Interleukin-4-mediated downregulation of cytotoxic T lymphocyte activity is associated with reduced proliferation of antigen-specific CD8+ T cells

During virus infection, exogenous IL-4 strongly downregulates expression of antiviral cytokines and cytotoxic T lymphocyte (CTL) responses. In this study, we have employed a T cell receptor (TCR) transgenic system to more closely investigate the effect of IL-4 on CTL activity. This system involves mice transgenic for an H2-Kb restricted TCR recognising an ovalbumin (OVA)-specific peptide (OT-I mice), and recombinant vaccinia viruses expressing the gene for OVA (VV-OVA), or OVA together with IL-4 (VV-OVA-IL-4). Spleen cells from OT-I mice were adoptively transferred to irradiated C57BL/6 mice infected with VV-OVA or VV-OVA-IL-4. Five days following transfer, markedly stronger CTL activity was detected in VV-OVA- than in VV-OVA-IL-4-infected recipients. The reduction in CTL activity was associated with a reduction in the number of OVA-specific CD8+ T cells. Proliferation of cells from VV-OVA-IL-4-infected recipients was dramatically reduced, and this is a likely explanation for the IL-4-mediated reduction in the total number of OVA-specific cells and the reduced cytotoxic activity. On a per cell basis, the production of IFNgamma and cytotoxic activity of OVA-specific CD8+ cells was not influenced by IL-4. Taken together, our results indicate that the reduction in CTL activity by exogenous IL-4 is due to a reduced number of antigen-specific effectors, and does not involve a downregulation of effector function of these cells.     

Mechanisms of lysis by cytotoxic T lymphocyte clones. Lytic activity and gene expression in cloned antigen-specific CD4+ and CD8+ T lymphocytes.

Cloned murine Th having properties of either Th1 or Th2 cells as well as CD8+ CTL were tested for the capacity to lyse: 1) nucleated target cells bearing Ag or coated with anti-CD3 mAb, or 2) SRBC target cells coated with anti-CD3 mAb in a short term 51Cr-release assay. The lysis of SRBC occurs by a mechanism that does not involve nuclear degradation but presumably does involve membrane damage. Three patterns were observed: CTL and some Th2 cells lysed efficiently nucleated target cells and SRBC coated with anti-CD3 mAb. Th1 and some Th2 T cells lysed nucleated target cells but did not lyse efficiently the SRBC coated with anti-CD3 mAb. Finally, some Th2 cells failed to lyse efficiently either nucleated or SRBC targets. We also examined these clones for their expression of N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity, and for the expression of perforin or CTLA-1 (granzyme B) mRNA. Total N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity expressed by CTL and Th2 clones tended to be higher than that of Th1 cells. Perforin mRNA and CTLA-1 mRNA were readily detectable in CTL and some Th2 clones. Expression of perforin and CLTA-1 mRNA correlated well with the capacity of these clones to lyse SRBC coated with anti-CD3 mAb. Our results show that some but not all Th2 clones have lytic characteristics similar to those of CD8+ CTL. Two mechanisms appear to contribute to their lytic process, one mechanism of lysis involves membrane damage that correlates with the expression of perforin mRNA; a second mechanism involves the induction of DNA degradation in the target cells. In contrast, some CD4+ effector cells appear to lack the capacity to lyse efficiently via the mechanism involving membrane damage and may only have the lytic activity associated with the capacity to induce DNA degradation.     

Dominant induction of vaccine antigen-specific cytotoxic T lymphocyte responses after simian immunodeficiency virus challenge

Cytotoxic T lymphocyte (CTL) responses are crucial for the control of human and simian immunodeficiency virus (HIV and SIV) replication. A promising AIDS vaccine strategy is to induce CTL memory resulting in more effective CTL responses post-viral exposure compared to those in natural HIV infections. We previously developed a CTL-inducing vaccine and showed SIV control in some vaccinated rhesus macaques. These vaccine-based SIV controllers elicited vaccine antigen-specific CTL responses dominantly in the acute phase post-challenge. Here, we examined CTL responses post-challenge in those vaccinated animals that failed to control SIV replication. Unvaccinated rhesus macaques possessing the major histocompatibility complex class I haplotype 90-088-Ij dominantly elicited SIV non-Gag antigen-specific CTL responses after SIV challenge, while those induced with Gag-specific CTL memory by prophylactic vaccination failed to control SIV replication with dominant Gag-specific CTL responses in the acute phase, indicating dominant induction of vaccine antigen-specific CTL responses post-challenge even in non-controllers. Further analysis suggested that prophylactic vaccination results in dominant induction of vaccine antigen-specific CTL responses post-viral exposure but delays SIV non-vaccine antigen-specific CTL responses. These results imply a significant influence of prophylactic vaccination on CTL immunodominance post-viral exposure, providing insights into antigen design in development of a CTL-inducing AIDS vaccine.     

