Induction Of Immunoglobulin Secretion Research Articles

Induction of immunoglobulin secretion and DNA synthesis in human lymphocytes in vitro by cytomegalovirus preparations.

Immunoglobulin secretion as evidenced by plaque-forming cells (PFC) in an indirect haemolysis-in-gel assay, and DNA synthesis were induced in human blood lymphocytes by the following preparations of cytomegalovirus (CMV): Nucleocapside Nc-CMV antigen, membrane M-CMV antigen, crude C-CMV preparations and CMV-incubated adherent cells. Peak stimulations occurred around day 6 in culture. Nc-CMV and M-CMV only stimulated PFC and DNA synthesis in lymphocytes from CMV seropositive individuals. C-CMV also stimulated lymphocytes from CMV seronegative individuals but gave better responses in lymphocytes from CMV seropositive individuals. CMV-incubated adherent cells occasionally stimulated lymphocytes from CMV seronegative individuals but always in CMV seropositive blood donors. Nc-, M-, and C-CMV gave high numbers of PFC in B cells enriched by sheep erythrocyte sedimentation and in co-cultures of enriched T and B cells. Almost no PFC were induced in enriched T cells. DNA synthesis induced by all the three antigenic CMV preparations increased after removal of adherent cells. Strong DNA synthesis was induced in enriched T cells compared to almost none in enriched B cells. It is concluded that some pure CMV preparations act as antigens and more crude preparations induce polyclonal activation. Different CMV preparations may be used to diagnose CMV, to study immune reactivity against CMV and as a model for CMV infections in vitro.

Mechanisms of B cell activation in patients with acquired immunodeficiency syndrome and related disorders. Contribution of antibody-producing B cells, of Epstein-Barr virus-infected B cells, and of immunoglobulin production induced by human T cell lymphotropic virus, type III/lymphadenopathy-associated virus.

Patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) have hyperimmunoglobulinemia and increased numbers of circulating immunoglobulin-secreting cells. In this paper, we studied the basis for this B cell hyperactivity. Limiting dilution studies of B cells from seven patients with ARC and four with AIDS revealed that some B cells spontaneously produced antibodies to human T cell lymphotropic virus, type III/lymphadenopathy-associated virus (HTLV-III/LAV) (39:10(6) and 7:10(6) B cells, respectively), suggesting that chronic antigenic stimulation by HTLV-III/LAV was one contributing factor. The patients also had an increased number of spontaneously outgrowing B cells than did normals (6:10(6) vs. less than 2:10(6) B cells), suggesting that they had an increased number of Epstein-Barr virus (EBV)-infected B cells. However, fewer B cells from patients were immortalized by exogenously added EBV than were B cells from normals. In additional studies, HTLV-III/LAV induced immunoglobulin secretion (mean 2,860 ng/ml) by peripheral blood mononuclear cells from normals; this HTLV-III/LAV-induced immunoglobulin secretion required the presence of both B and T cells. Thus, antigenic stimulation by HTLV-III/LAV, increased numbers of EBV-infected B cells, and HTLV-III/LAV-induced T cell-dependent B cell activation all contribute to the B cell hyperactivity in patients with HTLV-III/LAV disease.

Phorbol ester enhances both interleukin 2 receptor expression and immunoglobulin secretion in human Epstein-Barr virus-immortalized B cells.

The enhanced expression of interleukin 2 (IL2) receptors by 12-O-tetradecanoylphorbol 13-acetate (TPA) on sublines of an Epstein-Barr virus-immortalized human B17 B cell line (M. Steinitz et al., Immunobiology 1979. 156: 41.) was studied by immunofluorescence using the anti-Tac monoclonal antibody and by binding studies with purified radiolabeled IL2. These studies show that TPA at a final concentration of 5 ng/ml greatly increased Tac antigen expression on a number of sublines of B17. IL2-binding studies revealed that TPA induced an increase in not only the number of IL2 receptors per cell but also the affinity of the receptors for IL2. The number and affinity of IL2 receptors on the C76 subline treated with TPA appear to be similar to those of activated normal human peripheral T cells. Furthermore, TPA-induced differentiation of these B cell lines was measured by induction of immunoglobulin secretion using an enzyme-linked immunosorbent assay. The capacity of TPA to induce differentiation in human B cells and the biological significance of IL2 receptor expression by activated B cells are discussed.

