Stimulation of Subpopulations of Human Lymphocytes by a Vaccine Strain of Francisella tularensis

When purified T lymphocytes from individuals vaccinated with a viable, attenuated strain of Francisella tularensis were incubated in vitro in the presence of heat-killed bacteria or a membrane preparation of the vaccine strain, they were stimulated to form blast cells and to synthesize deoxyribonucleic acid. The blast cells had the characteristics of T cells, being devoid of surface immunoglobulin and able to form rosettes with sheep erythrocytes. The stimulation occurred only when monocytes were present. A lymphocyte preparation enriched in B lymphocytes did not respond to the heat-killed bacteria or to the membrane preparation. In a stimulated mononuclear leukocyte preparation, about 70% of the blast cells formed rosettes with sheep erythrocytes, and 10 to 20% of them had surface immunoglobulin. The results show that there is an enlarged population of specifically committed T lymphocytes after tularemia vaccination. It is suggested that the lymphocyte stimulation test measures mainly T-lymphocyte reactivity when membranes or whole bacteria of F. tularensis LVS are used as antigen, and that the stimulation of human T lymphocytes by whole bacteria or bacterial membranes is completely monocyte or macrophage dependent. The present experimental procedure may provide a model for study of antigen-induced stimulation of human lymphocytes under controlled conditions. The technique used gave a reproducible, extremely purified preparation of T lymphocytes and a preparation of monocytes especially suitable for microcultures.     

Interaction of human lymphocytes with fluid phase human C3b detected by immunofluorescence

Human lymphocytes obtained from tonsils and peripheral blood were found to bind human fluid phase C3b, obtained by trypsin treatment. This binding was detected by indirect immunofluorescence (IIF) using specific anti-C3 antisera. Lymphocytes isolated from thymus tissue scored low percentages in IIF, indicating that the main population of thymus-derived lymphocytes are T cells. The distribution pattern of C3b-binding cells was compared with that of cells forming rosettes with sheep erythrocytes coated with antibody and complement (EAC) and with sheep erythrocytes (E) only, as well as with that of Ig- bearing lymphocytes, as detected by direct immunofluorescence. It appeared that the distribution pattern of lymphocytes which can bind fluid phase C3b is similar to that of EAC rosette-forming and of Ig-bearing lymphocytes. Pre- incubation of the lymphocytes with C3b and pretreatment of the cells with trypsin decreased the capacity to form rosettes and to bind C3b to their sur- face. Human monocytes, granulocytes and erythrocytes did not bind fluid phase C3b, as judged by IIF. Therefore, the selective binding of fluid phase C3b to lymphocytes provides a specific method for the detection of complement-reactive lymphocytes in lymphoid cell preparations.     

T- and B-lymphocyte subpopulations following radiotherapy for invasive squamous cell carcinoma of the uterine cervix.

Lymphocyte subpopulations in patients with cervical carcinoma were studied before and after radiotherapy. T lymphocytes were recognized by their ability to form spontaneous rosettes with sheep erythrocytes (E rosettes). Two surface marker characteristics were used to detect B lymphocytes: receptors for activated complement responsible for erythrocyte--antibody--complement (EAC) rosette formation, and surface membrane immunoglobulin (SmIg), readily stainable by immunofluorescence. We have demonstrated a significant depression of total lymphocytes after radiotherapy which persists for more than 5 years. This reduction in lymphocytes is due to a loss of E rosette-forming T cells, SmIg-bearing B cells and null cells. Absolute numbers of EAC rosette-forming B cells are not altered by treatment, and there is a rise in this cell type when the results are expressed as percentages of the total lymphocyte count. The possible functional immunological significance of these changes is discussed.     

Localization of human T lymphocytes in tissue sections by a rosetting technique.

A technique for the formation of sheep erythrocyte (E) rosettes in frozen human tissue sections is reported. The labile nature of the receptor for E rosettes on lymphocytes requires the use of controlled conditions for tissue processing and the reaction with indicator cells. The distribution of E rosettes in sections of normal human thymus, lymph nodes, tonsils, and spleens was comparable to that of the T marker-positive cells identified by immunofluorescence with the specific anti-human T cell serum. There with no overlap with areas positive for 19S EAC and 7S EA rosettes. Erythrocytes treated with a sulfhydryl reagent, 2-aminoethylisothiuronium bromide (AET), and with neuraminidase formed better rosettes in sections than did untreated erythrocytes. E rosettes in tissue sections can determine changes in the distribution of T cells in different lymphoproliferative and infiltrating disorders.     

