Invariant NKT cells rapidly activated via immunization with diverse contact antigens collaborate in vitro with B-1 cells to initiate contact sensitivity

In cutaneous contact sensitivity there is an early elicited innate cascade of complement, mast cells, and platelets activated via IgM Abs. This response is required to initiate the elicitation of acquired classical contact sensitivity by leading to local recruitment of effector T cells. We recently performed in vivo experiments showing that collaboration is required between innate-like invariant Valpha14+ NKT cells (iNKT) and the innate-like B-1 B cell subset to induce this initiation process. Contact sensitization triggers iNKT cells to produce IL-4 to coactivate the B-1 cells along with specific Ag for production of the initiating IgM Abs. We now describe in vitro collaboration of iNKT and B-1 cells. Normal peritoneal B-1 cells, incubated in vitro with soluble Ag, and with 1-h in vivo immune iNKT cells producing IL-4, are activated to mediate the contact sensitivity-initiation cascade. The three components of this process can be activated by different Ag. Thus, 1-h iNKT cell activation, B-1 cell stimulation, and generation of immune effector T cells can be induced by sensitization with three different Ag to respectively generate IL-4 and Ag-specific IgM Abs, to recruit the Ag-specific effector T cells. These findings have relevance to allergic and autoimmune diseases in which infections can trigger exacerbation of T cell responses to allergens or to autoantigens.     

Subunits of IgM reconstitute defective contact sensitivity in B-1 cell-deficient xid mice: kappa light chains recruit T cells independent of complement

The elicitation of contact sensitivity (CS) to local skin challenge with the hapten trinitrophenyl (TNP) chloride requires an early process that is necessary for local recruitment of CS-effector T cells. This is called CS initiation and is due to the B-1 subset of B cells activated at immunization to produce circulating IgM Ab. At challenge, the IgM binds hapten Ag in a complex that locally activates C to generate C5a that aids in T cell recruitment. In this study, we present evidence that CS initiation is indeed mediated by C-activating classic IgM anti-TNP pentamer. We further demonstrate the involvement of IgM subunits derived either from hybridomas or from lymphoid cells of actively immunized mice. Thus, reduced and alkylated anti-TNP IgM also initiates CS, likely due to generated H chain-L chain dimers, as does a mixture of separated H and L chains that still could weakly bind hapten, but could not activate C. Remarkably, anti-TNP kappa L chains alone mediated CS initiation that was C-independent, but was dependent on mast cells. Thus, B-1 cell-mediated CS initiation required for T cell recruitment is due to activation of C by specific IgM pentamer, and also subunits of IgM, while kappa L chains act via another C-independent but mast cell-dependent pathway.     

Peritoneal cavity B cells are precursors of splenic IgM natural antibody-producing cells

Peritoneal cavity B-1 cells are believed to produce IgM natural Abs. We have used alpha1,3-galactosyltransferase-deficient (GalT(-/-)) mice, which, like humans, produce IgM natural Abs against the carbohydrate epitope Galalpha1,3Gal (Gal), to demonstrate that peritoneal cavity B-1b cells with anti-Gal receptors produce anti-Gal IgM Abs only after LPS stimulation. Likewise, peritoneal cavity cells of GalT(-/-) and wild-type mice do not produce IgM Abs of other specificities without LPS stimulation. Development of Ab-secreting capacity is associated with loss of CD11b/CD18 (Mac-1) expression. In contrast, there are large numbers of cells producing anti-Gal and other IgM Abs in fresh splenocyte preparations from GalT(-/-) and (for non-Gal specificities) wild-type mice. These cells are Mac-1(-) but otherwise B-1b-like in their phenotype. We therefore hypothesized a pathway wherein peritoneal cavity B cells migrate into the spleen after activation in vivo and lose Mac-1 expression to become IgM Ab-producing cells. Consistent with this possibility, splenectomy reduced anti-Gal Ab production after immunization of GalT(-/-) mice with Gal-positive rabbit RBC. Furthermore, splenectomized B6 GalT(-/-), Ig micro -chain mutant ( micro (-/-)) (both Gal- and B cell-deficient) mice produced less anti-Gal IgM than nonsplenectomized controls after adoptive transfer of peritoneal cavity cells from B6 GalT(-/-) mice. When sorted GalT(-/-) Mac-1(+) peritoneal cavity B cells were adoptively transferred to B6 GalT(-/-), micro (-/-) mice, IgM Abs including anti-Gal appeared, and IgM-producing and Mac1(-) B cells were present in the spleen 5 wk after transfer. These findings demonstrate that peritoneal cavity Mac-1(+) B-1 cells are precursors of Mac-1(-) splenic IgM Ab-secreting cells.     

