Characteristics of lymphoid subpopulations isolated from healthy subjects and those suffering from nonspecific ulcerative colitis

Results of the studies of functional activity of lymphocyte subpopulations T+G, T-G, "O" subpopulations isolated peripheral blood of healthy persons and those with nonspecific ulcerative colitis (NUC) have been presented in this paper. The functional activity of the cells was assessed by means of local xenogeneic graft-vs-host reaction performed on CBA mice and by determination of nonspecific esterase enzyme. It has been established that analogous lymphocyte subpopulations influence the graft-vs-host reaction development in different ways depending on the condition of cell donor state: health/pathology. "O" subpopulations isolated from healthy persons hampered the graft-vs-host reaction development, and on the contrary stimulated it in patients with NUC. The assay of nonspecific esterase in lymphocytes showed that the least activity was noted in T+G and "O" cells in nonspecific ulcerative colitis.     

Lymphatic tracing and T cell responses following oral vaccination with live Mycobacterium bovis (BCG)

Oral vaccination of mice with lipid-encapsulated Mycobacterium bovis bacille Calmette-Guérin (BCG) expands a subset of interferon-gamma (IFN-gamma)-secreting T cells and mediates protection against aerosol mycobacterial challenge. We have traced the movement of the live vaccine through the regional lymphatics of mice and monitored the resultant immune response. Six hours after oral vaccination BCG was detected in low numbers systemically and in draining lymphatic tissue. However, after 48 h, BCG was predominantly associated with alimentary tract lymphatic tissues, such as the cervical and mesenteric lymph nodes and Peyer's patches. Lymphocytes that produced IFN-gamma in response to PPD-B or BCG-pulsed dendritic cells predominated in the spleen and were almost exclusively CD4(+), CD44(+) and CD62L(-), thus resembling an effector memory T cell population. Despite the fact that an oral route was used for immunization, splenic IFN-gamma-secreting T cells in vaccinated mice did not express the mucosal homing antigens alpha(4)beta(7) integrin or alphaIEL (CD103). However, a proportion of BCG-specific CD4(+) T cells expressed the CD29 integrin (beta(1)) chain, potentially involved in lung homing function. Thus, oral priming with M. bovis BCG appears to induce a subset of spleen-resident CD4(+) T cells with the potential to provide protective immunity in the lung.     

Natural killer (NK)-like cytotoxic activity of allospecific T cell receptor-gamma,delta+ T cell clones. Distinct receptor-ligand interactions mediate NK-like and allospecific cytotoxicity

We recently generated a series of human alloantigen-specific, CD3+,TCR-gamma,delta+ clones by stimulating CD3+,CD4-,CD8- T cells from normal individuals with allogeneic lymphoblastoid cell lines (LCL). As reported previously, these clones display cytotoxic activity against their specific stimulators but not against irrelevant LCL. Further studies of these and other TCR-gamma,delta+ clones, described in this report, indicate that most but not all of these clones express the NK cell associated marker, NKH-1 or Leu-19, and kill NK-sensitive targets such as the K562 and Molt 4 lines, but not an irrelevant LCL or NK-resistant line, Raji. TCR-gamma,delta+ clones which lacked expression of Leu-19 lysed their allospecific targets but had no detectable NK activity. The allospecific cytotoxicity of Leu-19+ and Leu-19- clones was inhibited by mAb to CD3 or the TCR delta-chain. In contrast, the NK-like activity of the Leu-19+ clones was enhanced by these antibodies over a wide range of antibody concentration. Although mAb to LFA-1 markedly inhibited both the allospecific and NK-like activity of these clones, an HLA class I framework specific mAb (W6/32) had no effect on NK-like cytolysis but did inhibit allospecific killing, suggesting that the target structures on the surface of allospecific and NK-sensitive cells are distinct. The receptors utilized by the TCR-gamma,delta+ clones to recognize NK-sensitive and allospecific targets are also distinct, since killing of NK-sensitive targets was blocked by the presence of cold (unlabeled) NK-sensitive cells but not by cold allospecific targets, whereas allospecific cytolysis was inhibited by cold allospecific targets but not by NK-sensitive cells. We conclude that some CD3+,TCR-gamma,delta+ clones exhibit NK-like as well as allospecific killing and that these two activities are mediated by distinct receptor-ligand interactions.     

