Interleukin 3 augments the murine primary cytolytic T lymphocyte response to allogeneic tumor cells

The primary anti-H-2k allospecific cytolytic T lymphocyte (CTL) response by BALB/c (H-2d) spleen cells in vitro to x-irradiated RDM4 (H-2k) tumor cells is weak. This response has been shown to be augmented by CTL helper factor (CHF), a factor present in supernatants of spleen cells cultured with Sendai virus (SC-CM). Conditioned medium from WEHI-3 cells (WEHI-CM) also contains activity that augments the BALB/c anti- RDM4 CTL response. Attempts to separate the CHF activity from interleukin 3 (IL 3), also present in WEHI-CM, were unsuccessful. Purified IL 3 was then tested, and was found to increase the BALB/c anti- RDM4 CTL response by five- to 10-fold. IL 3 is apparently the only material in WEHI-CM that is active in this assay. The response is apparently a classical CTL response because: 1) the effector cells are sensitive to monoclonal anti-Thy-1.2 antibody plus C; 2) the response is dependent on antigen stimulation, and it peaks on day 5 or 6 of culture; and 3) the effector cells are specific for H-2k targets. IL 3 must be added very early during the in vitro culture period for maximal augmentation of the response, consistent with possible action of IL 3 as a differentiation factor.     

Mapping the fine specificity of a cytolytic T cell response to HIV-1 nef protein.

Epitope mapping of a MHC class I-restricted cytotoxic T cell response to nef, a regulatory protein of HIV, was performed with fresh PBMC from HIV-seropositive donors and target cells pulsed with a panel of overlapping peptides of the nef protein. These nef-specific CTL recognized a synthetic peptide of 10 residues derived from a nonamphipathic, highly conserved region of the nef protein in association with the HLA A3.1 molecule. Using human cell transfectants expressing mutations of the A3 molecule, we demonstrated that the amino acid at position 152 of the A3.1 molecule appears to be critical for detection of this response. Thus, rapid analysis of the epitopes of HIV proteins stimulating CTL responses can be achieved using a combination of fresh donor PBMC and target cells pulsed with synthesized peptides.     

The cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells. I. Evidence for antigen-specific helper T cells.

Mice were primed subcutaneously with trinitrophenyl (TNP)-modified syngeneic spleen cells. Seven days later, spleen cells from these in vivo primed mice, or spleen cells from naive mice, were co-cultured with TNP-modified syngeneic cells. Spleen cells from the in vivo primed mice demonstrated augmented cytolytic T lymphocyte (CTL) activity. The spleens of these in vivo primed mice contained a population of radioresistant, antigen-specific, helper T cells. Specifically, spleen cells from these mice, after x-irradiation, were able to augment the in vitro CTL response of normal spleen cells to TNP-modified syngeneic cells.     

Kinetics of cytolytic T lymphocyte binding to target cells in suspension

Cytolytic T lymphocytes (CTL) were able to specifically bind and lyse allogeneic P815 tumor cells and LPS blast cells in suspension. An assay was developed to measure the rate of target cell binding in suspension independent of the rate of lysis. Target cell binding was found to plateau within 3 hr in suspension. The presence of free, functional CTL and targets at these plateaus was demonstrated, indicating that target cell binding was an equilibrium process. Scatchard plots were used to derive values for Kd (apparent affinity) and bmax (maximum binding). Target cell binding in suspension could not be blocked by purified plasma membranes. Target cell binding was compared for CTL generated by secondary in vitro stimulation with intact cells or with purified membranes. These 2 CTL populations yielded distinct values for Kd and bmax. Implications of this kinetic difference for CTL recognition of purified plasma membranes are discussed.     

The cytolytic T lymphocyte response to trinitrophenyl-modified syngeneic cells. II. Evidence for antigen-specific suppressor T cells.