Lymphocyte subpopulations in peripheral blood of healthy persons. Characterization by surface markers and lack of selection during purification

Lymphocytes were isolated at 99% purity from peripheral blood of healthy persons by defibrination, gelatine sedimentation, treatment with carbonyl iron powder and centrifugation on Ficoll–Isopaque. Subpopulations were identified by three surface markers: cells forming rosettes with sheep red blood cells (SRBC) (E-binding lymphocytes) as a measure of T lymphocytes; lymphocytes with surface immunoglobulin identified by indirect immunofluorescence (B lymphocytes); lymphocytes with receptors for C3 observed by the rosette method using SRBC treated with rabbit antiserum and human complement (EAC-binding lymphocytes).

The yield of lymphocytes after purification varied from 15 to 65%. No selection of lymphocytes was observed either by counting immunoglobulin-bearing and EAC-binding lymphocytes in whole blood and in purified cells from the same sample, or by statistical analysis of lymphocytes in subpopulations as a function of the yields from twenty-six experiments. In the absence of selection during purification the total numbers of T and B lymphocytes could be calculated from the percentages and the total numbers of lymphocytes. Our normal values are close to those reported using other non-selective methods of purification.

When lymphocytes were simultaneously stained for immunoglobulin and rosetted with EAC, cells bearing either or both markers were found. In total, 27–35% cells were identified by these markers. Since about 70% of the cells were E-binding, practically all lymphocytes could be identified. A small overlap between E-binding and immunoglobulin-bearing/EAC-binding lymphocytes may occur.

Either the IgM or the IgG-containing fractions obtained after fractionation of rabbit anti-SRBC serum on Sephadex G-200 could be used for sensitization of SRBC with complement. Formation of rosettes was not prevented by pretreating the lymphocytes with aggregated IgG, while rosettes formed with EA prepared by high concentrations of IgG antibody (Fc-binding lymphocytes) were abolished. It is concluded that rosettes formed with IgG-EAC (or whole serum EAC) using diluted antiserum identify complement-reactive lymphocytes and are not caused by synergism with Fc receptors. When SRBC were sensitized with varying dilutions of whole antiserum or its IgG fraction identical plateaus for the percentages of EAC-binding lymphocytes were found. Subagglutinating concentrations of the IgM fraction was insufficient to reach the plateau and also consistently resulted in lower values for EAC-binding lymphocytes.

     

In vitro immunoglobulin synthesis by lymphocytes from patients with rheumatoid arthritis. I. Effect of monocyte depletion and demonstration of an increased proportion of lymphocytes forming rosettes with mouse erythrocytes.

We have investigated B cell function in nine patients with rheumatoid arthritis (RA) compared to sex and age matched controls in a pokeweed mitogen driven system. Levels of IgG and IgM synthesized in the supernatant were measured by a competition ELISA. We have found that cultured mononuclear cells from RA patients showed a defective Ig synthesis when depleted of monocytes. In contrast RA mononuclear cells not depleted of monocytes produced substantial levels of Ig after stimulation by the mitogen. The percentages of T and B lymphocytes in the peripheral blood of RA patients were normal; however, an increased number of lymphocytes formed rosettes with mouse erythrocytes indicating an abnormality in the B cell pool. These results demonstrate defective in vitro immunoglobulin synthesis by RA lymphocytes and show the importance of monocytes in this culture system.     

Conditions for Cytomegalovirus Stimulation of Lymphocytes

Lymphocytes from healthy donors or from patients with chronic lymphocytic leukaemia were subjected to live or inactivated cytomegalovirus (CMV) or the mitogen phytohaemagglutinin. No early or late CMV antigens could be demonstrated in the lymphocytes, indicating that neither abortive nor replicative CMV infection takes place. Only cells from CMV antibody-positive leukaemic and non-leukaemic donors were stimulated by CMV to DNA synthesis, with a maximum on day 5. Cells from all individuals responded to phytohaemagglutinin stimulation, the peak of activity occurring on day 3. The stimulation with CMV occurred in T cells and was independent of early CMV antigen production, viral DNA synthesis, or viral replication. CMV is thus not an in vitro lymphocyte mitogen like Epstein-Barr virus but is a very potent antigen for memory T cells.     

Generation of activated lymphocytes. Analysis of giant SRBC rosettes.

