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.     

Vbeta-dependent stimulation of bovine and human T cells by host-specific staphylococcal enterotoxins.

Staphylococcus aureus isolates from bovine and ovine species produce unique molecular variants of type C staphylococcal enterotoxin (SEC). The SEC animal variants have greater than 98% amino acid sequence identity with SEC1, a human-associated SEC. The two SEC animal variants have been designated SEC(bovine) and SEC(ovine) according to their corresponding host species. We showed previously that these toxins induce quantitatively different levels of T-cell stimulation in several animal species. The present study compared the abilities of these closely related host-specific SEC variants to stimulate Vbeta-bearing T cells from bovine and human donors. All three toxins expanded human T cells bearing T-cell receptor Vbeta elements (huVbeta) 3, 12, 13.2, 14, 15, 17, and 20. However, SEC1 resulted in greater expansion of hyVbeta12 than either SEC(bovine) or SEC(ovine). In addition, bovine T cells proliferate in a Vbeta-dependent manner in response to these superantigens (SAgs). All three toxins induced the proliferation of bovine T cells bearing the previously sequenced Vbeta element (boVbeta) from the bovine T-cell clone BTB13 (boVbetaBTB13). SEC1 and SEC(ovine) also were able to induce proliferation of bovine T cells bearing boVbetaBTB35, which SEC(bovine) failed to stimulate. The species-specific differences in T-cell proliferation exhibited by these closely related SEC variants may reflect the evolutionary adaptation of S. aureus, presumably to increase its host range by the manipulation of the immune system in a host-specific manner.     

四聚体技术检测人外周血巨细胞病毒抗原特异性OTL

为深入了解抗病毒免疫机制,探索人在健康状态及巨细胞病毒（CMV）急性感染时抗原特异性细胞毒T淋巴细胞（CTL）数量的动态变化.以CMV抗原肽、HLA-A＊0201重链和轻链制备CMV四聚体;分离并检测12名健康被检者外周血单个核细胞（PBMC）中CMV抗原特异CTL数量;PBMC体外培养建立CTL细胞系,于末次刺激的不同时相进行四聚体染色,流式细胞仪（FACS）检测抗原特异CTL数量.结果发现9名被检者PBMC中均检出抗原特异CTL;细胞系中CMV特异性CTL数量急剧增多,细胞系与PBMC相比,差异有显著的统计学意义（P＜0.001）;研究结果提示,9名被检者感染过CMV,血液中存在少量免疫记忆性T细胞,当再次遭遇同一抗原后发生克隆扩增.     

Antigen-independent acquisition of MHC class II molecules by human T lymphocytes

We report here that human T lymphocytes have the capacity of acquiring large amounts of MHC class II molecules from various types of antigen-presenting cells (APC) in an antigen-independent manner. The transfer of MHC class II molecules from APC to T cell required direct cell-to-cell contact and appeared to involve the interaction of numerous adhesion molecules between these cells. Depletion of cholesterol from the plasma membrane reduced the amount of MHC class II transferred onto the T cells. Most significantly, the newly acquired MHC class II molecules were capable of efficiently presenting antigen to T helper cells. These results suggest that T cells are able to interact with other T cells to regulate immune responses by presenting MHC peptide complexes that have been snatched away from nearby APC.     

Generation of cytotoxic T lymphocytes by MHC class I ligands fused to heat shock cognate protein 70

Immunization with gp96 and heat shock cognate protein 70 (hsc70) purified with in vivo bound naturally occurring peptides or bound to synthetic peptides by in vitro reconstitution has been shown to induce peptide-specific cytotoxic T lymphocytes (CTL). In addition, mycobacterial heat shock protein 70 covalently fused to ovalbumin (OVA)-derived fragments has been shown to generate MHC class I-restricted CTL responses. Here, we genetically fused five different CTL epitopes, including peptides derived from Plasmodium yoelii circumsporozoite protein, tumor antigens, HY antigen and OVA, to either the N- or C-terminus of murine hsc70 and expressed the resulting proteins in Escherichia coli. Vaccination with all five fusion proteins induced peptide-specific CTL, indicating that no cognate flanking regions of CTL epitopes are necessary for the immune response. The point of injection was crucial for CTL induction. CD4(+) T cells were not required for the priming of CD8(+) T cells and vaccination with bone marrow-derived dendritic cells pulsed with hsc70 fusion proteins also elicited CTL responses. Furthermore, by using deletion mutants of hsc70, we identified amino acid residues 280-385 of hsc70 as the region most critical for inducing the CTL response.     