Mac-1-negative B-1b phenotype of natural antibody-producing cells, including those responding to Gal alpha 1,3Gal epitopes in alpha 1,3-galactosyltransferase-deficient mice

Human natural Abs against Galalpha1-3Galbeta1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21(-/low) IgM(high) B220(low) CD5(-) Mac-1(-) 493(-) cells) in the spleens of alpha1, 3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of alpha1, 3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21(-/low) IgM(high) CD5(-) CD43(+) Mac-1(+)). However, these became Mac-1(-) and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1(+) B-1b cells and Mac-1(-) B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1(-) splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.     

Programmed cell death 1 suppresses B-1b cell expansion and long-lived IgG production in response to T cell-independent type 2 antigens

B-1b cells play a key role in producing Abs against T cell-independent type 2 Ags. However, the factors regulating Ab production by this unique B cell subset are not well understood. In this study, a detailed analysis of the B cell response to 2,4,6-trinitrophenol (TNP)-Ficoll was performed using normal mice. TNP-Ficoll delivered i.p. or i.v. induced rapid Ag-specific B-1b cell activation, expansion, isotype switching, and plasmablast/plasma cell differentiation. Ag-specific B-1b cell numbers peaked at day 5 and then gradually declined in the spleen but remained elevated in the peritoneal cavity beyond 40 d postimmunization. In addition to expressing CD43, CD44, and CD86, Ag-activated B-1b cells transiently expressed programmed cell death 1 (PD-1), which functionally suppressed BCR-induced B-1b cell in vitro proliferation when additional costimulatory signals were lacking. Inhibiting PD-1:PD-1 ligand interactions during TNP-Ficoll immunization significantly enhanced Ag-specific B-1b cell expansion and the frequency of IgG isotype switching and plasmablast/plasma cell differentiation. Remarkably, PD-1 mAb blockade during the first week following immunization resulted in significantly increased numbers of both splenic and bone marrow Ag-specific IgG3-secreting cells, but not IgM-secreting cells, at both early (day 5) and late (week 6) time points. Moreover, Ag-specific serum IgG3 levels, as well as IgG2c, IgG2b, and IgA levels, remained significantly elevated in PD-1 mAb-treated mice relative to control Ab-treated mice for ≥6 wk postimmunization. Thus, PD-1:PD-1 ligand interactions occurring shortly after initial T cell-independent type 2 Ag encounter play a critical role in suppressing Ag-specific B-1b cell expansion and the development of long-term IgG-producing bone marrow and spleen cells.     

B-1 B cells mediate required early T cell recruitment to elicit protein-induced delayed-type hypersensitivity

We define the initiation of elicited delayed-type hypersensitivity (DTH) as a series of processes leading to local extravascular recruitment of effector T cells. Responses thus have two sequential phases: 1) 2-h peaking initiation required for subsequent recruitment of T cells, and 2) the late classical 24-h component mediated by the recruited T cells. We analyzed DTH initiation to protein Ags induced by intradermal immunization without adjuvants. Ag-spceific initiating cells are present by 1 day in spleen and lymph nodes. Their phenotypes, determined by depletion of cell transfers by mAb and complement, are CD5(+), CD19(+), CD22(+), B220(+), Thy1(+), and Mac1(+), suggesting that they are B-1 B cells. DTH initiation is absent in micro MT B cell and xid B-1 cell deficient mice, is impaired in mice unable to secrete IgM, and is reconstituted with 1 day immune serum, suggesting that early B-1 cell-derived IgM is responsible. Study of complement C5a receptor-deficient mice, anti-C5 mAb neutralization, or mast cell deficiency suggests that DTH initiation depends on complement and mast cells. ELISPOT assay confirmed production of Ag-specific IgM Abs at days 1 and 4 in wild-type mice, but not in B-1 cell-deficient xid mice. We conclude that rapidly activated B-1 cells produce specific IgM Abs which, after local secondary skin challenge, form Ag-Ab complexes that activate complement to generate C5a. This stimulates C5a receptors on mast cells to release vasoactive substances, leading to endothelial activation for the 2-h DTH-initiating response, allowing local recruitment of DTH-effector T cells.     