Modification of Epstein-Barr virus replication by tunicamycin.

The effect of tunicamycin, which inhibits N-linked glycosylation, on the replication of Epstein-Barr virus was examined. Tunicamycin markedly reduced the yield of virus from producing cells. At concentrations of 1 to 2 micrograms of tunicamycin per ml, there was a buildup of intracellular virus in P3HR1-Cl13 cells but not in MCUV5 cells; at a concentration of 5 micrograms of tunicamycin per ml in P3HR1-Cl13 cells, viral DNA synthesis was inhibited as well. Viral glycoproteins lacking N-linked sugars were apparently inserted into the cell membrane, and the small amount of virus made in the presence of drug was able to bind specifically to its receptor on B cells. However, the ability of the virus to induce immunoglobulin secretion by fresh human lymphocytes was impaired. This implies a role for viral glycoproteins in the penetration as well as the attachment of virus.

Induction of Immunoglobulin Secretion in Follicular Non-Hodgkin’s Lymphomas: Role of Immunoregulatory T Cells

B cell neoplasms are clonal expansions of B lymphocytes thought to be frozen at various points along the normal B cell differentiation pathway. We studied cell suspensions from lymph nodes involved by follicular (nodular) non-Hodgkin's lymphoma to determine the capacity of the malignant B cells to secrete immunoglobulin (Ig). Neoplastic B cells from all 14 follicular lymphomas secreted monoclonal immunoglobulin in culture when appropriate signals were provided. In most cases, maximal Ig secretion occurred when autologous T cells were removed by E rosette depletion, replaced with allogeneic normal T cells, and the cultures were exposed to 12-O-tetradecanoylphorbol-13-acetate. Autologous T cells exerted a suppressor effect on Ig secretion in 8/8 cases studied, diminishing the response of the malignant B cells to allogeneic T cells. This suppressor effect did not correlate with the percentage of cells staining with anti-Leu-2a or with "helper-suppressor" (Leu-3a-Leu-2a) ratios of the lymph node T cells. Our findings demonstrate that the arrested differentiation of most follicular lymphomas is reversible and implicate a T cell-mediated host immunoregulatory mechanism affecting Ig secretion in vivo. An additional contribution of our results is the demonstration of a cell culture system for synthesis of sufficient monoclonal Ig for use as an immunogen in production of anti-idiotype antibodies.

Induction of immunoglobulin secretion in follicular non-Hodgkin's lymphomas: role of immunoregulatory T cells

B cell neoplasms are clonal expansions of B lymphocytes thought to be frozen at various points along the normal B cell differentiation pathway. We studied cell suspensions from lymph nodes involved by follicular (nodular) non-Hodgkin's lymphoma to determine the capacity of the malignant B cells to secrete immunoglobulin (Ig). Neoplastic B cells from all 14 follicular lymphomas secreted monoclonal immunoglobulin in culture when appropriate signals were provided. In most cases, maximal Ig secretion occurred when autologous T cells were removed by E rosette depletion, replaced with allogeneic normal T cells, and the cultures were exposed to 12-O-tetradecanoylphorbol-13- acetate. Autologous T cells exerted a suppressor effect on Ig secretion in 8/8 cases studied, diminishing the response of the malignant B cells to allogeneic T cells. This suppressor effect did not correlate with the percentage of cells staining with anti-Leu-2a or with “helper- suppressor” (Leu-3a-Leu-2a) ratios of the lymph node T cells. Our findings demonstrate that the arrested differentiation of most follicular lymphomas is reversible and implicate a T cell-mediated host immunoregulatory mechanism affecting Ig secretion in vivo. An additional contribution of our results is the demonstration of a cell culture system for synthesis of sufficient monoclonal Ig for use as an immunogen in production of anti-idiotype antibodies.

Mycoplasma mimicry of lymphokine activity in T-cell lines.

