CD103+CD11b+ mucosal classical dendritic cells initiate long-term switched antibody responses to flagellin.

Antibody responses induced at mucosal and nonmucosal sites demonstrate a significant level of autonomy. Here, we demonstrate a key role for mucosal interferon regulatory factor-4 (IRF4)-dependent CD103+CD11b+ (DP), classical dendritic cells (cDCs) in the induction of T-dependent immunoglobulin G (IgG) and immunoglobulin A (IgA) responses in the mesenteric lymph node (MLN) following systemic immunization with soluble flagellin (sFliC). In contrast, IRF8-dependent CD103+CD11b- (SP) are not required for these responses. The lack of this response correlated with a complete absence of sFliC-specific plasma cells in the MLN, small intestinal lamina propria, and surprisingly also the bone marrow (BM). Many sFliC-specific plasma cells accumulating in the BM of immunized wild-type mice expressed α4ß7+, suggesting a mucosal origin. Collectively, these results suggest that mucosal DP cDC contribute to the generation of the sFliC-specific plasma cell pool in the BM and thus serve as a bridge linking the mucosal and systemic immune system.

Tracking the response of Xid B cells in vivo: TI-2 antigen induces migration and proliferation but Btk is essential for terminal differentiation.

X-linked immunodeficient (Xid) mice carry a Bruton's tyrosine kinase (Btk) mutation and exhibit a selective failure to produce antibodies against bacterial capsular polysaccharides. Studies in vitro point to a fundamental survival defect of Xid B cells after receptor cross-linking by thymus-independent type-2 (TI-2) antigen because B cells undergo apoptosis without proliferating. We describe results from a novel model, which we have used to investigate the impact of the Xid mutation on migration, proliferation and differentiation of B cells after polysaccharide immunization in vivo. Immunoglobulin knock-in mice, in which a large proportion of B cells express transgene-encoded receptors specific for (4-hydroxy-3-nitrophenyl)-acetyl (NP), were crossed with CBA/N mice. The male progeny contain NP-specific Xid B cells, while the female progeny contain NP-specific B cells with normal Btk. After immunization with the TI-2 antigen NP-Ficoll, NP-specific Xid B cells migrate to the T zones and proliferate. Despite transient up-regulation of blimp-1 and survival beyond the time when terminal differentiation is normally underway, Btk-defective B cells fail to differentiate to plasmablasts or germinal center cells. CD40 ligation partially restores their ability to form plasma cells in response to TI-2 antigen.

Changing responsiveness to chemokines allows medullary plasmablasts to leave lymph nodes.

During T cell-dependent antibody responses lymph node B cells differentiate either to plasmablasts that grow in the medullary cords, or to blasts that proliferate in follicles forming germinal centers. Many plasmablasts differentiate to plasma cells locally, but some leave the medullary cords and migrate to downstream lymph nodes. To assess the basis for this migration, changes in the responsiveness of B cells to a range of chemokines have been studied as they differentiate. Naive B cells express high levels of CCR6, CCR7, CXCR4 and CXCR5. When activated B cells grow in follicles the expression of these chemokine receptors and the responsiveness to the respective chemokines is retained. During the extrafollicular response, plasmablast expression of CXCR5 and responsiveness to B-lymphocyte chemoattractant (CXCR5) as well as to secondary lymphoid tissue chemokine (CCR7) and stromal cell-derived factor (SDF)-1 (CXCR4) are lost while a weak response towards the CCR6 chemokine LARC is maintained. Despite losing responsiveness to SDF-1, extrafollicular plasmablasts still express high levels of CXCR4 on the cell surface. These results suggest that the combined loss of chemokine receptor expression and of chemokine responsiveness may be a necessary prerequisite for cells to migrate to the medullary cords and subsequently enter the efferent lymph.

Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival.

B cells recruited into splenic antibody responses grow exponentially, either in extrafollicular foci as plasmablasts, or in follicles where they form germinal centers. Both responses yield plasma cells. Although many splenic plasma cells survive <3 d, some live much longer. This study shows that early plasma cell death relates to a finite capacity of the spleen to sustain plasma cells rather than a life span endowed by the cell's origin or the quality of antibody it produces. Antibody responses were compared where the peak numbers of plasma cells in spleen sections varied between 100 and 5,000 cells/mm(2). In each response, plasmablast clones divided some five times, with the peak numbers of plasma cells produced relating directly to the number of B cells recruited into the response. The spleen seems to have the capacity to sustain between 20 and 100 plasma cells/mm(2). When this number is exceeded, there is a loss of excess cells. Immunoglobulin variable region gene sequencing, and 5-bromo-2'-deoxyuridine pulse-chase studies indicate that long-lived splenic plasma cells are a mixture of cells derived from the extrafollicular and germinal center responses and cells derived from virgin and memory B cells. Only a proportion has switched immunoglobulin class.

