Killing of mouse blastocyst stage embryos by cytotoxic T lymphocytes directed to major histocompatibility complex antigens

A new assay, mixed embryo leukocyte interaction assay, in which the ability of cytotoxic T lymphocytes (CTL) to kill preimplantation mouse embryos could be investigated, is described. CTL were generated both in vitro and in vivo to the H-2b and H-2d haplotypes. The specificity of the CTL was verified by using EL-4 (H-2b) and P815 (H-2d) target cells in a 51Cr-release assay. The cytolytic effect of the CTL on mouse blastocysts was measured by assessing blastocoel retention and inhibition of [3H]thymidine incorporation by the embryos. It was shown that CTL kill blastocyst stage embryos from C57BL/6J (H-2b) and B10.D2 (H-2d) mice with the zona pellucida removed, but not with the zona pellucida intact. These results demonstrate that the H-2 antigens present on mouse blastocysts can be recognized by CTL. It is suggested that one biologic role for the zona pellucida is the prevention of cell-mediated destruction of preimplantation embryos in utero.     

The role of major histocompatibility complex expression on head and neck cancer cells in the induction of autologous cytotoxic T lymphocytes

Using head and neck tumors, we studied the role of HLA class I and DR antigens on tumor cells in cytotoxic T lymphocyte (CTL) induction. Expression of major histocompatibility complex (MHC) antigens was investigated by two-color flow cytometry analysis and for this study we used the tumor cells, over 50% of which expressed both HLA class I and DR antigens on their surface. In seven cases, tumor cells were divided into three groups according to the specificity of monoclonal antibodies (mAb) to MHC to study the role of MHC antigens on tumor cells in CTL induction: one was not blocked (MHC double-positive tumor), a second was blocked by anti-class I mAb (class-Ingative DR-positive tumor) and third was blocked by anti-DR mAb (class-I-positive DR-negative tumor). Subsequently, these tumors were used to stimulate an autologous mixed lymphocyte/tumor cell culture for 5 days (MLTC) followed by further cultivation with interleukin-2 for 12 days. The induced autologous tumor killer cells were most cytotoxic when non-treated tumors, which consist mainly of cells that are both HLA-class I and DR-positive, were used as stimulator cells. When the tumor cells blocked by anti-DR mAb were used as stimulators, autologous tumor killer activity was lower than that induced by tumor cells blocked by anti-class-I mAb. Moreover, cytolysis by autologous tumor killer cells induced by stimulation of non-treated tumor cells was blocked during the effector phase, 26.6%–42.3% and 32.7%–53.8% by anti-class-I and anti-DR mAb respectively, suggesting that majority of the autologous tumor killer cells are MHC-restricted CD8⁺ or CD4⁺ CTL. These results suggest that both MHC class I and class II antigens on head and neck tumor cells play a critical role in inducing CTL.     

Acidification of internalized class I major histocompatibility complex antigen by T lymphoblasts

It has previously been shown that activated murine T lymphocytes express intracellular vesicles containing the class I major histocompatibility complex (MHC) antigen H-2K. Evidence has also been provided that such vesicles may be part of a cellular pathway of spontaneous H-2K antigen internalization and recycling, which is specific to T-lymphoid cells. Dual fluorescence flow cytometry has now been used to establish that H-2K antigen is acidified upon internalization in concanavalin A-stimulated but not lipopolysaccharide-stimulated murine splenocytes, thus providing further support that in T lymphoblasts this class I MHC antigen may travel intracellular routes similar to those reported for other cell surface receptors.     

A third class I major histocompatibility complex antigen encoded by a gene in the D region of the H-2 d haplotype recognized by cytotoxic T lymphocytes

The D region of the H-2 ^d haplotype contains five class I genes: H-2D ^d , D2 ^d , D3 ^d , D4 ^d and H-2L ^d . Although previous studies have suggested the presence of D-end encoded class I molecules in addition to H-2D^d and H-2L^d, segregation of genes encoding such molecules has not been demonstrated. In this report we have used cãtotoxic T lymphocytes (CTL) to examine the D region of the H-2 ^d haplotype for the presence of additional class I molecules. CTL generated in (C3H × B6.K1)F₁ (K ^k D ^k , K ^b D ^b ) mice against the hybrid class I gene product Q10^d/L^d expressed on L cells cross-react with H-2L^d but not H-2D^d molecules, as determined by lysis of transfected cells expressing H-2L^d but not H-2D^d. Although H-2L^d-specific monoclonal antibodies (mAb) completely inhibit H-2L^d-specific CTL from killing B10.A(3R) (K ^b D ^d L ^d ) target cells, only partial inhibition of anti-Q10 CTL-mediated lysis was observed, suggesting the presence of an additional D-end molecule as a target for these latter CTL. To identify the region containing the gene encoding the Q10 cross-reactive molecule, we show that anti-Q10 CTL lyse target cells from a D-region recombinant strain B10.RQDB, which has H-2D ^d , D2 ^d , D3 ^d , D4 ^d , and H-2D ^b but not the H-2L ^d H-2 ^d , and H-2L ^d (including D2 ^d , D3 ^d , and D4 ^d , lacks this anti-Q10 CTL target molecule. Together, these data demonstrate that a class I gene mapping between H-2D ^d and H-2L ^d encodes an antigen recognozed by anti-Q10 CTL. A likely candidate for this gene is D2 ^d , D3 ^d or D4 ^d .     

