We developed a method for production of antigen-specific, H-2-restricted T cell hybrids. The tumor cell partner in the fusions was itself a T cell hybrid, FS6-14.13.AG2 (or its derivatives), which could be induced to produce the growth factor, interleukin-2 (IL-2), in response to a challenge with concanavalin A, but had no known antigen specificity. The normal T cell partner in the fusions was a population of lymph node T cell blasts that had been highly enriched in antigen-specific, H-2-restricted T cells by in vivo immunization, followed by in vitro challenge with antigen and clonal expansion in IL-2-containing medium. These fusions produced hybrids that grew constitutively in culture. A sizable proportion of the hybrids demonstrated the ability to produce IL-2 in response to a challenge with specific antigen presented by irradiated spleen cells of the appropriate H-2 type. Four cloned antigen/H-2-specific hybrid lines were produced. AO-40.10 responded to chicken ovalbumin (OVA) when presented by I-A(k)-bearing cells. DC1.18.3 responded to the apo form of beef cytochrome c when presented with I-A(d). AODK-10.4 responded to keyhole limpet hemocyanin (KLH) presented with I-A (d). AODK-1.16 also responded to KLH presented by a product of the I region of H-2(d), but the data were consistent with either a product of the I-J-I-E(d) region or a combinatorial molecule with elements from both I-A(d) and I-E(d)/I-C(d). Coincidentally, AO-40.10 was shown to have an unexpected alloreactivity with a product of H-2(b) mapping to the K-I-A region. These hybrids should prove invaluable as sources of monoclonal material for the study of the receptor(s) on T cells with H-2-restricted antigen specificities. We also generated T cell hybrids with two antigen/H-2 specificities by fusing an azaguanine-resistant clone of AO-40.10 to normal T cells with a different antigen/H-2 specificity. Many of the hybrids retained reactivity to OVA plus H-2(a) and to the second antigen/H-2 combination. None reacted to either OVA plus the second H-2 type or to the second antigen plus H-2(a). One of these hybrids was successfully cloned to produce the line AOFK- 11.11.1. It retained the ability to recognize OVA plus I-A(k) inherited from one parent, and KLH plus IA(f) inherited from the other. It did not recognize OVA plus IA(f) or KLH plus I-A(k). These results have some bearing on models describing the nature of T cell receptors for antigen recognized in association with H-2 products. They do not support models in which antigen and H-2 are recognized separately by two independent T cell receptors.

/pdf/antigen-inducible-h-2-restricted-interleukin-2-producing-t-47suwvpjk5.pdf

Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas. Lack of independent antigen and H-2 recognition

III Antibodies A Monoclonal antibodies against determinants of the HLA-A,-B,-C glycoproteins and pjmicroglobulin 1. Antibodies against species specific, non-polymorphic determinants 2. Antibodies against polymorphic HLA-A,-B,-C determinants B Monoclonal antibodies against HLA-DRw determinants 1. Antibody against a species specific HLA-DRw determinants 2. Antibodies against polymorphic HLA-DRw determinants

Citation-classic - monoclonal-antibodies for analysis of the hla system

Since the first report of hybridomas producing monoclonal antibodies by Kohler and Milstein in 1975, this technique has spread to nearly all areas of biological, biochemical, and biomedical research. Watching the use of these methods spread from immunologists to cell biologists, developmental biologists, biochemists and to other biological disciplines and observing the nearly logarithmic increase in publications using these reagents has been in itself fascinating and informative. An overview of the development of this technology and its applications is presented including the use of monoclonal antibodies to study cell surface molecules, differentiation antigens, receptors, and histocompatibility antigens. The use of these antibodies to analyze microorganisms and parasitic antigens as well as their use in the genetic analysis of human cell surface antigens and the detection of polymorphic variation in enzymes and other proteins is discussed. Examples of the application of monoclonal reagents to the study of tumor cell biology including the labeling of metastatic tumor cells and the detection of cell surface molecules implicated in the regulation of growth control and cell division are provided.

Hybridomas: a new dimension in biological analyses.