Interplays between mouse mammary tumor virus and the cellular and humoral immune response.

Mouse mammary tumor virus has developed strategies to exploit the immune response. It requires vigorous immune stimulation to achieve efficient infection. The infected antigen-presenting cells present a viral superantigen on the cell surface which stimulates strong CD4-mediated T-cell help but CD8 T-cell responses are undetectable. Despite the high frequency of superantigen-reactive T cells, the superantigen-induced immune response is comparable to classical antigen responses in terms of T-cell priming, T-cell-B-cell collaboration as well as follicular and extra-follicular B-cell differentiation. Induction of systemic anergy is observed, similar to classical antigen responses where antigen is administered systemically but does not influence the role of the superantigen-reactive T cells in the maintenance of the chronic germinal center reaction. So far we have been unable to detect a cytotoxic T-cell response to mouse mammary tumor virus peptide antigens or to the superantigen. This might yet represent another step in the viral infection strategy.

T-independent type 2 antigens induce B cell proliferation in multiple splenic sites, but exponential growth is confined to extrafollicular foci.

During the primary splenic response to the T-independent type 2 (TI-2) antigen (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll, small numbers of antigen-specific B cells have entered S phase of the cell cycle 24 h after intraperitoneal immunization. These are distributed in all splenic compartments (T zones, marginal zones, follicles, and red pulp), indicating early proliferation induced by NP-Ficoll does not require accessory signals delivered in a particular splenic microenvironment. Subsequently B blasts accumulate selectively in the outer T zone areas, but exponential growth leading to plasma cell production occurs only in extrafollicular foci. This growth peaks after 5 days, but 20% of peak numbers of antibody-containing cells are still present 3 months after immunization and 9% of these cells are proliferating. It is unclear if these late plasmablasts are sustained by self-renewal or continued recruitment of virgin cells into the response. Unlike TD and TI-1 responses NP-specific memory cells do not accumulate in the splenic marginal zones. The level of Cgamma3 switch transcripts increases during the first 24 h of the response, but does not increase further during exponential plasmablast growth.

Defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.

Vav, a guanine nucleotide exchange factor for members of the Rho family of small GTPases, is activated through engagement of B and T lymphocyte antigen receptors. It is important for establishing the signaling threshold of the TCR, as mice lacking Vav display defective thymocyte selection. Here, conventional B cells are shown to develop normally in Vav-deficient mice but these mice have few B-1 B cells. The threshold for inducing B cell proliferation through BCR engagement in vitro is greater in Vav-deficient B cells. Nevertheless, in vivo the mutant mice have normal antibody responses to haptenated Ficoll. In contrast, Vav-/- mice show defective class switching to IgG and germinal center formation when immunized with haptenated protein. Interestingly, this defect is reversed in chimeras where normal T cells are present. Antigen-specific proliferation of T cells in the T zone was found to be similar in wild-type and Vav-/- mice but the induction of IL-4 mRNA and switch transcripts was specifically impaired. These results suggest that defective immunoglobulin class switching in Vav-deficient mice is attributable to compromised T cell help.

CD4 T cell cytokine differentiation: the B cell activation molecule, OX40 ligand, instructs CD4 T cells to express interleukin 4 and upregulates expression of the chemokine receptor, Blr-1.

