Early recognition of transplantation antigens by T lymphocytes. I. Test system and cellular parameters

Within 2-4 h of interaction of parental spleen cells from naive mice or of their supernates with alloantigen-bearing F1 hybrid spleen cells, a factor called soluble early product of immune recognition (SEPIR) is secreted. SEPIR could be revealed by its ability to enhance mixed leukocyte cultures (MLC) set up in suboptimal conditions. The factor appears to be generated by parental strain T but not B lymphocytes, is active at low concentration and acts in a pulse-like fashion. Its formation is triggered by unstimulated T cells reacting with H-2 antigens; no cytokine activity of IL 1, IL 2 or interferon character could be detected. It is suggested that the formation of SEPIR within the first few hours of MLC interaction is critically related to the further development of alloantigen-driven T cell proliferation. SEPIR might thus be the earliest discernible product of alloimmune recognition.     

Epitope recognition of conserved HIV envelope sequences by human cytotoxic T lymphocytes.

CTL constitute an essential part of the immune response against the HIV. CTL recognize peptides derived from viral proteins together with the MHC class I molecules on the surface of infected cells. The CTL response could be important in prevention or control of infection with HIV by destroying virus-producing cells. In this study we have attempted to identify peptide epitopes recognized by HIV-specific CTL. Using synthetic peptides, we have identified six conserved peptidic epitopes on the gp120 envelope glycoprotein recognized by polyclonal human CTL in association with HLA-A2 class I transplantation Ag. These results were confirmed by two approaches: i) blocking of CTL activities with antibodies specific for three of these conserved peptides; and ii) construction of doubly transfected P815-A2 target cells, using deletions of the HIV env gene. Vaccination or immunotherapy in HLA-A2 individuals can thus be considered using highly conserved HIV env peptidic sequences.     

Early plasma creatinine values in discordant twins.

It has been suggested that impairment of placental perfusion prior to delivery may manifest in early postnatal increase of creatinine values. We hypothesized that the smaller of a discordant set of twins would have a higher initial plasma creatinine value and decided to measure early plasma creatinine levels in discordant twins in order to evaluate whether this value may serve as an index of impaired placental perfusion. Plasma creatinine, urea nitrogen and blood hematocrit values were simultaneously measured in 35 sets of twins during the first day of life. The sets of twins were divided into 2 groups according to birth weight difference. Thus, 18 sets of discordant twins with birth weight difference greater than 15% comprised the GT group and 17 sets of twins with birth weight difference less than or equal to 15% comprised the LE group. The differences between the values obtained within each group were analyzed using the Wilcoxon Signed Rank test. In the GT group the mean plasma creatinine level of the smaller twins was significantly higher than the level of the larger ones (p = 0.03), but there was no statistically significant difference between values obtained in twins of the LE group. The mean plasma urea level was higher in the larger twins of both groups, however only the difference in the GT group was statistically significant (p = 0.01). The mean hematocrit of the smaller twins was higher in both groups, but only the difference in the LE group was statistically significant (p = 0.02). Generally, there was a negative correlation between gestational age and early creatinine values. These results apparently support the notion that prenatal exposure to impaired placental perfusion may compromise the creatinine clearance of the fetus and result in higher early creatinine values. Since the creatinine values in our growth-retarded twins were within the normal range, no distinguishing line for evidence of a uterine-placental compromise could be drawn. Whether a certain early plasma creatinine value is suggestive or indicative of an intra-uterine hypoxic-ischemic insult, should be determined by documented instances of severe fetal compromise prior to delivery.     

Insulin modulation of pyruvate dehydrogenase in human circulating lymphocytes

1. In human circulating lymphocytes pyruvate dehydrogenase (PDH) complex is present in the active (PDHa) and inactive (PDHi) forms. 2. PDHi conversion into PDHa is stimulated when intact lymphocytes are incubated with 5 microU/ml insulin at pH 7.4, for 15 min at 37 degrees C in a medium supplemented with 50 microM Ca2+-Mg2+. 3. The generation of a mediator is strongly suggestive since a cell free preparation from circulating lymphocytes, treated as above described, still stimulates PDHi----PDHa conversion, when combined with either disrupted or intact lymphocytes.     