TLR-dependent IL-4 production by invariant Valpha14+Jalpha18+ NKT cells to initiate contact sensitivity in vivo

LPS stimulated B-1 cell polyclonal in vivo IgM responses depend on IL-4 release by invariant Valpha14+Jalpha18+ NKT (iNKT) cells. The IgM Abs can recruit effector T cells to mediate contact sensitivity. LPS activates the B-1 cell response just 1 day later, and depends on CD1d, iNKT cells, IL-4, TLR4, and MyD88. LPS in vivo and in vitro stimulates rapid preferential production of IL-4 in hepatic iNKT cells within 2 h. TLR4 were demonstrated in iNKT cells by flow cytometry and functional studies. Thus, innate microbial stimulation via TLR can activate iNKT cell and B-1 cell collaboration. The result is polyclonal IgM Ab responses capable of recruiting Ag-specific T cells into tissues. This may be involved in the promotion of autoimmunity by infectious agents.     

IL-9-induced expansion of B-1b cells restores numbers but not function of B-1 lymphocytes in xid mice

Mice expressing the X-linked immunodeficiency (xid) mutation lack functional Bruton's tyrosine kinase and were shown to be specifically deficient in peritoneal B-1 lymphocytes. We have previously shown that IL-9, a cytokine produced by TH2 lymphocytes, promotes B-1 cell expansion in vivo. To determine whether IL-9 overexpression might compensate the xid mutation for B-1 lymphocyte development, we crossed xid mice with IL-9-transgenic mice. In this model, IL-9 restored normal numbers of mature peritoneal B-1 cells that all belonged to the CD5(-) B-1b subset. Despite this normal B-1 lymphocyte number, IL-9 failed to restore classical functions of B-1 cells, namely, the production of natural IgM Abs, the T15 Id Ab response to phosphorylcholine immunization, and the antipolysaccharide humoral response against Streptococcus pneumoniae. By using bromelain-treated RBC, we showed that the antigenic repertoire of these IL-9-induced B-1b lymphocytes was different from the repertoire of classical CD5(+) B-1a cells, indicating that the lack of B-1 function by B-1b cells is associated with distinct Ag specificities. Taken together, our data show that B-1b cell development can restore the peritoneal B-1 population in xid mice but that these B-1b cells are functionally distinct from CD5(+) B-1a lymphocytes.     

Transgenic overexpression of G5PR that is normally augmented in centrocytes impairs the enrichment of high-affinity antigen-specific B cells, increases peritoneal B-1a cells, and induces autoimmunity in aged female mice

To investigate signals that control B cell selection, we examined expression of G5PR, a regulatory subunit of the serine/threonine protein phosphatase 2A, which suppresses JNK phosphorylation. G5PR is upregulated in activated B cells, in Ki67-negative centrocytes at germinal centers (GCs), and in purified B220(+)Fas(+)GL7(+) mature GC B cells following Ag immunization. G5PR rescues transformed B cells from BCR-mediated activation-induced cell death by suppression of late-phase JNK activation. In G5PR-transgenic (G5PR(Tg)) mice, G5PR overexpression leads to an augmented generation of GC B cells via an increase in non-Ag-specific B cells and a consequent reduction in the proportion of Ag-specific B cells and high-affinity Ab production after immunization with nitrophenyl-conjugated chicken γ-globulin. G5PR overexpression impaired the affinity-maturation of Ag-specific B cells, presumably by diluting the numbers of high-affinity B cells. However, aged nonimmunized female G5PR(Tg) mice showed an increase in the numbers of peritoneal B-1a cells and the generation of autoantibodies. G5PR overexpression did not affect the proliferation of B-1a and B-2 cells but rescued B-1a cells from activation-induced cell death in vitro. G5PR might play a pivotal role in B cell selection not only for B-2 cells but also for B-1 cells in peripheral lymphoid organs.     

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.     

Identification of initiator B cells, a novel subset of activation-induced deaminase-dependent B-1-like cells that mediate initiation of contact sensitivity