Studies in our laboratory and elsewhere have shown that it is possible to propagate antigen-specific murine T cells in vitro with resultant specific stepwise enrichment of antigen-induced proliferative cells. The proliferative responses of these T cells are antigen specific and dependent upon the presence of antigen-presenting cells (spleen cells) that share the I-A subregion with the proliferating T cell. Using techniques of soft-agar cloning, it has been further possible to isolate clones of antigen-reactive T lymphocytes from such long-term cultures. Data suggesting that these were clones of antigen-reactive T cells were obtained by studying the recognition of antigen in association with antigen-presenting cells with a panel of such clones of antigen-reactive T cells. Proof of clonality was obtained by subcloning. Clones derived from F1-immune mice can be divided into three separate categories: one clone recognizes antigen in association with antigen-presenting determinants of parent A and the F1; the second type recognizes antigen in association with antigen-presenting determinants of parent B and the F1; and the third type recognizes antigen only in association with antigen-presenting determinants of the F1 mouse. Genetic studies on the major histocompatibility complex requirements for antigen presentation to such F1-reactive T cell clones suggests that the hybrid antigen-presenting determinant in this system results from transcomplementation of products of the I-A region of haplotypes a and b. These studies support the concept developed in our laboratory that there exist unique F1 hybrid determinants on (A/J X C57BL/6) F1 cells and suggest that these determinants can be utilized physiologically by hybrid mice in immunocompetent cellular interactions.

https://rupress.org/jem/article-pdf/152/4/759/1091091/759.pdf

Antigen-reactive T cell clones. I. Transcomplementing hybrid I-A-region gene products function effectively in antigen presentation.

Lymphocyte function associated antigen 1 (LFA-1) is a leukocyte cell adhesion protein. We have studied a novel human immunodeficiency disease in which LFA-1 and two other proteins which share the same beta subunit are lacking from the surface of leukocytes. The basis of the inherited defect in cell surface expression of both the alpha and beta subunits of LFA-1 was determined by somatic cell fusion of patient or normal human cells with an LFA-1+ mouse T cell line. Human LFA-1 alpha and beta subunits from normal cells could associate with mouse LFA-1 subunits to form interspecies hybrid alpha beta complexes. Surface expression of the alpha but not the beta subunit of patient cells was rescued by the formation of interspecies complexes. The findings show that the LFA-1 alpha subunit in genetically deficient cells is competent for surface expression in the presence of an appropriate beta subunit, and suggest that the genetic lesion affects the beta subunit. The human LFA-1 alpha and beta subunits were mapped to chromosomes 16 and 21, respectively. The genetic defect is inferred to be on chromosome 21.

https://rupress.org/jem/article-pdf/164/3/855/1096337/855.pdf

LFA-1 immunodeficiency disease. Definition of the genetic defect and chromosomal mapping of alpha and beta subunits of the lymphocyte function-associated antigen 1 (LFA-1) by complementation in hybrid cells.

THE immune system is now recognised as a network of interacting cells with some interactions augmenting, and others suppressing immune responses. Regulatory interactions are often mediated by T cells, and do not always require cell-to-cell contact between appropriate cells, but may be mediated by soluble transmitters, commonly termed ‘factors’. Both antigen-specific1–7 and nonspecific8–10 factors have been described. Biochemical characterisation of these factors is hampered by the often minute quantities required to mediate biological effects and by the multiplicity of regulatory pathways involved in immune responses. Our knowledge of these factors is derived from functional tests, often combined with serological analysis of the factors. One approach to the analysis of monoclonal cell products is the somatic cell hybridisation technique described by Milstein et al.11,12. Using this method, specific antibody-forming cells have been hybridised with myeloma cells, and have yielded clones producing specific antibody11. This antibody is particularly useful as it is monoclonal and large quantities can be obtained. Although attempts to produce T-cell hybrids have been successful13–15, the products have not been functional. However, only failures in expression of cytotoxic activity have been described13–15. We describe here one antigen-specific suppressor factor (SF)-producing line S1-41 and its products.