Immunoreceptor transmembrane peptides and their effect on natural killer (NK) cell cytotoxicity

Short peptides derived from the transmembrane sequence of NK activating receptors and associated molecules were tested in vitro for inhibition of NK cell cytotoxicity using a standard (51)Cr release assay in the absence or presence of peptides. NKL23 cell line was used as the NK effector and the target was the NKL23 sensitive 721.221 cell line. NKp46, NKp30, NKG2D and CD3-zeta peptides inhibited NK activity at higher concentration (100 microM) compared to controls by 6-13% (p<0.05). Modification of one non-effective peptide (NKP44) significantly enhanced inhibition by 30%, 17% and 11% at 100 microM, 50 microM and 10 microM respectively compared to controls. A T-cell antigen receptor-alpha chain transmembrane sequence derived peptide (CP) significantly inhibited NKL cell activation by 20-30% (p<0.05) at 50 microM and 100 microM concentrations compared to the control. The structural similarities between these immuno-receptors, and in particular the need for transmembrane electrostatic interactions for receptor function, provides the basis for current and future targeted therapeutic strategies.     

Suppression of BCG cell wall-induced delayed-type hypersensitivity by pretreatment with killed BCG: induction of nonspecific suppressor T cells by the adjuvant portion (MDP) and of specific suppressor T cells by the antigen portion (TAP)

We showed previously that antigen-nonspecific suppressor T cells induced by i.v. injection of heat-killed bacillus Calmette-Guérin (BCG) were involved in suppression of delayed-type hypersensitivity (DTH). We suggested that the adjuvant portion of BCG might be involved in the induction of these cells. In this report, we show that BCG cell wall-induced DTH responses in mice pretreated with muramyl dipeptide (MDP), a minimum adjuvant constituent of BCG, were suppressed nonspecifically. In addition, we show that pretreatment with tuberculin active peptide (TAP), the antigenic peptide from Mycobacterium tuberculosis, induces antigen-specific suppression of DTH responses. In both instances, suppression was shown to be due to non-adherent cells that act to inhibit elicitation of DTH. Furthermore, using the macrophage migration inhibition assay, an in vitro correlate of DTH, we found that antigen-nonspecific and antigen-specific suppressor T cells were induced by the injection of MDP and TAP, respectively. Thus, suppressor T cells induced by the adjuvant and antigen portions of BCG may act by interfering with the lymphokine-dependent mechanisms by which DTH effector T cells elicit DTH.     

K562 killing by K, IL 2-responsive NK, and T cells involves different effector cell post-binding trigger mechanisms

The monoclonal antibody 13.3 specifically blocks the trigger process of the NK-K562 cytolytic sequence at a post-binding effector cell level. This antibody was used to define differences in the lytic trigger processes of NK and other mechanisms of K562 lysis. Monoclonal antibody 13.3 inhibited lysis of K562 target cells by freshly isolated peripheral blood lymphocytes (PBL) and purified large granular lymphocytes (LGL), but had no inhibitory effect on antibody-dependent cell-mediated cytotoxicity to K562 by these effectors. Lectin-dependent cellular cytotoxicity (LDCC) to this target cell was also unresponsive to 13.3. The 13.3-induced inhibition of NK-K562 lytic activity persisted when PBL were activated in culture with interleukin 2 (IL 2) for periods up to 48 hr. After 48 hr of culture, the degree of inhibition diminished progressively in medium containing fetal calf serum but not in medium containing autologous serum. This 13.3-unresponsive lytic activity in cultured PBL could be attributed to more than one cell type and was present in both the LGL and Fc gamma receptor-depleted T cell fraction. Thus, K562 lysis by freshly isolated human lymphocytes via NK, K, and LDCC mechanisms is characterized by heterogeneity of the post-binding effector cell trigger mechanism. K562 lysis by lymphocytes cultured with IL 2 is similarly heterogeneous.     

Natural cell-mediated cytotoxicity in vitro and inhibition of tumor growth in vivo by murine lymphoid cells cultured with T cell growth factor (TCGF)

