European Journal of Immunology

A rapid method is described for effectively removing immunoglobulin‐bearing cells from either primed or unprimed mouse spleen and lymph node cell suspensions. Incubation of cell suspensions in nylon wool columns for 45 min at 37 °C resulted in a 9 to 100‐fold depletion of immunoglobulin‐bearing cells and a complementary 1.5 to 2‐fold enrichment of T cells in the column effluent populations.

The effluent population, derived from passage of spleen cells through these columns, was virtually devoid of B precursor and memory cell activity, but contained all of the helper cell and cytotoxic effector cell precursor activity when compared to unfractionated spleen cells.

Cell fusion techniques have been used to produce hybrids between myeloma cells and antibody‐producing cells. The hybrid lines derived are permanently adapted to grow in tissue culture and are capable of inducing antibody‐producing tumors in mice.

Spleens from mice immunized against sheep red blood cells (SRBC) were fused to an 8‐azaguanine‐resistant clone (X63‐Ag8) of MOPC 21 myeloma. Over 50 % of the derived hybrid lines produce and secrete immunoglobulins different from the MOPC 21 myeloma. About 10 % of the hybrid lines exhibit anti‐SRBC activity. The high proportion of antibody‐producing hybrids suggests that the fusion involves a restricted fraction of the spleen cell population, probably cells committed to antibody production.

In order to avoid the presence of the MOPC 21 heavy chain in the specific hybrids, another myeloma cell line (NSI/1‐Ag4–1) has been used. This is a nonsecreting variant of the MOPC 21 myeloma which does not express heavy chains.

Three anti‐SRBC (probably of the μ, γ_2b and γ₁ classes, respectively) and two anti‐2,4,6‐trinitrophenyl (of the μ class) antibody‐producing hybrids have been repeatedly cloned. By random selection and by selection of specific clones according to their lytic activity (clone plaque selection), a number of different lines have been constructed. Such lines express different combinations of the four possible chains of each hybrid line: the myeloma γ and k chains and the specific antibody heavy and light chains. In three cases (Sp1, Sp2 and Sp7) it is shown that only the specific H and L combination has activity and that the myeloma chains are unable to substitute for them. In most cases lines have been derived which no longer express the MOPC 21 chains but only the specific antibody chains.

A hybridoma clone which secretes a macrophage (MΦ)‐specific monoclonal antibody, F4/80, was produced by fusing spleen cells from a rat hyperimmunized with cultured thioglycollate‐induced mouse peritoneal MΦ with a mouse myeloma, NS1. Binding of antibody to primary cells and cell lines was detected by radioimmune indirect binding assay, autoradiography or fluorescence‐activated cell sorter analysis. F4/80 binds to mouse MΦ from the peritoneal cavity or other sources, blood monocytes, MΦ derived from bone marrow precursors in culture and MΦ‐like cell lines, but not to other cells, including polymorphonuclear leukocytes, lymphocytes or fibroblasts. F4/80 does not bind to MΦ via Fc receptors, is not cytotoxic and is of the rat IgG_2b subclass. Since F4/80 binds to all MΦ defined by adherence, morphology and immune phagocytosis, it provides a new marker to define the MΦ in the mouse. Large differences in expression of antigen F4/80 were found, depending on intraperitoneal stimulation, time in culture and stage of maturation. Immunoprecipitation experiments demonstrated that the antigen F4/80 is part of a component of Mr 160000 which is synthesized by the MΦ and, at least in part, exposed on the cell surface.

IL‐6 is a pleiotropic cytokine involved in the physiology of virtually every organ system. Recent studies have demonstrated that IL‐6 has a very important role in regulating the balance between IL‐17‐producing Th17 cells and regulatory T cells (Treg). The two T‐cell subsets play prominent roles in immune functions: Th17 cell is a key player in the pathogenesis of autoimmune diseases and protection against bacterial infections, while Treg functions to restrain excessive effector T‐cell responses. IL‐6 induces the development of Th17 cells from naïve T cells together with TGF‐β; in contrast, IL‐6 inhibits TGF‐β‐induced Treg differentiation. Dysregulation or overproduction of IL‐6 leads to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA), in which Th17 cells are considered to be the primary cause of pathology. Given the critical role of IL‐6 in altering the balance between Treg and Th17 cells, controlling IL‐6 activities is potentially an effective approach in the treatment of various autoimmune and inflammatory diseases. Here, we review the role of IL‐6 in regulating Th17/Treg balance and describe the critical functions of IL‐6 and Th17 in immunity and immune‐pathology.