The subcutaneous administration of trinitrophenyl (TNP)-coupled syngeneic cells 7 days before co-culture with TNP-coupled syngeneic stimulator cells results in increased cytolytic activity. This augmented cytotoxic response has been shown to be dependent, at least partially, on radioresistant "helper" T cells. In this paper we have demonstrated that TNBS-generated suppressor T cells that are capable of suppressing contact sensitivity can specifically suppress the augmented response seen after subcutaneous priming. The i.v. administration of TNP-coupled cells results in priming of the recipient; however, if cells from these animals are transferred to a second recipient, there is evidence of suppressor activity. Thus, the cytotoxic T lymphocyte response is controlled by the same type of complex interactions previously demonstrated for humoral and delayed-type hypersensitivity responses.     

Loss of specificity in cytolytic T lymphocyte clones obtained by limit dilution culture of Ly-2+ T cells

Nonspecific cytotoxicity developed reproducibly and with high frequency in limit dilution cultures consisting of low numbers of murine cells stimulated with concanavalin A in the presence of growth factors and irradiated filler cells. The individual clones in cultures showing nonspecific killing were all derived from single, Thy-1+, Ly-2+ cells. At early times of culture (day 5 or 6), clones appeared to be specific in their lytic activity, as expected of cytolytic T lymphocytes (CTL). On continued culture (day 8 or 9), most of the originally specific CTL clones became nonspecific, killing a range of murine target cells, both syngeneic and allogeneic. The lack of specificity was observed at all effector cell doses. The effector cells responsible for the nonspecific cytolysis were Thy-1+ and Ly-2+, as were most cells in the cultures. The effector cells had the normal DNA content for a dividing T cell population, and most cells in the cultures had a normal chromosome complement. In mixed cultures in which the responder cells and the irradiated filler cells were from different mouse strains, the nonspecific killers displayed the Thy-1 and H-2 allotypes of the responder, and not of the filler cells. The development of a broad cytotoxic potential appears to be a normal and rapid event when Ly-2+ T cell-derived CTL-clones are grown under these conditions; this is a caveat for the use of limit dilution cultures to determine the T cell specificity repertoire. The relationship between these nonspecific CTL, activated lymphocyte killers, and natural killer cells is discussed.     

Identification of endothelial cell junctional proteins and lymphocyte receptors involved in transendothelial migration of human effector memory CD4+ T cells

Human effector memory (EM) CD4(+) T cells can rapidly transmigrate across an endothelial cell (EC) monolayer in response either to chemokine or to TCR-activating signals displayed by human dermal microvascular EC under conditions of venular shear stress. We previously reported that the TCR-stimulated transendothelial migration (TEM) depends on fractalkine (CX3CL1), PECAM-1 (CD31), and ICAM-1 (CD54) expression by the EC, whereas chemokine-stimulated TEM does not. In this study, we further analyze these responses using blocking mAb and small interfering RNA knockdown to show that TCR-stimulated TEM depends on CD99 on EC as well as on PECAM-1 and depends on nectin-2 (CD112) and poliovirus receptor (CD155) as well as EC ICAM-1. ICAM-1 is engaged by EM CD4(+) T cell LFA-1 (CD11a/CD18) but not Mac-1 (CD11b/CD18); nectin-2 and poliovirus receptor are engaged by both DNAX accessory molecule-1 (CD226) and Tactile (CD96). EC junctional adhesion molecule-1 (JAM-1), an alternative ligand for LFA-1, contributes exclusively to chemokine-stimulated TEM and ICAM-2 appears to be uninvolved in either pathway. These data further define and further highlight the differences in the two pathways of EM CD4(+) T cell recruitment into sites of peripheral inflammation.     

Intercellular adhesion molecule-1 (ICAM-1) is involved in the cytolytic T lymphocyte interaction with a human synovial cell line