This report investigates the role of OX40 ligand (OX40L) and its receptor, OX40, expressed on activated B and T cells, respectively, in promoting the differentiation of T helper type 2 (Th2) CD4 T cells. These molecules are expressed in vivo by day 2 after priming with T cell- dependent antigens. Their expression coincides with the appearance of immunoglobulin (Ig)G switch transcripts and mRNA for interleukin (IL)-4 and interferon (IFN)-gamma, suggesting that this molecular interaction plays a role in early cognate interactions between B and T cells. In vitro, we report that costimulation of naive, CD62Lhigh CD4 T cells through OX40 promotes IL-4 expression and upregulates mRNA for the chemokine receptor, blr-1, whose ligand is expressed in B follicles and attracts lymphocytes to this location. Furthermore, T cell stimulation through OX40 inhibits IFN-gamma expression in both CD8 T cells and IL-12-stimulated CD4 T cells. Although this signal initiates IL-4 expression, IL-4 itself is strongly synergistic. Our data suggest that OX40L on antigen-activated B cells instructs naive T cells to differentiate into Th2 cells and migrate into B follicles, where T cell-dependent germinal centers develop.

T helper 1 (Th1) and Th2 characteristics start to develop during T cell priming and are associated with an immediate ability to induce immunoglobulin class switching.

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cgamma2a and Cgamma1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon gamma in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone. The addition of killed Bordetella pertussis to the hapten-protein induces nonhapten-specific IgG2a and IgG1 plasma cells, whereas the anti-hapten response continues to be IgG1 dominated. This indicates that a Th2 response to hapten-protein can proceed in a node where there is substantial Th1 activity.

Memory B-cell clones and the diversity of their members.

Memory B-cell clones develop from virgin B cells that take up processed antigen, make cognate interaction with primed T cells and then grow in germinal centres. Within the germinal centre the proliferating B cells undergo Ig variable-region mutation and are subsequently selected on their ability to bind antigen held on follicular dendritic cells and then to make cognate interaction with germinal centre T cells. The selected cells emerge as memory B cells or plasmablasts. Although many of the memory B cells and most of the plasma cells emerging from follicles have undergone Ig class switch recombination a substantial minority of the memory B cells have not switched. These non-switched memory cells can be induced to switch on re-exposure to antigen. Affinity maturation following a single immunization ceases as germinal centres wane some 3-4 weeks after immunization - memory cells and antibody production, on the other hand, persist for months and even years.

Properties of multinucleated giant cells in a new in vitro model for human granuloma formation.

Multinucleated giant cells (MGCs) are a key feature of granulomas. They have been studied with respect to the mechanism and regulation of their formation, but the function of these cells still remains elusive. A new method for the in vitro generation of granulomas was developed and characterized in which L3 larvae of Nippostrongylus brasiliensis, as a target for the cellular response, were co-incubated with human mononuclear blood cells. The development of epithelioid cells and MGCs was observed and single isolated MGCs were analysed by the reverse transcriptase polymerase chain reaction method. The presence of tumour necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) transcripts in MGCs was demonstrated. It is proposed that MGCs in the granuloma model may in part represent an active cellular constituent involved in granuloma formation and turnover and in the destruction of the irritant.

The changing preference of T and B cells for partners as T-dependent antibody responses develop.

Recirculating virgin CD4+ T cells spend their life migrating between the T zones of secondary lymphoid tissues where they screen the surface of interdigitating dendritic cells. T-cell priming starts when processed peptides or superantigen associated with class II MHC molecules are recognised. Those primed T cells that remain within the lymphoid tissue move to the outer T zone, where they interact with B cells that have taken up and processed antigen. Cognate interaction between these cells initiates immunoglobulin (Ig) class switch-recombination and proliferation of both B and T cells; much of this growth occurs outside the T zones B cells migrate to follicles, where they form germinal centres, and to extrafollicular sites of B-cell growth, where they differentiate into mainly short-lived plasma cells. T cells do not move to the extrafollicular foci, but to the follicles; there they proliferate and are subsequently involved in the selection of B cells that have mutated their Ig variable-region genes. During primary antibody responses T-cell proliferation in follicles produces many times the peak number of T cells found in that site: a substantial proportion of the CD4+ memory T-cell pool may originate from growth in follicles.

Immunoglobulin switch transcript production in vivo related to the site and time of antigen-specific B cell activation.

