Evaluation of accessory cell heterogeneity. III. Role of dendritic cells in the in vitro activation of the antibody response to soluble antigens

Dendritic cells and macrophages obtained from spleen and peritoneal exudate were tested as accessory cells for the activation of lymphokine production by T cells, for supporting T-B cooperation and for the induction of antigen-specific T helper cells. Dendritic cells as well as macrophages were able to activate T cells for interleukin-2 secretion and functioned as accessory cells in T-B cooperation, but only macrophages induced T helper cells, which cooperate with B cells by a linked recognition interaction, to soluble antigens. Dendritic cell- and antigen-activated T cells also did not help B cells in the presence of Con A supernatants which contained various T cell- and B cell-stimulatory factors. The failure of dendritic cells to differentiate memory into functional T helper cells, but their efficient accessory cell function in T-B cooperation, where functional T helper cells are already present, can be best explained by a differential accessory cell requirement for T helper cell activation dependent on the differentiation stage of the T helper cell.     

Evaluation of accessory cell heterogeneity. I. Differential accessory cell requirement for T helper cell activation and for T-B cooperation

Several Ia+ tumor cell lines and peritoneal exudate macrophages were tested as accessory cells (AC) for the activation of antigen-specific T cells and for T-B cooperation. The macrophages and all the Ia+ tumor lines tested induced the release of lymphokines from T cells in a major histocompatibility complex (MHC)-restricted fashion and reconstituted the antibody responses of AC-depleted spleen cells or of purified T and B cells. However, only the normal macrophages but none of the tumor lines induced carrier-specific T helper (Th) cells which help B cells for specific antihapten antibody responses by linked recognition. For T-B cooperation accessory cells were also required, but in contrast to Th cell activation any type of Ia+ AC (e.g. macrophage or tumor line) was effective. Strong MHC-restriction between the lymphocytes and the AC was seen if antigen-pulsed AC were added into the AC-depleted T-B cooperation cultures. If the AC and antigen were concomitantly added to the AC-depleted T-B cultures, MHC-restriction was less obvious. Concanavalin A supernatant reconstituted the response of AC-depleted T-B cultures provided antigen-specific Th cells and the hapten-carrier conjugate were present. If, however, tumor line-activated T cells were added instead of macrophage-induced Th cells, no cooperation with B cells took place even in the presence of Con A supernatant. The results obtained demonstrate a differential AC requirement for the induction of Th cells depending on the differentiation stage of the Th cells.     

Bacterial CpG-DNA activates dendritic cells in vivo: T helper cell-independent cytotoxic T cell responses to soluble proteins

Receptors for conserved molecular patterns associated with microbial pathogens induce synthesis of co-stimulatory molecules and cytokines in immature dendritic cells (DC), as do antigen-reactive CD4 T helper cells via CD40 signaling. Once activated, antigen-presenting DC may activate CD8 T cell responses in a T helper cell-independent fashion. Using immunostimulatory CpG-oligonucleotides (ODN) mimicking bacterial CpG-DNA, we tested whether CpG-DNA bypasses the need for T helper cells in CTL responses towards proteins by directly activating antigen-presenting DC to transit into professional APC. We describe that immature DC in situ constitutively process soluble proteins and generate CD8 T cell determinants yet CD8 T cell responses remain abortive. Induction of primary antigen-specific CD8 cytotoxic T lymphocyte (CTL)-mediated responses becomes initiated in wild-type as well as T helper cell-deficient mice, provided soluble protein and CpG-ODN are draining into the same lymph node. Specifically we show that CpG-ODN trigger antigen-presenting immature DC within the draining lymph node to acutely up-regulate co-stimulatory molecules and produce IL-12. These results provide new insights for generating in vivo efficient CTL responses to soluble proteins which may influence vaccination strategies.     

