Interactions between primed and unprimed cells in the regulation of in vitro antibody responses. I. Role of “plasma cells” as inducers of suppression

Specific immune suppression has been shown to be activated in culture by the interaction of primed and unprimed T cell subsets. The primed cell involved is found 8 days after immunization in spleen but not in lymph node or thymus cell populations. When the primed spleen cells were fractionated by nylon wool passage or anti-Thy-1 plus complement (C) treatment, prior to culture with unseparated unprimed cells, suppression was detectable only with primed B cells present in the co-cultures. Treatment of the primed spleen cells with anti-PC. 1 (an antiserum specific for plasma cells) plus C eliminated their ability to cooperate with either unseparated or T cell-enriched populations of unprimed cells in suppressing the antibody response of the co-cultures. These data are consistent with the hypothesis that antibody-secreting plasma cells activate suppressor T cell precursors in cell populations not previously exposed to antigen.     

Antigen-specific helper T cells are essential for cytotoxic T cell responses to metabolically inactivated stimulator cells.

Primed spleen cells respond well to metabolically inactivated stimulator cells while normal spleen cells do not. This observation has been interpreted as showing that cytotoxic T cell precursors are different from unprimed precursors in their antigen recognition requirements for induction. A different model is proposed here which accounts for these observations as due to enhanced helper cell levels in primed populations. Experiments are described in this study which test several predictions of this model. These experiments show that in the presence of in vitro primed helper T cells, normal cells are able to respond efficiently to glutaraldehyde-fixed stimulator cells. The helper effect is antigen-specific. Since unprimed spleen cells can be efficiently induced by metabolically active stimulators (γ-irradiated cells) and can respond to glutaraldehyde-fixed antigen (metabolically inactive cells) only in the presence of specific helper cells, it seems reasonable to propose that helper cell signals are enhanced by a nonantigenic property of γ-irradiated stimulator cells requiring metabolic activity. It is also clear that glutaraldehyde-fixed cells are anti- genically intact as helper cells, primed to antigens on γ-irradiated stimulator cells, efficiently and specifically help a response to fixed stimulators. Conversely, helper cells primed in vitro to glutaraldehyde-fixed stimulators recognize antigen on γ-irradiated stimulator cells. The level of help generated in response to glutaraldehyde- fixed stimulator cells is at least 10-fold higher in primed cells than in normal cells. In addition, primed spleen cells can be induced in vitro to yield helper function by both fixed or unfixed stimulator cells. Normal helper cell precursors are induced at least 100-fold more efficiently by γ-irradiated as compared to glutaraldehyde-fixed stimulator cells. This work supports the idea that a major effect of priming, which allows primed cells to respond to metabolically inactive stimulators, is to enhance levels of helper T cells in the primed population.     

Effects of adjuvants on the function of antigen presenting cells in the production of IgE in mice

In an examination of the effects of adjuvants on the production of IgE, amounts of mRNAs for cytokines in antigen presenting cells(APCs) were assayed by RT-PCR and expressions of surface molecules on the cells were analyzed by flowcytometry when primed with antigen plus adjuvant. When mice were primed with ovalbumin plus alum, the levels of total and specific IgE were higher than those of mice primed with ovalbumin plus CFA. The APCs from mice primed with ovalbumin plus alum expressed higher levels of IL-1 mRNA than those in APCs from mice primed with ovalbumin plus CFA. B7 molecules were more expressed on the surface of the APCs from mice primed with ovalbumin plus CFA. The results suggested that these modulations of the functions of APCs affected the induction of helper T cell subsets in mice primed with different adjuvant.     

Major histocompatibility complex-restricted and unrestricted interactions in the T cell-dependent activation of hapten-binding B cells