Listeriolysin O-liposome-mediated cytosolic delivery of macromolecule antigen in vivo: enhancement of antigen-specific cytotoxic T lymphocyte frequency,activity, and tumor protection

Cytotoxic T lymphocytes (CTLs) are primed by peptide antigens that are endogenously processed in the cytosol and presented in the context of major histocompatibility complex I (MHC I) molecules of antigen-presenting cells (APCs). Exogenous soluble protein antigens do not gain efficient entry into the cytosol of APCs, and therefore requires a special cytosolic delivery method. We have developed such a delivery strategy adopting the well-elucidated cytosol-invading listerial endosomal escape mechanism, and report here an efficient delivery of exogenous whole protein antigen into the cytosol in a mouse model. Co-encapsulation of listeriolysin O (LLO) inside liposome (LLO-liposome) was required for delivery of ovalbumin (OVA) into the cytosol of APCs in primary cultures. LLO-liposome-mediated OVA immunization in mice engendered significantly higher OVA-specific CTL activity and increased antigenic peptide-specific CTL precursor (CTLp) frequency as compared to non-LLO-liposome or soluble OVA immunizations. Interferon-gamma (IFN-gamma) production upon specific stimulation by MHC I-restricted peptide was also significantly stronger by the inclusion of LLO in the liposomes. Rerouting of antigen into the cytosol by LLO-liposomes, however, did not reduce the extent of anti-OVA antibody responses. Moreover, LLO-liposome-antigen vaccination was robust in conferring protection to mice from lethal challenges with antigen-expressing tumor cells. Our study demonstrates a novel delivery system for efficient introduction of exogenous protein into the cytosol in vivo, priming cellular immune responses, which are protective in nature.     

Induction of TNP-specific cytotoxic T lymphocyte memory in vivo in the absence of T helper cell activity

The question of whether TH cells are required for the priming of CTL precursors (CTLp) in vivo was studied by using Txbm mice (Thymectomized, irradiated, and stem cell-reconstituted mice). In these mice, TNP-specific CTL could be induced in vitro with TNP-coupled spleen cells only if the cultures were supplemented with an IL 2-containing supernatant (ConAsup). In contrast to normal mice, TNP-specific Lyt-2-TH cells could not be induced by skin painting with trinitrochlorobenzene (TNCB) (as tested by the ability to help CTL formation from thymocyte or normal spleen precursors). These data confirm previous findings that Txbm mice possess CTLp but that their TH compartment is deficient. TNCB skin painting had, however, a clear priming effect on the CTLp population: spleen cells from TNCB-painted mice could give rise to specific CTL with a lower amount of ConAsup than spleen cells from unprimed mice. In addition to this, priming changed the CTLp so that stimulation with lightly coupled cells (0.1 mM trinitrobenzene sulfonic acid [TNBS] instead of 10 mM TNBS) became effective. These changes took place without a significant increase in the frequency of TNP-specific CTL precursors. The data obtained are consistent with the concept that at least with some antigens, CTLp proliferation (clonal expansion), which is probably caused by activated TH cells, is not required for the induction of immunologic memory in vivo.     

