Gerald N. Callahan

Flow cytometric analysis of transport activity in lymphocytes electroporated with a fluorescent organic anion dye

Organic anion transport in polarized epithelia and macrophages has previously been studied by monitoring the efflux of fluorescent organic anion dyes from cells. We adapted this strategy to the study organic anion transport in lymphocytes. Cloned lymphoma cells and normal and activated human T cells were loaded with a membrane-impermeant, organic anion dye (Lucifer Yellow) by electroporation. Dye efflux in lymphocytes was rapid, energy-dependent, and inhibitable by organic anion transporter inhibitors. Dye efflux could not be attributed to the effects of electroporation. In addition, electroporated, dye-loaded T helper cells retained the ability to properly respond to specific antigen. Thus, dye loss occurred in viable, functionally competent cells. These experiments demonstrate that electroporation is an effective means of loading cells with Lucifer Yellow, and that lymphocytes possess organic anion transporters that are functionally similar to those previously described for secretory epithelia and macrophages.

Biochemical comparison of h-2k antigen isolated from c3hfb/hen and c3h/hen mice.

The H-2K glycoproteins were isolated from spleen cells of C3H/HeN and C3HfB/HeN mice and compared by tryptic peptide mapping techniques. The two antigens were found to be very similar in that more than 90 percent of detectable peptides appeared identical. However, two lysine-containing peptide present in tryptic digests of H-2K antigens isolated from C3H mice were absent from tryptic digests of H-2K antigens isolated from C3Hf mice. This was probably not the result of altered glycosylation since neuramindase digestion demonstrated that the disparate peptides were not glycopeptides but most probably resulted from substitution of one or two amino acids in the H-2K molecule of C3HfB/HeN mice. These differences were small but significant and demonstrated that H-2Kk (C3H) and H-2Kkv1 (C3Hf) antigens are structurally distinct. This is compatible with the observed reciprocal skin-graft rejection, MLR, and generation of cytotoxic T lymphocytes between the two strains. The significance of this finding in conjunction with what is known about properties of 1-ethyl-1-nitrosourea-induced tumors of C3Hf mice is discussed.

Selective impairment of humoral immunity in feline leukemia virus-induced immunodeficiency

We used a panel of in vitro assays to investigate the nature of immune dysfunction in cats infected with FeLV-FAIDS, a naturally occurring, molecularly cloned feline leukemia virus (FeLV) isolate which induces a fatal immunodeficiency syndrome in infected cats. During the asymptomatic period preceding immunodeficiency disease, we were unable to detect any deficits in concanavalin A-induced blastogenesis, xenogeneic mixed-lymphocyte reaction assays, stimulation of lymphocytes by soluble protein antigen, and cytotoxic T lymphocyte assays. However, during this period humoral immune responses in the FeLV-FAIDS-infected cats were dramatically impaired. As early as 9 weeks after virus inoculation, the ability to mount either an IgM or IgG response to soluble protein antigens was lost. Neither B cell function, as assessed by lipopolysaccharide-induced blastogenesis or circulating B cell numbers, as assessed by immunofluorescence, differed between infected and control cats. These results suggest that FeLV-FAIDS infection may impair a subpopulation of T helper cells, that provides help for the production of antibody. Consistent with earlier observations of cats naturally infected with FeLV, our results indicate that early impairment of humoral immunity is an important component of the immunodeficiency syndrome induced by FeLV in cats.

The nucleotide sequence of the H-2K gene of C3Hf/HeN mice.

The C3Hf/HeN strain of mice was derived from C3H/He mice in 1945 by cesarean delivery and foster-nursing of the young on C57BL/6 female mice (reviewed by Martin et al. 1977). Because of the nature of their origin, C3Hf/HeN mice were originally assumed to be fully syngeneic with C3H/HeN mice. However, Imamura and coworkers (1979) later found that a full range of immunological responses including mixed lymphocyte responses, cytolytic T cells, and skin graft rejection could be reciprocally generated between C3Hf/HeN and C3H/HeN mice. Gipson and colleagues (1978) further found that the immunological disparity between C3Hf/HeN and C3H/HeN mice was due to genetic differences at or near the K locus of the major histocompatibility complex (MHC). Based on this observation, these investigators proposed that a mutation had arisen in the K locus gene of C3Hf/HeN mice, and that this mutation was the basis of the immunological reactivity between C3Hf/HeN and C3H/HeN mice. To investigate the relationship between C3Hf/HeN and C3H/HeN mice, as well as to characterize the rates of divergence of H-2K genes, we have isolated and sequenced a genomic clone containing the H 2 K gene of C3Hf/HeN mice. The C3Hf/HeN mice used in this study were obtained directly from Harlan Sprague Dawley via The Animal Production Unit of the National Cancer Institute (Frederick, Maryland). These mice were derived from the only colony of C3Hf/He mice ever maintained by the National Cancer Institute and, thus, the only colony of C3Hf/HeN mice (personal communication, Clarence Reeder, NCI). Genomic DNA was isolated from C3Hf/HeN spleen cells, cloned into the cosmid vector pTCF (Grosveld et al. 1982), and packaged into lambda phage, and these