The requirements for linked recognition and major histocompatibility complex-restricted interactions in helper T cell-dependent activation of purified unprimed and primed hapten-binding B cells have been investigated. The activation of unprimed hapten-binding B cells required specific antigen and restricted interactions between helper T cells and B cells. As expected, specific hapten-carrier conjugates were essential for the activation of B cells at low antigen doses. Interestingly, however, supraoptimal concentrations (125 micrograms/ml) of carrier protein alone could substitute for specific conjugates in the activation of unprimed B cells. The requirement for restricted helper T cell-B interactions was unchanged under these conditions. These results are discussed in terms of the signalling requirements for B cell activation. In contrast to the results using hapten-specific B cells from unprimed mice, the further stimulation of B cells prepared from mice primed 5 to 7 days earlier with immunogenic forms of the hapten was limited only by the requirements for helper T cell activation. The transition in the activation properties of B cells following in vivo stimulation was not solely a result of the binding of B cell membrane immunoglobulin to specific antigen, since the interaction of unprimed B cells with hapten during their purification, even for extended periods of time, did not alter the requirements for their further stimulation. These results demonstrate that the recent antigenic experience of B cells determines their activation state which, in turn, dictates the further requirements for helper T cell function in B cell stimulation. This implies that specificity of the immune response is determined in the early stages of B cell activation.     

Primed B cells present type-II collagen to T cells

Development of type‐II collagen (CII)‐induced arthritis (CIA) is dependent on a T‐cell mediated activation of autoreactive B cells. However, it is still unclear if B cells can present CII to T cells. To investigate the role of B cells as antigen‐presenting cells (APCs) for CII, we purified B cells from lymph nodes of immunized and nonimmunized mice. These B cells were used as APC for antigen‐specific T‐cell hybridomas. B cells from naïve mice did present native, triple‐helical, CII (nCII) but also ovalbumin (OVA) and denatured CII (dCII) to antigen‐specific T‐cell hybridomas. In addition, B cells primed with nCII or OVA, but not dCII, activated the antigen‐specific T‐cell hybridomas two to three times better than naïve B cells. We conclude that antigen‐primed B cells have the capacity to process and present CII to primed T cells, and antigen‐primed antigen‐specific B cells are more efficient as APC than naïve B cells. We further conclude that B cells have the potential to play an important role as APC in the development of CIA.     

Allotype suppression induced in the adoptive transfer system: the variables of the system and an apparent absence of a role for T cells.

A study has been made of the variables concerned in allotype suppression of adult spleen cells in the adoptive transfer system. These are; SRBC (antigen) dose; the dose and timing of injection of anti-allotype serum IgG; the number of spleen cells transferred and whether these cells were taken from primed or unprimed donors. Adoptively transferred primed cells are considerably less susceptible to suppression by concomitantly injected anti-allotype serum IgG than are unprimed spleen cells. Injection of anti-allotype serum during the period after adoptive transfer, has shown that primed cells loose their susceptibility sooner (2 days) than the unprimed cells (4 days). Allotype heterozygous CBA spleen cells are less susceptible heterozygous CBA spleen cells are less susceptible to allotype suppression than either allotypically homozygous or heterozygous non-H-2k cells (H-2b,d, or s). Allotype suppression of the TI IgG response to DNP-Ficoll was measured 7 days after adoptive transfer of allotype-homozygous cells from both normal and nude CBA mice (unprimed). The results indicate that T cells do not play a role in the initiation of short-term allotype suppression in the adoptive transfer system.     

Frequency of precursor cells against the enzyme beta-galactosidase: an estimate of the BALB/c strain antibody repertoire.

A method has been developed for detecting anti-beta-galactosidase antibodies after isoelectric focusing in thin layers of polyacrylamide gel. By "staining" with wild-type enzyme, all antibodies against beta-galactosidase are detected, while a subset of antibodies able to activate a mutant enzyme is detected by staining with that enzyme. Limiting dilutions of beta-galactosidase-primed or unprimed spleen cells of BALB/c mice were transferred together with antigen into sublethally irradiated syngeneic hosts. The limiting role of the precursor B cells has been judged by the analysis of the clonal distribution of galactosidase-specific antibodies in recipient sera. The frequency of anti-wild-type beta-galactosidase precursor cells was one in 0.42 x 10(6) in the primed and one in 0.93 x 10(6) in the unprimed spleen. The frequency of precursor cells for antibodies activating the mutant enzyme was one in 1.5 x 10(6) in the primed and one in 4.6 x 10(6) in the unprimed spleen. Therefore four and five times less anti-M (mutant) than anti-B (wild-type) precursor cells exist in the spleens of primed and unprimed BALB/c mice, respectively. Comparing 51 clones derived from one primed donor mouse, it was possible to demonstrate that at least 43 different mutant-beta-galactosidase-activating antibodies can be produced in one mouse. Comparing these 43 clones with 27 clones derived from another donor mouse, only one clone seemed to be common to both mice. From this the repertoire of the BALB/c strain has been estimated to consist of over 1000 different mutant enzyme-activating antibodies.     