Immunoglobulin (Ig) class switch recombination is associated with the production and splicing of germline IgCH messenger RNA transcripts. Levels of gamma 1 transcripts in mouse spleen sections were assessed by semiquantitative analysis of reverse transcriptase polymerase chain reaction (PCR) products during primary and secondary antibody responses to chicken gamma globulin (CGG). This was correlated with the appearance of CGG-specific B cells and their growth and differentiation to plasma cells. After primary immunization with CGG, gamma 1 switch transcripts appeared after 4 d, peaked at a median of six times starting levels between 10 and 18 d after immunization, and returned to background levels before secondary immunization at 5 wk. By contrast, after secondary challenge with CGG, a sevenfold increase in transcripts occurs during the first d. The level again doubles by day 3, when it is six times that which is seen at the peak of the primary response. After day 4, there was a gradual decline over the next 2-3 wk. Within 12 h of secondary immunization, antigen-specific memory B cells appeared in the outer I zone and by 24 h entered S phase, presumably as a result of cognate interaction with primed T cells. Over the next few hours, they migrated to the edge of the red pulp, where they grew exponentially until the fourth day, when they synchronously differentiated to become plasma cells. The same pattern was seen for the migration, growth, and differentiation of virgin hapten-specific B cells when CGG-primed mice were challenged with hapten protein. The continued production of transcripts after day 3 indicates that switching also occurs in germinal centers, but in a relatively small proportion of their B cells. The impressive early production of switch transcripts during T cell-dependent antibody responses occurs in cells that are about to undergo massive clonal expansion. It is argued that Ig class switching at this time, which is associated with cognate T cell-B cell interaction in the T zone, has a major impact on the class and subclasses of Ig produced during the response.

The use of reverse transcription polymerase chain reaction to analyse large numbers of mRNA species from a single cell.

A PCR method is described for determining the expression of multiple heterogeneous mRNAs from single cells. The total mRNA pool of a single selected cell is subjected to reverse transcription and subsequent tailing with poly(dA). This cDNA is preamplified by a sequence non-specific PCR protocol using oligo(dT)-containing primers. The single cell cDNA library obtained permits the analysis of virtually unlimited numbers of mRNA species per cell using sequence-specific PCR. This procedure of multiple mRNA analysis enables phenotyping of any cell for its mRNA composition and could be used to study the cytokine mRNA expression of individual human T cells ex vivo. The method should greatly facilitate the analysis of combinatorial expression of known genes in any cell.

CD26 expression in leprosy and other granulomatous diseases correlates with the production of interferon-gamma.

BACKGROUND: Leprosy represents a spectrum of clinical manifestations that reflect the immune response to antigens of Mycobacterium leprae. The tuberculoid form of leprosy, which is characterized by an organized development of granulomas, has recently been correlated with a Th1-like immune response. The lepromatous form of leprosy, with a characteristic lack of cellular immunity, has been correlated with a Th2-like immune response to mycobacterial antigens. Dipeptidylpeptidase IV (CD26) is an ectopeptidase that is expressed in various tissues; in the hemopoietic system, it is predominantly expressed by T cells. EXPERIMENTAL DESIGN: We stained frozen sections of skin biopsies obtained from patients with different forms of leprosy, sarcoidosis, and Piringer's lymphadenitis. Sections were stained for interferon-gamma (IFN-gamma) and CD26 with the alkaline phosphatase anti-alkaline phosphatase technique and in two-color stainings by immunofluorescence. RESULTS: We found strong signals for IFN-gamma and for CD26 in all investigated cases of tuberculoid leprosy. In contrast, in all biopsies taken from patients with lepromatous leprosy, we found no or very weak signals for these antigens. By immunofluorescence double-labeling, we could show that IFN-gamma and CD26 were expressed by the identical cell population. We confirmed this correlation of CD26 expression and IFN-gamma production in other granulomatous inflammatory reactions such as sarcoidosis and Piringer's lymphadenitis. CONCLUSIONS: From our results, we conclude that a high expression of CD26 may be suggestive of Th1-like immune reactions.

Follicular dendritic cells productively infected with immunodeficiency viruses transmit infection to T cells.

Lymphoid organs have been proposed to function as the major reservoir for the human immunodeficiency virus type 1 (HIV-1). Within lymphatic tissues germinal centers represent foci of rapidly proliferating B cells governed by the interaction between B and T cells and follicular dendritic cells (FDC). Accumulating evidence suggests an important role of FDC in the pathophysiology of the acquired immunodeficiency syndrome. Direct proof for the infectibility of FDC with HIV-1 has been lacking until recently when we were able to demonstrate a CD4-independent infection of FDC in vitro. Here we report that in vitro HIV-1-infected human FDC do not only contain proviral DNA, but also produce the virus, and transmit the infection to T cells. Furthermore, electron microscopical studies on ex vivo isolated FDC from simian immunodeficiency virus (SIV)-infected rhesus monkeys revealed typical virus budding. In addition, FDC from SIV-infected rhesus monkeys transmitted the infection to T cells in vitro. Due to this central role within the immune response FDC may serve as preferential targets for HIV both by trapping of virions on their surfaces and by productive infection. During disease FDC become productively infected and may, thus, be regarded as crucial elements in viral dissemination.