Summary Lymphoid cells obtained from the spleen, thymus, bone marrow, peripheral blood, and peritoneal exudate of normal mice (BALB/c, BALB/c nude, C57BL/6, C3H) and from spleens of mice bearing a transplantable lung carcinoma or primary mammary carcinoma were expanded in culture for 1–9 months, with an increase in cell number of 10⁵- to 10⁶-fold per month, in crude or lectin-depleted medium containing T cell growth factor (TCGF). All these cultured lymphoid cell (CLC) lines exhibited strong cytotoxic activity in vitro (assessed by ⁵¹Cr-release assays) toward a variety of freshly harvested and cultured syngeneic, allogeneic, and xenogeneic tumor target cells, both lymphoid and solid (including metastatic growths) in origin. Extensive killing was observed against tumor targets that were resistant to lysis by natural killer (NK) cells as well as to NK-sensitive tumor lines. Low levels of cytotoxic reactivity were also demonstrated against fresh and cultured normal lymphoid cells. The CLC had some characteristics of NK cells but also expressed some typical T cell markers. In local Winn-type neutralization assays, CLC delayed or completely inhibited the growth of lymphomas and carcinomas in syngeneic and allogeneic recipients. In mice with metastatic growth of a second-generation transplant of mammary carcinoma, CLC were shown to have some therapeutic effect when administered IV 1 day after cyclophosphamide. No significant beneficial action of IV administered CLC was observed in the absence of chemotherapy in mice implanted with a lung carcinoma. The possibilities of employing TCGF-propagated cytotoxic effector cells in adoptive immunotherapy of human malignancies are discussed.     

Total leukocytes, T cell subpopulation and natural killer (NK) cell activity in rats exposed to restraint stress

Rats were stressed by immobilization for 3 hrs daily for 11 days and either sacrificed immediately after the last stress session (chronic stress group) or allowed to recover for 12 days and then sacrificed (recovery group). After 11 days of stress, leukocytes and lymphocytes were significantly decreased and neutrophils and large granular lymphocytes were markedly increased. The number of total, helper and suppressor T cells was significantly decreased but the percentage of T cells remained unchanged. Natural Killer (NK) cell activity was unaffected. After a 12 day recovery period from stress, the number of leukocytes returned to normal but the percentage of neutrophils was now below baseline whereas the percentages of lymphocytes and large granular lymphocytes had increased significantly. The number and percentage of total T cells and helper T cells was enhanced and NK cell activity tended to be increased. Thus, chronic stress as well as recovery from stress can affect individual components of the cellular immune system quite selectively and differently. In addition, a comparison of the effects of stress seen in this study with healthy rats with those seen under identical conditions in tumor bearing rats shows that stress or recovery from stress can affect the immune system differently in healthy or tumor bearing animals.     

Mechanism of defective NK cell activity in patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex. II. Normal antibody-dependent cellular cytotoxicity (ADCC) mediated by effector cells defective in natural killer (NK) cytotoxicity

Our studies and other investigations have shown that NK effector cells can also mediate antibody-dependent cellular cytotoxicity (ADCC) through the use of the Fc gamma receptor on the NK cell membrane. Peripheral blood lymphocytes (PBL) derived from patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex exhibit a poor NK activity due to a defective "trigger" required for activation in the lethal hit stage of the NK lytic pathway. Consequently, it was important to delineate whether the defect in AIDS NK cells affected the ADCC function. By using the 51Cr-release assay, the ADCC cytotoxic activity of AIDS PBL was found to be within the normal range, despite the absence of significant NK activity. Several experiments corroborated that the same effector cells mediate both NK CMC and ADCC. Depletion of Fc gamma R-bearing cells resulted in elimination of both the ADCC and NK cytotoxic functions. Single cell analyses, using one- and two-target cell conjugates, revealed that the frequency of ADCC effector:target conjugates and the frequency of killer cells from AIDS PBL were comparable to the frequencies seen in the normal controls. However, when mixtures of NK and ADCC targets were used to form mixed two-target conjugates, the AIDS effector cells lysed only the bound ADCC target, whereas the normal effector cells lysed both the bound NK and ADCC targets. These results demonstrate clearly that the same NK/K effector cells from AIDS PBL, defective in NK activity, are not impaired in mediating ADCC activity. These findings were supported by the demonstration that AIDS PBL stimulated with ADCC targets, but not with NK targets, released NK cytotoxic factors, postulated mediators of the NK CMC reaction. These findings indicate that the NK/K cells in AIDS are triggered normally for ADCC activity but are not triggered for NK activity. Furthermore, the results indicate that the lytic machinery is not impaired in the AIDS NK/K cells.     

Changes of lymphoid cell cytotoxicity, shown in vitro, of mice bearing a syngeneic tumor after injection of killed tumor cells]

Lymphoid cells from mice bearing a syngeneic tumour show a cytotoxic activity in vitro, occuring in two succesive waves. Normally inactive during the second week after grafting, lymphoid cells have a marked killing ability during this period when heavily irradiated tumour cells have been injected on the 6th day. On the contrary, the cytotoxicity due to the graft, which is important from the 20th to the 30th day, is inhibited by injection of killed tumour cells 15 days before the test. Quantitative variations of the tumour antigen liberated in the animal may be responsible for those effects.     