Correlated expression of VH framework and VH idiotypic determinants on T helper cells and on functionally undefined T cells binding group A streptococcal carbohydrate

Antibodies to framework determinants of the VH and V lambda fragments of MOPC 315 and antisera to the VH idiotype determinants of the A 5 A antibody were used to analyze the antigen receptors of mouse T (and B) cells. This was done by using the antibodies as inhibitors in (a) an assay in which the binding of radiolabeled streptococcal carbohydrate (A-CHO) antigen by primed and unprimed T and B cells is determined and (b) an assay in which the helper activity of group A streptococcal vaccine-primed T cells is determined. The results suggest that the major proportion of primed and unprimed T cells binding A-CHO (70-90%) exhibit VH framework and VH idiotypic determinants. This population appears to include the helper T cells. A minor proportion of T cells (10-30%) express V lambda-related framework determinants and lack VH framework and VH idiotypic determinants. This population does not include T helper cells. Taken together, the data suggest that a subpopulation of T cells, including the helper cells, uses entire Ig VH regions as part of their antigen receptor system.     

Dendritic cells primed with HPV positive cervical tumor lysate are superior to unprimed DCs in migratory capacity and induce a potent Th1 response

In this study, we assessed the efficacy of tumor lysate primed and unprimed monocyte derived mature dendritic cells (DCs) to trigger an effective anti-tumor immune response in cervical cancer patients who tested positive for human papilloma virus (HPV) DNA. Lysate primed and unprimed DCs were assessed for the expression of CD80, CD86, CD40, HLADR and CD83. The ability of DCs to migrate in response to the chemokines CCL19 and 21 as well as their ability to secrete IL12p40 was investigated. Mixed lymphocyte proliferation assays were used to assess DC stimulatory capacity and their ability to generate a Th1 response. Our results showed no difference in phenotypic expression between primed and unprimed DCs but both had significantly increased expression of the activation marker CD83 when compared to immature DCs. Importantly, the primed DCs showed significant (P value = 0.03) IL-12p40 secretion and a superior migratory capacity towards CC19 and CCL21 (P value = 0.04) compared to unprimed DCs even after cytokine withdrawal. Primed DCs showed superior stimulation of T cell proliferation (allogeneic and autologous) and secretion of IFN gamma (IFN-γ) than the unprimed DCs. Hence whole tumor lysate primed mature DCs could be potent immunotherapeutic adjuvants to standard treatment for cervical cancer.     

Suppressor T cells in low zone tolerance. I. Mode of action of suppressor cells.

Low zone tolerance (LZT) to bacteriophage fd seems to be a type of tolerance which is primarily caused by suppressor T cells. The aim of this paper is to analyze their mode of action. For the induction of antigen-specific suppressor cells in hydrocortisone pretreated CBA mice, we use tolerogenic and immunogenic doses of antigen. Suppressor activity can be demonstrated upon transfer of spleen cells into normal syngeneic mice. After immunization these animals are unable to produce IgG antibody against phage fd, whereas the IgM response is not suppressed; The half-life of transferred suppressor cells in nonimmunized animals is 5--6 weeks. The target of suppression are unprimed T helper cells, whereas primed helper cells cannot be blocked. T helper cells become "resistant" to suppression 18--36 h after contact with antigen. Differentiation from unprimed B into B memory cells is unaffected, yet under suppression conditions persisting B memory cells are blocked in IgG production. The experimental data are incorporated into a model of LZT.     

