Lysis of nonnucleated red blood cells by cytotoxic T lymphocytes

To establish the role of direct membrane damage in cytotoxic T lymphocytes (CTL)-mediated lysis we investigated whether CTL would be capable of lysing nonnucleated target cells. By antibody-mediated targeting we show that CTL are indeed capable of lysing sheep and human red blood cells.     

Characteristics of cytotoxic T lymphocytes eluted from allogeneic target cells

The increase in the specific cytotoxic effect of immune lymphocytes after their adsorption on the corresponding target cells (TC) and subsequent elution with pronase is due, not to increased cytotoxic activity of individual cells, but to a quantitative increase in the proportion of effector T cells in the population. The eluted and original immune lymphocytes are indistinguishable in the kinetics of their adsorption on TC. The fraction of eluted lymphocytes contains twice as many T cells and four to five times as many cells synthesizing DNA, on account of an increase in the percentage of medium-sized and large lymphocytes.Laboratory of Immunochemistry and Diagnosis of Tumors, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR G. V. Vygodchikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 1, pp. 50–53, January, 1977.     

Noncodominant expression of target antigens recognized by human cytotoxic T lymphocytes

Human CTL that recognize MHC-controlled target determinants distinct from HLA-A, B, C, and D/DR antigens were tested in a family with nine siblings. Segregation analysis of positive CML reactions showed strong lysis of target cells of three HLA-identical siblings, inheriting the b and c MHC haplotypes ($\text{blc}$), but not of the parents or siblings inheriting only one of these haplotypes. Some cytotoxicity was seen against parental target cells, although it seemed to be qualitatively distinct from that directed against the $\text{blc}$ targets. Studies using the competitive inhibition technique showed that cells inheriting only the $\text{b}$ or $\text{c}$ haplotypes were not effective in decreasing the specific cytotoxicity. Furthermore, simultaneous inclusion of inhibitor cells of both the $\text{b}$ and $\text{c}$ haplotypes did not hinder the specific cytotoxicity. Induction of CTL recognizing this new determinant did not occur when either antigens of the $\text{b}$ or $\text{c}$ haplotypes were used as MLC stimulating cells, or when antigens of both haplotypes were presented simultaneously, but on separate stimulating cells, in a three-cell MLC. These results suggest that the target determinant recognized by these unusual CTL is complex; it may be formed through interactions of two surface molecules or by genetic complementation yielding a hybrid antigen.     

The use of hybrid hybridomas to target human cytotoxic T lymphocytes

This study describes a general strategy to produce hybrid monoclonal antibodies that are capable of targeting human cytotoxic T lymphocytes (CTL) against any cell carrying the appropriate target antigen. This is done by fusing a HAT-sensitive, G418-resistant anti-T3 hybridoma with immune spleen cells (or with other hybridomas) that produce antibodies against the desired target antigen. In the hybrid hybridomas the reassortment of Ig heavy and light chains results in the production of bifunctional antibody molecules. Because of their double specificity, these antibodies are able to bridge human CTL to target cells and trigger cytotoxic function. We have isolated several stable hybrid hybridomas in which one specificity is against T3 and one either against HLA antigens (class II, DC-1, A3), human Ig (IgM, IgE, kappa), Toxoplasma gondii or an ovary carcinoma-associated antigen. In all of these cases we show that culture supernatants can efficiently and specifically target any CTL clone against any target cell that possesses the relevant surface antigen recognized by the antibody. Furthermore, the killing requires as little as 0.1 ng/ml of antibody, occurs at effector to target ratios comparable to those used in conventional cytotoxic assays and does not affect bystander cells. Nonspecific killing of Fc receptor-positive cells can be avoided using F(ab')2 fragments. As an example, we show that it is possible to change the major histocompatibility complex class and allospecificity of a CTL clone and target CTL against non-major histocompatibility complex antigens such as Ig, parasites and tumor-associated antigens.     

