Role of individual chains of HLA-DR antigens in activation of T cells induced by alloantigens

The role of HLA-DR antigens in the activation of T cells in the allogeneic mixed lymphocyte reaction (MLR) was studied by using antibodies raised against the alpha, beta or the complex of both chains of the HLA-DR antigens. Antisera directed against the alpha or the beta chain strongly inhibited the T-cell proliferative response when added at the begining of MLR cultures but not 72 h later. T cells from MLR cultures treated with either alpha-chainor beta-chain-specific antibodies did not respond to interleukin-2 (IL-2) by proliferating, whereas T cells from non-anti-DR-treated cultures showed a proliferative response to IL-2 stimulation. However, neither the anti-alpha chain nor the anti-beta chain serum was able to inhibit continuous proliferation of already activated, IL-2-reactive T cells supported by IL-2. In MLR, OKT4⁺ but not OKT8⁺ lymphocytes synthesized IL-2. This function was abrogated by the alpha-chain-specific antibody but not by the anti-beta chain serum. Interleukin-1 (IL-1) did not reverse the inhibitory activity on IL-2 synthesis of the alpha-chain antibody, while IL-1 promoted the production of IL-2 in MLR cultures not exposed to the anti-DR sera. In addition, nonstimulated OKT4⁺ cells were unresponsive to IL-1 and did not produce IL-2. From these results, it is concluded that HLA-DR antigens participate actively in the activation of T cells by allogeneic non-T cells. Thus, both the alpha and beta chains of HLA-DR antigens render resting T cells sensitive to IL-2. In addition, the alpha but not the beta chain participates in the production of IL-2 by enabling OKT4⁺ lymphocytes to respond to IL-1 and subsequently to synthesize IL-2. Once T cells have acquired responsiveness to IL-2 and this growth factor has been produced there is no further requirement for HLA-DR antigens. Continuous proliferation and growth of IL-2-reactive T cells depends on the availability of interleukin-2.     

Influence of sex hormones on Coxsackie B-3 virus infection in Balb/c mice

Background and “spontaneous” proliferation are terms often used for the proliferative activity normally exhibited by peripheral blood mononuclear cells (MNC) in vitro. In this report, we show that Interleukin-2 (IL-2) added to unfractionated MNC but not to isolated T or non-T cells significantly increased their proliferative activity. The cells responding to IL-2 stimulation from MNC were OKT3 positive lymphocytes. In addition, treatment of MNC with either a monoclonal anti-HLA-DR antibody (in the absence of C′) or Cyclosporin-A strongly suppressed the “background” whereas treatment of MNC with the 3A1 monoclonal anti-human T cell antibody did not modify “spontaneous” proliferation of these cells. IL-2 could not restore or increase the proliferative activity of MNC exposed to the anti-HLA-DR antibody or Cyclosporin-A while the T cell growth factor significantly enhanced proliferation of MNC cultured in the presence of the OKT4 antibody. Taken together these results strongly suggest that IL-2 responding T cells from MNC become sensitive to IL-2 by interacting with HLA-DR antigens on B lymphocytes and/or monocytes contained in MNC (resting T cells are Dr^?). By a similar mechanism we have previously shown that T cells acquire responsiveness to IL-2 in the autologous mixed lymphocyte reaction (AMLR). Since all the cells that participate in AMLR are present in MNC, we postulate that a “mini” AMLR taking place within MNC may explain the “spontaneous” proliferation of peripheral blood mononuclear cells.     

Improvement of decreased interleukin 2 (IL-2) responsiveness and IL-2 production in autologous mixed-lymphocyte reaction of cord blood lymphocytes by interferon-gamma and IL-2 and the role of HLA-DQ antigen