The cell surface molecules involved in the human cytolytic T lymphocyte (CTL)-synovial cell interaction may play an important role in T cell interactions with connective tissue mesenchymal cells. To examine the molecular basis for the CTL-synovial cell interaction, we immortalized synovial cell explants to establish the cell line SYN.SPP. The SYN.SPP cell line was compared to the established B lymphoblastoid cell line JY. Cell surface immunofluorescence demonstrated significantly different levels of the immunologically relevant cell surface molecules ICAM-1 and LFA-3. Both cell lines were used to stimulate CTL precursors. After several months in culture, CTL lines stimulated by the SYN.SPP and JY cell lines demonstrated HLA class I-directed cytolytic activity. The cell surface molecules utilized by the anti-SYN.SPP and anti-JY CTL lines were identified by monoclonal antibody (MAb) inhibition. MAb recognizing the CTL cell surface molecules CD3, CD8 and LFA-1 (CD11a) significantly inhibited CTL-mediated lysis of both target cells. An interesting observation was that the anti-SYN.SPP CTL line appeared to utilize the ICAM-1 and not the LFA-3 target cell molecule. In contrast, the anti-JY CTL line utilized the LFA-3 and not the ICAM-1 membrane molecule. These results indicate that CTL interactions with connective tissue mesenchymal cells may be regulated by a unique pattern of antigen nonspecific cell-cell interaction molecules.     

Fine specificity and antigen receptor expression among influenza virus-specific cytolytic T lymphocyte clones

Influenza virus stimulates a vigorous cytolytic T lymphocyte (CTL) response in the mouse that is directed to several virion polypeptides. This report examines the fine specificity of a panel of murine influenza-specific CTL clones restricted by MHC class I products of the H-2d haplotype. Ten of 22 A/JAPAN/305/57-specific CTL clones analyzed were directed to the A/JAPAN/305/57 hemagglutinin protein as detected by using target cells infected with a recombinant vaccinia virus containing hemagglutinin gene. Based on their fine specificity of hemagglutinin recognition, these clones defined four functional epitopes on the hemagglutinin. The remaining 12 cytolytic clones exhibited cross-reactivity for type A influenza viruses of the major human subtypes, and approximately 60% of these clones were directed to the nucleocapsid protein. KJ16-133 monoclonal antibody analysis of the utilization of the T cell receptor V beta 8 gene segment subfamily revealed that members of this V beta gene subfamily are expressed by both hemagglutinin- and nucleocapsid-specific MHC class I-restricted CTL (and by influenza-specific MHC class II-restricted T lymphocytes as well). These results suggest that CTL detect several distinct antigenic sites on the hemagglutinin. In addition, these results reveal no direct correlation between viral antigenic specificity and V beta gene expression by these virus-specific CLT clones.     

Avidity dictates the lytic capacity of human cytolytic T lymphocyte clones with similar fine specificity against murine cells expressing HLA-B27 antigen

The role of the avidity of human CTL in the recognition and lysis of murine P815 cells expressing HLA-B27.1 Ag has been examined. Seven B27-specific alloreactive CTL clones were tested for their ability to lyse a B27.1+-P815 transfectant clone 1-7E, obtained after cotransfection of P815-HTR cells with HLA-B27.1 and human beta 2-microglobulin genes. The expression level of HLA-B27.1 on 1-7E cells was comparable to that on a human lymphoblastoid cell line, as determined by flow cytometry. Of the seven CTL clones used, CTL 1, 26, and 29 displayed the same fine specificity as established with a panel of target cells expressing six structurally different HLA-B27 variants. However, CTL 1 and 29 were of higher avidity than CTL 26, in that the lysis of human target cells by only this latter clone was inhibited by an anti-CD8 mAb. Based on the same criteria, CTL 2, 15, and 48 possessed the same or very similar fine specificity, but CTL 48 was of higher avidity than CTL 2 or 15. The seventh clone, CTL 40, was of a different fine specificity and its lysis of human target cells was also inhibited by the same anti-CD8 mAb. Only those clones whose lysis of human targets could not be inhibited by anti-CD8 antibody were able to lyse the 1-7E murine transfectants. These results indicate that, for human CTL clones with identical or very similar fine specificity, only those of higher avidity are able to lyse P815 murine cells expressing the HLA-B27 antigen. The lysis of HLA-B27.1+-murine transfectants by relevant clones was inhibited by anti-CD8 antibody. This result strongly suggests that the relative contribution of CD8 in stabilizing the interaction between human CTL and HLA-B27+-murine target cells is more significant than with human target cells.     