Human mononuclear leukocytes produce a growth factor (HGF) for hybridoma and plasmacytoma cells. HGF has recently been proven to be identical to IFN‐β2, 26‐kDa protein and BSF‐2. HGF can be quantitated in a proliferation assay with the HGF‐dependent hybridoma cell line B13.29. By selection of an extremely sensitive variant of this cell line, we were able to measure HGF production of single cells. Limiting dilution analysis of the producing cells in combination with size, density and adherence characteristics showed that HGF is produced by monocytes and not by lymphocytes. There was no need for the monocytes to be stimulated but the cells did require the presence of serum. This serum requirement could be met by purified bovine serum albumin, but not by other proteins like ovalbumin or human γ‐globulin. HGF production in vitro by monocytes starts after 2 h of incubation and is completed within 24 h.

Mouse cell lines of different lineages have been established which constitutively secrete large quantities of recombinant mouse interleukins (mIL2, mIL3, mIL4 or mIL5). An existing bovine papilloma virus‐based expression vector, pBV‐lMTHA, was modified to allow transformed X63Ag8‐653 myeloma cells, NIH 3T3 fibroblasts and C127 mammary tumor cells to stably carry multiple copies of the vector, to express the inserted cDNA encoding a single interleukin constitutively, and to secrete the interleukin in high quantities.

Cell lines transformed with mIL2 cDNA stably carried 30–100 copies of the plasmid per cell and constitutively secreted biologically active mIL2 in quantities similar to those produced by murine EL4 thymoma cells or rat spleen cells stimulated with mitogens. Deletion of the 3′ untranslated region containing AT‐rich sequences from the mIL2 cDNA resulted in a 100‐fold increase in the constitutive production and secretion of mIL2 by the transformants. Addition of a heavy metal further increased the production 2 to 6‐fold. Cells transformed with 3′‐deleted mIL3 cDNA constitutively secreted 300–1000 times higher activities of mIL3 than the myelomonocytic leukemia line WEHI 3. mIL 4 produced by the similar transformants induced [³H]thymidine uptake of a T cell line, a mast cell line and B leukemia cells, and enhanced the production of IgG₁ by B cells. IL 4 liters were 150 times higher than those produced by the concanavalin A‐stimulated T cell line 2.19. mIL 5 was secreted by similar transformants at 10‐fold higher titers than those produced by concanavalin A‐stimulated 2.19 T cells, as judged by the proliferation and maturation of B cell leukemia BCL1. The expression vectors should be useful in establishing eukaryotic cell lines producing proteins from full length cDNA clones at higher rates. The established cell lines secreting IL2, 3, 4 or 5 at high rate should be useful sources for these interleukins in the investigation of their function in the immune system.

Antibody specificity is determined by structural v‐genes that code for the amino acid sequences of the variable regions of antibody polypeptide chains. The present hypothesis proposes that the germ‐cells of an animal carry a set of v‐genes determining the combining sites of antibodies directed against a complete set of a certain class of histocompatibility antigens of the species to which this animal belongs. The evolutionary development of this set of v‐genes in phylogeny is traced back to the requirements for cell to cell recognition in all metazoa. The hypothesis leads to a distinction between two populations of antigen‐sensitive cells. One population consists of cells forming antibodies against foreign antigens; these lymphocytes have arisen as mutants in clones descending from lymphocytic stem cells which expressed v‐genes belonging to the subset (subset S) coding for antibody against histocompatibility antigens that the individual happens to possess. The other population consists of allograft rejecting lymphocytes that express v‐genes of the remaining subset (subset A) coding for antibody against histocompatibility antigens of the species that the individual does not possess. The primary lymphoid organs are viewed as mutant‐breeding organs. In these organs (e. g. in the thymus), the proliferation of lymphocytes expressing the v‐genes of subset S and the subsequent suppression of the cells of these “forbidden” clones, leads to the selection of mutant cells expressing v‐genes that have been modified by spontaneous random somatic mutation. This process generates self‐tolerance as well as a diverse population of antigen‐sensitive cells that reflects antibody diversity. The proliferation in the primary lymphoid organs of lymphocytes expressing v‐genes of subset A generates the antigen‐sensitive cell population that is responsible for allo‐aggression.