Cord blood T cells did not produce interleukin 2 (IL-2) nor acquire responsiveness to it in autologous mixed-lymphocyte reaction (AMLR) as they do when activated by phytohemagglutinin (PHA). The ability of the cells to respond to IL-2 was restored either by the addition of recombinant IL-2 to the AMLR culture or by the preculture of non-T stimulator cells with recombinant interferon-gamma (IFN-gamma). IL-2 production was also induced when the T cells were added with recombinant IL-2 at the initiation of the AMLR culture, preceded by the treatment of non-T cells with recombinant IFN-gamma. IL-2-producing cells of cord blood induced in the above-mentioned condition were defined to be OKT4+ T cells, because the deletion of OKT4+ T cells from T-cell population abrogated the reaction, while that of OKT8+ T cells did not. Acquisition of IL-2 responsiveness and IL-2 production of T cells seemed to be mediated by HLA-DR and HLA-DQ molecules of non-T cells because these reactions were blocked by the treatment of non-T cells either with monoclonal anti-HLA-DR or with anti-HLA-DQ antibody. The HLA-DR and HLA-DQ densities of cord blood non-T cells were low as compared with those of adult, but the expression of HLA-DQ was remarkably improved by IFN-gamma treatment. In regard to IL-2, both IFN-gamma and IL-2 were needed to enable the lymphocytes to produce. This may suggest that some functional maturation by IL-2 of responder T cells is further required. These combined data suggested that cord blood non-T cells are defective as a stimulator in AMLR and this could be corrected by enhancing the expression of HLA-DQ antigen.     

HLA-DR antigens induce proliferation and cytotoxicity of T cells against haptenated (TNP and FITC) self structures

Antisera directed against the heavy, the light, or reactive against the complex of both chains of HLA-DR antigens strongly inhibited proliferation of T cells induced by TNP- or FITC-labeled autologous cells when added at initiation of the cultures, but not 72 h later. T cells from cultures treated with the anti-DR sera were unresponsive to interleukin-2 (IL-2). Nonetheless, the anti-DR sera did not inhibit proliferation of T cells that had already acquired sensitivity to IL-2. The DR antibodies abrogated the synthesis of IL-2 induced by both TNP- and FITC-conjugated autologous cells. Treatment of TNP- and FITC-labeled autologous cell cultures with the four different types of anti-DR sera significantly inhibited the induction of cytotoxic T cells. However, DR antibodies added at the effector phase of cytotoxicity assays did not inhibit the cytotoxic activity. Effector T cells from cultures treated with the anti-DR sera were unresponsive to IL-2 and addition of IL-2 to these cultures did not restore the cytotoxic activity. In contrast, effector T cells from cultures performed in the absence of the anti-DR sera proliferated to IL-2 stimulation and addition of IL-2 to these cultures significantly increased the generation of killer cells specific for hapten-labeled self structures. From these results we concluded the following: (1) Both the heavy and the light chains of DR antigens participate actively in the activation of T cells by rendering resting T cells sensitive to IL-2 and by inducing production of the growth factor in TNP- and FITC-conjugated autologous cell cultures. (2) The heavy and light chains of the DR antigens play an essential role in the induction of cytotoxic T cells specific for hapten-labeled self structures, most likely by enabling cytotoxic T cells to respond to IL-2 and by inducing the IL-2 producer T cells to synthesize the growth factor.     

The alpha chain,not the beta chain of HLA-DR antigens participates in activation of T cells in autologous mixed lymphocyte reaction

Using antisera against the alpha, the beta or the complex of both chains of HLA-DR antigens, we have studied the role of individual chains of HLA-DR antigens in activation of T cells in autologous mixed lymphocyte reaction (AMLR). Alpha chain-specific antibody, not anti-beta chain serum prevented T cells from acquiring responsiveness to interleukin-2 (IL-2), suppressed the production of 1L-2, and inhibited the T cell proliferative response in both primary and secondary AMLR cultures. However, proliferation of already activated IL-2 reactive T cells supported by IL-2 was not affected by any of the four different types of anti-DR sera used. Fifty to sixty percent of T cells activated by AMLR or by PHA possessed DR antigens and functioned well as stimulator cells in secondary AMLR cultures. Moreover, the stimulatory activity of these DR-positive T cells was suppressed by the anti-alpha chain, not by the beta chain-specific antibody. Since continuous proliferation of T cells requires IL-2 and since nonactivated T cells are not sensitive to IL-2 and are unable to absorb this growth factor, we conclude the following: (1) The alpha, not the beta chain of HLA-DR antigens seems to be the structure responsible for enabling resting T cells to respond to IL-2 and induce production of IL-2 in AMLR. (2) Once T cells have acquired responsiveness to IL-2 and the growth factor has been produced, there is no further requirement for HLA-DR antigens, but the availability of IL-2 determines the level and extent of proliferation of IL-2 sensitive T cells.     