During screening of T-T hybridomas for B-cell-stimulating factors (BSF) we found in the conditioned medium of such a line (TUH-14) an activity that turned out to cosediment with a contaminating strain of Mycoplasma arginini on centrifugation. Since it only affected B blasts and only to a minor extent small cells and since it did not induce immunoglobulin secretion, the mycoplasma strain seemed to mimic a BSF. M. arginini was also found in two other T-T hybridoma producer lines but not in a clone of the EL-4 line. Three other M. arginini isolates were not stimulatory for B cells. The presented results point towards the importance of careful control for mycoplasma contamination in established cell lines.

Age-related changes in mechanisms accounting for low levels of polyclonally induced immunoglobulin secretion in humans

The mechanisms accounting for low levels of T-cell-dependent polyclonal IgG secretion in old and young human adults were compared. In elderly donors, in contrast to young donors, low levels of IgG secretion by unfractionated mononuclear cells (MNCs) did not correlate with functional suppressor activity of “panning”-enriched T8 + cells. Levels of IgG secretion by MNCs did not correlate either with proportions of T8 + or T4 + cells in the MNC population or with the functional helper activity of panning-enriched T4 + cells, in either old or young age groups. In young donors suppression mediated by either 2 or 5 × 10 4 T8 + cells of “low” IgG secretors (80.2 ± 4.8 and 90.2 ± 1.9%) was significantly greater than that induced by T8 + cells of “high” secretors (41.8 ± 5.7 and 71.0 ± 4.0%). Among elderly donors, suppression mediated by either 2 or 5 × 10 4 T8 + cells was reduced in both the low IgG secretion (31.9 ± 5.3 and 52.3 ± 7.0%) and high IgG secreting (14.9 ± 7.2 and 46.8 ± 5.7%) compared to the corresponding young donor subgroup. Levels of suppression mediated by 2 × 10 4 T8 + cells were suggestively lower in the high IgG secreting elderly subgroup compared to the low secreting subgroup ( P < 0.1). Our data suggest that decreased suppressor activity is a generalized occurrence in aging and that low polyclonal T-dependent MNC IgG secretion in the elderly, in contrast to young adults, cannot be attributed to high T-suppressor activity; nor does it usually reflect defective T-helper activity.

Transferrin receptor induction is required for human B-lymphocyte activation but not for immunoglobulin secretion.

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. In the experiments reported here we have examined the regulation of transferrin receptor expression on activated human B cells and whether or not these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We have determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogen-stimulated T cells (probably B-cell growth factor). This expression is required for proliferation to occur, since antibody to transferrin receptor (42/6) blocks B-cell proliferation. Induction of immunoglobulin secretion, however, although dependent on PHA-treated T-cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen-stimulated cells whose proliferation has been blocked by antitransferrin receptor antibody. In addition, we have demonstrated that IgM messenger RNA induction following mitogen stimulation is unaffected by antitransferrin receptor antibody. These findings support a model for B-cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B-cell growth factor and transferrin receptor expression, for proliferation, and a second, dependent on T cell-derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.

The role of the transferrin receptor in human B lymphocyte activation.

Transferrin receptors are expressed on proliferating cells and are required for their growth. Transferrin receptors can be detected after, but not before, mitogenic stimulation of normal peripheral blood T and B cells. T cells demonstrate a functional requirement for transferrin receptors in the activation process. These receptors, in turn, are induced to appear by T cell growth factor (interleukin 2). In the experiments reported here, we examined the regulation of transferrin receptor expression on activated human B cells and whether these receptors are necessary for activation to occur. Activation was assessed by studying both proliferation and immunoglobulin secretion. We determined that transferrin receptor expression on B cells is regulated by a factor contained in supernatants of mitogen-stimulated T cells (probably B cell growth factor). This expression is required for proliferation to occur, because antibody to transferrin receptor (42/6) blocks B cell proliferation. Induction of immunoglobulin secretion, however, although dependent on phytohemagglutinin-treated T cell supernatant, is not dependent on transferrin receptor expression and can occur in mitogen-stimulated cells whose proliferation has been blocked by anti-transferrin receptor antibody. These findings support a model for B cell activation in which mitogen (or antigen) delivers two concurrent but distinct signals to B cells: one, dependent on B cell growth factor and transferrin receptor expression, for proliferation; and a second, dependent on T cell-derived factors and not requiring transferrin receptors, which leads to immunoglobulin secretion.

