Detection of antigen-specific T cells in experimental immune-mediated blepharoconjunctivitis in DO11.10 T cell receptor transgenic mice

Antigen (Ag)-specific T cells are thought to play a key role in pathogenesis of chronic allergic conjunctivitis (AC) such as atopic keratoconjunctivitis (AKC) and vernal keratoconjunctivitis (VKC). In order to investigate the trafficking of Ag-specific T cells in experimental immune-mediated blepharoconjunctivitis (EC), we established a novel AC model in DO11.10 T cell receptor (TcR) transgenic (Tg) mice. DO11.10 TcR-Tg mice were challenged with eye drops of whole OVA protein, OVA peptide 1-15, 321-335, or 323-339. Their eyes were histologically examined. Conventional proliferation assay was performed against each Ag. Phenotypes of infiltrating cells and kinetics of Ag-specific T cells were investigated by immunohistochemistry. Adoptive transfer of CD4(+) Ag-specific T cells from DO11.10 TcR-Tg to WT mice was performed. The distribution of KJ1-26(+) cells was investigated in recipient mice. The challenge of OVA peptide 323-339 induced infiltration of inflammatory cells in conjunctivae in a dose dependent manner, accompanied by the proliferative responses of splenocytes. Immunohistochemical analysis revealed Agspecific/ non-Ag-specific T cells, macrophages, and eosinophils in conjunctivae. Infiltration of Ag-specific T cells increased 24 hr later. Transfer of CD4(+) cells from DO11.10 TcR-Tg to WT mice induced EC depending on the number of transferred cells. Ag-specific T cells were detected in the conjunctivae and spleens of recipient mice, though its numbers were significantly smaller compared to DO11.10 TcR-Tg mice. The challenge of OVA peptide 323-339 induced EC in DO11.10 TcR-Tg mice without prior sensitization. The response was mediated by CD4(+) Ag-specific T cells. The trafficking of Ag-specific T cells in EC was clearly visualized.     

Generation of antigen-specific, Foxp3-expressing CD4+ regulatory T cells by inhibition of APC proteosome function

We tested the hypothesis that immature APC, whose NF-kappaB-signaling pathway and thus maturation was blocked by the proteosome inhibitor benzyloxycarbonyl-isoleucyl-glutamyl(O-tert-butyl)-alanyl-leucinal (PSI), could be a source of Ag-specific regulatory T (Treg) cells. DO11.10 CD4(+) T cells that were incubated with Ag- and PSI-pulsed APC proliferated poorly, produced less IL-2, IFN-gamma, and IL-10 in secondary cultures, and inhibited the response of both naive and memory CD4(+) T cells stimulated by Ag-pulsed APC. The generation of PSI-APC Treg cells required IL-10 production by APC. PSI-APC Treg cell inhibition required cell-cell contact but not IL-10 or TGF-beta. Addition of IL-2 did not reverse, but Ab to CTLA-4 did reverse partially the inhibitory effect. Depletion of CD25(+) T cells before initial culture with PSI-APC did not affect Treg generation. PSI-APC Treg cells expressed high levels of Foxp3, inhibited proliferation of naive DO11.10 T cells in vivo, and abrogated colitis driven by a memory Th1 response to bacterial-associated Ag. We conclude that NF-kappaB-blocked, immature APC are able to induce the differentiation of Treg cells that can function in vitro and in vivo in an Ag-specific manner.     

Peripheral deletion of antigen-specific T cells leads to long-term tolerance mediated by CD8+ cytotoxic cells

Peripheral deletion is one mechanism by which potentially self-reactive clones are removed whether they escape thymic deletion. We have examined the consequences of deleting Ag-specific T cells by i.v. injection of soluble Ag. Deletion of DO11.10 T cells by peptide was mediated predominately via a Fas/FasL mechanism. Animals that underwent deletion were tolerant to subsequent immunization with Ag, even when tolerant mice were given fresh Ag-specific DO11.10 T cells before immunization. Tolerance was mediated by CD8(+) T cells that killed the DO11.10-transgenic T cells in vivo. These data demonstrate that the programmed cell death of large numbers of T cells leads to peripheral tolerance mediated by CD8(+) CTLs.     