Lysis of CD4+ lymphocytes by non-HLA-restricted cytotoxic T lymphoe from HIV-infected individuals

Individuals infected with HIV have elevated numbers of total and activated CD8⁺ lymphocytes in peripheral blood. CD8⁺ lymphocytes from HIV-infected individuals have been shown to mediate non-human histocompatibility-linked antigen (HLA)-restricted suppression of viral replication, HLA-restricted killing of cells expressing HIV antigens, and killing of uninfected lymphocytes. We studied CD8⁽ T lymphocytes that lysed autologous CD4+ lymphocytes, hetcrologous CD4¹ lymphocytes from HIV-infected individuals and uninfected CD4⁺ lymphocytes. Killing in all cases required T cell receptor (TCR)-mediated recognition or triggering. However, these CD8 cytotoxic T lymphocytes (CTL) killed HLA class I mismatched CD4* lymphocytes and CD4⁴ lymphocytes treated with a MoAb against HLA-A, B and C antigens (PA2.6) which blocks HLA class I-restricted killing. HLA class H-negativc CD4* T lymphoma cells (CEM.NK^R) were also killed by anti-CD3 inhibited CTL. Stimulation of peripheral blood lymphocytes (PBL) from HIV-infected individuals, but not uninfected controls, with concanavalin A (Con A) and IL-2, induced non-HLA-restricted TCR aft¹, CD8^f CTL which lysed CD4⁺ lymphocytes. Activation ofCD4’lymphocytes increased their susceptibility to CD8^f CTL-mediated lysis. In HIV infection, a population of non-HLA-restricted CTL which lyse activated CD4⁺ lymphocytes is expanded. The expansion of CTL with unusual characteristics is interesting, because the stimulus for this expansion is unknown. CTL which recognize activated CD4⁺ cells could play a role in immune regulation and the pathogenesis of A IDS.     

Lysis of enucleated tumor cells with allogeneic and syngeneic cytotoxic thymus-derived lymphocytes.

These studies were initiated to understand the minimal requirements for lysis of target cells by cytotoxic thymus-derived lymphocytes (CTL). In particular, the significance of the nucleus to the susceptibility of the target cell to be lysed by CTL have been studied. P815 mastocytoma cells and EL4 lymphoma cells were enucleated by cen- trifugation through a discontinuous Ficoll gradient containing 10 μg/ml of cytochalasin B. The resultant vesicles were then analyzed for susceptibility to lysis by different CTL specific for the H-2K and H-2D gene products, minor histocompatibility antigens, trinitrophenyl groups, and Sendai virus-specific antigens. The results indicate that enucleated vesicles obtained from both EL 4 and P 815 cells are susceptible to lysis by these CTL. The lysis of EL 4-enucleated vesicles (unlike P 815-enucleated vesicles), however, required 2–4 times more effector cells than were required for equivalent lysis of intact EL 4 cells. This reduced susceptibility to lysis of enucleated EL 4 vesicles was not the result of an inability of the CTL to recognize and bind the vesicles. This conclusion was suggested by the fact that both enucleated EL 4 vesicles and intact cells inhibited equally well the lysis of ⁵¹Cr-labeled concanavalin A-stimulated BALB. B spleen cells by anti-H-2^b CTL. Although enucleated vesicles obtained from both P 815 and EL 4 cells were susceptible to lysis by CTL, they exhibited a reduced capacity to “cap” their serologically defined H-2 antigens. Lysis of these enucleated vesicles by CTL has the same requirements as lysis of intact cells. This conclusion was based upon the requirement for Ca⁺⁺ and Mg⁺⁺ and also because lysis of the modified vesicles (trinitrophenyl or viral antigens) occurred in an H-2-restricted fashion. Moreover, the enucleated vesicles were susceptible to antibody and complement-mediated lysis and also lectin-dependent cell-mediated cytotoxicity.     

Stimulus-dependent triggering or inhibition of cytotoxicity in human cytotoxic T lymphocytes by activators of protein kinase C.