γ-Interferon is one of several direct B cell-maturing lymphokines

Two classes of molecules often released after the interaction of T lymphocytes, macrophages and antigen are B-cell maturation factors (BMF)1-3 and immune (gamma) interferon (IFN-gamma)4-7. BMFs directly induce the maturation of resting B lymphocytes to the state of active immunoglobulin secretion, while IFN-gamma is defined by the reduction of viral infectivity in vitro. However, interferons have been shown to have a variety of effects and they have also been reported both to increase and decrease B-cell differentiation in intact animals and complex cellular mixtures in vitro. Here we show that murine IFN-gamma produced by recombinant DNA technology shows similar biological effects to BMFs from two other sources. All three preparations induce immunoglobulin secretion by both normal resting murine splenic B cells and the comparable B-cell tumour line WEHI-279.1 (refs 1, 3). IFN-gamma and the other two BMFs are not identical, however, as anti-IFN-gamma antibodies block the effects on B cells of IFN-gamma, but not those of the other two lymphokines. IFN-gamma may be one of several molecules with a direct role in driving the maturation of resting B cells to active immunoglobulin secretion.

Spontaneous and induced immunoglobulin secretion by synovial fluid B lymphocytes in rheumatoid arthritis.

The functional properties of B lymphocytes in synovial fluid (SF) from patients with rheumatoid arthritis (RA) were analysed by means of a reverse haemolytic plaque forming cell (PFC) assay. SF mononuclear cells spontaneously secreted IgG, but little IgM or IgA. The SF cells failed to respond to the polyclonal B cell activators pokeweed mitogen (PWM) and Epstein-Barr virus. However, SF B cells cocultured with autologous T lymphocytes from the blood and stimulated with PWM secreted IgG but little IgM or IgA. The PFC responses of blood B cells cocultured with autologous SF T cells in the presence of PWM were low; irradiation of the T cells increased the blood B lymphocyte responses, but the differences were not statistically significant. It is concluded that suppressor SF T cells may be partly responsible for the poor response of SF B cells to PWM.

Characterization of a long-term T-helper-cell line which produces IL-2 and induces immunoglobulin secretion

Six OKT4 + human T-cell lines that require continuing PHA stimulation and TCGF for continuous growth were established. The cells from all six of these T-cell lines became smaller in size and lost the cell surface Ia antigen when they were grown in phytohemagglutinin (PHA)-depleted growth factor. These cells were unable to survive in the absence of PHA even if exogenous factor was present in great abundance. One of the cell lines (FL) was capable of providing helper functions. In the presence of PHA and phorbol myristate acetate, FL cells produced a growth factor, tentatively identified as Interleukin 2 (IL-2) by its ability to promote the proliferation of an IL-2-dependent murine T-cell line. Moreover, when FL cells were cocultured with B cells, pokeweed mitogen-induced immunoglobulin production was enhanced.

T cell regulation of polyclonally induced immunoglobulin secretion in humans.

We measured the pokeweed mitogen (PWM)-induced secretion of IgG by the unfractionated mononuclear cells (MNC) of young adult donors, and correlated the results with the functional activity of cell suspensions enriched for T helper (T4+) and T suppressor/cytotoxic (T8+) cells. The distribution of IgG levels secreted by MNC differs from a Gaussian curve, implying that the group is composed of distinct heterogeneous populations. When donors were compared who were judged to be very low responders or very high responders on the basis of IgG secretion levels by MNC (less than 700 ng/ml or greater than 2500 ng/ml), no differences were found in the capacity of T4+-enriched cells to support PWM-driven IgG secretion by a common B cell pool. In contrast, the addition of 0.2 X 10(5) T8+ cells from these low responders to PWM-stimulated cultures of 0.5 X 10(5) T4+ cells plus 0.5 X 10(5) B cells resulted in significantly less IgG secretion (389 +/- 121 ng/ml) than did the addition of the same number of T8+ cells from the high responders (2241 +/- 548 ng/ml, p less than 0.01). Normalized percent suppression by T8+ cells was higher in low responders than in high responders (77.0 +/- 9.9% vs 33.0 +/- 8.5%, p less than 0.01). Both high and low responders markedly suppressed IgG secretion when 0.5 X 10(5) T8+ cells were added. No correlation was found either between proportion of T3+, T8+, T4+, or M1+ cells within the MNC population and levels of IgG secretion by MNC or between T8+ numbers and levels of suppression induced by a constant number of T8+-enriched cells. Our data indicate that differences in the functional activity of T8+ cells, rather than quantitative differences, account for the wide range of PWM-induced IgG secretion by MNC.