Suppressor T cells regulate the cytolytic T lymphocyte response to syngeneic tumors induced by murine sarcoma virus (MSV) in the mouse.

Studies were designed to analyze the immune activities of spleen cells from mice previously injected with murine sarcoma virus (MSV) and undergoing the processes of MSV tumor growth and rejection. Fractionation of MSV-primed spleen cells according to cell size by velocity sedimentation at unit gravity showed that MSV-specific cytolytic T lymphocytes (CTL) generated in vivo underwent an apparent transition in size from large to small cells as the tumor regressed. The majority of CTL precursors, however, were invariably recovered among small to medium-sized MSV-immune cells, as revealed to CTL generation in vitro in secondary mixed leukocyte-tumor cell cultures (MLTC). Evidence was obtained for the existence in MSV-immune spleens of two suppressor cell populations capable of inhibiting CTL generation in vitro: one population probably consisted of macrophages and could be removed by treatment with carbonyl iron; the second population was comprised of T cells and inhibited the differentiation of tumor-immune CTL precursors in a selective manner. These results provide a preliminary overview of the mechanisms regulating the generation, differentiation, and activity of tumor-specific CTL in a syngeneic model system.     

Ligation of the lymphocyte homing receptor CD44 triggers T-helper and cytolytic functions of human T cells

We show that antibodies to the CD44 molecule trigger proliferation of human CD3+/CD4+ T-cell clones. Such effect is IL2-dependent, as shown by IL2 production induced by anti-CD44 mAb and by inhibition of cell proliferation in the presence of anti-IL2 antibodies or cyclosporin A (CsA). Moreover, anti-CD44 mAb triggered human cytolytic CD4+ and CD8+ TCR /+ clones, and V1 or V2 TCR Y/+ clones to lyse Fc-gamma-R+ P815 cells and to release granule trypsin-like esterase enzymes. Anti-CD44 mAb-triggered proliferation and cytotoxicity were blocked by the PTK-inhibitor, genestein. In addition, ligation of the CD44 molecule induced tyrosine phosphorylation of proteins identical, by molecular weight, to those phosphorylated following anti-CD3 mAb-stimulation. Notably, anti-CD44 mAb does not induce tyrosine phosphorylation of a 21 kD protein (the phosphorylated zeta chain of the TcR molecular complex) typically observed upon anti-CD3 mAb stimulation.     

CD40 ligation restores cytolytic T lymphocyte response and eliminates fibrosarcoma in the peritoneum of mice lacking CD4+ T cells

Absence of CD4⁺ T cell help has been suggested as a mechanism for failed anti-tumor cytotoxic T lymphocytes (CTL) response. We examined the requirement for CD4⁺ T cells to eliminate an immunogenic murine fibrosarcoma (6132A) inoculated into the peritoneal cavity. Immunocompetent C3H mice eliminated both single and repeat intraperitoneal (IP) inoculums, and developed high frequency of 6132A-specific interferon-γ (IFNγ)-producing CTL in the peritoneal cavity. Adoptive transfer of peritoneal exudate cells (PEC) isolated from control mice, protected SCID mice from challenge with 6132A. In contrast, CD4 depleted mice had diminished ability to eliminate tumor and succumbed to repeat IP challenges. Mice depleted of CD4⁺ T cells lacked tumor-specific IFNγ producing CTL in the peritoneal cavity. Adoptive transfer of PEC from CD4 depleted mice failed to protect SCID mice from 6132A. However, splenocytes isolated from same CD4 depleted mice prevented tumor growth in SCID mice, suggesting that 6132A-specific CTL response was generated, but was not sustained in the peritoneum. Treating CD4 depleted mice with agonist anti-CD40 antibody, starting on days 3 or 8 after initiating tumor challenge, led to persistence of 6132A-specific IFNγ producing CTL in the peritoneum, and eliminated 6132A tumor. The findings suggest that CTL can be activated in the absence of CD4⁺ T cells, but CD4⁺ T cells are required for a persistent CTL response at the tumor site. Exogenous stimulation through CD40 can restore tumor-specific CTL activity to the peritoneum and promote tumor clearance in the absence of CD4⁺ T cells.Supported in part by grants from Children’s Hospital of Wisconsin Foundation, Society of University Surgeons Foundation, Florence and Marshall Schwid Foundation, Elsa Pardee Foundation, Kathy Duffy Fogarty Fund of the Greater Milwaukee Foundation (JS) and NIH grant RO1-CA-37156 (HS); Andrew Lodge and Ping Yu have contributed equally to this work.     