Concanavalin A triggers T lymphocytes by directly interacting with their receptors for activation

Anti-HLA-DR antibodies did not inhibit concanavalin A-(Con A) induced T cell proliferation or the generation of suppressor cells capable of inhibiting immunoglobulin synthesis in autologous mononuclear cells after pokeweed mitogen stimulation. Nylon-wool purified T cells (pretreated with anti-HLA-DR antibody and C) exposed to Con A acquired responsiveness to interleukin 2 (IL 2) and were able to absorb this growth factor, whereas nonlectin-treated cells did not respond to IL 2 and could not absorb it. In the presence of interleukin 1 (IL 1), Con A stimulated the synthesis of IL 2 in purified OKT4+ lymphocytes but not OKT8+ cells. However, in the absence of IL 1, neither resting OKT4+ nor Con A-treated OKT4+ cells produced IL 2. Con A by itself did not directly stimulate macrophages to synthesize IL 1, although it could do so in the presence of OKT4+ but not OKT8+ lymphocytes. In addition, Con A induced proliferation of purified T cells provided IL 1 was supplied to the cultures. Cyclosporin A rendered Con A-treated T cells unresponsive to IL 2, made lectin-stimulated OKT4+ lymphocytes unable to respond to IL 1, and inhibited the synthesis of IL 2. Furthermore, this drug abrogated the Con A-stimulated synthesis of IL 1 by acting on OKT4+ lymphocytes and not on macrophages. Finally, cyclosporin-A suppressed the proliferative response and the generation of suppressor T cells induced by Con A. The following are concluded: 1) HLA-DR antigens do not seem to play any role in the triggering of T cells by Con A, and macrophages participate in lectin-induced activation of T cells mainly by providing IL 1. 2) Cyclosporin-A inhibits activation of T cells by interfering with the mechanism by which Con A stimulates T lymphocytes. 3) Con A triggers T lymphocytes by directly interacting with their receptors for activation.     

Ia-positive T lymphocytes are the producer cells of interferon gamma

The production of γ-interferon (IFNγ) in human peripheral blood T lymphocytes was induced by stimulation with PHA. For identification of the producer cell of IFNγ, double fluorescence studies were undertaken and titers of interferon were determined in preparatively separated T-cell subpopulations reactive with one of the monoclonal antibodies OKT3, OKT4, OKT8, and OKIa1. Production of IFNγ was found in OKT3⁺, OKT4⁺, and OKT8⁺ cells. However, IFNγ production occurred only in T cells also reactive with the monoclonal antibody OKIa1. Addition of macrophages had no substantial effect on interferon titers in these subpopulations. It is suggested that the T cell subset producing IFNγ is characterized by its reactivity with the monoclonal antibodies OKT3, OKT4 or OKT8, and OKIa1.     

Interferon gamma modulates the ability of autologous non-T cells to stimulate T cells to produce and respond to interleukin 2

The interactions of T-cell receptor with self-Ia antigen on non-T cells induced IL-2 production and IL-2 receptors on the cell surface and thus responsiveness to IL-2 of T cells in autologous mixed-lymphocyte reaction (AMLR). Four-day-cultured autologous non-T cells lost their ability to stimulate T cells to produce and respond to IL-2 with concurrent decrease of HLA-DR and HLA-DQ antigen expressed on the cell surface. Culturing of non-T cells with 500 U/ml of recombinant interferon gamma (IFN-gamma) maintained their stimulating ability which was otherwise lost. Treatment of non-T cells with monoclonal anti-HLA-DR or anti-HLA-DQ antibody before mixture with T cells abrogated their ability to induce IL-2 production and IL-2 responsiveness of T cells. The combined data suggested that Ia antigen expressed on non-T cells is modulated by IFN-gamma, which increases the ability of non-T cells to stimulate autologous T cells to produce and respond to IL-2.     