The influence of activators of protein kinase C (PKC) on the delivery of the lethal hit by human cytotoxic T lymphocyte (CTL) clones was studied. In the absence of other signals, short-term incubation with two structurally unrelated activators of PKC, but not with a non-activating phorbolester, resulted in significant triggering of CTL, whereas overnight incubation with PKC activators led to reduction of cytotoxic activity. Furthermore, activation of PKC had an inhibitory effect on simultaneous triggering by anti-T3 monoclonal antibodies or by phytohaemagglutinin, but strongly enhanced the activating effect of anti-T11 antibodies. These results suggest that PKC is part of the cascade of signals transmitted within a CTL after triggering.     

Activation of human alloreactive cytotoxic precursor T lymphocytes

The requirements for allogeneic T-cell activation have been studied in experiments with T and/or B cells as stimulator. Although target determinants (TDs, defined by CTL effectors in CML) are present on B and T cells used as target cells, this study indicates that TDs are functionally different when expressed on B and T cells used as stimulator cells, as only B cells can activate CTL precursors. Further, the study confirms that inducing TDs and strong lymphocyte-activating determinants (LADs, defined by proliferation in MLC) can be distinct structures found on two different stimulator B cells. The study suggests that binding of cytotoxic precursor T cells to TDs per se does not allow any detectable activation or start of proliferation and differentiation but requires another function of the stimulator cells in the non-T-cell compartment. The nature of this function is unknown, but it is the background for the first signal received by the TD-specific clones of CTL precursors, resulting in the expression of growth receptors for T-cell growth factor or interleukin 2 which is the second signal necessary for clonal expansion and differentiation.     

Demonstration of direct xenorecognition of porcine cells by human cytotoxic T lymphocytes.

It is not known whether human cytotoxic T cells can recognize porcine major histocompatibility antigens directly, or whether recognition occurs by co-operation with syngeneic human antigen-presenting cells (APC). Limiting dilution assays were used to quantify human anti-pig precursor cytotoxic T-cell (CTLp) frequencies and to analyse the 'kinetics' of the interaction between human lymphoid cells and porcine splenic cells. Single-hit kinetics are demonstrative of direct recognition, as only one cell type, the CTLp, is diluted out, whereas multi-hit kinetics indicate that more than one cell is limiting and provide evidence for co-operative recognition of xenoantigens. Initial assays indicated that the frequency of CTLp reactive with alloantigens on human splenic targets (mean 1/1845; n = 3) was approximately sixfold greater than the frequency of CTLp reactive with porcine splenic cells (1/12,082; n = 3). However, not all of the assays performed using the xenogeneic combination produced single-hit kinetics. Subsequent assays were performed by mixing limiting numbers of human peripheral blood mononuclear cells (PBMC) or APC-depleted PBMC preparations with porcine splenocytes. There was a significant difference in the frequency of xenospecific CTLp between PBMC and APC-depleted preparations (P = 0.034). The overall frequency increased in the APC-depleted group. Variation between the seven human donors was also significant (P = 0.006). There was no significant difference in frequency between the two cell preparations after correction for the proportion of CD3+ cells (P = 0.13). There was, however, a significant departure from single-hit kinetics in the PBMC group (P = 0.004) which was not observed in the APC-depleted group (P = 0.052). It is concluded that human cytotoxic T cells can be activated by porcine xenoantigens directly. However, the direct recognition mechanism can be altered in the presence of human APC.     

Lysis of hybridoma cells bearing anti-clonotypic surface immunoglobulin by clonotype-expressing alloreactive cytotoxic T cells

A B cell hybridoma (Désiré-1) was derived which secreted and expressed at its cell surface immunoglobulin (Ig) specific for the antigen-specific T cell receptor (Ti) of an H-2Kb-specific alloreactive cytotoxic T lymphocyte (CTL) clone (KB5-C20). It was found that the CTL clone could lyse hybridoma Désiré-1, whereas it could not lyse hybridoma which expressed surface Ig (sIg) binding to other cell surface structures of clone KB5-C20 such as the H-2Kk molecule. Blocking of CTL-target cell interactions using monoclonal antibodies (mAb) indicated that the CTL-target cell interaction was inhibited with appropriate anti-H-2 mAb and by anti-Lyt-2 mAb when CTL-H-2Kb interaction was involved but not when CTL-sIg interaction was involved. The two types of interactions were inhibited by anti-LFA-1 mAb. The involvement of the CTL-Ti structure was necessary to obtain a lytic interaction between CTL and target cells, but a major histocompatibility complex product on the target cells did not need to be involved. Comparison of CTL-target cell inhibition with cold target cells or with anti-clonotypic mAb indicated that the Ti-sIg cellular interaction was of much higher apparent affinity than the Ti-H-2Kb cellular interaction. These results further suggest potential regulatory effects of CTL-B cell cross-idiotypic interactions.     