Giant SRBC rosette-forming cells were detected in samples of mitogen-stimulated human peripheral blood lymphocytes. When the concentrations of mitogens were varied, the amount of [3H]TdR incorporated by the lymphocytes also varied. In general, the higher the amount of [3H]TdR incorporated, the higher were the percentages of giant rosettes. Hence the percentages of rosettes constituted a reliable index of mitogenic responses. Lymphocytes were stimulated by mitogens and cultured in the presence of cardiac glycosides or inhibitors of the synthesis of DNA, RNA or protein. The generation of giant SRBC rosette-forming cells was found to be dependent on RNA and protein synthesis and the integrity of membrane Na+ K+ ATPase, but not on DNA synthesis.     

Rosette formation with goat erythrocytes. A marker for human T lymphocytes.

We demonstrate the use of goat erythrocytes in a rosette procedure for the classification of human lymphocytes. The population is almost perfectly overlapping with the lymphocytes which form rosettes with sheep red blood cells. 70·2 ± 7·5% of peripheral lymphocytes form rosettes with goat erythrocytes and less than 1% of these cells have surface immunoglobulins. Enrichment of goat rosette-forming cells results in a population with an increased percentage of both goat and sheep rosettes. This population retains activity to the T-cell mitogens Con A and PHA, while the cells depleted of goat rosettes have greatly diminished responses to these same mitogens. Tonsil and spleen lymphocytes form 50·2 ± 6·8% and 24% of goat rosettes respectively, while peripheral blood lymphocytes from patients with CLL rarely form goat rosettes. Cell lines maintained in vitro rosetted with goat cells in a parallel fashion to sheep cells. Thus T-cell lines, such as Molt-3, which form rosettes with SRBC also rosette with GRBC, while sheep rosette-negative lines, i.e. Molt-4, are negative for both erythrocytes. B-lymphoid cell lines were negative, as were several lymphoma cell lines. There was a slight variation in the binding of goat cells, depending on the source of the goat. Thus, as in sheep rosettes, some animals were better sources than others, although all the animals tested formed rosettes.Human lymphocytes are capable of binding goat red cells. The cells which bind to the erythrocytes seem identical to those binding sheep red blood cells, and should be considered as a T-cell population. Preliminary inhibition data suggests that the receptor on T cells is the very same structure for both erythrocytes.     

Human T cell receptors for monkey erythrocytes.

To test the specificity of T cell receptors, erythrocytes and lymphocytes of man and rhesus monkey (Macaca mulatta) and erythrocytes of sheep were mixed in four different combinations to observe the rosette formation. During the study, a major proportion of human T cells formed spontaneous rosettes with the erythrocytes of rhesus monkey. A small number of monkey lymphocytes formed rosettes with human group O Rh-negative cells while T cells both of man and rhesus monkey formed rosettes with sheep red blood cells.     

Immunoglobulin secretion by human B lymphocytes exposed to herpes simplex type I antigen

Herpes simplex type I (HSV I) antigen was studied for its capacity to induce immunoglobulin(Ig)-secreting cells in human peripheral-blood lymphocytes. Ig-secreting cells were detected using an indirect hemolytic plaque assay. These results were compared to those obtained by using pokeweed mitogen (T-dependent) and Epstein-Barr virus (T-independent) to induce Ig-secreting cells. The HSV I induction of Ig synthesis was T-dependent, similar to the pokeweed-mitogen system. Peripheral-blood mononuclear cells depleted of T cells did not produce immunoglobulin. In contrast, the same T-depleted cell populations stimulated with Epstein-Barr virus (another member of the Herpes group) produced Ig. Lymphocytes in the HSV-I-stimulated cultures were found to secrete immunoglobulins of the IgA, IgG, and IgM classes. It was the lymphocytes from seropositive individuals that made Ig after HSV I stimulation. Such responses suggest that this system may be antigen-specific. This T-dependent virally induced Ig synthesis system may be useful in studying immunodeficient and immunocompromised individuals.     

Stimulation of human lymphocytes by a vaccine strain of Francisella tularensis.

An immune response to Francisella tularensis was demonstrated in man by the lymphocyte stimulation test. Peripheral blood lymphocytes were obtained from 26 individuals vaccinated with a viable tularemia vaccine, from 29 unvaccinated individuals, and from two patients who had recently undergone tularemia. The lymphocytes were incubated in the presence of various dilutions of heat-killed bacteria of the vaccine strain. The bacteria induced a deoxyribonucleic acid synthesis in the lymphocytes from 18 of the vaccinated individuals and from the two patients which was higher than that in the lymphocytes from any of the unvaccinated individuals. The deoxyribonucleic acid synthesis was maximal after about 6 days of incubation irrespective of concentration of bacteria. Lymphocytes from vaccinated and unvaccinated individuals were stimulated by two unrelated agents, tuberculin purified protein derivative and pokeweed mitogen. Lymphocytes from the vaccinated individuals did not show a higher response to these agents than did those of the unvaccinated. This suggests that the lymphocyte response to the Francisella bacteria was not due to a nonspecific activation. The vaccine-induced lymphocyte stimulation did not correlate with serum antibodies agglutinating F. tularensis antigen.     