Functional analysis of monkey lymphocyte subsets defined by OKT4 and OKT8 monoclonal antibodies

The percentages of rhesus monkey blood lymphocytes (PBL) reactive with OKT4 and OKT8 antibodies and the $\text{OKT}\text{4}\text{OKT}\text{8}$ ratio showed significant correlations with the log of the immunoglobulin plaque-forming cell (PFC) response after stimulation with pokeweed mitogen (PWM). These correlations suggested that monkey OKT4⁺ cells function as “helper” cells and OKT8⁺ cells function as “suppressor” cells for the PFC response. This was confirmed by separation and study of enriched T- and B-cell subpopulations. OKT8-depleted (OKT4⁺) and OKT4-depleted (OKT8⁺) cells were obtained by treatment of purified T cells with antibody and complement. OKT4⁺ cells augmented the PWM-induced B-cell differentiation into PFC but OKT8⁺ cells did not. OKT8⁺ cells suppressed the PFC response by mixtures of B cells and OKT4⁺ cells. OKT8 antibodies also detected a suppressive cell subset in African green monkeys since the percentage of OKT8⁺ cells showed a negative correlation with the log PFC response. OKT4 antibodies failed to bind to African green monkey PBL.     

Monocyte-independent interleukin-2 production and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody: interleukin-1 is not required for T-cell proliferation

We report a new, monocyte-independent system for the induction of activation and proliferation of human T cells in response to murine hybridomas expressing the OKT3 monoclonal antibody (OKT3 hybridomas). Incubation of nylon-wool-nonadherent (NA) lymphocytes or purified T cells with OKT3 hybridomas resulted in interleukin-2 (IL-2) production, expression of IL-2 receptor, modulation of the CD3 antigen, and proliferation. In contrast, murine hybridomas (OKT4, OKT8, anti-HLA-DR, and others) expressing monoclonal antibodies (mAb) other than OKT3 did not induce T-cell activation and proliferation. T cells did not respond to OKT3 mAb alone. OKT3 hybridomas alone did not produce interleukin-1 (IL-1) or other soluble factors that might be involved in the induction of IL-2 production by T cells, and they did not contain membrane-bound IL-1. In addition, IL-1 activity was not detected in cultures of NA-lymphocytes and OKT3 hybridomas, clearly demonstrating that IL-1 was not required, at least in this system, for T-cell activation and proliferation. Direct cell-cell contact between T cells and OKT3 hybridomas was required for IL-2 production. Thirty to fifty percent of T cells formed conjugates with the OKT3 hybridomas but not with the OKT4 or OKT8 hybridomas. Both conjugate formation and IL-2 production were significantly inhibited by the OKT3 mAb and by the anti-LFA-1 mAb. The cells responsible for IL-2 production were found to be of the T3+ T4+ T8- Leu 7- Leu 11- phenotype. IL-2 activity produced by NA-lymphocytes in response to OKT3 hybridomas became detectable as early as 1 hr and reached a maximum by 8 hr, preceding IL-2 receptor expression, modulation of the CD3 antigen, and [3H]thymidine incorporation of T cells. T cells produced higher concentrations of IL-2 in response to OKT3 hybridomas than in response to equal numbers of monocytes and OKT3 mAb. Addition of monocytes to cultures of T cells and OKT3 hybridomas resulted in suppression of IL-2 production in a concentration-dependent manner, suggesting that monocytes regulate the levels of IL-2 production. This monocyte-independent system may be useful for further dissection of T-cell activation and proliferation and its regulation by monocytes.     