Skin-resident murine dendritic cell subsets promote distinct and opposing antigen-specific T helper cell responses

Skin-resident dendritic cells (DCs) are well positioned to encounter cutaneous pathogens and are required for the initiation of adaptive immune responses. There are at least three subsets of skin DC- Langerhans cells (LC), Langerin(+) dermal DCs (dDCs), and classic dDCs. Whether these subsets have distinct or redundant function in?vivo is poorly understood. Using a Candida albicans skin infection model, we have shown that direct presentation of antigen by LC is necessary and sufficient for the generation of antigen-specific T helper-17 (Th17) cells but not for the generation of cytotoxic lymphocytes (CTLs). In contrast, Langerin(+) dDCs are required for the generation of antigen specific CTL and Th1 cells. Langerin(+) dDCs also inhibited the ability of LCs and classic DCs to promote Th17 cell responses. This work demonstrates that skin-resident DC subsets promote distinct and opposing antigen-specific responses.     

Antigen-presenting cell function of dendritic cells and macrophages in proliferative T cell responses to soluble and particulate antigens

The capacity of dendritic cells (DC) and macrophages (MΦ) to present soluble and particulate antigen was tested in an ovalbumin (OVA)-specific T cell proliferation assay. In a comparative investigation we found that both DC and MΦ, were able to present soluble OVA, but that only MΦ, could present insolubilized OVA to T cells. DC were found to be able to present OVA in collaboration with MΦ. The failure of DC to present insolubilized OVA is probably caused by their inability to endocytose these antigens. DC appeared not to endocytose substantial amounts of soluble OVA either. In contrast to MΦ, antigen presentation by DC is not blocked by lysosomotropic drugs. Taken together, these observations suggest that DC can present soluble protein antigens without intracellular degradation.     

The fraction 1 and V protein antigens of Yersinia pestis activate dendritic cells to induce primary T cell responses

The F1 and V antigens of Yersinia pestis, despite acting as virulence factors secreted by the organism during infection, also combine to produce an effective recombinant vaccine against plague, currently in clinical trial. The protective mechanisms induced by rF1 + rV probably involve interactions with dendritic cells (DC) as antigen uptake, processing and presenting cells. To study such interactions, naive ex vivo DC from bone marrow, spleen and lymph node were cultured with rF1, rV or combined antigens and demonstrated to secrete interleukin (IL)-4 and IL-12 into the culture supernatant. Cytokine production in response to pulsing was dependent on the maturity of the bone marrow-derived DC culture, so that pulsed 8-day-old cultures had accumulated significantly more intracellular IL-4 and IL-12 than unpulsed cells. DC, pulsed with rF1 + rV for 2-24 h, were able to prime naive autologous lymph node T cells to proliferate in an antigen dose-dependent manner, with an order of potency of 3d bone marrow-derived DC (BMDC) > 7d BMDC > splenic DC. Significantly, cell-free supernatants from rF1 + rV-pulsed BMDC and splenic DC were also able to induce specific primary responses effectively in naive T cells, suggesting that these supernatants contained stimulatory factor(s). This study suggests an important role for DC, or factors secreted by them, in the induction of protective immunity to plague by the rF1 and rV antigens.     

Lymphoid reservoirs of antigen-specific memory T helper cells

How vaccines control the development of antigen-specific effector and memory T helper cells is central to protective immunity but remains poorly understood. Here we found that protein vaccination selected high-affinity, CXCR5+ICOS(hi) follicular B-helper T cells (T(FH) cells) that developed in draining lymphoid tissue to regulate B cell responses. In the memory phase, reservoirs of antigen-specific CXCR5+ICOS(lo) T(FH) cells persisted with less effector activity but accelerated antigen-recall ability. This new compartment of memory T(FH) cells was retained in draining lymphoid sites with antigen-specific memory B cells, persistent complexes of peptide and major histocompatibility complex class II and continued expression of CD69. Thus, protein vaccination promotes B cell immunity by selecting high-affinity effector T(FH) cells and creating lymphoid reservoirs of antigen-specific memory T(FH) cells in vivo.     

Specificity of helper T cells for different antigens.

