A novel role for interleukin 2 in antibody responses: down-regulation of T helper cell activation

This report provides new insights into the role of the T cell growth factor interleukin 2 (IL 2) for the regulation of antibody responses. Evidence is presented that IL 2 down-regulates T helper cell (Thc) activation but not Thc effector function, i.e., Thc-B cooperation. Thus, reagents which block the IL 2 pathway, e.g., cyclosporin A (CsA) or IL 2 receptor monoclonal antibodies (IL 2R Mab) enhanced Thc activation although T cell proliferation was blocked. In contrast, CsA or IL 2R Mab blocked Thc-B cooperation, suggesting that IL 2 is required for this step. The regulatory role of IL 2 was reconfirmed by the addition of exogenous IL 2 into the cultures which reversed the enhancing or blocking effect of CsA.     

Graft-versus-host induced immunosuppression: depressed T cell helper function in vitro.

The cause of graft-versus-host (GVH) induced suppression of the plaque forming cell (PFC) response to sheep erythrocytes (SRBC) was investigated by in vitro restoration experiments employing a double compartment culture vessel. The two culture compartments were separated by a cell impermeable membrane. Restoring cells were placed in one chamber and responding GVH spleen cells plus SRBC were placed in the other chamber. It was demonstrated that thymus, lymph node, and spleen cells restored the PFC response whereas bone marrow cells did not. Treatment of the restoring cells with anti-theta serum plus complement abrogated restoration. Supernatants obtained from antigen free cell cultures restored nearly as well as whole cell suspensions. The degree of restoration was not increased by allogeneic or xenogeneic antigenic stimulation of the restoring cells. Thymus and lymphoid cells obtained from animals experiencing a GVH reaction restored as well as normal cells, however spleen cells were unable to restore by day 5 post-GVH induction. The results suggest that GVH induced immunosuppression of the PFC response is due, at least in part, to a depressed T cell factor production by splenic T cells.     

T4+ T helper cell function in vivo: differential requirement for induction of antiviral cytotoxic T-cell and antibody responses.

This study documents the differential requirements of T4+ T helper cells in the induction of virus-specific cytotoxic T-lymphocyte (CTL) and antibody responses during acute lymphocytic choriomeningitis virus infection. Two monoclonal antibodies (GK1.5 and RL172.4) directed against the L3T4 (T4) molecule were used for depleting T helper cells from mice. Depletion of T4+ cells caused a pronounced suppression of antiviral antibody response (20-fold decrease) but had minimal effect on virus-specific CTL response (less than 2-fold reduction). Despite the elimination of greater than 90% of T helper cells, anti-L3T4-treated mice were able to generate a CTL response of sufficient magnitude to control the viral infection. In contrast, depletion of Lyt2+ T cells abrogated the CTL response and the ability to eliminate virus. Thus, our results underscore the importance of the Lyt2+ T-cell subset in controlling infection with this virus and show that a deficiency of T4+ T cells is likely to have a more severe effect on antibody production than on CTL responses.     

Immune deficiency in chronic Trypanosoma cruzi infection. Recombinant IL-1 restores Th function for antibody production

Mice with chronic Trypanosoma cruzi infections are unable to mount primary responses to T-dependent Ag, such as SRBC. Responses to SRBC were restored in vitro and in vivo with rIL-1. The cellular basis of the immunodeficiency and the mechanism of IL-1 action were investigated. B cells from infected mice were capable of normal levels of PFC production when provided with the appropriate signals, IL-2 plus IL-1. T cells from infected mice were unable to provide Th function to normal B cells. However, Th activity was provided by these cells if IL-1 was added to the cultures. Furthermore, T-depleted spleen cells from infected mice did not make antibody in the presence of normal T cells unless IL-1 was added to the cultures. Neutralizing antibody against IL-2 greatly reduced the augmentation by IL-1 of the antibody response of cells from infected mice. Together these results indicate that splenic B cells from infected mice are capable of antibody production, but that Th function is lacking in the spleens of infected mice. These results suggest that the inability of mice with T. cruzi infection to mount primary antibody responses to T-dependent Ag may be due to a macrophage defect lending to impairment of Th function. These results document the potential of IL-1 in restoring immune competence in an infectious disease model.     

Antigen concentration determines helper T cell subset participation in IgE antibody responses.