Interleukin 7 Up-regulates CD95 Protein on CD4+ T Cells by Affecting mRNA Alternative Splicing: PRIMING FOR A SYNERGISTIC EFFECT ON HIV-1 RESERVOIR MAINTENANCE*

Interleukin-7 (IL-7) has been used as an immunoregulatory and latency-reversing agent in human immunodeficiency virus type 1 (HIV-1) infection. Although IL-7 can restore circulating CD4⁺ T cell counts in HIV-1-infected patients, the anti-apoptotic and proliferative effects of IL-7 appear to benefit survival and expansion of HIV-1-latently infected memory CD4⁺ T lymphocytes. IL-7 has been shown to elevate CD95 on CD4⁺ T cells in HIV-1-infected individuals and prime CD4⁺ T lymphocytes to CD95-mediated proliferative or apoptotic signals. Here we observed that through increasing microRNA-124, IL-7 down-regulates the splicing regulator polypyrimidine tract binding protein (PTB), leading to inclusion of the transmembrane domain-encoding exon 6 of CD95 mRNA and, subsequently, elevation of CD95 on memory CD4⁺ T cells. Moreover, IL-7 up-regulates cellular FLICE-like inhibitory protein (c-FLIP) and stimulates c-Jun N-terminal kinase (JNK) phosphorylation, which switches CD95 signaling to survival mode in memory CD4⁺ T lymphocytes. As a result, co-stimulation through IL-7/IL-7R and FasL/CD95 signal pathways augments IL-7-mediated survival and expansion of HIV-1-latently infected memory CD4⁺ T lymphocytes. Collectively, we have demonstrated a novel mechanism for IL-7-mediated maintenance of HIV-1 reservoir.     

Surface antigen changes occurring in short-term cultures of activated human T lymphocytes: analysis by flow cytometry

Surface antigens of activated and cultured human T cells were studied using peripheral blood lymphocytes activated with conditioned medium from phytohemagglutinin-activated leukocytes and maintained in liquid culture for 2 weeks with conditioned medium containing Interleukin 2. The ensuing cell population was tested for kinetic changes in cell size and for the expression of surface antigens by immunofluorescence staining with a panel of monoclonal antibodies and analysis by flow cytometry. Upon activation, the cell population progressively increased in size to large blasts, with the rapid appearance on all of the large dividing cells of the antigen recognized by OKT9, the transferrin receptor. Cells within the population continued to express the common peripheral T-cell antigens bound by OKT3 and UCHT1, and also the antigen bound by 3A1, but never the antigen bound by OKT6, a thymic cell marker. From the time of activation an increasing proportion of the T cells, up to 80%, expressed the antigen detected with OKIa and FMC4, which recognise nonpolymorphic Ia determinants. This sequence of events was followed by a general decrease in size of the cell population, a process accompanied by further phenotypic changes. The percentage of cells expressing Ia antigens decreased, but most striking was the rapid change in the OKT4:OKT8 ratio of cells within the population, from 60:40 to 40:60. Thereafter the proportions of OKT4⁺ to OKT8⁺ cells within the cultures remained relatively stable and it is suggested that these data provide evidence for a possible change in phenotype of cultured human T lymphoblasts, from OKT4 to OKT8.     

Characterization of interleukin-2-initiated versus OKT3-initiated human tumor-infiltrating lymphocytes from glioblastoma multiforme: Growth characteristics,cytolytic activity,and cell phenotype

Summary Outgrowth of tumor-infiltrating lymphocytes (TIL) from the human primary brain tumor glioblastoma multiforme was achieved by OKT3 initiation (10 ng/ml), followed by sustained expansion by interleukin-2 (IL-2; 200 U/ml). Tumor-infiltrating lymphocyte (TIL) initiation by this process was performed in parallel with the standard IL-2-only method. Of ten tumors, seven yielded TIL in response to OKT3/IL-2, whereas only three of these seven grew after initiation with IL-2 alone. On the basis of cell doubling times, at least 60 doublings, resulting in (hypothetically) up to 10²³ TIL from as few as 2 × 10⁵ cells in tumor suspensions, could be achieved using OKT3/IL-2. OKT3-initiated TIL proliferated in culture for as long as 288 days, although senescence of some cultures occurred at as early as 73 days. Significant heterogeneity of lymphocytes infiltrating the fresh tumors and heterogeneity of resultant TIL phenotype and function were apparent, yet several common trends were noted. In all cases after OKT3 initiation, significant net growth was not apparent until approximately 14 days. In contrast, in the three samples that grew in response to IL-2 alone, log-phase growth was always observed earlier. During the early phase of the cultures, all TIL expressed some killing activity toward a broad spectrum of tumors, including the autologous tumor. No consistent preference of TIL for lysis of autologous tumor was observed. Glioblastoma multiforme TIL cultures contained a mixture of CD8⁺ and CD4⁺ cells, with few CD16⁺ or NKH-1⁺. Of the six TIL examined in detail for population phenotype in relationship to time in culture, four eventually became exclusively CD4⁺. Further analysis of these CD4⁺ TIL indicated that all were of the helper-inducer class, 4B4⁺ and 2H4^–. Concurrent with the decline in CD8⁺ cells, a decline in the cytolytic activity of these TIL cultures occurred. Furthermore, in two TIL that remained CD8⁺, a decline in the cytolytic activity also occurred. Therefore, loss of killing activity was not merely a reflection of the major cell phenotype changes. These results indicate that the OKT3/IL-2 process provides an alternative to IL-2 alone for TIL initiation and growth, as well as providing a novel system for further analysis of tumorderived lymphocyte and accessory cell functional potential.This work was supported by a grant from the Dunn Foundation, Houston, Texas     