Inhibition of polyclonally induced immunoglobulin secretion by aurothiomalate.

The influence of sodium aurothiomalate on the secretion of immunoglobulins by normal human lymphocytes in vitro was investigated by means of a reverse hemolytic plaque forming cell (PFC) assay. Aurothiomalate inhibited the PFC response induced by pokeweed mitogen (PWM) and by Epstein-Barr virus (EBV) in a dose dependent manner. The inhibition was irreversible, as pre-incubation for 2 h with the drug followed by extensive washing and further culture in gold salt-free medium still caused an inhibition of the PFC response to PWM and to EBV. Cell proliferation was not significantly affected, suggesting that the inhibition of PFC formation was not due to cytotoxicity. Pre-incubation of monocytes/macrophages (Mø's), T lymphocytes and B lymphocytes with the gold compound prior to culture with PWM showed that Mø's and B cells were highly sensitive, whereas T lymphocytes were resistant to the drug. The findings indicate that aurothiomalate inhibits the polyclonally induced PFC response by interfering with accessory Mø function and by affecting the B lymphocyte itself.

Mitogen‐Induced Switching of Immunoglobulin Heavy‐Chain Class Secretion in Chronic B‐Lymphocytic Leukaemia and Immunocytoma Cell Populations

Lymphocytes from peripheral blood, bone marrow, and lymph nodes from six patients with chronic B-lymphoproliferative disease were studied. Two of the patients had a polymorphic immunocytoma with surface membrane immunoglobulin phenotype mu kappa. Four patients had chronic B-lymphocytic leukaemia (CLL) with the phenotypes mu kappa, mu delta kappa, mu lambda, and mu delta lambda, respectively. In no case did the monoclonal cell populations express gamma chains on the surface, and none produced monoclonal immunoglobulin in vivo. The malignant B lymphocytes were cultured in the presence of polyclonal B-cell activators. Immunoglobulin secretion was detected with the protein A plaque assay and with a modification of the reversed haemolysis-in-agar plaque assay. Immunoglobulin secretion was induced in four of the cell populations. In both the immunocytomas and in one of the CLL cell clones the heat-inactivated Staphylococcus aureus strain Cowan I induced secretion of IgG, as well as IgM, associated only with the same light chain as was expressed on the surface of the unstimulated cells. Thus, mitogens can induce immunoglobulin secretion in vitro in malignant B lymphocytes, and Cowan I bacteria also have the ability to induce a switch of immunoglobulin production from IgM to IgG in a fraction of the leukaemic cell populations.

Induction of immunoglobulin secretion by a simple nucleoside derivative.

8 bromoguanosine (8BrGuo), a low m.w. nucleoside derivative that rapidly traverses the lymphocyte membrane to the interior of the cell, is an extremely potent inducer of immunoglobulin production by B lymphocytes. It has recently been demonstrated that this same agent can initiate high level lymphocyte proliferation in vitro. Data in the current report demonstrate that 8Br-cGMP and other cGMP analogues are at best weak inducers of immunoglobulin secretion, being more than two orders of magnitude less effective (per molecule taken up) than 8BrGuo. Incubation of lymphocytes with 8BrGuo fails to elevate their intracellular concentrations of cGMP. Moreover, at equimolar concentrations, underivatized cGMP actually antagonized the induction of antibody production by 8BrGuo. These data, in concert with observations that many lymphocyte activators fail to alter cGMP content, that many agents that elevate cGMP fail to induce immunoglobulin synthesis, and that some cGMP-elevating agents even inhibit it, suggest that the induction of immunoglobulin production, like the induction of proliferation in B cells, is not primarily dependent upon cGMP. A simple nucleoside derivative is described, the use of which should prove to be a powerful probe for investigating the triggering mechanisms underlying the proliferative and differentiative B lymphocyte pathways at the molecular level.