A recently developed in vitro system for antigen-stimulated primary and secondary murine IgE antibody responses has been used to define (a) the relative participation of the Th1 and Th2 cell-derived lymphokines IFN-gamma and IL-4, respectively, in such responses, and (b) the role of antigen concentration in determining functional helper T cell activity. These studies confirm that IL-4 and IFN-gamma exert regulatory effects on IgE synthesis, but the nature and extent of their respective effects on primary and secondary IgE responses differ. Thus, primary IgE responses are considerably more sensitive to and dependent on IL-4 than are secondary IgE responses since (1) anti-IL-4 monoclonal antibody totally inhibited primary IgE responses, but only partially affected secondary responses; and (2) exogenously added IL-4 could stimulate primary IgE responses to optimal antigen concentrations, but had no effect on secondary IgE production. Likewise, antigen-stimulated primary IgE responses are about eightfold more sensitive than are secondary responses to the inhibitory effects of IFN-gamma. Studying the effect of antigen dose on the quantity of IgE antibody produced revealed that although IFN-gamma could be detected by ELISA in cultures exhibiting high-dose antigen-dependent diminution of IgE production, anti-IFN-gamma monoclonal antibody could not reverse this phenomenon. Thus, IFN-gamma is not solely responsible for decreased IgE synthesis associated with high-dose antigen exposure. IL-4 activity was detected in the fluid from cultures stimulated with low, but not high, levels of antigen. Moreover, addition of exogenous IL-4 restored IgE production to normal levels in cultures exposed to high antigen concentrations. Therefore, it appears that high levels of antigen result in selective stimulation of Th1 cells which produce IFN-gamma, and diminished activation of IL-4-producing Th2 cells. These results help explain observations regarding the influence of antigen dose on the generation of experimental and clinical IgE antibody responses in vivo.     

T cell regulation of antibody responses: an I-A-specific, autoreactive T cell collaborates with antigen-specific helper T cells to promote IgG responses

In earlier studies we showed that hapten-specific inducer T cell clones specifically induce B cells from immunized donors to secrete IgM antibodies. However, IgG responses were not observed, suggesting that an additional signal(s) was required. In this report, we show that an autoreactive T cell clone produces a factor(s) that collaborates with antigen-specific inducer T cells to promote specific IgG responses. This factor is not restricted by antigen or MHC determinants and promotes IgG production both in vivo and in vitro. These findings suggest that autoreactive cells may play an important role in the regulation of isotype expression.     

Trypanosoma cruzi: homocytotrophic antibody response in mice immunized with unrelated antigens.

The homocytotropic antibody response to unrelated antigens of mice with acute or chronic infection with Trypanosoma cruzi was studied. The production of IgG1 and IgE antibodies was observed in animals immunized with ovalbumin. The levels of IgG1 and IgE antibody were determined by passive cutaneous anaphylaxis. There was a depression in both IgG1 and IgE when infection and immunization were simultaneous. This depression was more intense when the animals were immunized 3 days after infection. A depression of IgG1 but not of IgE was observed in the animals with chronic infection and in the animals infected during the course of the humoral antibody response (12 days after immunization). It is suggested that a loss of T-cell regulatory mechanism may explain these results.     

Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleukin 1 in proliferative responses

Mice were immunized with either poly(Glu, Arg, Ala) or poly(Glu, Lys, Phe) contained in two different adjuvant preparations, alum (A1K(SO4)2) or complete Freund's adjuvant (CFA), and in vitro antigen-driven proliferative responses of lymph node cells were assayed 4-16 days later. After immunization with antigens on alum, endogenous interleukin 1 (IL-1) as well as interleukin 4 (IL-4) production was required for the proliferation of the elicited T cells as inclusion of either polyclonal goat anti-mouse IL-1 alpha or monoclonal anti-mouse IL-4 (11B11) in the cultures inhibited proliferative responses to antigen. In contrast, proliferative responses of cells elicited by antigen in CFA were not inhibited by either anti-IL-1 or anti-IL-4. Monoclonal antimouse CD4 (GK 1.5) inhibited proliferative responses regardless of which adjuvant was used to elicit antigen-reactive cells. These data indicated that phenotypically different subpopulations of CD4+ cells were elicited by the same antigen administered in different adjuvant preparations, Th2-like cells after immunization with polymers on alum and Th1-like cells after immunization with antigens in CFA. An examination of the isotypes of polymer-specific antibodies present in the sera of immunized mice also supported this conclusion.     