Generation of MHC class I-restricted cytotoxic T lymphocytes by expression of a viral protein in muscle cells: antigen presentation by non-muscle cells.

Expression of reporter genes in muscle cells has been achieved by intramuscular (i.m.) injection of plasmid DNA expression vectors. We previously demonstrated that this technique is an effective means of immunization to elicit both antibodies capable of conferring homologous protection and cell-mediated immunity leading to cross-strain protection against influenza virus challenge in mice. These results suggested that expression of viral proteins by muscle cells can result in the generation of cellular immune responses, including cytotoxic T lymphocytes (CTL). However, because DNA has the potential to be internalized and expressed by other cell types, we sought to determine whether or not induction of CTL required synthesis of antigen in non-muscle cells and if not whether transfer of antigen to antigen-presenting cells from muscle cells may be involved. In the present study we demonstrate that transplantation of nucleoprotein (NP)-transfected myoblasts into syngeneic mice led to the generation of NP-specific antibodies and CTL and cross-strain protective immunity against a lethal challenge with influenza virus. Furthermore transplantation of NP-expressing myoblasts (H-2k) intraperitoneally into F1 hybrid mice (H-2d x H-2k) elicited NPCTL restricted by the MHC haplotype of both parental strains. These results indicate that NP expression by muscle cells after transplantation was sufficient to generate protective cell-mediated immunity and that induction of the CTL response was mediated at least in part, by transfer of antigen from the transplanted muscle cells to a host cell.     

Selective expression of class II MHC isotypes by MLC-activated human T lymphocytes

Although activated human T cells express class II MHC molecules, the biologic significance of this event is not understood. Using two-color flow cytometry, we have analyzed the expression of HLA-DR, -DQ, and -DP isotypes by T cells following activation by allogeneic lymphoblastoid B-cell lines. Within the CD3+ population, transient expression was observed at 1 day following initiation of culture, which preceded a dramatic and sustained increase around 6-7 days. DR expression was always highest, followed by DP and DQ with DP expression usually somewhat higher than DQ. At day 8, three populations were observed consisting of DR+DP+DQ+ (60%), DR+DP+ (69%), and DR+ (75%) T cells. Interestingly, DQ+ or DP+ but DR- T cells were not observed. These patterns of class II isotype expression were similar in CD2+, CD4+, and CD8+ subgroups and suggest that class II molecules are selectively expressed on T cells and may play a role in the regulation of T-cell responses to alloantigens.     

Effective and selective immune surveillance of the brain by MHC class I-restricted cytotoxic T lymphocytes

Cytotoxic CD8(+) T cells are abundantly present in human virus-induced or putative autoimmune diseases of the central nervous system (CNS). Their direct role in the induction of inflammatory brain damage is, however, poorly understood. We have studied CD8(+) T cell-mediated brain inflammation by transferring MHC class I-restricted hemagglutinin (HA)-reactive T cells from a TCR transgenic mouse line into transgenic mice, which express HA in astrocytes. We show that activated CD8(+) T cells alone can induce monophasic brain inflammation in immunocompetent recipient animals. Similar to previous studies, involving transfer of CD4(+) cells, brain inflammation peaks after 5-7 days and then declines. The pathology of brain inflammation, however, differs fundamentally from that induced by CD4(+) cells. The inflammatory reaction is dominated by T cells and activated microglia in the virtual absence of hematogenous macrophages. This is associated with exquisitely specific destruction of antigen-containing astrocytes in the absence of any bystander damage of myelin, oligodendrocytes or neurons. Furthermore, in contrast to CD4(+) T cells, some CD8(+) cells accumulate in the brain and activate microglia in recipient animals, even in the absence of the specific antigen in the CNS. These data indicate that CD8(+) T cells are prime candidates for immune surveillance of the CNS.     

Specific receptor binding of staphylococcal enterotoxins by murine splenic lymphocytes.

We describe a reliable assay to measure the specific binding of 125I-labeled staphylococcal enterotoxin A (SEA) by murine spleen cells. Toxin binding by lymphocytes was specific in that it was inhibited by unlabeled SEA but not by unrelated proteins. The biological activity of SEA (T-lymphocyte mitogenesis) correlated with toxin binding to splenic lymphocytes. In the presence of high concentrations of [125I]SEA, specific binding increased rapidly and approached saturation after 2 h. Toxin binding was sensitive to temperature and pH and was directly proportional to the concentration of spleen cells in the incubation mixture. We estimated that there was a single class of toxin-binding sites, which had an apparent equilibrium dissociation constant (Kd) of 8 x 10(-7) M and numbered 3,600 sites per cell. SEA and the antigenically distinct compounds staphylococcal enterotoxins B and E in excess competitively inhibited binding of [125I]SEA to mouse spleen cells. Our data suggest a common class of binding sites for the three staphylococcal enterotoxins.     