Enhanced cytotoxic T cell activity in IL-4-deficient mice

CD8+ effectors are critical components of type 1 responses against viral infections as well as for antiviral vaccines. IL-4 plays a clear role as an inhibitor of CD4+ Th1 cells; however, its role in CD8+ T cell regulation appears to be more complex. Thus, IL-4 may augment CD8+ T cell growth, but also limit effector function. Moreover, abundant IL-4 is inhibitory for viral clearance, but the lack of IL-4 appears not to affect CTL-mediated immunity. This report investigates these disparate roles of IL-4 in CD8+ T lymphocyte regulation by comparing T cell responses specific for a single HIV-IIIIB gp120-derived epitope in BALB/c mice deficient in IL-4 to those in wild-type controls. CTL activation was monitored during the acute and memory phases following immunization with recombinant vaccinia virus. Similar frequencies of gp120-specific CTL precursors in splenocytes from both groups indicated that IL-4 plays no significant role in either CTL priming or the establishment of memory. However, cytolytic activity in cultures derived from IL-4-deficient mice developed earlier and was strikingly enhanced following in vitro restimulation, an effect exhibited by both primary and memory T cells. Secretion of IL-2 and IFN-gamma by CD8+ T cells from IL-4-deficient mice was also elevated, reflecting their enhanced activation. Thus, IL-4 appears to limit the activation, expansion, and differentiation of CD8+ T cells with high cytolytic potential.     

Measurement of thymus weight, lumbar node weight and progesterone levels in syngeneically pregnant, allogeneically pregnant, and pseudopregnant mice.

Female CBA mice were mated to fertile CBA males, to vasectomized CBA males, to fertile C57BL males or to vasectomized C57BL males. After allogeneic or syngeneic mating the extent of thymic involution on the 10th day of pregnancy and pseudopregnancy was similar. Lumbar lymph node weight was not affected by pseudopregnancy but increased similarly in allogeneic and syngeneic pregnancies. Serum progesterone levels on the 10th day of pseudopregnancy were similar to those of non-pregnant females, and significantly lower than those of pregnant females. On the 4th to 7th days progesterone levels in pseudopregnant animals were equal to those in pregnant animals. Progesterone levels and thymic involution were similar in syngeneically and allogeneically pregnant females. Progesterone levels were negatively correlated with thymus weight but reached significance only when the mating was allogeneic. It is suggested that there is an interaction between progesterone concentrations and the degree of thymic involution during pregnancy.     

Composition of splenic T lymphocytes in allophenic mice.

Guinea pig peritoneal macrophages were activated in vitro by culturing with MAF (macrophage activating factor)-containing fractions from stimulated lymphocytes. These macrophage preparations demonstrate a 60% increase in the production of prostaglandins of the E series (PGE) when compared with macrophages cultured with fractions from unstimulated lymphocytes. PGE accumulation in macrophage cultures is maximal after 24 hr with MAF; tumor cytotoxicity is also maximal at this time. The final PGE concentration in cultures of activated macrophages averaged 3 × 10^?8M.     

Soluble granzyme B and cytotoxic T lymphocyte activity in the pathogenesis of systemic lupus erythematosus

Activated cytotoxic T lymphocyte (CTL) mediated target cell death has been implicated in the development of systemic autoimmune disease like SLE. However, the role of soluble granzyme B and its relationship with CTL activity and disease activity is still unknown. In this study, we evaluated role of soluble granzyme B and cytotoxic T lymphocyte activity in SLE patients. The soluble granzyme B was measured in the serum by an enzyme-linked immunosorbent assay while cytotoxic T lymphocyte activity was measured by flow cytometry. The disease activity was determined by using SLE Disease Activity Index (SLEDAI) score. Cytotoxic T lymphocyte activity was increased and strongly associated with disease activity. The soluble granzyme B levels were higher in SLE patients and associated with various clinical features like reduced complement components; C3 & C4 and skin lesion. The soluble granzyme B levels were also sturdily related with severity of the disease. The findings of this study suggest that excessive secretion of soluble granzyme B and enhanced activity of cytotoxic T lymphocyte may play a vital role in the pathogenesis of SLE and organ damage. Also, evaluation of soluble granzyme B may be helpful in monitoring the clinical features associated with activated CTL in SLE.     

Cellular interactions in the cytotoxic T lymphocyte response to herpes simplex virus antigens: differential antigen activation requirements for the helper T lymphocyte and cytotoxic T lymphocyte precursors