Heterogeneity of complement receptor expression on surface immunoglobulin-bearing cells from neonatal and adult mice

The ontogeny of acquisition of complement receptors (CR) on splenic B lymphocytes from mice of varying ages was examined. Reagents used for the identification of CR included antibody- and complement-coated erythrocytes (EAC), bacteria (BAC) and bacteria treated with complement alone (BC). When whole mouse serum was used as the source of Complement both EAC and BAC failed to bind to human erythrocytes (C3b receptor-negative). showed binding to Raji and Daudi cells (C3d receptor) and formed rosettes with human neutrophils (C3bi receptor). Therefore these reagents bore C3d and C3bi, but not intact C3b. Although EAC and BAC detected nearly equal percentages of CR lymphocytes in adult mice, BAC bound to a higher percentage of neonatal lymphocytes (8-1296) than did EAC (1-395). The ability of BAC to detect EAC-negative lymphocytes among neonatal spleen lymphocytes appeared to be due to the increased sensitivity of this bacterial reagent for binding to CR. Further evidence of this enhanced sensitivity was that BAC were less inhibitable than EAC by soluble antigen-antibody-complement complexes.     

Direct activity of human T lymphocytes and natural killer cells against Cryptococcus neoformans.

Lymphocytes constitute a critical component of host defenses against cryptococcosis. Previously, we demonstrated that human lymphocytes cultured with interleukin-2 formed conjugates with, and directly inhibited the growth of, Cryptococcus neoformans. Here, we explore the anticryptococcal activity of freshly isolated, highly purified populations of human peripheral blood lymphocytes. Lymphocytes were incubated with encapsulated C. neoformans for 24 h, after which the lymphocytes were lysed, dilutions and spread plates were made, and CFU were counted. Fungistasis was determined by comparing growth in wells with and without lymphocytes. Nylon wool-nonadherent peripheral blood mononuclear cells (NWNA PBMC) were highly fungistatic, even if either T cells or natural killer (NK) cells were depleted by panning. A mixed population of T cells and NK cells, obtained by rosetting NWNA PBMC with sheep erythrocytes, completely inhibited cryptococcal growth, whereas the nonrosetting cells had little fungistatic activity. CD4+, CD8+, and CD16/56+ lymphocytes, isolated by positive immunoselection, had potent growth-inhibitory activity. In contrast, purified B cells had no activity. Fungistasis was seen even in the absence of opsonins. Antifungal activity was markedly diminished when surface receptors on NWNA PBMC were cleaved by treatment with trypsin or bromelain. Supernatants from stimulated lymphocytes or concentrated lymphocyte sonicates were not active. Lymphocyte-mediated fungistasis was seen with two different strains of C. neoformans. CD4+, CD8+, and CD16/56+ lymphocytes formed conjugates with C. neoformans, as observed under Nomarski differential interference contrast microscopy and videomicroscopy. These data demonstrate that freshly isolated peripheral blood T cells and NK cells have the capacity to bind and directly inhibit the growth of C. neoformans.     

Spontaneous and PHA-induced rosetting of human blood, tonsil lymphocytes and MLC blasts with sheep, human and horse erythrocytes.

Compared to peripheral blood lymphocytes the ability of human tonsil T cells and MLC blasts to bind sheep, human and horse erythrocytes was found to be increased. Tonsil and MLC T cells were able to bind sheep red blood cells without any cold incubation, i.e. they were 'early' rosettes, and higher percentage of human and horse erythrocyte rosettes were formed by these cells. Low doses of phytohaemagglutinin increased the proportion of rosettes between peripheral blood, tonsil, MLC cells and human and horse erythrocytes. PHA acted only on T cells, and not on B cells, lymphoblastoid B and other cell lines. On the ground of the stronger rosetting property of MLC blasts and tonsil cells, it is likely that the T cells responsible for binding of horse and human erythrocytes after PHA treatment are 'early' or 'active' rosetting cells.     

Cytochemical reactions of normal and neoplastic lymphocytes.