Disruption of innate-mediated proinflammatory cytokine and reactive oxygen species third signal leads to antigen-specific hyporesponsiveness

Successful Ag activation of naive T helper cells requires at least two signals consisting of TCR and CD28 on the T cell interacting with MHC II and CD80/CD86, respectively, on APCs. Recent evidence demonstrates that a third signal consisting of proinflammatory cytokines and reactive oxygen species (ROS) produced by the innate immune response is important in arming the adaptive immune response. In an effort to curtail the generation of an Ag-specific T cell response, we targeted the synthesis of innate immune response signals to generate Ag-specific hyporesponsiveness. We have reported that modulation of redox balance with a catalytic antioxidant effectively inhibited the generation of third signal components from the innate immune response (TNF-alpha, IL-1beta, ROS). In this study, we demonstrate that innate immune-derived signals are necessary for adaptive immune effector function and disruption of these signals with in vivo CA treatment conferred Ag-specific hyporesponsiveness in BALB/c, NOD, DO11.10, and BDC-2.5 mice after immunization. Modulating redox balance led to decreased Ag-specific T cell proliferation and IFN-gamma synthesis by diminishing ROS production in the APC, which affected TNF-alpha levels produced by CD4(+) T cells and impairing effector function. These results demonstrate that altering redox status can be effective in T cell-mediated diseases such as autoimmune diabetes to generate Ag-specific immunosuppression because it inhibits the third signal necessary for CD4(+) T cells to transition from expansion to effector function.     

Transgenic CD4 T cells (DO11.10) are destroyed in MHC-compatible hosts by NK cells and CD8 T cells

During an immune response a small number of rare Ag-specific clones proliferate extensively and decline, leaving a residual population for long-term memory. TCR transgenic (tg) CD4 T cells have been used widely to study the primary and memory response in vivo. We show here that naive TCR tg CD4 T cells from the DO11.10 strain transferred into wild type (wt) BALB/c recipients and not stimulated declined rapidly at the same rate as those primed in vivo by Ag. In the same recipients wt CD4 T cells survived. There was no evidence of an inherent defect in the tg T cells, which survived well when returned to DO11.10 recipients. Surprisingly, wt CD4 T cells declined rapidly in the same DO11.10 hosts. By depleting wt recipients of NK cells or CD8+ cells, the speed of reduction was slowed by half; rapid destruction was prevented completely by combing the two treatments. In contrast, preimmunization accelerated the loss of tg T cells. The results suggested that tg CD4 T cells were actively rejected by both NK and CD8 T cell responses. We consider whether, despite extensive backcrossing, tg T cells may retain genetic material (minor histocompatibility Ags) flanking the construct that compromises their survival in wt recipients.     

The state of CD4+ T cell activation is a major factor for determining the kinetics and location of T cell responses to oral antigen

Current models suggest that inductive immune responses to enteric Ag are initiated in Peyer's patches (PP) and mesenteric lymph nodes (MLN) followed by migration of activated, memory-like CD4(+) T cells to extralymphoid sites in the intestinal lamina propria (LP). The resultant immune system contains both naive and activated T cells. To examine the differential responses of naive and memory-like T cells to oral Ag, bone marrow chimeras (BMC) were generated. Irradiated BALB/c hosts were reconstituted with a mix of DO11.10 x RAG-1(-/-) and BALB/c bone marrow. In unprimed DO11.10 and BMC models, LP and PP DO11.10 T cells responded to oral Ag with similar kinetics. Responses of activated, memory-like T cells to oral Ag were examined in thymectomized BMC 60 days after i.p. immunization with OVA peptide in Freund's adjuvant (OVA(323-339)/CFA). Results indicate that i.p. OVA(323-339)/CFA generated a high proportion of memory-like CD45RB(low) DO11.10 T cells in peripheral lymphoid (40%) and intestinal LP (70%) tissue. Previously activated DO11.10 T cells in the LP responded to oral Ag earlier and at 50% higher levels compared with memory CD4(+) T cells localized to PP tissue. These data indicate that responses to oral Ag in antigenically naive animals are initiated in PP whereas in Ag-experienced animals LP T cells respond earlier and more vigorously than cells in PP. Taken together, these data suggest that previous activation alters the hierarchy of T cell responses to oral Ag by enhancing the efficiency of LP T cell activation.     

IL-2 and autocrine IL-4 drive the in vivo development of antigen-specific Th2 T cells elicited by nematode parasites

The intestinal nematode parasite, Nippostrongylus brasiliensis, triggers potent type 2 immunity. Using OVA peptide as a model Ag, we have examined the adjuvant effects of this parasite on the in vivo development of Ag-specific Th2 cells from naive DO11.10 T cells. Our findings show that Th2 cells can develop from transferred naive OVA-specific DO11.10 T cells in recipient IL-4-/- mice inoculated with N. brasiliensis plus OVA. However, autocrine IL-4 is required for in situ Th2 cell differentiation since transferred IL-4Ralpha-deficient DO11.10 T cells showed greatly reduced Th2 cell development in inoculated IL-4-/- recipient mice. Surprisingly, we also found that IL-2 blockade promoted B7-dependent T cell cycling, but inhibited the development of OVA-specific Th2 cells. Furthermore, the effects of IL-2 occurred independently of CD25+ T regulatory cells. These studies establish a previously unrecognized requirement for autocrine IL-4 and IL-2 in Th2 responses elicited by nematode parasites.     