Ethanol: an enhancer of major histocompatibility complex antigen expression

Ethanol enhances expression of cell surface class I major histocompatibility complex (MHC) antigens in a variety of cell lines; up to an eightfold increase is observed in an embryonic cell line. In ethanol-treated L cells, increased cell surface expression of MHC antigens occurs with a concomitant increase in steady-state RNA levels. This effect is promoter dependent and restricted, because not all gene products are elevated. The effective ethanol concentration (1%) is physiologically attainable, leading to speculations about the role of elevated MHC antigens in alcohol-related diseases.     

Recognition and lysis of target cells by cytotoxic T lymphocytes

A single cytotoxic T lymphocyte (CTL) is capable of performing the two most fundamental functions of an immune response, recognition and elimination of foreign antigens. It is now clear that in a CTL these two functions are linked via the antigen-specific, heterodimeric receptor. We review here some experimental approaches that justify this conclusion and provide the means for further examination of the mechanisms by which CTLs lyse their target cells. When antireceptor antibodies serving as antigen substitutes are attached to various cells, they trigger the lytic activity of particular CTLs, which results in lysis of the antibody-modified cell. In the process, a novel serine esterase, which is located within cytolytic granules of the CTL, is released. The presence of this enzyme and a complement-like protein, perforin, in granules of a CTL has led to the suggestion that CTLs and complement have similar cytolytic mechanisms. However, the resistance of some CTLs to lysis by other CTLs, but not to lysis by antibody-activated complement, suggests fundamental differences between cytolytic mechanisms of CTLs and complement.     

Aberrant expression of the hematopoietic-restricted minor histocompatibility antigen LRH-1 on solid tumors results in efficient cytotoxic T cell-mediated lysis

CD8(+) T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate effective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a significant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be effectively targeted by LRH-1-specific cytotoxic T lymphocytes under inflammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-specific immunotherapy after SCT.     

Genetically encoded and post-translationally modified forms of a major histocompatibility complex class I-restricted antigen bearing a glycosylation motif are independently processed and co-presented to cytotoxic T lymphocytes

The mechanisms by which antigenic peptides bearing a glycosylation site may be processed from viral glycoproteins, post-translationally modified, and presented by major histocompatibility complex class I molecules remain poorly understood. With the aim of exploring these processes, we have dissected the structural and functional properties of the MHC-restricted peptide GP92-101 (CSANNSHHYI) generated from the lymphocytic choriomeningitis virus (LCMV) GP1 glycoprotein. LCMV GP92-101 bears a glycosylation motif -NXS- that is naturally N-glycosylated in the mature viral glycoprotein, displays high affinity for H-2D(b) molecules, and elicits a CD8(+) cytotoxic T lymphocyte response. By analyzing the functional properties of natural and synthetic peptides and by identifying the viral sequence(s) from the pool of naturally occurring peptides, we demonstrated that multiple forms of LCMV GP92-101 were generated from the viral glycoprotein and co-presented at the surface of LCMV-infected cells. They corresponded to non-glycosylated and post-translationally modified sequences (conversion of Asn-95 to Asp or alteration of Cys-92). The glycosylated form, despite its potential immunogenicity, was not detected. These data illustrate that distinct, non-mutually exclusive antigen presentation pathways may occur simultaneously within a cell to generate structurally and functionally different peptides from a single genetically encoded sequence, thus contributing to increasing the diversity of the T cell repertoire.     

Target cell lysis by cytotoxic T lymphocytes redirected by antibody-coated polystyrene beads

Cytotoxic T lymphocytes were found to mediate rapid lysis of target cells not normally recognized in the presence of small polystyrene beads coated with a combination of anti-T3 and antitarget cell antibodies. Lysis was not seen with beads bearing one of these antibodies alone, nor with a mixture of two types of beads each coated with a single antibody. The effector cells mediating this lysis include long term allospecific human CTL, and both human and mouse CTL clones recognizing mouse class I MHC Kb Ag. TNP-modified mouse tumor cells, a human lymphoblastoid line, and human red cells were found to be good targets for this cytotoxicity. Polystyrene beads with diameters of 3 to 15 mu caused target lysis, with a dose-response curve which typically went through a maximum and declined at high bead numbers. Maximal bead-redirected lysis by CTL was less efficient than that mediated by soluble antibody heteroconjugates of the same two antibodies. Bead-redirected target lysis was calcium dependent. These results are interpreted as a form of bystander lysis induced by the beads, since the target cell membrane is not directly crosslinked to the region of CTL activation. These observations thus favor a mechanism of lysis involving the polarized secretion of a locally acting lytic agent by CTL.     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