Rosetting and non-rosetting lymphocytes collected from normal individuals were stained for the presence of beta-glucuronidase, periodic-acid Schiff activity, gamma glutamyl transpeptidase, acid phosphatase, and alpha-naphthyl butyrate esterase. Lymphocytes which formed rosettes with sheep erythrocytes and non-rosette forming lymphocytes contained cytochemical reaction products for all five stains. Beta-glucuronidase (P less than 0-02) and acid phosphatase (P less than 0-01) were more frequently found in rosette forming lymphocytes. However, non-rosetting cells were more frequently periodic-acid Schiff positive (P less than 0-001). Gamma-glutamyl transpeptidase and alpha-naphthyl butyrate esterase were present equally in rosette and non-rosette forming lymphocytes. In addition, 33 non-Hodgkin's lymphomas were studied for cell surface markers and cytochemical reactions. In 17 of 19 B cell lymphomas, there was a paucity of lymphocytes containing beta-glucuronidase. However, in three of four T cell proliferations, there were numerous lymphoid cells positive for beta-glucuronidase. The periodic-acid Schiff and acid phosphatase reactions varied greatly within B, T, and null cell lymphomas and thus were of little diagnostic value in determining the cell of origin of these neoplastic lymphoid cells.     

T and B lymphocytes in South American pemphigus foliaceus.

T and B lymphocytes were quantified in peripheral blood of thirty patients with South American pemphigus foliaceus according to their ability to form rosettes with sheep erythrocytes (E) or sheep erythrocytes sensitized with antibody an complement (EAC). When compared with the counts obtained from thirty normal subjects, a decrease was found in the total T-lymphocyte count (32-33+/-7-7 versus 46-7+/-8-7) and in the T functional lymphocyte count as detected by the active rosette test. The mean percentage of B lymphocytes within the total number of lymphocytes was not significantly different from that of normal subjects (24-0 +/- 8-2 versus 25-3+/-8-0). Lymph node sections from three pemphigus patients examined for E or EAC adherence showed depletion of T cells in the paracortical areas. The low percentage of E cells in the peripheral blood and the depletion of E cells in paracortical areas of lymph nodes from patients with South American pemphigus foliaceus may reflect an impaired cellular immunity.     

E and EAC rosetting lymphocytes in patients with carcinoma of bronchus I. Some parameters of the test and of its prognostic significance.

Using peripheral blood lymphocytes separated by a Ficoll method and suspended in saline, means of 77-1% (s.d. 5-2) E rosettes (T lymphocytes) and 20-1% (s.d. 6-7) EAC rosettes (B lymphocytes) have been obtained with normal healthy donors. Poorer E-rosette formation resulted from higher centrifugation speeds during the washing of lymphocytes or erythrocytes, insufficient chilling, or rough handling. The presence of 5% albumin in the final mixture stabilized the rosettes and brought a constant subpopulation of B lymphocytes into rosetting. In patients with bronchial carcinoma who, at the time of diagnosis, had E-rosette percentages below 1 s.d. of the mean for normal donors, the length of survival was significantly shorter than in those with normal or high values. The same was true for those in whom null cells were detected. In each case the correlation effect was mainly found in the group of patients with squamous carcinoma.     

Granulocytes are more sensitive than lymphocytes to changes in EA and EAC indicator cell quality

Granulocytes were more sensitive to age variation of EAC3b indicator cells than lymphocytes, and showed a significant decrease in rosette formation with EAC3b indicator cells more than 2 weeks old; whereas lymphocytes showed unchanged EAC3b rosette formation with indicator cells up to 4 weeks old. EAIgG rosette formation by granulocytes was less affected by the age of the indicator cells than by their degree of sensitization, with a 25% loss of rosettes after 1 dilution of the sensitizing antibody. Thus optimal sensitization is essential for granulocyte EAIgG rosette formation, and relatively fresh indicator cells for EAC3b rosette formation. Hemolysis of contaminating erythrocytes did not influence EAIgG or EAC3b rosette formation.     

Enzymatic modification of the lymphocyte surface

Lymphocytes were treated with hydrolytic enzymes primarily to assess whether such modified cells would give improved cytotoxicity reactions during tissue typing. Papain-treated and alpha-chymotrypsin-treated lymphocytes were approximately twice as sensitive as untreated cells in the microcytotoxicity test used, and this finding might be usefully exploited by immunological laboratories for purposes of cross-matching, HLA antibody screening and HLA-DR typing. Trypsin treatment promoted massive cell clumping, while neuraminidase treatment was responsible for indiscriminate cell death after exposure to rabbit serum. The capacity of lymphocytes to form rosettes with sheep erythrocytes was abolished after treatment with trypsin or alpha-chymotrypsin, but enhanced by papain or neuraminidase.