Contact sensitivity (CS) is related to delayed-type hypersensitivity and is a well-characterized prototype of T cell-mediated inflammation. However, the inflammatory response associated with CS is additionally dependent on Ag-specific IgM produced by a subpopulation of B cells in response to sensitization. Upon re-exposure to hapten, this IgM mediates rapid vascular activation and subsequent recruitment of proinflammatory T cells to the local site. Interference with this pathway prevents the full development of the classic delayed inflammatory response and is therefore termed the "CS initiation" pathway. In this study, we show that CS initiation is defective in mice deficient in activation-induced deaminase, an enzyme central to the process of somatic hypermutation. Using adoptive transfer experiments, we demonstrate that the defect is specific to a B-1-like population of B cells and that transfer of WT cells reconstitutes CS initiation mechanisms in deficient recipients. We went on to identify a novel subpopulation of Ag-binding B cells in the spleens of sensitized mice that possess initiation activity (CD19(+)CD5(+)Thy-1(int)IgM(high)IgD(high)) that we name "initiator B cells." Analysis of BCR H chain genes isolated from these cells revealed evidence of activation-induced deaminase-mediated somatic hypermutation. The sensitivity of CS initiation to very low amounts of sensitizing hapten suggests that the responsible B cells have increased IgM receptor gene mutations enabling selection to generate Abs with sufficient affinity to mediate the response.     

Isolated lymphoid follicles are not IgA inductive sites for recombinant Salmonella

In this study, we investigated whether isolated lymphoid follicles (ILF) play a role in the regulation of intestinal IgA antibody (Ab) responses. The transfer of wild type (WT) bone marrow (BM) to lymphotoxin-alpha-deficient (LTalpha(-/-)) mice resulted in the formation of mature ILF containing T cells, B cells, and FDC clusters in the absence of mesenteric lymph nodes and Peyer's patches. Although the ILF restored total IgA Abs in the intestine, antigen (Ag)-specific IgA responses were not induced after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin. Moreover, Ag-specific cell proliferation was not detected in the ILF. Interestingly, no IgA anti-LPS Abs were detected in the fecal extracts of LTalpha(-/-) mice reconstituted with WT BM. On the basis of these findings, ILF can be presumed to play a role in the production of IgA Abs, but lymphoid nodules are not inductive sites for the regulation of Ag-specific intestinal IgA responses to recombinant Salmonella.     

TLR9 in peritoneal B-1b cells is essential for production of protective self-reactive IgM to control Th17 cells and severe autoimmunity

The role of TLR9 in the development of the autoimmune disease systemic lupus erythematosus is controversial. In different mouse models of the disease, loss of TLR9 abolishes the generation of anti-nucleosome IgG autoantibodies but at the same time exacerbates lupus disease. However, the TLR9-dependent tolerance mechanism is unknown. In this study, we show that loss of TLR9 is associated with low peritoneal B-1b cell numbers and low levels of protective self-reactive IgM serum autoantibodies in lupus-prone FcγRIIB-deficient mice leading to the uncontrolled accumulation of proinflammatory CD4(+) cells and exacerbated autoimmunity. TLR7 signaling was not able to compensate for the loss of TLR9 signaling in peritoneal B-1b cells to induce IgM Abs. Transfer of TLR9-expressing peritoneal B-1b cells from FcγRIIB-deficient mice or of recombinant monoclonal self-reactive IgM Abs was sufficient to reduce the frequency of proinflammatory Th17 cells and lupus disease in FcγRIIB/TLR9 double-deficient mice. Taken together, these data provide evidence for a TLR9-dependent tolerance mechanism of peritoneal B-1b cells generating protective self-reactive IgM in lupus-prone mice to control Th17 cell development and severe autoimmunity.     

Functional activity of natural antibody is altered in Cr2-deficient mice

The major source of natural IgM Abs are B-1 cells, which differ from conventional B cells in their anatomic location, cell surface phenotype, restricted usage of particular V(H) genes and limited use of N-region addition during V-D-J rearrangement. The origin of B-1 cells is unclear. However, they are capable of self-renewal and their development is sensitive to signaling via the B cell receptor, as genetic defects that impair the strength of the signal often result in limited development. These findings suggest that B-1 cells require either an intrinsic signal, or contact with Ag, for positive selection and expansion and/or maintenance in the periphery. In support of interaction with cognate Ag, deficiency in the complement receptors CD21/CD35 results in a 30-40% decrease in the CD5(+) B-1 population. To determine whether this reduction reflects a loss of certain specificities or simply a proportional decline in the repertoire, we examined peritoneal B cells isolated from Cr2(+) and Cr2(def) mice for recognition of a B-1 cell Ag, i.e., phosphatidylcholine, and assayed for injury in an IgM natural Ab-dependent model of reperfusion injury. We found a similar frequency of phosphatidylcholine-specific CD5(+) B-1 cells in the two strains of mice. By contrast, the Cr2(def) mice have reduced injury in the IgM-dependent model of reperfusion injury. Reconstitution of the deficient mice with pooled IgM or adoptive transfer of Cr2(+) peritoneal B cells restored injury. These results suggest that complement receptors CD21/CD35 are important in maintenance of the B-1 cell repertoire to some, but not all, specificities.     