MHC-unrestricted recognition of bacteria-infected target cells by human CD8+ cytotoxic T lymphocytes.

A CD8+ alpha beta TCR+ T cell clone (A35) was isolated from the synovial fluid of a patient with post-enteric reactive arthritis caused by Yersinia enterocolitica. This clone efficiently killed autologous and allogeneic target cells that had been preincubated with live but not with heat-killed bacteria. There was no restriction by polymorphic parts of HLA-A, -B, or -C molecules and a HLA class II-deficient mutant cell line was lysed as efficiently as its normal counterpart, whereas infected HLA class I-deficient cells (Daudi cells) were not. The clone showed crossreaction between Yersinia enterocolitica, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes, but did not lyse target cells preincubated with Staphylococcus epidermidis. MAb to CD2, CD3, and CD8 efficiently blocked A35, whereas the addition of mAb to HLA class II or to HLA class I did not. This clone apparently represents a novel effector mechanism against bacteria-infected or -modified cells that could be involved in the immunopathology of reactive arthritis.     

Conversion of human lymphocytes to antitumor immune reactivity by xenogeneic and allogeneic imune RNA detected by leukocyte-adherence inhibition tests

Summary Using leukocyte adherence inhibition (LAI) tests, we studied the activity of xenogeneic immune RNA (I-RNA) extracted from the spleen and lymph nodes of sheep after immunization with human breast carcinoma tissue or keyhole limpet hemocyanin (KLH) in inducing lymphocytes from normal healthy donors to mediate immune responses in vitro. Mononuclear cells isolated from venous blood of normal donors, depleted of monocytes and, in some experiments, separated into T cells and non-T cells, were incubated with and without anti-breast carcinoma I-RNA or anti-KLH I-RNA for 20 min at 37° C. Then, lymphocyte adherence was determined by a Coulter counter method in the presence of 3 M KCl extracts of breast carcinoma tissues, control tissue, or KLH. Following incubation with anti-breast carcinoma I-RNA, the adherence of lymphocytes from normal donors was found to be inhibited only in the presence of breast carcinoma extracts. Following incubation with anti-KLH I-RNA, lymphocyte adherence was inhibited only in the presence of KLH. The principal effector cells involved appeared to be T lymphocytes. I-RNA treatment with RNase, but not with DNase or pronase, completely abrogated the LAI responses. In a blind study utilizing coded samples of xenogeneic and allogeneic I-RNA of unknown origin, samples containing activity against breast cancer extracts were identified correctly by LAI. Abbreviations used: I-RNA, immune RNA; LAI, leukocyte adherence inhibition; KLH, Keyhole limpet hemocyanin; PBS, phosphate buffered saline; RNase, ribonuclease; DNase, deoxyribonuclease     

Differential cytotoxicity of activated lymphocytes on allogeneic and xenogeneic target cells. II. Activation by phytohemagglutinin

In the preceding paper it has been shown that human or mouse lymphocytes stimulated by a variety of agents, damaged allogeneic target cells while damage of xenogeneic target cells was weak or absent. In this study, the species specificity of the cytotoxicity of PHA activated lymphocytes has been studied in greater detail. Effector cells were purified lymphocytes either from human peripheral blood, or from spleen or lymph nodes of inbred mice. Target cells were ⁵¹Cr-labeled human Chang liver cells or mouse L cells.PHA stimulated human or mouse lymphocytes were significantly more cytotoxic to allogeneic than to xenogeneic target cells. At low PHA doses at which damage of allogeneic target cells was significant, damage of xenogeneic target cells was very weak or absent. At higher PHA doses, damage of xenogeneic target cells became also significant but always remained at a lower level than that of allogeneic target cells.Prestimulation of human lymphocytes with PHA for 3 days increased their cytotoxic efficiency. Furthermore, damage of human Chang cells by human lymphocytes had a dose-response relationship similar to that valid for stimulation of DNA synthesis. However, damage of mouse L cells by human lymphocytes increased at PHA-doses at which stimulation of DNA-synthesis declined. For mouse lymphocytes, these doseresponse relationships were less clear-cut, probably due to differences in origin and survival of the effector cells. This confirms previous observations that cytotoxicity and DNA-synthesis are different but probably interdependent expressions of lymphocyte activation.     