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.     

Demonstration of MHC class II-restricted cytotoxic T lymphocytes in mice against herpes simplex virus

In humans CD4+ major histocompatibility complex (MHC) class II-restricted T cells dominate cytotoxic T-lymphocyte (CTL) responses to herpes simplex virus (HSV), whereas in the mouse only CD8+ MHC class I-restricted CTL have been reported. In this study, we demonstrate that a minor fraction (around 30%) of the response in draining lymph nodes of acute local HSV infections is attributable to CD4+ CTL mice. Such CTL were identified on the basis of antiserum inhibition studies, negative depletion approaches, as well as their differing antigen processing requirements to CD8+ MHC class I-restricted CTL. A possible role for CD4+ CTL as immunoregulators in local infections is discussed briefly.     

Human major histocompatibility complex class II-negative colon carcinoma cells present staphylococcal superantigens to cytotoxic T lymphocytes: evidence for a novel enterotoxin receptor.

The staphylococcal enterotoxins (SE) bind to major histocompatibility complex (MHC) class II molecules on target cells and activate T cells expressing particular T cell receptor V beta sequences. In this report we demonstrate that SE bind to the MHC class II- SW620, Colo320DM and WiDr human colon carcinoma cell lines and direct cytotoxic T lymphocytes (CTL) to mediate strong target cell killing. Flow cytometry analysis, immunoprecipitation and Northern blotting experiments failed to demonstrate any surface expression of HLA-DR, HLA-DP and HLA-DQ isotypes on the SW620 colon carcinoma cell line, whereas abundant expression of these isotypes was seen on Raji cells, SEB and SEC1 were efficiently presented at picomolar concentration by the MHC class II- colon carcinoma cells and MHC class II+ Raji cells, whereas SEA and SED were preferentially presented on the MHC class II+ Raji cells. An anti-HLA-DR monoclonal antibody inhibited SEB-induced CTL targeting to Raji, but did not influence the killing of SW620 cells. Our data suggests the existence of functionally active SE-binding structures on human colon carcinoma cells which are distinct from the conventional MHC class II molecules. The possibility that these putative new SE receptors play a role in the enterotoxin action of SE must be considered.     

The lysis of cytotoxic T lymphocytes and their blasts by cytotoxic T lymphocytes.

After binding to their targets, cytotoxic T lymphocytes (CTL) deliver a lethal hit signal, ultimately leading to target cell lysis, and then can recycle to lyse additional targets, without themselves being destroyed. If non-specific secreted lytic mediators are involved in such lysis. CTL survival would not be expected unless the effectors are immune to CTL-mediated lysis. Therefore the lytic susceptibilities of alloimmune peritoneal exudate lymphocytes (PEL), containing up to 50% CTL, and of the cytolytic PEL blasts (PEB), obtained by culturing with interleukin-2 (IL-2), were examined. 51Cr-labelled BALB/c (H-2d) anti-EL4 (H-2b) (d alpha b) PEL were lysed 88%, 78%, and 48% by C3H/eb (H-2k) anti-P815 (H-2d) (k alpha d) PEL, C57BL/6 (H-2b) anti-P815 (b alpha d) PEL and b alpha d PEB, respectively. Similarly, b alpha d PEL were lysed 82% and 21% by d alpha b PEL and PEB, respectively. b alpha d PEB were lysed 82%, 28-39% and 39-51% by k alpha d PEL, b alpha d PEL and b alpha d PEB, respectively, b alpha d PEB were lysed 29-55% by d alpha b PEL. Furthermore, the CTL-containing populations were no less susceptible to lysis than normal lymphocytes. Since the majority (80-90%) of cells in these two types of CTL-containing populations can be directly and specifically lysed by appropriately immunized PEL CTL, we conclude that both the lytic granule and perforin lacking (PEL) and containing (PEB) CTL are not a priori immune to CTL-mediated lysis. These findings are in accord with theories proposing lysis to be induced by receptor-mediated contact between effector CTL and target cells, and challenge those suggesting the involvement of secreted lytic mediators.     

Enrichment by counterpart centrifugal elutriation of human lymphocytes cytotoxic to human tumour cells.