A randomized trial to evaluate the immunorestorative properties of thymostimulin in patients with Hodgkin's disease in complete remission

Summary A total of 19 Hodgkin's disease (HD) patients (12 male, 7 female) aged 26–67 years, who had been in complete unmaintained remission for 6 months or more when the study was initiated, were randomly given 50 mg thymostimulin (TS) i.m. daily (G1) or every other day (G2) for 35 days. A third group (G3) was not treated. Then TS, at the same dose was administered twice a week for the following 22 weeks in patients both initially receiving loading or intermittent TS treatment. When compared with age-and sex-matched controls, as a group, the patients' circulating OKT ₃ ⁺ , OKT ₄ ⁺ , OKT ₁₁ ⁺ and E-AETR⁺ cells were depressed (P<0.001 for both proportions and absolute numbers), whereas their OKT ₈ ⁺ cell population was not. Following 5 weeks of daily TS administration, the proportions and numbers of all T cell fractions significantly increased in G1 patients (P<0.03 for all the comparisons tested), while following intermittent TS treatment (G2) only the proportions of OKT ₃ ⁺ and OKT ₁₁ ⁺ cells (P<0.03), but not of other T cell fractions, significantly increased. In addition, no significant changes in the absolute numbers of T cell fractions were observed in this group of patients. Furthermore, no spontaneous variations in the T cell pool size occurred in untreated patients. TS maintenance therapy did not produce any further improvement in the size of overall T cells and T cell subsets but sustained percentage and absolute numbers of these cells during administration and the absolute number of T cells even after discontinuation of therapy. The TS-induced improvement in the T cell pool was not associated with any change in the size of circulating non-T lymphocytes and monocytes. In vitro phytohemagglutinin-induced interleukin-2 (IL-2) and gamma-interferon (IFN-) synthesis was assessed in 11 patients (3 G1, 4 G2, and 4 G3). Although it was not statistically significant, a rise in IL-2 and IFN- production was observed in TS-treated patients, but not in untreated controls. TS failed to exert any effect on the serum circulating levels of neopterin, type I and II IFN, beta-2 microglobulin (B₂-M) and immunoglobulins (Ig). TS can thus improve defective T cell frequences and numbers and may modulate IL-2 and IFN- production.     

Endothelial Cell Stimulation Overcomes Restriction and Promotes Productive and Latent HIV-1 Infection of Resting CD4+ T Cells

Highly active antiretroviral therapy (HAART) is able to suppress human immunodeficiency virus type 1 (HIV-1) to undetectable levels in the majority of patients, but eradication has not been achieved because latent viral reservoirs persist, particularly in resting CD4⁺ T lymphocytes. It is generally understood that HIV-1 does not efficiently infect resting CD4⁺ T cells, and latent infection in those cells may arise when infected CD4⁺ T lymphoblasts return to resting state. In this study, we found that stimulation by endothelial cells can render resting CD4⁺ T cells permissible for direct HIV infection, including both productive and latent infection. These stimulated T cells remain largely phenotypically unactivated and show a lower death rate than activated T cells, which promotes the survival of infected cells. The stimulation by endothelial cells does not involve interleukin 7 (IL-7), IL-15, CCL19, or CCL21. Endothelial cells line the lymphatic vessels in the lymphoid tissues and have frequent interactions with T cells in vivo. Our study proposes a new mechanism for infection of resting CD4⁺ T cells in vivo and a new mechanism for latent infection in resting CD4⁺ T cells.     