The recognition of HLA-B27 by human CD4(+) T lymphocytes

HLA-B27 transgenic animal models suggest a role for CD4(+) T lymphocytes in the pathogenesis of the spondyloarthropathies, and murine studies have raised the possibility that unusual forms of B27 may be involved in disease. We demonstrate that CD4(+) T cells capable of recognizing B27 can be isolated from humans by coculture with the MHC class II-negative cell line T2 transfected with B27. These CD4(+) T cells recognize a panel of B27-transfected cell lines that are defective in Ag-processing pathways, but not the nontransfected parental cell lines, in a CD4-dependent fashion. Inhibition of responses by the MHC class I-specific mAb w6/32 and the B27 binding mAb ME1 implicates the recognition of a form of B27 recognized by both of these Abs. We suggest that B27-reactive CD4(+) T cells may be pathogenic in spondyloarthropathies, particularly if factors such as infection influence expression of abnormal forms of B27.     

Preferential recognition of peroxynitrite modified human DNA by circulating autoantibodies in cancer patients

Peroxynitrite (ONOO⁻) has been vastly implicated in mutagenesis and cancer development. Present study probes the antigenicity of peroxynitrite damaged DNA (ONOO⁻-DNA) in cancer patients. Purified human placental DNA was damaged by the synergistic action of sodium nitroprusside (SNP) and Pyrogallol for 3 h at 37 °C. Binding characteristics of cancer autoantibodies as well as experimentally induced anti-peroxynitrite-DNA (anti-ONOO⁻-DNA) antibodies were assessed by ELISA and band shift assay. DNA modifications produced single strand breaks, decreased melting temperature (T_m), hyperchromicity in UV spectrum and decreased fluorescence intensity. The ONOO⁻-DNA induced high titre antibodies in experimental animals. Cancer autoantibodies exhibited enhanced binding with the modified DNA as compared to the native form. Lymphocyte DNA from cancer patients showed appreciable recognition of anti-ONOO⁻-DNA IgG as compared to the DNA from healthy subjects. The peroxynitrite modified DNA presents unique epitopes which may be one of the factors for the autoantibody induction in cancer patients.     

Inhibition of human antibody-dependent cellular cytotoxicity, cell-mediated cytotoxicity, and natural killing by a xenogeneic antiserum prepared against "activated" alloimmune human lymphocytes