The human germinal centre cells, follicular dendritic cells and germinal centre T cells produce B cell-stimulating cytokines.

Cytokine gene expression was investigated in the germinal centre constituents of follicular dendritic cells and germinal centre T cells and compared to the mRNA expression of nongerminal centre tonsillar cells. Cells were isolated from human tonsils by preenrichment with MACS and subsequent FACS sorting. Cytokine gene expression was investigated by intronspanning RT-PCR for IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-alpha, IFN-beta, IFN-gamma, and TNF-alpha. Frequency of cytokine-producing cells and the cytokine production pattern of single cells were determined by single cell PCR. Furthermore, cytokine protein expression was investigated by immunohistology. Using these methods, we found a strong production of IL-1 beta mRNA and protein in a small percentage of FDC. Germinal centre T cells showed production of IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha mRNA. The mRNAs of these cytokines could also be detected at the single cell level; as they were produced in a high percentage of germinal centre T cells, whereas immunohistological staining was negative, we conclude that a high percentage of germinal centre T lymphocytes produce IL-2, IL-4, IL-10, IFN-gamma, and TNF-alpha in low concentrations in contrast to T cells outside the germinal centre, in which strong cytokine production in few cells was shown earlier.

Endotoxin and lipid A stimulate proliferation of human T cells in the presence of autologous monocytes.

In this paper we describe a new activity of LPS and partial structures: the induction of DNA synthesis and lymphokine production of human T lymphocytes. The LPS-induced T cell proliferation is dose dependent and requires 100 to 10,000 ng/ml of LPS or synthetic lipid A (compound 506) for optimal stimulation. In contrast, the synthetic lipid A precursor Ia (compound 406) is not active but rather antagonizes LPS-induced proliferation. The proliferation is accompanied by the expression of mRNA for the Th1 cell-derived lymphokines IFN-gamma and IL-2, but not for the Th2 lymphokines IL-4, IL-5, or IL-10. Highly enriched T lymphocyte preparations with less than 0.1% monocytes are not stimulated by LPS, showing that monocytes are required for T cell proliferation. Reconstitution experiments show that only monocytes, but not B lymphocytes, are able to support induction of DNA synthesis. Separating LPS-stimulated monocytes from T lymphocytes by a membrane, permeable for cytokines but not for cells, abolishes T cell proliferation. Fixation of monocytes with paraformaldehyde also abrogates their accessory function for T cells. If the monocytes are preincubated for 2 h at 37 degrees C with LPS and then washed, they still are able to induce T cell proliferation in the absence of additional LPS. Our results indicate that human T cells respond in a monocyte-supported manner to LPS exposure by proliferation and lymphokine production. We hypothesize that this reactivity of T lymphocytes to LPS may be of clinical relevance.

Pentoxifylline: a potent inhibitor of IL-2 and IFN-gamma biosynthesis and BCG-induced cytotoxicity.

In the present study we investigated the influence of pentoxifylline (POF) on bacillus Calmette-Guérin (BCG)- and phytohaemagglutinin (PHA)-induced DNA synthesis and cytokine release, and BCG-induced cytotoxicity of human peripheral blood mononuclear cells (PBMC). DNA synthesis of PBMC stimulated with either BCG or PHA was inhibited by POF. We also demonstrated that the addition of POF led to a POF dose-dependent decrease of the release of the cytokines interleukin (IL)-2, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). The release of IL-6 remained unaffected. With respect to the inhibition of BCG-induced IL-2 and IFN-gamma release POF is active at the transcriptional (mRNA) level, as found by polymerase chain reaction (PCR). However, PHA-induced mRNA expression of these lymphokines is not affected by POF. Thus, the existence of a post-transcriptional regulation of PHA-induced cytokine release by POF can be assumed. The observed inhibition of cytokine release is correlated with a potent inhibitory effect of POF on BCG-induced cytotoxicity against bladder tumour cell lines. This effect is reversible.