Characteristics of Epstein-Barr virus activation of human B lymphocytes.

Epstein-Barr virus (EBV) will infect at least every third cell if exposed in vitro to an extensively purified B cell population from human peripheral blood. About 10% of such infected cells will be driven into immunoglobulin synthesis and secretion, as judged by the indirect protein A plaque assay. The appearance of EB nuclear antigen, de novo DNA synthesis, and immunoglobulin secretion are linked phenomena accompanying infection as judged by viral dilution experiments, which yield kinetics of a one-hit order. Induction of immunoglobulin secretion in B cells by EBV requires de novo synthesis of DNA, and consequently, nontransforming EBV (P3HR1) will not induce immunoglobulin secretion and will also specifically block such induction from subsequently added EBV. The termination of immunoglobulin induction by EBV in short-term cultures appears to be T cell dependent.

Induction of immunoglobulin-secreting cells in the allogeneic mixed leukocyte reaction: regulation by helper and suppressor lymphocyte subsets in man.

The existence of functionally distinct T lymphocyte subsets in man, initially demonstrated with heteroantisera, has been confirmed with monoclonal reagents. Two major subsets have been defined: Leu-2 cells, which effect cell mediated lymphocytotoxicity (CML), and Leu-3 cells, which amplify CML and other T cell functions. This study is an effort to determine the effects of these subsets on the immunoglobulin secretion induced in the human mixed leukocyte reaction (MLR). T cells were separated from non-T cells by rosetting with sheep erythrocytes and were fractionated into Leu-2 and Leu-3 subsets by solid phase immunoabsorption with monoclonal antibodies. T cell subsets were cultured with autologous non-T cells and irradiated allogeneic stimulator non-T cells. Secretion of IgM and IgG was measured by a reverse hemolytic plaque assay. MLR-induced antibody secretion was specifically dependent on the Leu-3 T lymphocyte subset. The Leu-2 subset was incapable of generating large numbers of IgM- or IgG-secreting cells, and, in fact, suppressed the Leu-3-induced response. Exposure of Leu-3 cells to a dose of irradiation sufficient to prevent their proliferation in MLR did not reduce induced immunoglobulin secretion. Leu-2-mediated suppression, however, was sensitive to low dose irradiation. Thus Ig secretion in human MLR is regulated by a balance of helper activity from the Leu-3 subset and suppressor activity from the Leu-2 subset.

B Lymphocyte: Activation by Insoluble Anti-Immunoglobulin: Induction of Immunoglobulin Secretion by a T Cell-Dependent Soluble Factor

Abstract Purified rabbit antibodies to mouse κ chain or Fab become strongly mitogenic for mouse splenic B lymphocytes when covalently attached to the surface of polyacrylamide beads. The same antibody preparations are weakly if at all mitogenic in soluble form. The proliferative response to these anti-Ig beads is not dependent on T lymphocytes, adherent cells, serum supplement, or Fc receptor interactions. The response to anti-Ig beads can be blocked by soluble F(ab′)2 anti-Ig at concentrations that modulate surface Ig, or by excess soluble mouse IgG. Hence, the response results from direct interaction of the beads with B cells via surface Ig. In the presence of a supernatant of Con A-treated spleen cells (Con A supernatant), B lymphocytes respond to anti-Ig beads by sustained proliferation and differentiation to polyclonal IgM and IgG secretion. The response is comparable in magnitude to the lipopolysaccharide response. Ig secretion is completely dependent on the Con A supernatant, which by itself has only a small, primarily differentiative effect on isolated B lymphocytes. It can be added 22 hr after the start of culture without changing the kinetics of the response; addition at later times up to 66 hr delays the appearance of secreting cells, but not the size of the peak response. Cells from 10-day-old mice and adult male (CBA/N♂ × BALB/c♀) F1 mice fail to respond. These experiments suggest that redistribution of surface Ig by the anti-Ig beads delivers a limited proliferative signal to a subset of B lymphocytes, but that sustained cell division and differentiation to Ig secretion by anti-Ig activated B lymphocytes require soluble substances produced by activated T lymphocytes and/or T lymphocyte-activated accessory cells.