Ea

MOLT-4 or Jurkat

Human leukemia

Soft agar

HGPRT , Deficiency of hypoxanthine-guanine-phosphoribosyl transferase (HGPRT); TK , deficiency of thymidine kinase (TK); HAT. hypoxanthine, aminopterin and thymidine; EA, emetine and actinomycin D.

This line, derived from BW5147, lacks the genes of both T cell receptor a and p chains.

HGPRT , Deficiency of hypoxanthine-guanine-phosphoribosyl transferase (HGPRT); TK , deficiency of thymidine kinase (TK); HAT. hypoxanthine, aminopterin and thymidine; EA, emetine and actinomycin D.

This line, derived from BW5147, lacks the genes of both T cell receptor a and p chains.

Enrichment of specific T lymphocytes

Since there are very few cells specific for any one given antigen in a population of immune-lymphocytes, it is crucial to enrich those T cells prior to fusion. Antigen-specific helper T cells recognize a processed antigen in the context of class II major histocompatibility complex (MHC) molecules on anti-gen-presenting cells (APCs), and proliferate. Therefore, they are enriched by restimulating immune T lymphocytes with the antigen and appropriate APC. Cytotoxic T lymphocytes (CTLs) are enriched by restimulation with the specific target cells in the presence of interleukin 2. Although the nature of the cell type that actually fuses is not known, activation of the immune lymphocytes appears to select cells that are favored for the fusion. Thus, some of the procedures described above can serve to enrich cells that are not only specific for antigens but also amenable to fusion.

Cell fusion

Cells are fused by means of biological, chemical or physical methods. Among them, the chemical method, which uses polyethylene glycol (PEG) as a fusogen, is the most common.

PEG method All procedures must be done at 37°C, and the medium must contain no serum because PEG precipitates protein. First, the mixture of normal and tumor T cells is washed in serum-free medium and pelleted. Next, 50% PEG solution is added dropwise, and the mixture is gently stirred. This maximizes cell contact with PEG, resulting in many fusion products. Then, warm serum-free medium is added slowly to dilute the PEG gradually without lysing the cells. Finally, the cells are centrifuged, resuspended in medium with 10% fetal calf serum (FCS), and dispersed in 0.2 ml aliquots into 96-well plates. Successful fusion is influenced by lot-to-lot variation in PEG and FCS, requiring tedious batch testing.

Electrofusion Cell fusion can also be induced by a physical method, which uses an electric pulse. This method, compared with PEG, is highly efficient, but needs a special device. Cells are suspended in fusion buffer (0.2 mM Tris-HCl, pH 7.2, containing manni-tol, CaCl2 and MgCl2) and transferred into a fusion chamber. The cells are brought together in a low-amplitude alternating electric field (300 V cm 1 for 5 s), and then subjected twice to a high-amplitude short pulse (3000 V cm-1 for 10 ps), which causes an electrical breakdown of cell membranes. Cell fusion results when the membrane breakdown occurs in areas of cell-cell contact. The cells are cultured overnight in a 60 mm dish, and then dispersed in 0.2 ml aliquots into 96-well plates.

Selection of hybrid cells

Fusion is a random process. Fusion of a population of normal and tumor T cells results in a mixture of normal-normal, normal-tumor and tumor-tumor hybrid cells together with unfused cells. Since normal T cells have a limited growth potential, most normal-normal hybrids (and normal unfused cells) die before 2 weeks of culture. Therefore the selection methods have only to kill tumor-tumor hybrids (and unfused tumor cells) and allow normal-tumor hybrids to keep growing. The most common method is HAT selection.

HAT selection When the parental tumor cells lack an enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) or thymidine kinase (TK), selection is accomplished by culturing the fusion mixture in HAT medium. Aminopterin blocks the de novo synthesis of DNA. To survive in HAT medium, cells must make DNA via the so-called salvage pathway, in which hypoxanthine and thymidine are incorporated into DNA by means of HGPRT and TK, respectively. Therefore, tumor cells lacking either of these enzymes cannot grow in HAT medium, unless they fuse with normal cells provided with the required enzymes. Thus, the only cells able to grow in HAT medium are the normal-tumor hybrids. Preparation of mutants lacking these enzymes involves first mutagenizing T cell rumor lines and then exposing them to toxic base analogs that are incorporated into DNA via the salvage pathway. The toxic agents used are 8-azaguanine (or 6-thioguanine) and bromodeoxyuridine for the mutants lacking HGPRT and TK, respectively. The drug-marked variants listed in Table 1 have been selected and isolated in this way. These cell lines, however, can possibly regain enzyme activity, resulting in revertant cells that are no longer susceptible to HAT medium. Therefore it is recommended to maintain periodically the tumor cells in the presence of the appropriate toxic base analog.

Emetine and actinomycin D selection Tumor cells are treated with emetine and actinomycin D before fusion. Emetine inhibits protein synthesis in mammalian cells at the trans-location that involves movement of mRNA along a ribosome, and actinomycin D inhibits RNA synthesis irreversibly. The tumor cells treated with these drugs therefore die of ribo-somal depletion, unless they fuse with normal untreated cells and are provided with the devices required for replication. Thus, only normal-tumor hybrids are permitted to grow. Although this method is applicable to any tumor cell line, the optimal conditions of the treatment must be carefully determined for each line. This method seems to yield hybrid cells more frequently than HAT selection (10 4 versus 10"6).

Soft agar selection An alternative method is cultur-ing cells in soft agar after fusion. This strategy is based on the ability of T cell hybridomas to form colonies in the soft agar, whereas neither fusion partner forms colonies. By using this selection, T cell hybridomas have been successfully established from the fusion between antigen-specific normal human T cells and HAT-resistant human T lymphoblastoid cell lines such as MOLT-4 or Jurkat.

Screening of specific hybridomas

The method of selecting a few specific hybridomas out of a great number of hybrid cultures must be fast and simple enough to allow screening of many samples, and it must be unequivocal. In principle, the methods that serve to enrich specific T cells mentioned above can also be used for selecting the antigen-specific hybridomas. Thus, antigen-specific helper hybridomas are screened by examining their lymphokine-producing capability in an antigen-specific and MHC-restricted activation. Cytotoxic hybridomas are selected by a standard CTL assay using an appropriate target.

Cloning

Cloning, the initiation of a cell line from a single progenitor, can be achieved either in soft agar, by limiting dilution, or by using a cell sorter. Cloning by limiting dilution is performed by culturing hybridoma cells in 96-well plates at <1 cell per well together with feeder cells. Even on this condition, a significant proportion of wells receive two or more cells and hence cannot initiate a true clone; therefore cloning must be repeated to ensure the homogeneity of the hybrid lines. To verify the clonality it should be demonstrated that their T cell receptors are homogeneous at the level of proteins, mRNA or genes.

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