presence of helper factors or Th cells responded vigorously to recombinant IL-2 with growth and Ig secretion, and this response was enhanced in the presence of anti-Ig. The B blasts activated directly by Th cells, but not by anti-Ig plus lymphokines, were primed to secrete high levels of IgG1 and IgA. Therefore, the phenotype and function of a B lymphoblast depends upon the manner in which it is primed. When primed by Th cells, IL-2 proves to be the predominant mediator of clonal expansion and antibody secretion.     

免疫比浊法检测血清游离轻链的方法学评价

目的对Beckman Immage 800全自动特种蛋白分析仪检测血清游离κ、λ轻链(sFLC)进行方法学评价。方法通过Beckman Immage 800定量测定sFLC,探讨其精密度、正确度、分析测量范围、干扰和携带污染率,同时对其参考区间进行验证。结果κFLC低、高值批内精密度的变异系数(CV)分别为7.84%、2.95%,批间精密度的CV分别为7.38%、5.57%;λFLC低、高值批内精密度(CV)分别为4.59%、3.94%,批间精密度CV分别为3.97%、2.01%;测定κFLC、λFLC定值质控血清与靶值的偏倚小于5%;κFLC、λFLC分析测量范围分别为10.8~128.0 mg/L、10.1~121.0 mg/L时,a值在0.95~1.05,r2≥0.98;κFLC、λFLC携带污染率分别为0.411%、0.216%;干扰试验结果显示游离胆红素≤342.0μmol/L、结合胆红素≤342.0μmol/L、血红蛋白≤5g/L和乳糜≤2 400浊度时,对sFLC结果无明显影响;40例健康体检者中有1例κFLC测定结果超出厂家推荐的参考区间,40例λFLC检测值均在厂家推荐的参考区间内。结论 Beckman Immage 800全自动特种蛋白分析仪检测sFLC的主要分析性能符合质量目标的要求,能够为临床提供科学、准确的诊疗依据。     

T helper 1 and T helper 2 cells are pathogenic in an antigen-specific model of colitis.

Dysregulated T cell responses to enteric bacteria have been implicated as a common mechanism underlying pathogenesis in rodent models of colitis. However, the bacterial species and T cell specificities that induce disease have been poorly defined. We have developed a model system in which target antigen, bacterial host, and corresponding T cell specificity are defined. OVA-specific T cells from DO11.RAG-2(-/-) TCR transgenic mice were transferred into RAG-2(-/-) recipients whose intestinal tracts were colonized with OVA-expressing or control Escherichia coli. Transfer of antigen-naive DO11.RAG-2(-/-) T cells into recipients colonized with OVA-E. coli resulted in enhanced intestinal recruitment and cell cycling of OVA-specific T cells; however, there was no development of disease. In contrast, transfer of polarized T helper (Th) 1 and Th2 populations resulted in severe wasting and colitis in recipients colonized with OVA-expressing but not control E. coli. The histopathologic features of disease induced by Th1 and Th2 transfers were distinct, but disease severity was comparable. Induction of disease by both Th1 and Th2 transfers was dependent on bacterially associated OVA. These results establish that a single bacterially associated antigen can drive the progression of colitis mediated by both Th1 and Th2 cells and provide a new model for understanding the immunoregulatory interactions between T cells responsive to gut floral antigens.     

Selective induction of rheumatoid factors by superantigens and human helper T cells.

Production of autoantibodies specific for the Fc region of autologous IgG, called rheumatoid factors (RF), is a characteristic finding in patients with rheumatoid arthritis (RA). To study the requirements regulating the synthesis of these autoantibodies, we have cloned human helper T cells and co-cultured them with purified B cells. To mimic cognate T-B cell interaction, we have used bacterial superantigens that function by cross-linking HLA molecules on the B cell with selected T cell receptor (TCR) molecules expressing a particular polymorphism of the V beta gene segment. Data presented here demonstrate that the staphylococcal enterotoxin D (SE D), but not other bacterial superantigens, exhibits an ability to induce IgM, IgG, and especially RF production, in B cells from RA patients and normal individuals. Comparison with the polyclonal antibody production in B cell cultures driven by anti-CD3-stimulated T cell clones confirmed that SE D shifted the repertoire of secreted antibodies toward immunoglobulins with Fc binding specificity, suggesting that SE D preferentially stimulates RF+ B lymphocytes. B cells with the potential to secrete RF were highly frequent in RA patients, requiring as few as 150 peripheral B cells/culture to detect RF in the culture supernatants. SE D-induced RF synthesis was strictly dependent on the presence of selected CD4+T helper cells and required a direct membrane contact between B cells and T helper cells. Here, we propose a model that SE D selectively induces RF production depending on the availability of SE D responsive T cells in the TCR repertoire of the responder.     

Turbidimetric measurement of Bence-Jones proteins using antibodies against free light chains of immunoglobulins. An artifact caused by different polymeric forms of light chains

The different polymeric forms of free kappa and lambda light chains from urines of two myeloma patients were separated by gel filtration. When samples containing kappa chains in equal protein concentration were quantified by turbidimetry with anti-free kappa chain antibodies, two out of three samples reacted much less sensitively than the third sample. When analysed with SDS-polyacrylamide gel electrophoresis in conditions dissociating non-covalent but not covalent linkages, these two samples contained monomeric light chains, whereas the third sample contained dimers.     

The estimation of free light chains of immunoglobulins in biological fluids.

Methods for the estimation of the free light chains of immunoglobulins in serum, urine and cerebrospinal fluid are divided into two groups, electrophoretic and immunological, and the analytical performance of each method described. The problems associated with the accurate and precise determination of free light chains by the different methods are discussed and their complementary clinical roles emphasized. It is proposed that an International Reference Preparation for free light chains is established.