Stimulator cell requirements for allospecific T cell subsets: specialized accessory cells are required to activate helper but not cytolytic T lymphocyte precursors

Murine cortisone-resistant thymocytes were separated by staining with monoclonal anti-Lyt-2 antibody and FMF into Lyt-2- and Lyt-2+ subsets in order to analyze the nature of stimulator accessory cells required to activate each of these functionally distinct T cell subpopulations. The Lyt-2- fraction was able to proliferate but not to generate cytotoxic cells when stimulated by irradiated allogeneic spleen cells. Fractionation of the stimulator population showed that low numbers of dendritic cells and splenic macrophages, but not equivalent numbers of whole spleen cells or peritoneal macrophages, were able to stimulate the Lyt-2- population. On the other hand, the Lyt-2+ population, which showed little if any proliferation in response to irradiated spleen cells, contained all the precursors of cytolytic T lymphocytes. In contrast to the highly specific stimulator requirement of the Lyt-2- fraction, allospecific cytotoxic cells were generated from Lyt-2+ cells by any alloantigen-bearing stimulator cell provided interleukin 2 was present. This was confirmed by limiting dilution analysis: alloreactive CTL-P frequencies in spleen and thymus were not influenced by the nature of the stimulator cell. These data collectively indicate that heterogeneous Ia+ accessory cells are required to stimulate helper but not cytolytic T cell precursors.     

Human T lymphocyte adhesion to endothelial cells and transendothelial migration. Alteration of receptor use relates to the activation status of both the T cell and the endothelial cell

An in vitro model of T cell adhesion to human umbilical vein endothelial cells (HUVEC) and transendothelial migration was used to determine whether the activation state of the T cell or cytokine exposure of the HUVEC altered T cell-HUVEC interactions or receptor utilization. Stimulation of T cells with the activator of protein kinase C, phorbol dibutyrate (PDB) alone or in combination with the calcium ionophore, ionomycin increased their binding to HUVEC. Much of the binding of control and activated T cells to HUVEC was mediated by leukocyte function-associated Ag-1 (LFA-1) (CD11a/CD18), because mAb to either chain of this molecule inhibited binding substantially, but not completely. Activation of HUVEC with IL-1 also increased binding of T cells. Binding of control T cells to IL-1-stimulated HUVEC, however, was found to be LFA-1 independent, because mAb to CD11a/CD18 failed to block the interaction. In contrast, binding of activated T cells to IL-1-stimulated HUVEC was partially inhibited by mAb to LFA-1. Binding of activated T cells to IL-1-stimulated HUVEC also involved CD44 because this interaction was partially blocked by mAb to this determinant. When T cell migration was analyzed, it was found that the migration of PDB-activated T cells was three to four-fold more than that of control T cells. Migration through HUVEC and random migration were both enhanced by PDB stimulation. However, when the T cells were costimulated with PDB and ionomycin, migration was not increased above that of control T cells. PDB-activated T cells appeared to use LFA-1 for migration regardless of the activation status of the HUVEC, because mAb to CD11a/CD18 partially blocked their migration after binding to HUVEC. There was also a modest inhibition of PDB-activated T cell migration by mAb to CD44. In contrast, migration of control T cells involved neither LFA-1 nor CD44. Finally, binding of control T cells to high endothelial venules of peripheral lymphoid tissue was found to be CD11a/CD18 and CD44 independent, and completely inhibited by activation with either PDB or the combination of PDB and ionomycin. These results demonstrate that T cells use LFA-1 and CD44 as well as other as yet unidentified adhesion receptors for interactions with HUVEC, and that use of these adhesion receptors is mutable and related to the activation state of the T cell and cytokine stimulation of the HUVEC.     