Cellular requirements for the in vitro induction of specific suppression of antibody responses by immune spleen cells

Feedback regulatory signals to an intermediary suppressor cell may provide the mechanism of the suppression of antibody responses which has been described in cocultures of primed (immune) and unprimed (normal) spleen cells. The active cell(s) in the unprimed spleen population is nonadherent to nylon wool, Sephadex G-10 and glass beads. Lymph node cells and cortisone-resistant thymocytes from normal animals act similarly to normal spleen cells in this coculture system. Spleen cells from homozygous nude mice, unlike their heterozygous thymus-bearing littermates, do not produce a high degree of suppression in coculture with immune spleen cells. These data strongly suggest that the normal cell which interacts with primed cells in the cocultures to produce suppression is of thymic origin. However, spleen cells from neonatally thymectomized mice are suppressive in the presence of spleen cells immune to sheep red blood cells. The unprimed cell(s) active in the suppression are sensitive (in the presence of complement) to antisera directed against the surface markers, Thy-1, Qa-1, Lyt-1, Lyt-2, and Ia. Most of the antiserum treatments abrogated the suppressive capacity of the normal spleen cells only partially. Only treatment with anti-Qa-1 and complement routinely eliminated the ability of these cells to suppress in the cocultures suggesting either low concentration of or inaccessible surface alloantigen on the active cells. Alternatively, more than one Qa-1-positive cell set from the unprimed population may be involved. It is postulated that there is at least one subset of Qa-1-positive T lymphocytes present in unprimed spleen and lymph node cell populations capable of participating in the suppression of specific secondary antibody responses when cocultured with spleen cells from specifically primed animals.     

Both CD45R(low) and CD45R(high) "revertant" CD4 memory T cells provide help for memory B cells

During a primary response to thymus dependent antigens, B cells undergo a number of qualitative changes to become memory B cells - processes that require co-stimulatory signals and cytokine help from CD4 T cells. The question of whether distinct, antigen-experienced memory CD4 T cells are subsequently needed to program memory B cells into antibody synthesis has not been clearly resolved.Using an adoptive transfer model in which memory but not naive B cells were stimulated, we evaluated CD4 T cell help using lymphocytes obtained from primed or unprimed thymectomized donors and expressing a naive (CD45R(high)) or a memory (CD45R(low)) phenotype. Memory B cells, most of which were committed to the IgG1 (Th2) subclass, could be stimulated to produce antibody using help transferred by the CD45R(high) naive subset of unprimed donors (slow onset of response), the CD45R(low) subset of 7 day primed donors (large, rapid antibody response) or by both the CD45R(low) and the CD45R(high) "revertant" subsets of 6 month primed donors. We found that antigen primed CD45R(low) CD4 T cells reverted (defaulted) with time to a CD45R(high) resting state, a change that was prevented by persisting antigen. The evidence suggests that CD4 memory T cells are partitioned into two different functional states (CD45R(high) and CD45R(low)) and that these determine the characteristics of the memory B cell response in terms of speed, size and longevity.     

Expression of Ia determinants on immunocompetent cells.

The expression of Ia (immune response region-associated) antigens on the surface of lymphocyte subpopulations with defined immunological function has been investigated by negative selection of subpopulations with anti-Ia sera and complement. Ia determinants were found on both unprimed (IgM) and on primed (IgG) antibody-forming precursor cells. No Ia antigens were detected on the surface of helper T cells. In contrast, suppressor T cells were sensitive to treatment with anti-Ia sera and complement demonstrating the presence of Ia determinants on this T cell subpopulation.     

Gamma interferon enhances internalization and early nonoxidative killing of Salmonella enterica serovar Typhimurium by human macrophages and modifies cytokine responses

Gamma interferon (IFN-gamma) is a critical cytokine in host defense against salmonella infections, but its role in phagocytic killing of intracellular Salmonella spp. has been investigated mainly in animal rather than human cells. We measured the effect of recombinant IFN-gamma (rIFN-gamma) priming on bacterial internalization, intracellular killing, oxidative burst, and cytokine release during phagocytosis of Salmonella enterica serovar Typhimurium by human monocyte-derived macrophages (MDM). Eleven-day-old MDM, primed for 72 h with rIFN-gamma (100 ng/ml) exhibited an increased proportion of cells with associated bacteria (31% versus 26%, P = 0.036) and a 67% increase in internalized bacteria per cell compared to unprimed cells (P = 0.025). Retrieval of viable bacteria following internalization was reduced 3.6-fold in 72-h primed versus unprimed MDM (interquartile range, 3.1 to 6.4) at 0.5 h due to enhanced early intracellular killing, and this difference was maintained up to 24 h. In contrast, cells primed for only 24 h exhibited no increase in early killing. MDM were competent to produce an early oxidative burst when stimulated with phorbol myristate acetate, which was fully abrogated by the respiratory burst inhibitor diphenyleneiodonium chloride (DPI), but infection of MDM with S. enterica serovar Typhimurium did not cause an increase in the early respiratory burst under unprimed or primed conditions, and DPI had no effect on the early killing of bacteria by primed or unprimed MDM. During 24 h following infection, rIFN-gamma-primed MDM released more interleukin-12 (IL-12) and less IL-10 relative to unprimed cells. We conclude that 72-h priming with rIFN-gamma increases the efficiency of internalization and nonoxidative early intracellular killing of S. enterica serovar Typhimurium by human macrophages and modifies subsequent cytokine release.     