Distinct classes of human T-cell activation antigens

The characterization of three groups of antigens expressed by activated human T lymphocytes and detected by monoclonal antibodies is reported. Antigens defined by OKT19, OKT21, and OKT22 do not appear on in vitro activated T cells until increases in DNA synthesis become apparent and are not detected on most Interleukin 2 (IL-2)-independent cell lines and normal peripheral blood lymphocytes, monocytes, and granulocytes. Cell surface molecules reactive with the monoclonal antibodies OKT23 and OKT24 are displayed prior to any notable increase in DNA synthesis and are present on IL-2 independent cell lines, irrespective of lineage. T23 and T24 do not appear on peripheral blood cells and their distribution more closely resembles that of the T9 antigen (the receptor for transferrin) than antigens of the other groups. The third group of antigens, T14 and T20, have been classified as "early" antigens relative to DNA synthesis. They are expressed by distinct populations of normal lymphoid cells as well as by some IL-2-independent cell lines. Display of each group of activation antigens on T lymphocytes can be induced by either phytohemagglutinin, purified protein derivative from tuberculin, or allogeneic non-T cells, is not restricted to the OKT4+ or OKT8+ subsets, and is predominant on cells exhibiting the light-scattering properties of blast cells. The relative lack of expression of these antigens among normal peripheral blood cells make them attractive candidates for identifying changes in the status of immune activation.     

Lysis of herpes simplex virus-infected targets: II. Nature of the effector cells

Peripheral blood lymphocytes (PBL) from patients with herpes simplex virus (HSV)-1 recurrences in the cornea only (Group I) exhibited reduced lysis of HSV-1-infected targets compared to PBL from patients with oral-facial and corneal HSV recurrences (Group II). The cytotoxic lymphocytes appeared to belong to a subpopulation of natural killer (NK-HSV) cells. Monoclonal antibodies to human lymphocyte differentiation antigens were used to define the surface phenotype of the NK-HSV cells. Most of the NK-HSV activity was mediated by lymphocytes expressing the surface markers Leu-7⁺ (HNK-1), OKT3⁺ (pan T), OKM1⁺ (myeloid and NK), Leu-2^? (cytotoxic/ suppressor T cell), and Leu-8^? (regulatory T cell). In contrast, lysis of K562 cells (NK-K562) was mediated by lymphocytes bearing the surface phenotype Leu-7⁺, OKT3^?, OKM1⁺, Leu-2^+/?, and Leu-8^?. The low level of NK-HSV activity in PBL from Group I donors appeared to be due to active suppression by suppressor T lymphocytes. Depletion of Leu-2⁺ cells from PBL of Group I donors resulted in significant augmentation of NK-HSV activity. Similar treatment of PBL from Group II donors either had no effect or slightly diminished the NK-HSV activity.     

Further characterization of the autologous mixed lymphocyte reaction: induction of double negative gamma delta T lymphocytes.