HUMAN AND MURINE TUMOURS: CHANGES IN CELL SURFACE STRUCTURES CODED BY THE MAJOR HISTOCOMPATIBILITY COMPLEX REGION

Serological and structural changes of surface markers involved in immune reactions may occur in human and murine tumour systems. Thus nine out of twenty‐one human tumour cell lines and SV40‐transformed fibroblasts differed from autologous lymphoblastoid cells or fibroblasts in their reactivity with HLA allonatisera. H‐2 antigens isolated from the murine tumour cells 6C3HED and TP 1422, undergo structural changes. An alien HLA‐B7 was detected in sera from two melanoma patients. The serologic activity on H‐2 antigens was significantly increased in the serum and ascites fluid of tumour bearing mice. Additionally, human SV40‐transformed fibroblatsts acquire receptors for monkey red blood cells and the murine lymphosarcoma cells 6C3HED express receptors for sheep red blood cells.

Products of the Major Histocompatibility Complex on Tumor Cells

The major histocompatibility complex (MHC) was originally defined as that region of murine genome that contained the genes that determined the fate of allografts (Gorer, 1938 and Gorer et al., 1948). A similar complex has since been identified in all mammalian species investigated. In addition, it is now known that the MHC contains a variety of genes whose products are intimately involved in the development and culmination of most, if not all, immune responses.

Cross-reactivity between human and murine lymphocyte antigens

The anti-H-2 alloantiserum D-32 [(BlO.A(2R) × C3H.SW) anti-C3H] is cytolytic to human lymphocytes. Fab2 blocking assays, indirect immunoprecipitation and sequential immunoprecipitation experiments showed that the anti-H-2 alloantiserum D-32 recognizes antigenic determinants which are expressed on the heavy chain of subpopulations of HLA-A, B antigens. These determinants are different from those defining the serological polymorphism of the HLA-A, B, C system, are the same as or spatially close to those recognized by the anti-HLA-A, B monoclonal antibody Q6/64 and are expressed on rabbit, rat or guinea pig lymphocytes.

Protective antitumor immunity induced by immunization with MHC class II gene-transfected tumor cells is unrelated to MHC class II expression.

A/JCr mice reject Sa1N fibrosarcoma cells genetically engineered to express major histocompatibility complex (MHC) class II molecules and are highly resistant to subsequent challenge with unmodified Sa1N cells. In this report we examine the mechanism by which this protective antitumor immunity is induced. We found that MHC class II antigen-positive tumor cells were no more effective than irradiated, MHC class II antigen-negative cells at inducing secondary protective immunity. Additionally, therapeutic immunization with MHC class II antigen-positive tumor cells had no effect on the growth of admixed Sa1N cells or preexisting Sa1N tumors. Based on these observations, we conclude that the MHC class II antigen-induced immune response is not related to Sa1N-specific antitumor immunity.

Cells of chemically induced tumors differentially express major histocompatibility complex class I antigens.

Several recent studies have indicated that alterations in expression of major histocompatibility complex (MHC) antigens by tumor cells affects the ability of the host to mount an effective antitumor immune response. To investigate whether newly induced tumors frequently exhibit altered MHC antigen expression, we used methylcholanthrene to induce a series of tumors and elevated MHC antigen expression by these cells. The tumors exhibited a variety of MHC phenotypes in vitro. The nature of their phenotypes suggested that these cells were, in fact, capable of independent and abnormal regulation of MHC class 1 genes. However, when maintained in vivo, these same tumor cells expressed measurable levels of all of the appropriate MHC class I antigens. Thus, newly induced tumor cells are capable of abnormal MHC class I antigen expression. However, there was no obvious correlation between the phenotypes exhibited by these tumor cells in vitro and either their phenotype or their tumorigenic potential in vivo.

Cross-reacting xenoantibodies to the heavy chain of H-2 and HLA-A,B antigens: serologic and immunochemical characterization.

H‐2 cross‐reactive antibodies present in HLA xenoantisera were purified by absorption on and elution from murine cells. Antibodies isolated from one of these antisera. 78‐E48, were found to mediate complement‐dependent lysis of both human and murine lymphoid cells but not of Daudi cells or of human lymphoid cells coaled with Fab2 fragments from a cow anti‐human β2‐microglobulin (β2m) antiserum. In indirect immunoprecipitation analyses 78‐E48 reacted only with proteins of approximately 45,000 and 12,000 MW present in NP‐40 extracts of both human and murine lymphoid cells. Sequential precipitation experiments with rabbit anti‐human β2m and allo‐anti‐H‐2Kk, Iak sera established that these proteins were in fact H‐2 and HLA‐A, B antigens. It was also found that 78‐E48 reacted only with the heavy chain of HLA‐A, B and H‐2 antigens, since this eluate was unreactive with β2m in a radioimmunoassay, and its capacity to immunoprecipitate the 45,000 and 12,000 MW proteins from human cell extracts was unaffected by prior reaction with purified human β2m. These data show for the first time that H‐2 and HLA‐A, B antigens share properties that probably depend upon their tertiary structure.