抗T细胞免疫毒素的T细胞清除效率及其对造血细胞的影响

抗T细胞免疫毒素主要用于同种异体骨髓移植中清除T细胞,减轻移植物抗宿主反应(GVHD),提高移植成功率,也可用于白血病自体骨髓移植中清除残留白血病细胞,减少移植后白血病复发的可能性。本实验结果表明抗T细胞免疫毒素在10~(-8)M浓度对靶细胞的杀伤达3.0log以上,即可清除99.99%的靶细胞,并可有效地抑制周血T细胞转化功能和骨髓T细胞分泌IL—2的功能,同时对骨髓粒单系和红系造血细胞的增殖无明显的抑制作用。     

Anchorage and lymphocyte function. Antibodies as adhesion and spreading factors for human T lymphocytes

When attached to a solid surface coated with protein A various antibodies reacting with lymphocyte membrane antigens (anti-beta 2m, OKT3, OKT8, Leu2, 3, 4 and certain patient sera) catalyse the formation of peripheral lamellar activity, i.e. an active spreading process in human T lymphocytes. In contrast, binding only of the same antibodies to the cells or allowing antibody-coated cells to settle and bind to a protein A-coated surface did not induce spreading although the number of cells attached to the solid surface was virtually the same as in the former case. The peripheral lamellar activity markedly facilitated short-range lymphocyte interactions and appeared to constitute the region of the lymphocyte that actively contacts other cells. These results show that antibodies can act as spreading factors, and indicate that this function is critically dependent on the presentation of the inducing ligand. The asymmetry in the induction of active cell edges may influence functional lymphocyte interactions with environmental surfaces.     

Function and distribution pattern of human T lymphocytes. II. Presence of T lymphocytes in normal humans and in humans with various immunodeficiency disorders

Using rabbit–anti-human T lymphocyte antisera in cytotoxic assays estimates of human T lymphocytes in normal or diseased lymphoid populations have been made. Lymphocytes from normal adult blood contained on the average 53% T cells, with slightly higher values for umbilical cord blood and lymph nodes. Close to 100% of human thymocytes were killed under the same conditions in one case analysed.

Blood lymphocytes from Bruton or CVH patients could be shown to contain very few lymphocytes with high surface concentrations of immunoglobulin (with one exception) and a corresponding increase of T cells as tested by the cytotoxic assay. Patients with selective IgA deficiency had normal B and T lymphocyte levels whereas a patient with cryomacroglobulinaemia had close to 90% B lymphocytes and no detectable T lymphocytes. Patients with Hodgkin's disease could be shown to have a highly significant reduction in the number of T cells in the peripheral circulation.

     

Antibodies specific for human T lymphocytes in cold agglutinin and lymphocytotoxic sera

Sera of patients with cold agglutinin disease (anti-I,i), systemic lupus erythematosus, infectious mononucleosis or cryoglobulinemia contained cold IgM antibodies cytotoxic for a subpopulation of normal lymphocytes. Evidence was obtained that the antibodies were specific for T lymphocytes. There was an enrichment of cells reacting with the sera after removal of Ig-bearing cells on immuno adsorbent columns. Absorption studies with adult and cord erythrocytes revealed a minimum of three antigenic determinants on the lymphocyte membrane, viz. i,I and a third specificity which was unique to T cells. Antibodies to this third specificity were present, at high titer, in sera which were monoclonal for the i antigen.     

Antibody-dependent direct cytotoxicity of human lymphocytes. I. Studies on peripheral blood lymphocytes and sera of patients with systemic lupus erythematosus.

Antibody-dependent direct cytotoxicity (ADDC) is generally believed to be unrelated to T-cell function in experimental animals. The role of ADDC in humans and its clinical usefulesss was evaluated in patients with systemic lupus erythematosus (SLE) and normal controls. Peripheral blood lymphocytes from patients with active SLE were unable to lyse antibody-coated target cells in vitro to the same degree as lymphocytes from patients with inactive SLE and controls. Sera from patients with active SLE suppressed ADDC by lymphocytes derived from normal controls and this abnormality was not corrected by overnight incubation or by extensive washing of lymphocyte preparations. Although there was poor correlation between ADDC and the proportions of B cells and null cells in effector lymphocyte populations from SLE patients and controls, it is concluded that this assay provides another means of determining immune competence in man.     