Murine epidermal antigen-presenting cells in primary and secondary T-cell proliferative responses to herpes simplex virus in vitro.

The role of epidermal Langerhans' cells in infection with herpes simplex virus (HSV) was investigated using a culture system that supports antigen-specific primary and secondary T-cell proliferative responses. Epidermal cell suspensions were capable of restimulating the response of in vivo primed T cells to UV-inactivated HSV. This capability was also present in cell suspensions enriched for Langerhans' cells, but was abrogated by the depletion of I-A-bearing cells. The magnitude, kinetics and phenotype of the responding cells were similar to those elicited when HSV was presented to primed T cells by antigen-presenting cells from the spleen. In marked contrast, whereas splenic antigen-presenting cells induced strong antigen-specific proliferation of unprimed T cells (primarily of the helper phenotype), Langerhans' cells failed to invoke any detectable reaction of such cells.     

Antigen-nonspecific T cell-derived factors in B cell activation: differences in the requirements for interleukin 2 in responses of unprimed and primed B cells

The requirements for interleukin 2 (IL2) and other T cell-derived helper factors in the responses of unprimed and antigen-primed B cells to sheep erythrocytes were investigated. Unprimed B cells required both IL2 and additional factor(s), hereafter referred to as T cell-replacing factor (TRF), in addition to specific antigen, for antibody production. IL2 was required only during the early stages (approximately equal to 24 h) of culture while TRF was necessary only after this time and was then required throughout the remaining culture period. IL2 stimulated the appearance of Thy-1+ cells in unprimed "B cell" populations which could substitute for the function of IL2, implicating an indirect role, at least in part, for IL2 in the TRF assay. Furthermore, in contrast to the results with unprimed B cells, primed B cells required only late-acting TRF for optimal antibody responses. We suggest that IL2 activates residual T cells or precursors of T cells in B cell populations which then function, in the presence of specific antigen, to render B cells receptive to T cell-derived factors which promote B cell growth and differentiation.     

Activation of cholera toxin-specific T cells in vitro.

Cholera toxin (CT) and its B subunit (CT-B) are potent oral immunogens in vivo, although both strongly inhibit polyclonal lymphocyte activation in vitro. In order to help understand this paradox, we have studied the activation and proliferation of CT-specific T cells in vitro, by using CT-B-primed lymph node T cells as responders, concanavalin A-stimulated peritoneal macrophages as antigen-presenting cells (APCs), and various forms of CT-B as antigen. The results indicate that in many ways CT-specific T cells respond in a manner similar to that of T cells specific for other protein antigens: the degree of proliferation was proportional to the dose of antigen and APCs in the cultures, was antigen specific, and was H-2 restricted. APCs from genetic high-responder strains to CT stimulated significantly more proliferation in F1 (high x low) responder T cells than did APCs from low responder strains. However, there was a marked difference in the activation of CT-specific T cells when different forms of CT-B were used. Native CT-B stimulated little or no T-cell proliferation, whereas denatured CT-B or CT-B blocked by its ligand, GM1 ganglioside, stimulated T cells well. Addition of native CT-B to cocultures of primed T cells, APCs, and these latter stimulatory forms of CT-B inhibited the specific proliferative response to CT-B to varying degrees, depending on the ratio of the two forms in culture. We conclude that the ability of CT-B to inhibit T cells extends even to T cells specific for CT itself. Because of these inhibitory properties, processing of CT to nonbinding molecular forms or fragments must be an important prerequisite for the immune response to CT to occur in vivo, and such processing is likely to be important in the immune response to a variety of other enterotoxins as well.     