Expression of nonspecific cross-reacting antigen species in myeloid leukemic patients and healthy subjects

The reactivity of two monoclonal antibodies recognizing NCA-95 and NCA-55 (MAb 47 and MAb 192, respectively) with a polyclonal anti-NCA serum in myeloid leukemic cells isolated by density gradient centrifugation was compared using an immunofluorescence test (IF). It was observed that the blood myeloid cells in 78.8% of the patients with different types of myelocytic leukemias and all granulocytes of 15 normal donors showed similar expression of the NCA species studied. The leukocytes of the remaining patients did not synthesize the NCA-95 species regardless of the maturation stage of the cells studied. In two patients, synthesis of this NCA form was limited to the fractions containing myelocytes and metamyelocytes. We have found that all anti-NCA antibodies studied recognized different antigenic epitopes in a myeloid cell series. A relationship between the patient's survival and the proportion of NCA-containing cells was also observed.     

Changes of dopamine-beta-hydroxylase activity during ontogenesis in healthy subjects and in an experimental model (rats)

In children and adolescents (250 healthy subjects) serum dopamine-beta-hydroxylase (DBH) activity (23.9+/-5.2 to 57.1+/-17.5 micromol/min/ml) increases with the age between 3-10 years, later it decreases approximately by the age of 10-14 years. At the age of 21 to 60 years DBH level is stable. Our study described decreasing DBH activity in adolescents at the age of 10-14 years in the studied sample of healthy persons. Experimental animals (200 Wistar rats, 5-120 days old) show the same trend of enzymatic activity, similarly as in humans. DBH activity in rats is between 0.85+/-0.1 to 2.8+/-0.05 micromol/min/ml. This activity is highest in 5-day-old rats; it decreases till the age of 14 days and increases mainly in 14- to 35-day-old animals. Decrease of DBH activity in rats between 35 to 40 days is significant and corresponds to the reduction of DBH activity in adolescent humans (10-14 years). Adult rats (aged 90-120 days) show a stable DBH activity. DBH activity intermediately decreases in 10- to 14-year-old children. This decrease corresponds to the intermediate developmental changes of electrophysiological parameters (decreasing EEG activity in healthy adolescents occurs in 10-14 years old children). Puberty is coupled with intermediate decreasing of DBH activity in man and also in experimental animals in the period of prominent psychological and physiological changes.     

Age related changes in T cell mediated immune response and effector memory to Respiratory Syncytial Virus (RSV) in healthy subjects

Respiratory syncytial virus (RSV) is the major pathogen causing respiratory disease in young infants and it is an important cause of serious illness in the elderly since the infection provides limited immune protection against reinfection. In order to explain this phenomenon, we investigated whether healthy adults of different age (20-40; 41-60 and > 60 years), have differences in central and effector memory, RSV-specific CD8+ T cell memory immune response and regulatory T cell expression status. In the peripheral blood of these donors, we were unable to detect any age related difference in term of central (CD45RA^-CCR7⁺) and effector (CD45RA^-CCR7^-) memory T cell frequency. On the contrary, we found a significant increase in immunosuppressive regulatory (CD4⁺25⁺FoxP3⁺) T cells (T_reg) in the elderly. An immunocytofluorimetric RSV pentamer analysis performed on these donors' peripheral blood mononuclear cells (PBMCs), in vitro sensitized against RSV antigen, revealed a marked decline in long-lasting RSV specific CD8+ memory T cell precursors expressing interleukin 7 receptor α (IL-7Rα), in the elderly. This effect was paralleled by a progressive switch from a Th1 (IFN-γ and TNF-α) to a Th2 (IL-10) functional phenotype. On the contrary, an increase in T_reg was observed with aging. The finding of T_reg over-expression status, a prominent Th2 response and an inefficient RSV-specific effector memory CD8+ T cell expansion in older donors could explain the poor protection against RSV reinfection and the increased risk to develop an RSV-related severe illness in this population. Our finding also lays the basis for new therapeutic perspectives that could limit or prevent severe RSV infection in elderly.     

A comparison of antibody-dependent cellular cytotoxicity (ADCC) mediated by murine and human lymphoid cell populations

Mouse lymphoid cells are known to lyse chicken red blood cells (CRBC) in the presence of antibody and in the absence of complement. They have also been reported to effect lysis of mouse tumor cells and other nucleated targets, although this has been disputed. Using a 4-hr ⁵¹Cr-release assay, we have compared the activity of mouse effector cells from the thymus, spleen, bone marrow, peritoneal cavity, and mesenteric and subcutaneous lymph nodes of many strains of mice to the activity of human lymphoid cell effectors against CRBC and a number of murine targets. Human effectors mediate lysis of all targets tested. Mouse effectors lyse CRBC, but usually less well than human effectors. Mouse cells from lymphoid organs were either very inefficient or completely inactive against nucleated mammalian targets under a range of test conditions. Interestingly, in experiments where cells from solid lymphoid organs or the peritoneal cavity were ineffective, peripheral blood lymphocytes from one subline of DBA/2 consistently gave significant lysis of EL4 targets, while cells from another subline of the same strain did not.     