Activation of human T lymphocytes I. Requirements for mitogen-induced proliferation of antigen-specific T lymphocyte clones

In a model system for accessory cell (AC)-dependent mitogen-induced T cell proliferation the response of several human antigen-specific, HLA-restricted helper T lymphocyte clones (HTLC) to mitogens was studied. It was found that the HTLC themselves did not or only weakly respond to various mitogens or to oxidation by galactose oxidase, but that the response could be strongly increased if certain tumor cell lines were added to the assay as AC. Pretreatment with lectins or oxidation of either HTLC or AC was effective in stimulating the proliferation of the T cells in this system. Reduction of the aldehydes formed during oxidation completely abolished the stimulatory activity of oxidized B lymphoblastoid cell line. This shows that crosslinking of T cell and AC is required to induce proliferation. When several established cell lines were tested for their capacity to function as AC in this system, profound differences in AC activity were detected. The inability of cells with poor AC activity to stimulate the HTLC was not due to trivial reasons, such as requirements for different mitogen concentrations, a decreased binding of mitogens or suppressive effects. Furthermore, AC activity was not dependent on the presence of Ia antigens on the AC. These findings are discussed with regard to the mechanism of mitogen-induced T cell activation.     

T lymphocyte induction of non-T cell-mediated nonspecific cytotoxicity. I. Introduction mechanisms.

Mononuclear cells (MNC) from normal humans consistently failed to give nonspecific cytotoxic responses. However, after removal of T cells by sheep erythrocyte (E) rosetting, the remaining non-RFC (rosette-forming cells) now gave significant nonspecific cytotoxic responses against both autologous and allogeneic target cells. Reconstitution experiments with T cell subpopulations failed to suppress these nonspecific non-E-RFC-mediated cytotoxic responses. There was also no evidence to indicate the involvement of antibody in this nonspecific cytotoxicity. The cytotoxic cells were characterized as non-E-rosetting, non-phagocytic, and glass adherent lymphocytes; no evidence of monocyte-macrophage participation was found. The inductive trigger of non-E-RFC-mediated cytotoxicity was found to be soluble factors released by T cells during E-rosette formation at 4 degrees C. Incubation of MNC with horse, marmoset and human erythrocytes under identical conditions failed to trigger cytotoxicity. The incubation of quiescent MNC with E-rosetting supernatants (ERS) induced nonspecific cytotoxic responses equivalent to those mediated by separated non-E-RFC. ERS-activated MNC destroyed both autologous and allogeneic target cells. The ERS supernatants themselves were not cytolytic. These findings suggested that cell separation procedures, and possibly in vivo events, which activate T cells may also induce non-T cell-mediated nonspecific cytotoxicity.     

Migration of human lymphocytes. II. Variation of lymphocyte distribution.

Variations in the distribution of 51Cr-labelled human lymphocytes from several donors and from one donor on several occasions have been determined after their injection into carbon-treated mice and reasons for the variation discussed. Natural alteration of the lymphocyte surface (leukaemic lymphocytes) or induced change (trypsin-treatment) reduced lymph node localisation as did treatment with formalin, sodium azide and antilymphocyte globulin. We have concluded that the presence of radioactivity in the lymph nodes is due to the presence of viable, metabolically active lymphocytes with normal surfaces capable of interacting with the endothelium of the post-capillary venules.     

自身骨髓瘤抗原致敏树突细胞介导特异性CTL反应的研究

目的：研究多发性骨髓瘤(MM)患者肿瘤冻融物致敏的树突细胞(DC)能否诱导特异性细胞毒T淋巴细胞(CTL)反应。方法：用MM患者骨髓CD34^ 细胞诱生DC。将MM患者骨髓瘤冻融物冲击致敏DC。MTT法检测骨髓瘤抗原致敏及非致敏DC诱导的自身T细胞对不同靶细胞(患者骨髓瘤细胞、K562细胞)的杀伤率。结果：骨髓瘤冻融物致敏DC诱导的自身T细胞对患者骨髓瘤细胞的杀伤远大于对K562细胞的杀伤。结论：患者骨髓瘤冻融物致敏的DC能有效诱导自身T细胞特异性抗瘤免疫。     

Studies of cultured human T lymphocytes. I. Production of the T cell growth-promoting lymphokine interleukin-2

Described herein is a large-scale procedure that has been successfully employed for producing 62 lots (800–3000 ml) of supernatants containing the T cell growth-promoting factor Interleukin-2 (IL-2). The efficiency of these crude, unconcentrated supernatants was documented in studies in which 70 human long-term (>100 days) IL-2-dependent T cell lines were established from 50 different donors. These included lines initiated from the peripheral blood healthy subjects (N = 54), blood of children with active acute lymphoblastic leukemia (N = 6) and the thymus of children undergoing surgery to correct congenital heart defects (N = 10). The underlying concept used in constructing this method emphasizes the requirement of the monocyte-derived macrophage and its Interleukin-1 (IL-1) product to mediate IL-2 production by activated T cells. The most salient feature of this technique is the utilization of buffy coat leukocytes that had been pooled from several blood donors and sustained in spinner cultures for several days prior to polyclonal activation with phytohemagglutinin and pooled B cells of established human lymphoblastoid lines.     