BALB/c nude mice have been injected with 10(6) congenic thymus cells, a number which allows some, but not all mice to respond to any particular T-dependent antigen. These mice have been tested for their ability to respond to three bacteriophages, T4, T7 and phiX, sheep and horse erythrocytes, and alloantigens of C3H and C57BL/6 mice. The number of mice able to respond to each of these antigens was of the same order of magnitude. Sheep and horse erythrocytes showed cross-reactivity at the T cell level, i.e. the responses to these two antigens were not independent. The same was observed for C3H and C57BL/6. Otherwise, the responses were independent showing that the antigens are recognize by different populations of specific helper T cells.     

Detection of helper and suppressor T cell lines to soluble antigens using a modified ELISA system

Helper and suppressor activity of T cell lines were investigated using an assay system which is based on measurement by the enzyme-linked immunosorbent assay (ELISA) of the antibody produced in vitro. T cell lines were established from spleens of BALB/c mice immunized with ovalbumin and human serum albumin. The T cells were expanded in culture with irradiated spleen cells and antigen or concanavalin A supernatant. The culture system used for assay of T cell activities contained antigen (Ag)-primed unfractionated spleen cells or Ag-primed B cells. Ag-primed cells and cultured T cells were incubated together with Ag (0.05 ng/ml-100 micrograms/ml) for various times, washed at varying intervals, and supernatants assayed for specific antibody activity by an ELISA adapted for this purpose. A total incubation time of 9 days was required for significant antibody production. Complete reconstitution of the antibody response was observed with Ag-primed B cells and Ag-primed T cells were combined. In one experiment, a helper cell line was shown to restore specific antibody production to approximately 50% of the normal response while a suppressor cell line suppressed antibody production by 90%. A linear response of between 0.2 and 1.4 OD units was observed in the ELISA allowing easy detection of help or suppression. As little as 80 ng of specific antibody could be detected. This technique allows quantitative determination of antibody production for a large number of individual microcultures.     

Anatomic location defines antigen presentation by dendritic cells to T cells in response to intravenous soluble antigens

In the spleen, exogenous antigen is preferentially presented by CD8alpha+CD11b- DC to CD8 T cells and by CD8alpha-CD11b+ DC to CD4 T cells. However, it is not yet clear whether the same rule applies to other secondary lymphoid organs. To address this issue, we first classified secondary lymphoid tissues into three categories based on the expression pattern of CD8alpha and CD11b in C57BL/6 and BALB/c mice: (a) spleen, (b) mesenteric lymph node (MLN) and (c) other peripheral lymph nodes (PLN). We then analyzed the OVA-specific T cell-stimulating capacity of each DC subset after intravenous injection with soluble OVA. Our results show that, regardless of tissue origin, CD8alpha-CD11b+ DC generally present OVA to CD4 T cells, a finding that held true as well for CD8alpha+CD11b+ DC in PLN. In striking contrast, CD8alpha+CD11b- DC in spleen, CD8alpha-CD11b+ DC in MLN and CD8alpha+CD11b+ DC in PLN mainly cross-present OVA to CD8 T cells in their respective tissues. Of note, CD8alpha-CD11b+ DC in MLN and CD8alpha+CD11b+ DC in PLN present OVA to both CD4 T and CD8 T cells. Therefore, the antigen-presenting capacity of each distinct DC subset is determined by its anatomic environment in combination with its surface phenotype.     