CD4 is involved in a post-binding event in the cytolytic reaction mediated by human CD4+ cytotoxic T lymphocyte clones

CTL lyse their target cells in discrete phases. First, the CTL bind to the target cell in a Mg2+-dependent manner followed by a Ca2+-dependent cytolytic phase. In the present study, we investigated the role of CD4 in the different phases of the cytolytic reaction mediated by human CD4+ class II MHC-specific CTL clones by using a single cell assay. It was found that the anti-CD4+ mAb OKT4A, which blocks cytotoxic reactions by CD4+ CTL clones as measured with a 51Cr release assay, only marginally affects the formation of conjugates. It appeared that OKT4A more strongly blocked the post-binding phase of the cytolytic reaction. In contrast, anti-leukocyte function-associated mAb strongly blocked the formation of conjugates but not the subsequent lytic phase. As was found previously with CD8+ CTL clones, anti-TCR mAb generally did not affect the formation of conjugates. One exception was noted. The activity of a CD4+ CTL clone, HY-640, could not be blocked by OKT4A, but was affected by an anti-TCR mAb. This anti-TCR mAb could partly reduce the formation of conjugates between HY-640 cells and their specific target cells. These results suggest that this clone has a high affinity TCR, which can contribute to the formation of conjugates. Although preincubation of the CTL clones with OKT4A only marginally affects the number of conjugates upon subsequent mixture with target cells, it was observed that incubation at 37 degrees C of preformed conjugates with OKT4A markedly reduced the number of conjugates. This dissociation of preformed conjugates was optimal only after 2 h of incubation. In contrast, an anti-leukocyte function-associated mAb induced a much more rapid dissociation of preformed conjugates.     

T lymphocyte activation by staphylococcal enterotoxins: role of class II molecules and T cell surface structures

The enterotoxins produced by Staphylococcus aureus (SE) are the most potent mitogens known. Triggering of proliferation or cytotoxicity by SE requires the presence of MHC class II molecules on accessory or target cells. In this study we have investigated the role of HLA class II molecules in the activation of human T cells by SE and the nature of the target structure on the responding T lymphocyte for SE. This dependence on class II molecules is not due to an immunological "recognition" of SE since there is no restriction by polymorphic determinants of HLA molecules and since even xenogeneic class II molecules can reconstitute the human T cell response to SE. Furthermore, HLA class II-positive but not -negative cells absorb the mitogenic activity from SE solutions and significant binding of 125I-labeled SE can be demonstrated to class II-positive but not to class II-negative cells. Enterotoxin molecules react directly with T cells since they cause an increase in cytosolic Ca2+ concentration similar to anti-CD3 mAb. This increase is abrogated by prior modulation of the TCR/CD3 complex. Antibodies to CD2, CD3 and the TCR that block antigen-specific activation also block T cell activation by SE. Moreover, preincubation of purified resting accessory cell-free T cells with SE leads to modulation of the TCR/CD3 complex. Taken together these data indicate that SE interact selectively with HLA class II molecules on accessory or target cells and with a TCR-associated structure on the T cell.     

Generation of cytolytic T lymphocytes in vitro. VIII. failure of anti-RS antisera to inhibit the generation of cytolytic T lymphocytes or cytolytic T lymphocyte activity.

Antibody reactive with "recognition structures" (RS) of mouse lymphoid cells for alloantigens (anti-RS) was prepared by immunization of F1 hybrid mice with parentalstrain lymphoid cells or with antibody produced in one parental strain against alloantigens of the other parental strain. Such antisera prevented generation of the "product of antigenic recognition" (PAR) that is produced within a few hours in cultures prepared with a mixture of lymphoid cells from genetically disparate mice. However, treatment of responding lymphoid cells with anti-RS sera and complement did not inhibit generation of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Treatment of cells obtained from MLC with anti-RS sera and complement failed to inhibit cytolytic activity of such cells for specific alloantigens.