T-cell lines producing antigen-specific suppressor factor

To investigate the histocompatibility requirements for the macrophage-T cell interaction in the secondary antibody response, splenic T cells from antigen (carrier)-primed F1 hybrid mice were restimulated in vitro with carrier-pulsed F1, parental or allogeneic macrophages. Surviving T cells were cocultured with hapten-primed F1 or parental “B cells” and restimulated with the appropriate hapten-carrier conjugate. The IgG antibody-forming cell response was then measured using a plaque assay.



Mapping of the genetic restriction was performed by use of different strain combinations. T helper cells could be restimulated in the presence of macrophages only provided they shared the I-A subregion of the major histocompatibility complex with the F1 T cells. T cells from F1 hybrids restimulated with parental or I-A-identical macrophages were shown to only cooperate with parental B cells of the same I-A haplotype as the macrophages used for restimulation. The defect was at the level of the macrophage, as addition of macrophages of the I-A haplotype used for the restimulation culture reconstituted the ability of F1 helper cells to cooperate with the I-A-nonidentical B cells.

Nature of macrophage-T cell interaction in secondary helper cell generation in vitro. Genetic restriction of macrophage-T cell interaction, which determines T-B genetic restriction.

Different T cell functions, e.g. help, suppression and cytotoxicity are associated with different T cell subsets, bearing distinctive differentiation markers. Thus effector T cells mediating help are Ly 1+2−3− and may bear Ia determinants, those mediating suppression are Ly 1−2+3+, IJ+, and cytotoxic cells are Ly 1−2+3+ and do not bear Ia determinants (1). At least the cytotoxic effectors are Ly 6.2 positive, but they arise from Ly 6.2 negative precursors (2). Thus expression of certain differentiation markers (Ly 123) is associated with acquisition of the ability to perform a particular function, even before encounter with antigen, whilst others are expressed as a result of further differentiation, including clonal expansion, following response to antigen (e.g. Ly 6.2 and possibly Ia antigens).

T cell hybrids with T cell functions.

The antibody response to murine alloantigens is thought to be thymus-dependent. In this study, we were using H-2 and H-minor antigens presented on the surface of cells as particulate carriers and 2, 4, 6-trinitrophenyl (TNP) as a hapten.



To study conditions for the induction of T helper and T suppressor cells directed against such antigens, T cell donor mice of a given inbred strain were primed with spleen cells of another inbred strain differing in distinct subregions of its H-2 haplotype and/or differing in its H-minor haplotype. Spleen T cells of mice in vivo allogeneically (carrier)-primed in this way were cocultured in vitro with splenic in vivo TNP (hapten)-primed B cells plus irradiated TNP-conjugated spleen cells (hapten-carrier conjugate) from the haplotype used for carrier priming.



The anti-hapten antibody response was taken as a quantitative indicator of helper activity. Suppression was tested by measuring the reduction of the antibody-forming cell response of in vivo TNP or sheep red blood cell (SRBC)-primed B cells restimulated in vitro with homologous antigen in the presence of allogeneically primed T cells.



Helper cell functions were clearly demonstrated when donors of T cells were primed to Ia antigens. Additional priming to K and/or D antigens yielded help and suppres- sion simultaneously, priming to K and/or D antigens alone and in some cases to H- minor antigens yielded no help but resulted in strong suppression. The effect of the helper cells was shown to be camer-specific occurring only in the presence of the relevant carrier, whereas the suppressor T cells were of the nonspecific type sup- pressing both anti-TNP and anti-SRBC responses in the absence of any restimulating carrier. The helper effect was seen not only when the hapten was conjugated to the appropriate (allogeneic) cells but also when it was presented on an unrelated carrier (syngeneic cells) provided that the specific nonhaptenated carrier was present. - Pos- sible recognition mechanisms are discussed.

In vitro evidence from anti-hapten antibody responses for T helper and suppressor cells directed against major histocompatibility antigens in the mouse. Participation of I region determinants in the induction of T helper cells.

Genetic control of T cell-macrophage interaction in helper cell induction in vitro.

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