Subsets of T lymphocytes in relation to T lymphocyte function in multiple sclerosis.

T lymphocyte control of Epstein-Barr virus (EBV) infection of autologous B lymphocytes was examined in parallel to the enumeration of subpopulations of mononuclear cells in 22 multiple sclerosis (MS) patients and in 22 healthy individuals. All were seropositive for EBV. The incidence of lack of T cell control was significantly higher in patients than in controls, confirming previous published work. In the present study, we have shown in addition a significantly reduced proportion of OKT8+ cells and a significantly increased ratio of OKT4/OKT8 cells in the group of patients with lack of control. The findings point to abnormal immunoregulation in MS.     

Peritoneal exudate T lymphocytes with specificity to sheep red blood cells. I. Production and characterization as to function and phenotype.

T lymphocytes which mediate DTH reactions to sheep red blood cells (SRBC) in mice enter casein-induced peritoneal exudates from which they can be recovered and assayed in a passive transfer system. Peritoneal exudates need not contain specific antigen for inducement of T-cell immigration. The amount (or biological activity) of DTH-transferring peritoneal exudate lymphocytes is enhanced by the previous use of immune modulating agents, such as cyclophosphamide (Cy) (200 mg/kg 2 days prior to sensitization), or BCG (10(7) live organisms i.v. 14 days prior to sensitization). SRBC-specific peritoneal exudate lymphocytes phenotypically are Thy 1+ and Ly 1+, 2-. In vivo, peritoneal exudate T cells from Cymodulated donors persist in circulation for a short period only and are subject to the suppressive mechanisms acting in anergic mice. Cells from BCG-plus-Cy-modulated donors, on the other hand, persist in circulation for a longer period and appear to be less susceptible to immune suppression.     

T and B lymphocytes. Exclusive role as responders and stimulators in human one-way mixed lymphocyte reaction.

Four different combinations of one-way mixed lymphocyte reactions between human peripheral blood T and B lymphocytes (at a ratio of 1:1), purified by the E-rosetting technique, were carried our. A significant mixed lymphocyte reaction was observed only in a combination in which T lymphocytes, as responding cells, and B lymphocytes, as stimulating cells, were utilized. No significant mixed lymphocyte reaction was noted in the other three combinations of cells, using T or B lymphocytes as responders and T lymphocytes as stimulators, and also B lymphocytes as both responders and stimulators. Mixed lymphocyte reactions between T lymphocytes as responders (at constant concentration) and T and B lymphocytes as stimulators (varying proportions) showed that the response decreased proportionately with decreasing numbers of B cells and increasing numbers of T cells used as stimulators. Addition of increasing numbers of stimulating T cells to a constant number of stimulating B cells did not suppress or enhance the T-cell response to B cells. These observations indicate that the human peripheral blood T and B lymphocytes play an exclusive role as responding cells and stimulating cells, respectively.     

Anchorage and lymphocyte function. Spreading-capacity distinguishes common thymocytes and peripheral T lymphocytes

Contact of T-enriched human blood lymphocytes with an adhesive surface in the presence of Concanavalin A (Con A) almost immediately induced a sequence of motile changes in virtually all cells. The initial event in this spreading process was the formation of filopodia distinct from the microvilli of lymphocytes in suspension. The filopodia were accompanied by lamellipodia, ruffles and flattening of the nucleus. Contact with a nonadhesive substratum in the presence of Con A did not trigger this sequence of changes. Cytochalasin B and D or low temperature inhibited the contact-induced changes. With the exception of a small number of cells (5-15%), T-enriched lymphocytes that were allowed to settle in the absence of Con A showed a radius of action (area occupied by the cells/translational movement per hr) of 39 micrometers 2/ less than 1 micrometer. The small 'motile' population showed a radius of action of 74 micrometers 2/8 micrometers. The Con-A-mediated spreading-process yielded a radius of action of the lymphocytes of 117 micrometers 2/6 micrometers. This augmented radius of action markedly facilitated cell-cell interaction in a high frequency of the cells and appeared to be a prerequisite for such interactions at 'low' cell density. Thymocytes reactive with OKT 6 antibodies or belonging to the 'high-density' fraction of cells attached to a Con-A-coated surface to the same extent as peripheral OKT 3 positive lymphocytes, but did not exhibit the morphological changes characteristic of a spreading-process. In contrast, OKT 6 negative thymocytes or thymocytes with a relatively low density showed spreading indistinguishable from that of OKT 3 positive peripheral lymphocytes. These results characterize the spreading-process in human T lymphocytes and demonstrate its functional importance for interactions with the environment. Spreading-capacity appears to reflect the stage of maturation of T cells.     