The role and induction requirements of helper T lymphocyte responses to herpes simplex virus type 1 (HSV-1) was examined. Splenocytes from mice that had been primed in vivo with infectious HSV-1 can be restimulated in vitro with live or partially UV-inactivated HSV-1 to generate high levels of herpes virus-specific cytotoxic T lymphocyte (CTL) activity. By comparison, naive splenocytes or splenocytes taken from mice primed with heat-inactivated HSV-1 failed to generate CTL after in vitro viral stimulation. In addition, infectious HSV-primed splenocytes can be rendered unresponsive to secondary in vitro restimulation by pretreatment with anti-Lyt-1 antiserum plus complement. Spleen cells were taken from mice that had been primed and restimulated in vivo with infectious HSV-1. Two days after the second priming, splenocytes were prepared and irradiated. These cells were capable of assisting in the generation of CTL to varying degrees in all of the above unresponsive populations of cells. The irradiated cells did not produce detectable levels of CTL activity when cultured alone with antigen. Also, if the irradiated splenocytes were treated with anti-Lyt-1 plus complement before their addition to cultures, all restorative activity was ablated. In contrast, irradiated splenocytes from mice that had been primed and restimulated in vivo with either heat-inactivated or UV-inactivated HSV-1 were unable to provide help to naive or helper-depleted cultures. The failure to supply helper activity appears not to involve the preferential activation of suppressor cells, as evidenced by cell mixing experiments and the addition of concentrated, antigen-stimulated spleen cell supernatant fluids to secondary anti-HSV-1 splenocyte cultures. Proliferative assays using interleukin 2- (IL 2) dependent cell lines as a measure of relative helper activity indicated that the inactivated forms of HSV-1 were incapable of effectively enlisting helper activity. These experiments therefore suggest that the observed failure of heat-inactivated or UV-inactivated HSV-1 preparations to induce anti-HSV CTL responses reflects the inability of the HSV-1-specific subset of helper T lymphocytes to recognize these forms of the antigen.     

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells

Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-gamma secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.     

A molecular view of cytotoxic T lymphocyte induced killing.

Cytotoxic T lymphocytes (CTLs) search out and destroy pathogenic cells, such as those infected with viruses. The biochemistry laboratory at the University of Alberta (Edmonton, Alta.) studies the molecular mechanisms used by these effectors, and this review covers research on this topic primarily from this group. Research there began with the discovery of the granzyme genes and the realization that granzyme B (GrB) had an unusual substrate specificity. Cleavage at aspartate residues gave us the clue that caspases, key regulators of apoptosis, were important substrates. However, it is now clear that mitochondria are also important in controlling granzyme-induced apoptosis. This led to the discovery that the proapoptotic member of the Bcl2 family, Bid, is also activated by GrB. Cleaved Bid then translocates to the mitochondria, resulting in the release of antagonists of inhibitors of apoptosis proteins. The evolution of our understanding of the molecular basis of CTL killing is presented.     

Inhibition of human cytotoxic T lymphocyte activity in vitro by autologous peripheral blood granulocytes

Peripheral blood granulocytes from normal healthy donors were found to reproducibly inhibit the cytolytic effector function of specifically sensitized cytotoxic T lymphocytes in vitro when co-incubated with these effector cells and target cells in 8 hr 51Cr release assays. Inhibition required intact granulocytes, was proportional to the number of granulocytes present, and was independent of granulocyte adherence, phagocytic function, and viability. Equivalent numbers of enriched normal or leukemic peripheral T lymphocytes did not cause inhibition of 51Cr release, and preincubation of granulocytes with effectors did not significantly alter viability or cytotoxic function. Because granulocytes can inhibit natural killer cell function in vitro, these data indicate that granulocytes can regulate diverse antigen-specific and spontaneous cytotoxic functions in vitro, suggesting that circulating granulocytes may have the potential for in vivo regulation of these cytotoxic effectors.     

Specificity repertoire of splenic Lyt-2+/F23+ cytotoxic lymphocyte precursors from B6 mice

As revealed by flow cytometric analysis, about 30% of nylon wool nonadherent Lyt-2+ B6 spleen cells were F23+, i.e., were stained with the monoclonal antibody F23.1 directed against an allotypic T-cell receptor determinant. The specificity repertoire of splenic Lyt-2+/F23+ cytotoxic lymphocyte precursors (CLP) from B6 mice was investigated in a limiting dilution (LD) system designed to support clonal expansion in vitro of a representative fraction of this T-cell subset: in highly purified Lyt-2+ responder cells cocultured with mitomycin-treated F23 hybridoma cells in the presence of (recombinant) interleukin 2 under LD conditions, one out of three Lyt-2+/F23+ CLP gave rise to a functional cytotoxic T lymphocyte (CTL) clone. The split-well analysis of individual CTL populations demonstrated a clear-cut segregation of the lytic reactivities toward different allogeneic Con A blast targets. A large fraction of B6-derived CTL clones (3-10%) specifically lysed fully H-2 allogeneic (H-2k, H-2d), or H-2K mutant (bm1) targets. Self-reactive and allorestricted lytic patterns were not found.     

Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.     

Aloe QDM complex enhances specific cytotoxic T lymphocyte killing in vivo in metabolic disease mice

We developed spontaneous diet-induced metabolic disease in mice by feeding them a high-fat diet for 23 weeks and administered Aloe QDM complex for 16 weeks to examine its restorative effect on immune disorders and metabolic syndrome. A series of immune functional assays indicated Aloe QDM complex enhanced lymphocyte proliferation and antigen-specific immunity as determined by the restored functions of cytotoxic T lymphocytes (CTL) and IgG production. The elevated serum TNF-α level was also regulated by Aloe QDM complex treatment, which suggested its complex therapeutic potential. As for metabolic phenotypes, oral administration of Aloe QDM complex significantly improved diabetic symptoms, including high fasting glucose levels and glucose tolerance, and distinctly alleviated lipid accumulation in adipose and hepatic tissue. The simultaneous restoration of Aloe QDM complex on metabolic syndrome and host immune dysfunction, especially on the specific CTL killing was first elucidated in our study.     

The role of T cells in anti-herpes simplex virus immunity. I. Induction of antigen-specific cytotoxic T lymphocytes.

Mice infected with herpes simplex virus develop little or no cytotoxic T lymphocyte (CTL) response. However, in lymph nodes (LN's) draining a local site infected with HSV, antigen-specific CTL precursors are sensitized, which upon transfer to in vitro culture conditions develop within 72 hr into effective CTL. The in vivo blockade of CTL differentiation can be overcome by cyclophosphamide, suggesting that a cyclophosphamide-sensitive mechanism blocks the in vivo generation of HSV-immune CTL. The cytolytic activity of HSV-immune CTL is H-2 restricted and antigen specific. Thus CTL sensitized toward HSV type 1 discriminate between syngeneic targets infected with either the immunologic HSV variant type 1 or type 2 (and vice versa). H-2-matched target cells exposed for 30 min to infectious HSV are lysed within 60 min of contact with CTL. Since HSV replication is believed to require more than 4 to 5 hr, the data suggest that either the expression of HSV-dependent "early proteins" takes place within 30 to 90 min or cell membrane-integrated HSV virion represents the target antigen of CTL.     

Augmentation of in vivo cytotoxic T lymphocyte activity and reduction of tumor growth by large multivalent immunogen.

Class I alloantigen incorporated into cell-size supported membranes provides an effective stimulus for in vitro stimulation of CTL responses. When alloantigen-bearing cell-size (5 microns) microspheres, termed large multivalent immunogen (LMI), were administered in vivo, no primary cytotoxic response to the Ag could be detected. However, coadministration of LMI and allogeneic tumor stimulator cells resulted in substantial augmentation of the resulting CTL response, compared with that obtained from mice that received just stimulator cells. Responses were augmented only when the same alloantigen was present on the LMI and on the stimulator cells, and the effector cells remained specific for the cognate alloantigen-bearing targets. The physical form of the alloantigen was critical for augmentation; alloantigen in liposomes had no effect on response levels. Tumor cell Ag in the form of purified plasma membrane vesicles can also be incorporated onto the surface of cell-size microspheres. As with allogeneic responses, tumor Ag on LMI specifically augmented the in vivo CTL activity generated in response to irradiated tumor cells in syngeneic mice. Administration of Ag-bearing LMI to mice inoculated i.p. with live P815, EL4, or RDM4 tumor cells resulted in a significant reduction in growth of the tumors in their syngeneic hosts. Similarly, LMI treatment significantly reduced growth of P815 as a solid s.c. tumor. LMI-mediated growth reduction occurred only when plasma membrane Ag from the cognate tumor was used to prepare the LMI, and Ag in the form of free plasma membrane vesicles was not effective. Although Ag has been used to manipulate in vivo humoral and Th responses, this has proven to be much more difficult for CTL responses. The ability of Ag-bearing LMI to affect significantly the in vivo levels of cytolytic response and to reduce syngeneic tumor growth has potential for application to tumor immunotherapy and, possibly, treatment of other diseases in which CTL can provide a protective effect.