Central memory CD4+ T cell responses in chronic HIV infection are not restored by antiretroviral therapy

A strong CD4(+) T cell response has been correlated with better control of HIV infection. However, the effect of HIV on the maintenance of Ag-specific memory CD4(+) T cells is not fully understood. We characterized the function and phenotype of memory CD4(+) T cells generated by mumps and influenza A or B viruses in HIV-infected individuals receiving highly active antiretroviral therapy (n = 21), HIV-infected long-term nonprogressors (n = 10), and HIV-seronegative volunteers (n = 10). We observed significantly decreased proliferation of the Ag-specific central memory CD4(+) T cell population (CD28(+)/CCR7(+)/CD45RA(-)) in the antiretroviral treated HIV-infected individuals compared with the seronegative controls. Restored CD4(+) T cell count and decreased HIV viral load while on highly active antiretroviral therapy did not result in increased proliferation, whereas nadir CD4(+) T cell count predicted the presence of Ag-specific proliferation. Our results indicate that HIV infection leads to impaired maintenance of virus-induced or vaccine-generated central memory CD4(+) T cells that is not restored by HAART.     

Altered antibody production and helper T cell function in mice lacking chemokines CCL19 and CCL21-Ser

The roles of chemokines CCL19 and CCL21 in Ab production were investigated using plt mutant mice, which lack expression of CCL19 and CCL21-ser in their lymphoid organs. In these mice, the Th response has been shown to tend towards the Th1 type because of accumulation of inflammatory dendritic cells. When plt mice were immunized with 100 μg OVA in CFA, the number of Ab-forming cells in the draining LN, and serum concentrations of OVA-specific IgM and IgG Ab, were very close to those of the control, yet IgG2a Ab in plt mice was increased. In vitro IFN-γ production by the draining LN cells of plt mice was increased. In addition, the ability of helper T cells from plt mice to stimulate Ab production in vitro was prolonged. Also, in the plt mice, in vivo challenge with OVA in incomplete Freund's adjuvant elicited a stronger IgG2a response and a weaker IgG1 response, which is suggestive of a Th1-dominant response. Similar findings were obtained when mice were immunized with 100 μg OVA in alum, except that with alum the increases observed in plt mice were IgG1 produced in vivo and IL-4 produced in vitro by draining LN cells. Furthermore, immunization with alum adjuvant also induced a prolonged in vitro recall response of IFN-γ and IL-4. These findings indicate that plt mice mount an anti-OVA Ab response, and suggest that CCL19 and CCL21 induce prompt Ab responses to antigen, and negatively regulate helper T cell responses in vivo.     

Role of interleukin 2, interleukin 4 and interleukin 5 in the T helper cell-driven B cell polyclonal differentiation in the elderly.

There is evidence for an impaired T cell-mediated B cell response during senescence. In thirty aged donors, pokeweed mitogen (PWM)-driven immunoglobulin (Ig) synthesis by B cells co-cultured with autologous enriched CD4+ lymphocytes and low amounts of monocytes, was evaluated. Under such experimental conditions, elderly cultures displayed a reduced IgG and/or IgM production when compared with the younger counterpart. Moreover, interleukin (IL)-2 and/or IL-5 addition to cultures led to an enhancement of Ig release. In contrast, IL-4 supplementation failed to positively modulate B cell differentiation. At the same time, aged cells cultured in the presence of IL-2 + IL-5 exhibited an increased Ig synthesis, while the addition of IL-2 + IL-4 or IL-4 + IL-5 mixtures did not induce any significant effect in comparison with homologous untreated samples. The results suggest a critical role for IL-2, IL-4 and IL-5 in the modulation of T helper cell-driven B cell polyclonal responsiveness in the elderly.     

T helper cell-dependent, microbial superantigen-induced murine B cell activation: polyclonal and antigen-specific antibody responses.

Microbial superantigens (SA), bound to human B cell surface MHC class II molecules, have been shown to promote direct, "cognate" interaction with SA-reactive autologous Th cells, resulting in polyclonal Ig production. To investigate the potential for microbial SA to support Th cell-dependent, Ag-specific antibody responses, we have extended our studies to the murine system. BALB/c Th cell lines (TCL), specific for either the Mycoplasma arthritis-derived SA or the Staphylococcus aureus-derived toxic shock syndrome toxin-1) were generated. These TCL cells are SA-specific, functionally noncross-reactive, and utilize distinct TCR V beta gene families. Coculture of SA-reactive TCL cells and syngeneic B cells bearing the relevant SA results in B cell proliferation and polyclonal IgM and IgG production. In contrast, Ag-specific (SRBC-specific) antibody-forming cells are only generated in cultures that also contain SRBC. Thus, microbial SA-mediated Th-B cell interactions induce both polyclonal B cell activation and provide selective help for the proliferation and/or differentiation of B cells that have encountered specific Ag. In additional studies, we determined that the in vivo administration of toxic shock syndrome toxin-1 to young, athymic (nude) BALB/c mice results in SA binding to splenic B cells, rendering these B cells effective stimulators of and targets for SA-reactive helper TCL cells. Taken together, these results demonstrate that microbial SA mediate productive Th-B cell interactions analogous to those that occur during allospecific Th-B cell interactions in vitro and GVHD in vivo. These findings are consistent with the hypothesis that microbial SA represent environmental factors that may trigger autoimmune disease in the genetically susceptible host.     