The theory explains how a functional immune system can develop through a selection pressure exerted by self‐antigens, starting during a period in early ontogeny that precedes clonal selection by foreign antigens. The hypothesis provides explanations for the variability of the N‐terminal regions of antibody polypeptide chains, for the dominant genetic control of specific immune responsiveness by histocompatibility alleles, for the relative preponderance of antigen‐sensitive cells directed against allogeneic histocompatibility antigens, for antibody‐idiotypes, for allelic exclusion, for the precommitment of any given antigen‐sensitive lymphocyte to form antibodies of only one molecular species and for the cellular dynamics in the primary lymphoid tissues.

The isolation and propagation of functional antigen‐specific lines of T lymphoblasts is described. These lines were found to recognize foreign or self antigens in association with accessory cells of syngeneic major histocompatibility complex genotype. Intravenous inoculation of a T cell reactive only against myelin basic protein led to development of clinical paralysis in syngeneic rats. Thus, it is possible to study biological function as well as antigen specificity using T cell lines.

Chúng tôi đã nghiên cứu các cơ chế dung nạp miễn dịch do khối u gây ra bằng cách so sánh các dòng tế bào khối u có khả năng sinh miễn dịch và dung nạp miễn dịch được tách ra từ ung thư đại tràng chuột. Khi được tiêm vào vật chủ cùng loài, các tế bào REGb sinh miễn dịch tạo ra khối u bị đào thải, trong khi các tế bào PROb dung nạp miễn dịch tạo ra khối u phát triển và ức chế sự thoái triển của các khối u REGb. Chúng tôi cho thấy rằng thể tích khối u PROb có tương quan với sự gia tăng của các tế bào T điều hòa CD4⁺CD25⁺ trong các mô bạch huyết. Những tế bào này làm chậm lại sự đào thải in vivo các khối u REGb và ức chế in vitro các phản ứng miễn dịch do tế bào T chống lại các tế bào REGb thông qua một cơ chế yêu cầu tiếp xúc giữa các tế bào T hiệu lực và tế bào T điều hòa và có sự tham gia của TGF-β. Trong khi các tế bào T tổng từ chuột mang khối u PROb không tạo ra phản ứng miễn dịch kháng khối u rõ ràng, việc loại bỏ các tế bào T CD25⁺ khôi phục phản ứng này. Một lần sử dụng cyclophosphamide làm suy giảm các tế bào T CD4⁺CD25⁺ trong động vật mang khối u PROb, trì hoãn sự phát triển của khối u PROb, và chữa khỏi chuột mang khối u PROb hiện hành khi được theo sau bằng liệu pháp miễn dịch mà không có hiệu quả chữa trị khi được áp dụng đơn lẻ. Những kết quả này chứng minh vai trò của các tế bào T điều hòa CD4⁺CD25⁺ trong việc dung nạp miễn dịch do khối u gây ra và sự quan tâm đến việc loại bỏ các tế bào T điều hòa để làm nhạy khối u hiện hành với liệu pháp miễn dịch.

High levels of interleukin 6 (IL 6/B cell stimulatory factor‐2) were detected in synovial fluids from the joints of patients with active rheumatoid arthritis (RA). The cells found in freshly isolated synovial fluid constitutively expressed IL6 mRNA. The synovial tissues obtained by joint biopsy were also found to produce IL6 in vitro. Immunohistochemical analysis demonstrated that CD2⁺ T cells as well as CD20⁺ blastoid B cells in the synovial tissues produce IL6. The data indicate that IL6 is generated constitutively in RA and its overproduction may explain the local as well as the generalized symptoms of RA, since IL6 can function as B cell growth and differentiation factor as well as hepatocyte‐stimulating factor.