Absence of Peyer's patches and abnormal lymphoid architecture in chronic proliferative dermatitis (cpdm/cpdm) mice

The chronic proliferative dermatitis (cpdm) mutation causes inflammation in multiple organs, most prominently in the skin. Examination of the immune system revealed severe abnormalities in the architecture of lymphoid tissues. Peyer's patches were absent. In contrast, the spleen, lymph nodes, and nasal-associated lymphoid tissues were present. The spleen had normal numbers of T and B cells, but the spleen, lymph nodes, and nasal-associated lymphoid tissues had poorly defined follicles and lacked germinal centers and follicular dendritic cells. The marginal zone in the spleen was absent. The total concentration of serum IgG, IgA, and IgE in cpdm/cpdm mice was significantly decreased, whereas serum IgM was normal. Fecal IgA was low to undetectable in mutant mice, and the concentration of fecal IgM was increased. The titer of DNP-specific Abs following immunization with DNP-keyhole limpet hemocyanin was significantly decreased for all IgG subclasses. In contrast, T cell function appeared normal as assessed by evaluation of the contact hypersensitivity response in cpdm/cpdm mice. The cpdm mutation causes a complex phenotype that is characterized by multiorgan inflammation and the defective development of lymphoid tissues. The cpdm/cpdm mouse may be a useful model to study the factors that control the development of lymphoid tissues, in particular the Peyer's patches, and the mechanisms that control the humoral immune response.     

Rapid induction of splenic and peritoneal B-1a cells in adult mice by thymus-independent type-2 antigen

We have produced a transgenic mouse (PV1TgL) that can only generate B lymphocytes with an Ig receptor specific for the synthetic polymer polyvinyl pyrrolidinone. Before immunization, bone marrow B cell numbers are very low, and peripheral lymphoid organs are almost devoid of B cells, confirming the role of positive selection by Ag in the development of mature B cell populations. The predominant population of B cells in the spleens of naive adult PV1TgL mice have most of the characteristics of marginal zone B cells, including anatomical location in the peripheral areas of the splenic white pulp. After immunization, a new population of B cells appears in the spleen with the characteristics of B-1 cells. Similar cells also appear somewhat later in the peritoneal cavity. Our findings suggest that immunization with a thymus-independent Ag can lead to the appearance and expansion of Ag-reactive B-1 cells in an adult mouse.     

Antigen-specific CD8+ T cells switch the immune response induced by antigen from an IgG to a cell-mediated mode.

We have developed an in vivo/in vitro immunization procedure with xenogeneic RBC as Ag that results in the generation of a lymphoid population that expresses potent delayed-type hypersensitivity (DTH) and produces little, if any, antibody. This lymphoid population contains Ag-specific CD8+ T cells that can inhibit the induction of a strong IgG response. These CD8+ T cells are shown to not only inhibit the antibody response in an Ag-specific manner but allow the Ag to induce cells of the target population to express DTH. Furthermore, the Ag-specific inhibition of the antibody response and the Ag-specific enhancement of the induction of DTH appear to be coordinately regulated, as the same number of CD8+ T cells cells is required to achieve both effects. Thus these CD8+ T cells are shown to switch the response induced by Ag from a humoral to a cell-mediated mode. These regulatory characteristics are consistent with a physiologic role for these cells of ensuring the absence of antibody production during a strong, cell-mediated response.     

Cutting edge: Langerin+ dendritic cells in the mesenteric lymph node set the stage for skin and gut immune system cross-talk

Topical transcutaneous immunization (TCI) presents many clinical advantages, but its underlying mechanism remains unknown. TCI induced Ag-specific IgA Ab-secreting cells expressing CCR9 and CCR10 in the small intestine in a retinoic acid-dependent manner. These intestinal IgA Abs were maintained in Peyer's patch-null mice but abolished in the Peyer's patch- and lymph node-null mice. The mesenteric lymph node (MLN) was shown to be the site of IgA isotype class switching after TCI. Unexpectedly, langerin(+)CD8alpha(-) dendritic cells emerged in the MLN after TCI; they did not migrate from the skin but rather differentiated rapidly from bone marrow precursors. Depletion of langerin(+) cells impaired intestinal IgA Ab responses after TCI. Taken together, these findings suggest that MLN is indispensable for the induction of intestinal IgA Abs following skin immunization and that cross-talk between the skin and gut immune systems might be mediated by langerin(+) dendritic cells in the MLN.     