Lysis of allogeneic human lymphocytes by nonspecifically activated T-like cells

In the generation of cytotoxic effector cells specific for influenza A virus-infected lymphocytes, three donors have given an unusual pattern of lytic activity, killing HLA-mismatched target cells. This has been analyzed in detail for one donor and one of the other two shows similar results. Activation only requires culture in medium between 1 and 4 days and parallels development of cell line K562-directed natural killer cells. Target lymphocytes do not need to be virus-infected and appear to be normal lymphocytes. The effector cells carry the surface markers T3 and T8 defined by OKT3/anti-Leu4 and OKT8/anti-Leu2a monoclonal antibodies, respectively. Unlike HLA class 1-restricted or -directed cytotoxic T cells, neither anti-Leu2a/nor anti-Leu4 blocked killing in the absence of complement. MHM23, a monoclonal antibody specific for the human lymphocyte function antigen, blocked lysis. The results indicate that these effector cells are related to cytotoxic T lymphocytes, but can lyse allogeneic target cells through a different recognition process. There is some specificity because autologous cells were not killed.     

Efficient priming of antigen-specific cytotoxic T lymphocytes by human cord blood dendritic cells

Previous studies have suggested that defective immune responses in early life may be related to the immaturity of neonatal antigen-presenting cells. To test this hypothesis, we assessed the capacity of neonatal dendritic cells (DC) to prime and polarize in vitro human naive antigen-specific T cells. We report that mature cord blood DC efficiently prime an oligoclonal population of antigen-specific CD8 T cells, capable of cytolytic activity and IFN-gamma secretion. In contrast, cells primed by immature cord blood DC do not acquire cytolytic activity and secrete lower amounts of IFN-gamma. Upon priming by either immature or mature DC, neonatal T cells acquire markers of activation and differentiation towards effector-memory cells. Our results demonstrate that, if appropriately activated, neonatal DC can prime efficient cytotoxic T lymphocyte (CTL) responses. Furthermore, these findings have important implications for the development of vaccine strategies in early life and for the reconstitution of a functional CTL repertoire after bone marrow transplantation.     

Characterization of primary cell cultures as potential target cells for analysis of bovine cytotoxic T lymphocytes

In domestic animal species, assessment of cell-mediated immune responses to virus infection is hampered by the requirement for class I MHC compatibility between target and effector cells. Additional complicating factors can include an inability to infect target cells in vitro, or virus-induced lysis of infected target cells. One way to circumvent these problems is to use virus-mediated gene transfer to deliver individual viral genes to autologous primary target cells. Several primary bovine cell cultures were assessed as potential target cells for cytotoxic T lymphocyte (CTL) assays by measuring their levels of class I MHC expression and susceptibilities to retroviral gene delivery. High levels in both class I MHC expression and susceptibility to gene delivery were seen in adherent cell cultures isolated from peripheral blood (PBAC). PBAC, which arose as an outgrowth of adherent peripheral blood mononuclear cell cultures, had morphology, protein expression patterns, and response to functional assays characteristic of high endothelial cells. Expression of viral vector-delivered genes in PBAC cells was confirmed with a recombinant retrovirus carrying the green fluorescent protein (GFP) gene. The use of vector-mediated delivery of viral genes to bovine high endothelial cells is a promising method for assessment of cell-mediated immunity in cattle.     