To investigate the specific nature of the autologous mixed lymphocyte reaction (AMLR), we applied a method in which mixtures of NY-nonadherent responder cells and NY-adherent stimulator cells were treated with neuraminidase before culture and then cultured to assay the AMLR. This method produced a marked enhancement of DNA replication in the responder cells and the results were reproducible, regardless of the individuals tested. Using this method, we were able to make the following observations regarding the specific nature of the AMLR. (i) The AMLR is an IL-2-independent reaction, as revealed by bioassay to detect the presence of IL-2 by a blocking test using anti-IL-2R sera and as shown by the absence of mRNA for IL-2 in Northern hybridization. (ii) It is also HLA-DR dependent as proven by the fact that anti-DR sera almost completely inhibited the reaction. (iii) The AMLR was also found to induce the generation of activated CD4+ helper T cells in direct response to stimulation by NY-adherent cells, in which HLA-DR antigens were involved. (iv) Also, it induced the generation of CD4-CD8- double-negative (DN) lymphocytes, including gamma delta T cells with a cytotoxic activity against NK-resistant target cells and with a variety of lymphocyte activation markers (CD56, HLA-DR, CD25, transferrin receptors, CD38, and LFA-1). However, the AMLR did not induce the generation of NK cell markers CD16 and CD57. (v) The DN lymphocytes and gamma delta T cells appeared to be generated from the precursors of CD4-CD8- DN cells, in direct response to the stimulator cells. These results strongly suggest that the AMLR may be a phenomenon which induces the proliferative response of gamma delta T cells and their precursors, in addition to that of alpha beta T cells, particularly of CD4+ helper T cells.     

Polyclonal activation of human lymphocytes and induction of cytotoxic lymphocytes by streptococcal preparations

Polyclonal activation of human peripheral blood lymphocytes (PBLs)in vitro by preparations ofStreptococcus pyogenes Su strain (OK-432) and other heat-killed strains was investigated. The streptococcal preparations tested induce a proliferative response of PBLs via interleukin-2 (IL-2)-independent pathways. The proliferative response is accompanied by the generation of lymphoblastic cells (LBCs), which consist of heterologous lymphocyte populations: CD4⁺ helper type of T cells, and CD4^–CD8^– double-negative (DN) lymphocytes, including both CD3⁺ TcR ⁺ T cells and CD2⁺CD3^– immature type of T or non-T cell type of lymphocytes. Almost all the LBCs express Leu19, TfR (transferrin receptor), LFA-1 and CD38 (OKT10) antigens, which are expressed on activated T cells, NK cells and some other lymphocytes. The proliferative response of human PBLs is also accompanied by the generation of potent cytotoxic activity against NK-sensitive and -resistant targets. C-dependent cytolysis and cell sorting experiments of OK-432-activated LBCs revealed that both CD3⁺ and CD3^– types of CD4^–CD8^– DN lymphocytes, but not CD4⁺ helper T cells, may be major populations responsible for the cytotoxicity induced. On the other hand, CD4^–CD8 T cells may be required for the proliferation of PBLs and generation of cytotoxic effector cells. These results suggest that the OK-432 and other streptococcal preparations stimulate the human PBLsin vitro to induce the proliferation/activation of CD4⁺ T cells, mediating the following generation of DN cytotoxic effector lymphocytes.     

Interleukin-13 secretion by normal and posttransplant T lymphocytes; in vitro studies of cellular immune responses in the presence of acute leukaemia blast cells

T lymphocyte secretion of interleukin-13 (IL-13) in response to different activation signals was characterized in vitro. IL-13 release was investigated when virus transformed B lymphocytes or acute myelogenous leukaemia (AML) blasts were used as accessory cells during T cell activation. First, a majority of both CD4⁺ and CD8⁺ TCRαβ⁺ T lymphocyte clones, derived from normal individuals and bone marrow transplant recipients, secreted IL-13 in response to a standardized mitogenic activation signal (phytohaemagglutinin+IL-2+ B lymphocyte accessory cells). The CD4⁺ cells showed significantly higher IL-13 levels than the CD8⁺ subsets. Second, when leukaemic accessory cells (more than 95% AML blasts) were used during T cell activation, IL-13 was released both during alloactivation of normal T lymphocytes and during mitogen activation of posttransplant T cells. Third, when normal T lymphocytes were stimulated with allogeneic AML blasts, addition of IL-13-neutralizing monoclonal antibodies decreased interferon γ levels. Although addition of IL-13-neutralizing antibodies did not alter granulocyte-colony-stimulating factor secretion by allostimulating AML blasts, altered blast proliferation was detected for certain patients. Thus, most T cell clones can release IL-13, and IL-13 can modulate cytokine responses during T cell recognition of allogeneic AML cells. Received: 24 April 1997 / Accepted: 24 July 1997