Tolerance induction by transcutaneous immunization through ultraviolet-irradiated skin is transferable through CD4+CD25+ T regulatory cells and is dependent on host-derived IL-10

UV radiation of the skin impairs immune responses to haptens and to tumor Ags. Transcutaneous immunization (TCI) is an effective method of inducing immune responses to protein and peptide Ag. We explore the effect of UV irradiation on TCI. The generation of Ag-specific CTL to OVA protein, but not class I MHC-restricted OVA peptide, is inhibited by TCI through UV-irradiated skin. Consequently, the induction of protein contact hypersensitivity and in vivo Ag-specific CTL activity following OVA protein immunization is prevented. Application of haptens to UV-exposed skin induces hapten-specific tolerance. We demonstrate that application of protein or class II MHC-restricted OVA peptide to UV-irradiated skin induces transferable Ag-specific tolerance. This tolerance is mediated by CD4(+)CD25(+) T regulatory (T(reg)) cells. These Ag-specific T(reg) cells inhibit the priming of CTL following protein immunization in the presence of CpG adjuvant. IL-10 deficiency is known to prevent hapten-specific tolerance induction. In this study, we demonstrate, using IL-10-deficient mice and adoptive T cell transfer, that IL-10 is required for the direct inhibition of CTL priming following immunization through UV-irradiated skin. However, IL-10 is not required for the induction of T(reg) cells through UV-irradiated skin as IL-10-deficient T(reg) cells are able to mediate tolerance. Rather, host-derived IL-10 is required for the function of UV-generated T(reg) cells. These experiments indicate that protein and peptide TCI through UV-irradiated skin may be used to induce robust Ag-specific tolerance to neo-Ags and that UV-induced T(reg) cells mediate their effects in part through the modulation of IL-10.     

Persistence, immune specificity, and functional ability of murine mutant ras epitope-specific CD4(+) and CD8(+) T lymphocytes following in vivo adoptive transfer.

Adoptive T-cell transfer has been shown to be a potentially effective strategy for cellular immunotherapy in some murine models of disease. However, several issues remain unresolved regarding some of the basic features involved in effective adoptive transfer, such as the influence of specific peptide antigen (Ag) boost after T-cell transfer, the addition of IL-2 post-T-cell transfer, the trafficking of transferred T cells to lymphoid and nonlymphoid tissues, and the functional stability of recoverable CD4(+) and CD8(+) T cells. We investigated several of these parameters, particularly as they relate to the persistence and maintenance of effector functions of murine CD4(+) and/or CD8(+) T lymphocytes after adoptive cellular transfer into partially gamma-irradiated syngeneic hosts. Our laboratory previously identified murine (H-2(d)) immunogenic CD4(+) and CD8(+) T-cell peptide epitopes reflecting codon 12 ras mutations as tumor-specific Ag. Therefore, the model system chosen here employed epitope-specific MHC class II-restricted CD4(+) T cells and MHC class I-restricted CD8(+) T cells produced from previously immunized BALB/c mice. Between 2 and 7 days after T-cell transfer, recipient mice received various combinations of peptide boosts and/or IL-2 treatments. At different times after the T-cell transfer, spleen and lung tissues were analyzed phenotypically to monitor the persistence of the immune T cells and functionally (via proliferation or cytotoxicity assays) to assess the maintenance of peptide specificity. The results showed that immune donor T lymphocytes (uncultured immune T cells or cloned T cells) were recoverable from the spleens and lungs of recipient mice after transfer. The recovery of Ag-specific T-cell responses was greatest from recipient mice that received peptide boosts and IL-2 treatment. However, mice that received a peptide boost without IL-2 treatment responded nearly as well, which suggested that including a peptide boost after T-cell transfer was more obligatory than exogenous IL-2 treatment to sustain adoptively transferred T cells in vivo. Ag-specific T-cell responses were weak in mice that either received IL-2 alone or did not receive the cognate peptide boost after T-cell transfer. The T-cell clones were also monitored by flow cytometry or RT-PCR based on expression of the T-cell receptor Vbeta-chain, which was previously characterized. Ag-specific T cells were recovered from both spleens and lungs of recipient mice, demonstrating that the T-cell clones could localize to both lymphoid and nonlymphoid tissues. This study demonstrates that both uncultured and in vitro-cloned T lymphocytes can migrate to lymphoid tissues and nonlymphoid (e.g., lung) tissues in recipient hosts and that their functional activities can be maintained at these sites after transfer, if they are exposed to peptide Ag in vivo.