Cryopreservation of human lymphocytes for assessment of lymphocyte subsets and natural killer cytotoxicity

Cryopreservation of lymphocytes has become increasingly important, especially when the cells are to be used in retrospective studies of selected and dwindling populations, such as A-bomb survivors. This report describes an efficient method for cryopreservation of human lymphocytes which does not significantly alter various immunological characteristics of these cells. The proportions of Leu-1⁺ cells (T cells), Leu-2a⁺ cells (suppressor-cytotoxic T cells), Leu-3a⁺ cells (helper-inducer T cells), HLA-DR⁺ cells, Mo2⁺ cells (monocytes), B1⁺ cells (B cells), and Leu-7⁺ cells (natural killer (NK) cells), as determined by monoclonal antibodies, were found to be stable following cryopreservation. NK cell activity against K-562 target cells showed a 40–60% decrease immediately after thawing, but recovered to approximate pre-freezing levels after preincubation for 18 h. Neither lymphocyte subsets nor cell viability significantly changed following preincubation after cryopreservation. However, the ratio of cells binding to K-562 cells increased after this preincubation and may account for the observed recovery of NK cell activity. NK cell activity remained relatively stable up to 14 months of storage which confirms that freezing damage depends on the freezing process rather than on the duration of cryopreservation.     

Increase of T.G lymphocytes in human one-way mixed lymphocyte culture

Human one-way mixed lymphocyte culture induces a significant increase of EA(7S) rosette-forming cells. Using fractionation procedures, an increased number of Fc receptor-bearing cells in the T high-enriched populations was found from alloactivated lymphocytes compared with similar fractions obtained after autologous control cultures. Additional experiments showed a parallel increase of E-rosette-forming cells in the Fc receptor-enriched alloactivated fractions. The results indicate that an increase of T.G lymphocytes occurs during in vitro alloactivation in man.     

Migration inhibition of T lymphocytes from human peripheral blood by specific antigen and lymphokines.

The inhibition of migration of human peripheral blood cells in the presence of PPD was studied. It was found that migration inhibition of peripheral blood mononuclear cells (MN) from Mantoux-positive donors was far greater than the migration inhibition of peripheral blood leucocytes (PBL). Moreover, MN cells and T lymphocytes showed larger and more uniform areas of migration. In contrast, the migration of B lymphocytes and monocytes was poor. Further analysis using purified subpopulations of MN cells showed that PPD inhibited the migration of T lymphocytes but not of B lymphocytes and monocytes. Corresponding to these findings, lymphokine-containing supernatants also inhibited the migration of purified T cells from Matoux-negative donors. It was concluded that the T lymphocyte was the predominant cell in the MN cell population, which migrated, and was subject to inhibition by PPD or lymphokines. These results imply that the movement of human T lymphocytes may be influenced by soluble factors from antigen-activated sensitized cells.     

Inhibition of lymphocyte proliferation by free fatty acids. I. Differential effects on mouse B and T lymphocytes

Saturated and unsaturated long-chain fatty acids were compared for their effects on mitogen-induced DNA synthesis in mouse B and T lymphocytes. At high concentrations (100-120 microM) all of the fatty acids tested were inhibitory to some extent, while at lower concentrations (20-60 microM) only the saturated fatty acids suppressed DNA synthesis. The inhibitory effects of the saturated acids could be reversed by the simultaneous addition of either a cis- or a trans-monounsaturated or a cis,cis-diunsaturated fatty acid. Compared to T cells stimulated with either phytohaemagglutinin or concanavalin A, B cells stimulated with either lipopolysaccharide or 8-bromoguanosine were considerably less susceptible to the inhibitory effects of the saturated fatty acids. These results demonstrate that free fatty acids may be useful tools for delineating the metabolic events involved in B cell and T cell activation.     

Mixed lymphocyte reactivity of human lymphocytes primed in vitro. I. Secondary response to allogenic lymphocytes.

In order to study the mixed lymphocyte culture reactivity of human lymphocytes primed in vitro, a nucleopore culture chamber technique allowing human lymphocytes to be cultured for a period of at least two weeks has been developed. During the primary culture period in nucleopore chambers, human lymphocytes were sensitized against mitomycin-treated allogenic stimulating cells. It was shown that the stimulated lymphocytes underwent a blastogenic reaction and the results suggest a reversion to the state of small, resting, primed lymphocytes. In vitro primed lymphocytes displayed allogenic memory. This was characteristic of a secondary response, which is shown by the following: 1) acceleration, the peak of thymidine incorporation occurring on day 4,2) specificity, the accelerated response was observed only when the primed lymphocytes were confronted with the cell used for priming. Contact with a third party cell did not produce this kind of activation. 3) Amplitude; the peak DNA synthesis response was greater than that of unprimed lymphocytes cultivated for the same length of time.