Helper cell function of primed T cells. II. T-T cell Synergism between Ig+ and Ig− subpopulations of primed thymocytes: a mechanism for amplification of helper cell function

Thymocytes collected from spleens of lethally irradiated and thymocyte-reconstituted mice, 6 h after “education” with SRBC, exert a markedly augmented helper cell function, especially for the 7 S response. Pretreatment of primed thymocytes with a rabbit anti-mouse Ig serum and complement abolished the augmented helper cell function, although it had no effect on unprimed cells. Treatment of 6 h primed T cells with an anti-Θ serum and complement also abolished the augmented helper cell function. However, in striking contrast with unprimed T cells where such treatment entirely abolished the helper cell function, the anti-Θ treated 6 h T cells are still able to provide a significant helper cell function of the same magnitude as untreated nonprimed cells. This result suggests that a T cell subpopulation 6 h after priming becomes resistant to lysis by anti-Θ serum and supports a substantial helper cell function. Since the sum of plaque-forming cells (PFC) given by anti-Ig and anti-Θ-treated 6 h primed cells was far smaller than the PFC given by untreated cells, a T-T synergism is implicated as a mechanism for the augmented helper cell function of 6 h primed thymocytes. The amplification was also eliminated following treatment with a mouse anti SRBC serum indicating that some primed cells carry antigen on their surface. The data from various recombinations of primed cells treated with one of these three antisera, as well as primed cells treated sequentially with various combinations of two of the three antisera, suggested that the Ig⁺ and antigen-carrying cells belong to the same subpopulation which is resistant to lysis by anti-Θ serum. These cells are distinct from the rest which remain Ig^? and still sensitive to lysis by anti-Θ serum (Θ⁺). The Ig and antigen most likely represent a cytophilic complex present on the surface of a subpopulation of T cells. Thymocytes collected 5 days after education show little or no synergistic effect. The T-T cell synergism described here may represent a basic regulatory function of T cells.     

Rapid loss of feedback suppressor T-cell activity after priming in vivo.

T cells from antigen-primed mice have a diminished capacity to mediate feedback suppression when compared to T cells from unprimed mice. This was demonstrated using an in vitro model of B-cell induced feedback suppression in which spleen cells from mice primed with sheep erythrocytes (SRBC) activate feedback suppressor T-cell precursors to mediate suppression of the primed spleen cell response. The addition of splenic T cells from unprimed mice to cultures of spleen cells from SRBC-primed mice resulted in suppression of the secondary IgM and IgG anti-SRBC response. In contrast, no suppression was detected when T cells from mice primed with SRBC were added to the primed spleen cell cultures. The loss of suppression by T cells from primed mice was antigen-specific and was detectable by 24 hr after priming, coinciding with the appearance after priming of T-cell enhancing activity. The reduced suppressive activity could be due to changes in the active T-cell subset itself or to the appearance of cells or factors within the T-cell population that block or mask detection of feedback suppression. In either case, the present finding suggests that priming of a host not only activates feedback suppression induction mechanisms, but also rapidly affects the ability of the T-cell population to develop effective feedback suppression.     

Naive CD4+ T Cells Can be Sensitized with IL-7

We addressed the question whether it is possible to lower the threshold for naive T cells to respond to antigens. Purified adult and cord blood derived CD4+ CD45RA+ naive T cells were incubated in the presence of various cytokines for two days ("primed T cells"), after which the cytokines were removed by extensive washing. Primed and unprimed cells were activated with solid phase-coupled anti-CD3 and soluble anti-CD28 monoclonal antibodies (MoAb). Naive T cells, primed with interleukin(IL)-7 proliferated more vigorously than unprimed cells. Primed cells required 6 h for antigenic stimulation, whereas unprimed cells required 20 h. The priming also shifted the threshold of naive T cells in order to stimulate the antigen concentration to a lower level. The addition of IL-10 almost completely abrogated the enhancing effect of IL-7 on naive T cells. Other cytokines (IL-1, IL-2, IL-6, IL-12, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha had less effect on the cell proliferation. However, priming of naive T cells with IL-7 had no impact on the proliferation to allogeneic immature or mature dendritic cells (DC). We conclude that the antigen-independent activation of naive T cells with IL-7 prior to antigen stimulation sensitizes cells, and may be of help in trying to stimulate immune responses against weak antigens. This approach, however, does not enhance proliferative responses stimulated by DC.