Synergy between recombinant interleukin 2 (rIL 2) and IL 2-depleted lymphokine-containing supernatants in facilitating allogeneic human cytolytic T lymphocyte responses in vitro

Supernatants from human mixed leukocyte cultures or lectin-depleted supernatants from cultures of PHA-activated human peripheral blood leukocytes were depleted of IL 2 by passage over an anti-human rIL2 immunoadsorbent column. The column eluates were concentrated, dialyzed, and tested for their ability to synergize with human rIL 2 in facilitating human cytolytic T lymphocyte (CTL) responses to allogeneic, uv-irradiated HT144 melanoma cells in vitro. CTL were generated in the presence of 1 X 10(-4) M hydrocortisone sodium succinate in order to minimize the generation of nonspecific lymphokine-activated killer (LAK) cells. IL 2-depleted lymphokine-containing supernatant (LKS), alone or in the presence of less than or equal to U/ml rIL 2 did not stimulate significant CTL responses. Recombinant IL 2 at greater than 2 U/ml stimulated weak CTL responses in the absence of LKS. However, strong synergistic CTL responses were observed when both IL 2-depleted LKS and greater than 2 U/ml rIL 2 were added to the cultures. CTL generated in these cultures could be distinguished from nonspecific LAK cells on the basis of their i) specificity, ii) T3 phenotype, and iii) kinetics of generation. Nevertheless, rIL 2 and IL 2-depleted LKS were sometimes observed to synergize in facilitating the generation of nonspecific LAK cells as well as the generation of specific CTL. When the times at which rIL 2 and IL 2-depleted LKS were added to the cultures were varied, IL 2 was found to be required early in CTL responses, whereas the synergistic factor(s) in LKS seemed to act later. Recombinant human interferon-gamma was unable to replace LKS in synergizing with rIL 2 to elicit CTL responses. In summary, these experiments suggest that LKS contains a late-acting factor(s), antigenically distinct from IL 2, which synergizes with IL 2 in facilitating human CTL responses.     

Graft versus host-induced immunosuppression: mechanism of depressed T-cell helper function in vitro.

The cellular basis of graft versus host (GVH)-induced immunosuppression was investigated. Results showed that thymus, lymph node, and splenic T cells from normal mice and thymus and lymph node T cells from GVH mice, when cultured on one side of a cell impermeable membrane, restored the plaque-forming cell (PFC) response to sheep erythrocytes of GVH-immunosuppressed spleen cells (GVH-SC) cultured on the other side of the membrane. The restoring ability of T cells present in GVH-SC was inhibited by splenic accessory (A) cells. A direct relationship was shown between the proportion of splenic A cells and the degree of suppression of the PFC response during the first 10 days of the GVH reaction. Normal or GVH A cells reconstituted the PFC response of normal cells and GVH-SC depleted of their A-cell fraction. An optimum ratio of A: nonadherent (NA) cells (1: 10) was required for maximum reconstitution. Larger proportions of A cells inhibited the PFC response. The results suggest that GVH-induced immunosuppression is due, at least in its initial phase, to a depressed T-cell helper function caused by a marked increase of A cells in the spleen.     

Independent analysis of T helper and T killer cell function in young and old NZB mice.

With age, NZB mice lose their ability to develop a cytotoxic response after alloimmunization in vitro. This decline is shown to coincide with a diminution of T-helper cell activity as assessed by proliferation in mixed lymphocyte culture or in response to PHA. When cytotoxic T cell precursors are activated with the polyclonal activator Con A, there is no reduction in the number of cytotoxic effector T cells that develop. No autoreactive cytotoxic cells are seen in Con A-activated cultures. These findings are related to previous work on cell-mediated immunity in NZB and B/W mice.