Xenogeneic antiserum (RH1) was prepared in Lewis rats by hyperimmunization with concanavalin A- (Con A) activated alloimmune human lymphocytes. The antiserum RH1 effectively inhibited human antibody-dependent cellular cytotoxicity (ADCC), cell-mediated cytotoxicity (CMC), and natural killing (NK) in the absence of complement (C). Inhibition by RH1 was dependent on the dilution of antiserum employed and the number of cytotoxic lymphocytes present during cytolysis. Pretreatment of lymphocytes with RH1 or the presence of RH1 in culture did not inhibit lymphocyte proliferation stimulated by Con A, phytohemagglutinin, or allogeneic cells; lymphokine production as measured by leukocyte-inhibiting factor production; antibody-dependent C lysis; or CMC mediated by murine cytotoxic T lymphocytes. Analysis of the mechanism of inhibition of cytotoxicity by RH1 revealed that 1) RH1 was not cytotoxic for human lymphocytes at 37 degrees C in the absence of C; 2) purified F(ab')2 fragments were equally inhibitory as whole serum; 3) pretreatment of lymphocytes with RH1 effectively inhibited their capacity to mediate ADCC, CMC, or NK, and this effect was reversible by culturing the cells overnight at 37 degrees C; 4) RH1 did not inhibit target cell binding by K cells, effector cells of ADCC, or alloimmune T cells, but did inhibit binding by NK cells; and finally, 5) the addition of RH1 to preformed lymphocyte-target conjugates in a single cell cytotoxicity assay inhibited killing of the bound target cells in all three systems without disrupting the conjugates. Collectively, these findings suggest that RH1 antiserum interacts with structures present on the surfaces of cytotoxic lymphocytes that are involved in the activation of the lytic mechanism(s) or with the actual lytic molecule or molecules themselves. Furthermore, the ability of RH1 to inhibit ADCC, CMC, and NK during the post-binding cytolytic phase of these reactions indicates that binding and cytolysis are distinct and separate events in all types of cell-mediated cytolysis.     

Pyruvate dehydrogenase activation by insulin in human circulating lymphocytes and the possible pathway involved

1. The incubation of human fresh circulating lymphocytes with insulin leads to modifications in the behaviour of the pyruvate dehydrogenase complex (PDH) when the contact medium is supplemented with 50 microM Ca2+ and Mg2+. 2. To investigate the mechanism involved in the PDH responsiveness to insulin in circulating lymphocytes and the role of Ca2+ and Mg2+ in this process, the PDH activity was assayed in lymphocytes combined with insulin and/or a number of substances whose mechanism of action is partially known. 3. Of these some have been seen to mimick insulin effects on PDH, whereas other were tested for the first time in this study.     

Conversion of normal human lymphocytes to tumor-specific immunoreactivity by xenogeneic Immune RNA: Blastogenic responses to soluble tumor antigens

Summary Lymphocyte blastogenesis was used as an assay of Immune RNA (I-RNA) activity. Normal, non-immune human lymphocytes following incubation with xenogeneic antitumor I-RNA extracted from the lymphoid organs of specifically immunized sheep underwent blastogenesis when exposed to solubilized human tumor antigens in vitro. Blastogenic responses were, unexpectedly, relatively specific for the tumor type used to immunize the I-RNA donor sheep. No significant blastogenic responses were elicited by the I-RNA extracts or by the antigen preparations themselves. This study suggests that normal, human lymphocytes incubated (sensitized) with I-RNA, in vitro, behave, in terms of antigen recognition, like lymphocytes which have previously been sensitized to tumor antigens and demonstrates that xenogeneic Immune RNA will mediate afferent limb immune responses to human tumor antigens.Supported, in part, by Public Health Service Grant CA-18321 from the National Institute of Health     

Immune recognition of AIDS virus antigens by human and murine cytotoxic T lymphocytes

The CTL response to HIV was analyzed in humans and in mice. By using a novel and strictly autologous lymphocyte culture system, human CTL lines were established with PBL from seropositive asymptomatic donors and from patients suffering from AIDS or presenting AIDS-related complex. CTL from HLA-A2 donors recognize and kill murine P815 mastocytoma cells doubly transfected with the human HLA-A2 gene and the HIV env gene; they also kill HLA-compatible human macrophages infected with HIV. CTL specific for the HIV env Ag were also generated in BALB/c mice by immunization with syngeneic murine cells transfected with the HIV env gene. Human and murine HIV-immune CTL populations belong to the CD8 subset of T lymphocytes and are restricted by class I HLA or H-2 transplantation Ag, respectively, in the recognition of HIV env Ag. The two different experimental systems presented here can be used to study CD8 lymphocyte immunity against HIV. The murine model of CTL immunity offers the additional advantage of avoiding the manipulation of infectious virus isolates.