Targeting cytotoxic T cells to antigen-specific B lymphocytes

A recent development in immunomanipulation involves the targeting of cytotoxic T lymphocytes (CTL) to cell-bound antigens using bispecific antibodies. These antibodies have been engineered such that specificity is directed against the T cell receptor (TCR) or TCR-associated T3 molecules, as well as against the chosen antigen. The present study was aimed to force interactions between T and B cells by bridging their receptors. F23.1 antibodies, which are specific for gene products of the TCR V beta 8 gene family, were conjugated with TNP (2,4,6-trinitrophenyl) and this construct was used to bridge the receptors of V beta 8+ T cells with the receptors of TNP-specific B cells. The bridging was demonstrated by direct killing of both a TNP-specific B hybridoma and of blast cells from mice transgenic for mu, kappa of the TNP-specific antibody Sp6. Further, F23.1-TNP constructs in conjunction with V beta 8+ CTL were shown to specifically deplete Ig-secreting B cells from Sp6 transgenic mice. Conjugates of TCR-specific antibodies and antigen are theoretically useful in vivo to either deplete or expand B cells of a given specificity by coupling their receptors to the TCR of CTL or T helper cells, respectively.     

Activation of regulatory T cells instigates functional down-regulation of cytotoxic T lymphocytes in human breast cancer

Regulatory T (Treg) cells are a subpopulation of T cells with the ability to control the responses of both CD4+ and CD8+ T cells. A case–control study was conducted in order to determine the functional attributes of Treg cells within the breast cancer milieu. Triple-color flow cytometry was utilized to study the phenotype expression of CD4+CD25+ Treg cells and CD8+ T cells in autologous tumor-infiltrating lymphocytes (TILs) and peripheral blood lymphocytes (PBLs) derived from 33 patients with stage I–III breast cancer. The prevalence of CD4+CD25+ T cells was significantly higher in TILs than in PBLs. The expressions of FOXP3 and GITR in CD4+CD25+ Treg cells were lower in PBLs than in TILs. Functional studies showed that both granzyme B and perforin were barely expressed in peripheral Treg cells but were highly expressed in Treg cells in the tumor microenvironment. On the contrary, down-regulation of both granzyme B and perforin expressed in the CD8+ cytotoxic T lymphocytes was significantly lower in TILs than in PBLs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules were synchronously up-regulated in CD8+ cytotoxic T cells. The in vitro kinetic study showed that adequate activation of TILs derived from breast cancer tissue could restore the appropriate antitumor immune response.     

Delayed kinetics of tumor necrosis factor-mediated bystander lysis by peptide-specific CD8+ cytotoxic T lymphocytes

Mouse CD8⁺ CTL reactive with an H-2D^b presented 9-mer peptide of the human papilloma virus 16 (HPV-16) protein E7^{49 – 57} (RAHYNIVTF) were generated from the splenocytes of wild-type C57BL/6 (B6), B6.perforin-deficient, B6. gld or B6.TNF-deficient mice. In short-term (4 h) assays, CTL from B6, B6.TNF-deficient and B6. gld mice displayed peptide-specific perforin- and/or Fas ligand (FasL)-mediated lysis of E7-transfected mouse RMA lymphoma cells (RMA-E7) or E7^{49 – 57} peptide-pulsed RMA-S cells, while CD8⁺ CTL from B6.perforin-deficient mice lysed via FasL exclusively. Rapid and efficient lysis of syngeneic bystander B6 spleen T cell blasts by B6, B6.TNF-deficient or B6.perforin-deficient antigen-activated CTL was mediated apparently exclusively by a FasL/Fas mechanism. By contrast CTL from B6. gld mice did not mediate rapid bystander lysis of B6 blasts. Rather B6. gld CTL delivered delayed bystander lysis after 36 – 48 h that was mediated by TNF. TNF-mediated bystander lysis of syngeneic blasts appeared to be independent of class I molecules and was mediated at least in part by soluble TNF. By contrast, there was no evidence that soluble FasL-mediated bystander lysis. For the first time, these data indicate that CD8⁺ CTL may use FasL or TNF in a kinetically and physically distinct fashion to mediate bystander killing.