T cell clones interacting with accessory and T helper cells for a proliferative response

The requirement for cell interactions in T cell activation has been studied with two continuously in vitro growing T cell clones. These clones are specific for minor histocompatibility antigens, are H-2K restricted, and one clone is functionally a cytolytic T lymphocyte. Both can proliferate when interleukin 2 is added to the cultures, but for continuous growth they require irradiated spleen cells carrying the specific minor histocompatibility antigen and the restricting H-2. In this study we show that for proliferation the clones require at least two cell populations in the stimulator spleen, one is a splenic-adherent cell (SAC), the other a T cell. The SAC are plastic adherent, Thy-1-, Ia+. The T cells are nylon wool nonadherent, Thy-1+, Lyt-1+2- and Ia-. Cell mixing experiments of stimulator cells (all were done with H-2-syngeneic cells), depleted of either SAC or T cells confirm the requirement for a specific interaction between these two cell types and the T clone. Neither SAC, syngeneic with the T clone when mixed with T cells of the stimulator type, nor T cells syngeneic with the clone added to stimulator SAC, can induce an optimal proliferative response. Such a response is obtained only if both cell types, SAC and T cells, are of the stimulating genotype. This suggests that, in addition to an interaction of clonal T cells with SAC, a specific recognition at the T cell level between T stimulator and T clone is necessary. The interaction of the T clones with stimulator SAC and T cells leads to an activation, mediated by antigen recognition, of all three cell populations. Since we also show that each of the stimulator cell types are impaired by ultraviolet light irradiation, we conclude that factor production by SAC and T helpers is the final prerequisite for clonal expansion.     

The molecular interactions with helper T cells which limit antigen-specific B cell differentiation

Helper T (Th) cell-dependent activation requirements for 2,4,6-trinitrophenyl (TNP)-specific resting B cells obtained from mice transgenic for Sp-6 mu, kappa genes were analyzed. Carrier-specific T cell help required linked recognition of TNP carrier and was functionally restricted by the B cell major histocompatibility complex. However, histoincompatible T cell-B cell conjugates formed by bridging surface immunoglobulin and Th cell receptor for antigen (TcR) through TNP-conjugated anti-TcR antibodies resulted in the efficient differentiation of TNP-specific B cells. Thus, Th cell-dependent cognate recognition of B cells is not obligatory. Specific conjugate formation could be obviated by using unconjugated fragments of anti-TcR antibodies. If dimeric, these fragments supported the Th cell-dependent differentiation of co-cultured histoincompatible resting B cells. Unconjugated monomeric fragments were ineffective, demonstrating the necessity for TcR cross-linking. Resting B cells from Sp-6+ mice rendered TNP-conjugated monomeric fragments of anti-TcR antibodies effectively multivalent, thereby satisfying conditions for the activation of co-cultured Th cells. The results demonstrate that Th cells do not transduce activation signals through TcR recognition of B cell membrane-associated ligand which limit the induction of B cell differentiation. Cross-linking of TcR on Th cells is required, sufficient and can be induced through interaction with the antigen-specific B cell surface.     

'Educated' dendritic cells act as messengers from memory to naive T helper cells.

Ingested antigens lead to the generation of effector T cells that secrete interleukin 4 (IL-4) rather than interferon-gamma (IFN-gamma) and are capable of influencing naive T cells in their immediate environment to do the same. Using chimeric mice generated by aggregation of two genotypically different embryos, we found that the conversion of a naive T cell occurs only if it can interact with the same antigen-presenting cell, although not necessarily the same antigen, as the effector T cell. Using a two-step culture system in vitro, we found that antigen-presenting dendritic cells can act as 'temporal bridges' to relay information from orally immunized memory CD4 T cells to naive CD4 T cells. The orally immunized T cells use IL-4 and IL-10 (but not CD40 ligand) to 'educate' dendritic cells, which in turn induce naive T cells to produce the same cytokines as those produced by the orally immunized memory T cells.     