The enriched fractions of cytotoxic cells responsible for natural killer (NK) activity against both human sarcoma and neuroblastoma (LA-N2) cell lines were readily obtained by countercurrent centrifugal elutriation (CCE). The NK cells were obtained in the larger lymphocyte fractions (fraction 6 +/- 1), having a mean cell volume of 180 u3. The cytotoxic-enriched fraction contained 51% large lymphocytes having cytoplasmic granules. On the other hand, monocytes were purified to greater than 90% and isolated in another fraction (final fraction) and these cells had the lowest NK activity against both human tumour cell lines. However, compared with the lymphocyte fractions, small and large monocytes displayed greater antibody-dependent cellular cytotoxicity (ADCC) activity against human B erythrocytes. These results indicate that NK found to have activity against both tumour cells lines were larger lymphocytes, not small monocytes. Thus, countercurrent centrifugal elutriation (CCE) can provide a sensitive method to obtain enriched fractions of large lymphocytes contained tumoricidal activity against human sarcoma and neuroblastoma cell lines.     

Equine T lymphocytes express MHC class II antigens

Six anti-HLA class II mouse monoclonal antibodies (mAbs) were used in conjunction with a rat monoclonal antibody raised against horse lymphocytes to define class II major histocompatibility complex (MHC) molecules in the horse. By utilizing an ELISA assay and complement dependent lymphocytotoxicity assay, five out of the six anti-HLA class II antibodies and the rat anti-horse monoclonal antibody were found to react with a high percentage of peripheral blood lymphocytes. Flow cytometry demonstrated a variable antigen density on peripheral blood lymphocytes and clear evidence for expression by lymphocytes that carried no detectable surface immunoglobulin. None of the antibodies reacted with equine platelets. The mAbs immunoprecipitated an antigenic complex of Mr 29,000-33,000 from horse lymphocytes. It appears that the distribution of MHC class II antigens in the horse is different from that in man but is similar to that in the dog, since MHC class II antigens are expressed on resting peripheral blood lymphocytes which lack membrane-bound immunoglobulins. Correlations between the distribution of MHC class II antigens on lymphocyte subpopulations and their role in immunological phenomena may contribute to our understanding of the functional properties of these molecules.     

Transection of major histocompatibility complex class I-induced neurites by cytotoxic T lymphocytes.

Damage to neurites with transection of axons and spheroid formation is commonly noted in the central nervous system during viral and autoimmune diseases such as multiple sclerosis, but it remains open whether such changes are caused primarily by immune mechanisms or whether they are secondary to inflammation. The present experiments explored whether neurites can be directly attacked by cytotoxic T lymphocytes (CTLs). Cultured murine neurons induced by interferon-gamma and tetrodotoxin to express major histocompatibility complex class I were pulsed with a dominant peptide of the lymphochoriomeningitis virus envelope glycoprotein (GP33) and then confronted with GP33-specific CD8(+) CTLs. Within 3 hours the neurites developed cytoskeleton breaks with adjacent solitary neuritic spheroids, as documented by confocal examination of the cytoskeletal marker beta-tubulin III. At the same time cytoskeleton staining of the neuronal somata showed no damage. The CTLs selectively attacked neurites and induced segmental membrane disruption 5 to 30 minutes after the establishment of peptide-specific CTL-neurite contact, as directly visualized by live confocal imaging. Thus, major histocompatibility complex class I/peptide-restricted CD8(+) T lymphocytes can induce lesions to neurites, which might be responsible for axonal damage during neuroinflammatory diseases.     

Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes

To demonstrate that antibody-guided targeting of antigenic MHC class I-peptide tetramer on tumor cells can render them susceptible to lysis by relevant cytotoxic T lymphocytes (CTL), biotinylated HLA-A*0201/Flu matrix peptide complexes were tetramerized on streptavidin molecules previously coupled to Fab' fragments from monoclonal antibodies (mAb) specific for cell surface markers such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. Flow cytometry analysis showed that coating of the HLA-A2-peptide complexes on the four HLA-A2-negative human cancer lines tested (including a CEA-positive colon carcinoma, an ErbB-2(+) breast carcinoma and two CD20(+) B lymphomas) was entirely dependent upon the specificity of the conjugated antibody fragments. More importantly, HLA-A2-restricted Flu matrix peptide-specific CTL were then found to lyse specifically and efficiently the MHC-coated target cells. These results open the way to the development of new immunotherapy strategies based on antibody targeting of MHC class I-peptide complexes.     

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.