On the mechanism of early recovery of specifically depleted lymphoid cell populations by nonspecific activation of T cells.

Specific depletion from normal CBA mouse spleen cells of those bound on pigeon erythrocyte (PRBC) immunoabsorbent columns before transfer of the depleted population into irradiated syngeneic recipients resulted in elimination of the anti-PRBC responsiveness as assessed by rosette (RFC) and hemolytic plaque (PFC) formation. The anti-sheep erythrocyte (SRBC) responses of cell populations treated in the same manner remained unimpaired. When, however, these populations were stimulated with both PRBC and muramyl dipeptide (MDP), an early recovery of specific anti-PRBC responsiveness was produced. PFC response in particular, suddenly increased between the fourth and fifth day after transfer and stimulation thus exhibiting a doubling time of only 4 to 6 hr. This effect of MDP was T-cell dependent since treatment of the depleted population with anti-θ antigen serum and complement hindered early recovery. Depleted populations stimulated with PRBC alone resumed their T-dependent RFC (but not PFC) responsiveness after the eighth day. In spite of the existence of these educated T cells, a second stimulation on the tenth day with PRBC was unable to elicit a specific PFC response. On the other hand stimulation with MDP alone on the day of cell transfer (Day 0) followed by stimulation with PRBC on Day 10 resulted in a specific PFC response on Day 15. Thus, MDP appeared to do more than simply promote education of T cells by antigen. In vitro cultures of depleted populations also recovered their specific reactivity when stimulated by antigen and MDP.     

Natural cell-mediated cytotoxicity in mice treated with total lymphoid irradiation (TLI)

Fractionated total lymphoid irradiation (TLI) of adult (BALB/c × C57BL/6)F₁ mice resulted in transiently augmented natural killer (NK) and natural cytotoxic (NC) cell activities. Thus, 1 day after completion of TLI, NK and NC activities in the spleens of treated mice were lower than controls but values increased and reached a maximum level of 23- to 190-fold above control at 6 days after irradiation, returning to normal levels 9 days later. Cytotoxicity was enhanced after removal of the plastic adherent population. No cytotoxicity was observed against P 815 target cells, which are sensitive to activated macrophages but not to NK. The significance of this modulation of natural cell-mediated cytotoxicity following TLI is discussed.     

Lymphocytes from children and infants mediate natural cell mediated cytotoxicity, (cytotoxicity)

Summary Lymphocytes from infants and young children were tested for natural cell mediated cytotoxicity (NCMC) against K562 and CCRF-CEM. NCMC by lymphocytes from pediatric donors of all ages was equivalent to that mediated by lymphocytes from adults. Since it has been suggested that the biological function of NCMC is to effect immunological surveillance against cancer, the appearance of NCMC effector cells early in development is consistent with early mobilization of the policing mechanism.This work was supported by Grant CA 25765 from the National Cancer InstitutePartially supported by a Grant from the Concern Foundation.     

Stimulated T cell and natural killer (NK) cell lines fail to synthesize leukotriene B4

The ability of leukotriene B4 (LTB4) to influence T cell and natural killer (NK) cell functions makes the question of LTB4 generation by these cells important to address. Consequently, LTB4 generation was evaluated in a human (Jurkat), and in a murine (EL-4) T cell line as well as in a rat NK cell line (RNK-16). Incubation of each of the 3 cell lines with [1-14C]arachidonic acid alone or in the presence of phytohemagglutinin (PHA), of calcium ionophore A23187, or of concanavalin A (Con A) plus the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) failed to generate radiolabelled LTB4 or other eicosanoids as determined by thin layer radiochromatography. Using two different radioimmunoassays for LTB4 also failed to demonstrate the generation of LTB4 under basal or stimulated conditions. These results support earlier studies that demonstrate that T cells are not capable of de novo synthesis of prostaglandins, thromboxanes, or leukotrienes and also provide evidence that NK cells also do not have the capacity to generate LTB4 or other eicosanoids. Our findings are also critically discussed in relation to studies claiming eicosanoid synthesis by T cells.