6 Murine T cell subsets and interleukins: Relationships between cytotoxic T cells,helper T cells and accessory cells

The aim of this review has been to present our view of the interaction of functionally distinct T cellsubsets involved in the generation of cytotoxic T cell respinses. The preactivation of CTL-P is induced bycomplex antigens, most frequently composites of viral and self- MHC class I(H-2) histocompaitbility antigens.The clonal expansion (growth) and differentiation into lytic effector cell is mediated by the CTL-class-specific lymphokines II-2 and CTDF, respectively. The II-2 producer cell is Lyt-1+ T helper cell which, byvirtue of its lymphokine secretion, tightly controls the CTL activation. Under conditions where the growthand diffrentiation-inducing lymphokines are supplied in excess, the frequency of antigen-inducible CTL-Ps orCon A-responsible CTL-Ps (total pool) can be enumerated directly. Thus, the repertoire of CTL-Ps in variouslymphocyte population is now amenable to quantitative studies at a clonal level. Furthermore, since largequantities of biochemically defined lymphokines such as II-2 may soon become available, the prospect ofanalysing the immunoregulatory effect of this growth factor under pathophysiogical conditions (with a potentialtherapeutic interest) mightsoon be realized.     

CD4+ T cells regulate the magnitude of the antibody response to several helper T cell independent antigens.

The effect of prior depletion of CD4+ T cells on the magnitude of the primary antibody response to several antigens considered to be helper T cell independent (TI) was examined. Treatment of mice with the monoclonal antibody GK1.5 to remove CD4+ T cells had little effect on the magnitude of the specific antibody response to the lipopolysaccharide (LPS) of Escherichia coli 0113; however, such treatment reduced the magnitude of the splenic IgM antibody response to E. coli 055 LPS, Serratia marcescens LPS, TNP-Ficoll, and Type III pneumococcal polysaccharide (SSS-III), compared to control untreated or rat IgG treated mice. Thus, CD4+ T cells positively influence of magnitude of the antibody response to the majority of helper T cell independent antigens tested.     

Xenogeneic recognition of soluble and cell surface HLA class II antigens by proliferative murine T cells

In order to characterize the murine anti-human xenogeneic mixed lymphocyte reactions (MLR), we studied T cell proliferative responses against various human lymphoid cells by immunization of mice either with cellular or purified HLA-DR antigens. Data presented here indicated that small amounts of soluble HLA-DR antigen were able to prime mice, and that the xenogeneic MLR depends on the expression of HLA class II antigens on the stimulating cells. Experiments using a mutant cell line clearly showed that HLA-DP molecules were also sufficient in eliciting a primary or a secondary xenogeneic MLR while no secondary proliferative response was obtained with cells expressing only HLA class I molecules. Using a large panel of human cells with various haplotypes, our results also showed that (a) nonpolymorphic determinants of HLA class II antigens trigger dominantly the murine T cells and (b) the xenogeneic response required I-E and L3T4 accessory molecules and was not inhibited with anti I-A and monomorphic anti-HLA class II antigen monoclonal antibodies. Altogether these results suggest that HLA class II antigens act as nominal antigens in triggering a murine anti-human proliferative response.     

Selected populations of alloreactive T cells contain helper T cells but lack ThId,an antigen-specific helper T cell required for dominant production of the T15 idiotype

Isolated, alloreactive T cell populations were primed with protein carriers in vivo and tested for their ability to provide help for an anti-phosphorylcholine (PC) antibody response and for production of the T15 idiotype. It was found that alloreactive T cell populations would support anti-PC antibody responses but did not selectively activate B cells capable of producing the T15 idiotype that normally dominates such responses. This failure to help for the production of the T15 idiotype was shown to be due to the absence of an antigen-specific helper T cell that is required for dominant idiotype production (ThId). These studies suggest that ThId cells have recognition structures for antigen and for self idiotype, but lack recognition structures for major histocompatibility complex-encoded antigens.     

Back-stimulation of B lymphocytes binding to helper T cell surface antigens

Helper T cells directed to B cell surface determinants activate their "targets" into polyclonal antibody production in vitro and in vivo, but B cells which bind to epitopes on the helper cell surface are preferentially induced. Furthermore, "anti-helper" B cell activation also occurs by "back-stimulation", that is even when the responding B lymphocytes are not specific targets for the inducing helper cells, as long as these are simultaneously activated by appropriate interactions with presenting cells. In our experimental systems, this is the only condition where "bystander" activation can be recorded. These findings suggest mechanisms of auto-antibody production associated with unrelated helper cell activity in vivo.