Developmental regulation of class I major histocompatibility complex antigen expression by equine trophoblastic cells

Between days 36-38 of pregnancy equine trophoblastic cells of the chorionic girdle migrate and form endometrial cups. Just prior to invasion, the chorionic girdle cells express high levels of polymorphic, paternally inherited, major histocompatibility complex (MHC) class I antigens. Their descendents, the mature, invasive trophoblast cells of the endometrial cups, however, express low or undetectable levels of MHC class I antigens by day 44 of pregnancy. Experiments with MHC compatible pregnancies, the study of residual chorionic girdle cells that had failed to invade the endometrium and remained on the surface of a conceptus, and the study of chorionic girdle cells recovered on days 34-36 of pregnancy and then maintained in vitro for up to 24 days strongly suggest that the reduction of MHC class I antigen expression by mature invasive trophoblast cells of the endometrial cups is developmentally regulated. This phenomenon does not appear to be induced by a maternal antibody response or by other uterine factors acting after the chorionic girdle trophoblast cells invade the endometrium.     

Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct viral epitopes restricted by a single major histocompatibility complex molecule.

Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.     

Two major histocompatibility complex class I-restricted epitopes of the Borna disease virus p10 protein identified by cytotoxic T lymphocytes induced by DNA-based immunization

Borna disease virus (BDV) infection of Lewis rats is the most studied animal model of Borna disease, an often fatal encephalomyelitis. In this experimental model, BDV-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a prominent role in the immunopathogenesis of infection by the noncytolytic, persistent BDV. Of the six open reading frames of BDV, CTLs to BDV X (p10) and the L-polymerase have never been studied. In this study, we used plasmid immunization to investigate the CTL response to BDV X and N. Plasmid-based immunization was a potent CTL inducer in Lewis rats. Anti-X CTLs were primed by a single injection of the p10 cDNA. Two codominant p10 epitopes, M(1)SSDLRLTLL(10) and T(8)LLELVRRL(16), associated with the RT1.A(l) major histocompatibility complex class I molecules of the Lewis rats, were identified. In addition, immunization with a BDV p40-expressing plasmid confirmed the previously reported RT1.A(l)-restricted A(230)SYAQMTTY(238) peptide as the CTL target for BDV N. In contrast to the CTL responses, plasmid vaccination was a poor inducer of an antibody response to p10. Three injections of a recombinant eukaryotic expression plasmid of BDV p10 were needed to generate a weak anti-p10 immunoglobulin M response. However, the antibody response could be optimized by a protein boost after priming with cDNA.     

Sequential proteolytic processing of the capsular Caf1 antigen of Yersinia pestis for major histocompatibility complex class II-restricted presentation to T lymphocytes

We studied the mechanisms of antigen presentation of CD4 T cell epitopes of the capsular Caf1 antigen of Yersinia pestis using murine bone marrow macrophages as antigen presenting cells and T cell hybridomas specific for major histocompatibility complex (MHC) class II-restricted epitopes distributed throughout the Caf1 sequence. The data revealed diversity in the pathways used and the degrees of antigen processing required depending on the structural context of epitopes within the Caf1 molecule. Two epitopes in the carboxyl-terminal globular domain were presented by newly synthesized MHC class II after low pH-dependent lysosomal processing, whereas an epitope located in a flexible amino-terminal strand was presented by mature MHC class II independent of low pH and with no detectable requirement for proteolytic processing. A fourth epitope located between the two regions of Caf1 showed intermediate behavior. The data are consistent with progressive unfolding and cleavage of rCaf1 from the amino terminus as it traverses the endosomal pathway, the availability of epitopes determining which pool of MHC class II is preferentially loaded. The Caf1 capsular protein is a component of second generation plague vaccines and an understanding of the mechanisms and pathways of MHC class II-restricted presentation of multiple epitopes from this candidate vaccine antigen should inform the choice of delivery systems and adjuvants that target vaccines successfully to appropriate intracellular locations to induce protective immune responses against as wide a T cell repertoire as possible.     

Equilibrium binding of cytotoxic T lymphocytes to class I antigen

Cloned cytotoxic T lymphocytes specifically bind to purified alloantigen that has been immobilized on a surface. When the time course was examined, it was found that binding reached a plateau level within about 1 h at 37 degrees C, at which time about 30% of the CTL were tightly adhered to the surface. Analysis of the properties of binding demonstrated that this does not simply result because only a fraction of the cells in the clonal population are capable of binding. Instead, the binding is shown to result from an equilibrium involving tightly bound and unbound (or weakly bound) cells. Thus, the cells cycle between a tightly bound and unbound state, despite continuous contact with the Ag-bearing surface. The results suggest that dissociation of the bound cells may be an actively signaled event. A model that could account for these results based on activated CD8 binding is discussed.