Regulation of lymphoid homeostasis by IL-2 receptor signals in vivo

High-affinity IL-2R signals are required for peripheral lymphoid homeostasis in vivo. We found that CD25 was required for regulation of peripheral T cells in mice bearing either the DO11.10 MHC class II-restricted TCR transgene or an Iabeta-null mutation, suggesting that MHC class I- and class II-dependent T cell subsets are regulated independently by IL-2R signals. In contrast, deregulation of serum IgG1 levels in CD25-/- mice was dependent on CD4+ T cells. T cell expansion in DO11.10 CD25-/- mice was not preferential for cells escaping allelic exclusion by the TCR transgene, but was suppressed by a Rag-2-null mutation. Together, these findings suggest that endogenous TCR are required to trigger T cell expansion, but that CD25 regulates T cells activated by low-specificity signals. Expansion of DO11.10 T cells in response to cognate Ag was modestly reduced in CD25-/- T cells transferred into the normal lymphoid compartments of BALB/c mice. Moreover, activation-induced clonal contraction and apoptosis in vivo were intact in the absence of CD25. These data indicate that the regulatory role of high-affinity IL-2R signals extends beyond the control of Ag-specific responses and suggest a role for these signals in control of bystander T cell activation.     

CD100 enhances dendritic cell and CD4+ cell activation leading to pathogenetic humoral responses and immune complex glomerulonephritis

CD100, a member of the semaphorin family, is a costimulatory molecule in adaptive immune responses by switching off CD72's negative signals. However, CD100's potential pathogenetic effects in damaging immune responses remain largely unexplored. We tested the hypothesis that CD100 plays a pathogenetic role in experimental immune complex glomerulonephritis. Daily injection of horse apoferritin for 14 days induced immune complex formation, mesangial proliferative glomerulonephritis and proteinuria in CD100-intact (CD100+/+) BALB/c mice. CD100-deficient (CD100-/-) mice were protected from histological and functional glomerular injury. They exhibited reduced deposition of Igs and C3 in glomeruli, reduced MCP-1 and MIP-2 intrarenal mRNA expression, and diminished glomerular macrophage accumulation. Attenuated glomerular injury was associated with decreased Ag-specific Ig production, reduced CD4+ cell activation and cytokine production. Following Ag injection, CD4+ cell CD100 expression was enhanced and dendritic cell CD86 expression was up-regulated. However, in CD100-/- mice, dendritic cell CD86 (but not CD80) up-regulation was significantly attenuated. Following i.p. immunization, CD86, but not CD80, promotes early Ag-specific TCR-transgenic DO11.10 CD4+ cell proliferation and IFN-gamma production, suggesting that CD100 expression enables full expression of CD86 and consequent CD4+ cell activation. Transfer of CD100+/+ DO11.10 cells into CD100-/- mice resulted in decreased proliferation demonstrating that CD100 from other sources in addition to CD100 from Ag-specific CD4+ cells plays a role in initial T cell proliferation. Although T cell-B cell interactions also may be relevant, these studies demonstrate that CD100 enhances pathogenetic humoral immune responses and promotes the activation of APCs by up-regulating CD86 expression.     

Early events in peripheral regulatory T cell induction via the nasal mucosa

Nasal application of soluble Ags leads to Ag-specific suppression of systemic immune responses. This tolerance can be transferred to naive mice by CD4(+) regulatory T cells (T(R) cells) from the spleen, but little is known about the induction of mucosal T(R) cells in vivo. To investigate the induction of T(R) cells in the nose-draining cervical lymph node (CLN), CD4(+) T cells from DO11.10 OVA TCR transgenic mice were transferred to BALB/c recipients. Within 48 h after nasal OVA application, CD4(+) DO11.10 T cells in CLN, but not in the peripheral lymph node, had divided. Similarly, nonmucosal (i.m.) OVA application also induced CD4(+) DO11.10 T cells to proliferate in the draining inguinal lymph node (ILN), yet more vigorously and with different kinetics than the CD4(+) DO11.10 T cells in CLN. Functional analysis revealed that only proliferating CD4(+) DO11.10 T cells from CLN, and not ILN, could transfer tolerance to naive recipients. CD4(+) DO11.10 T cells from CLN were phenotypically similar to CD4(+) DO11.10 T cells from ILN, however, in CLN a higher percentage of CD25(+) proliferating CD4(+) DO11.10 T cells were detected compared with ILN. CD25 is not a discriminative marker for mucosal T(R) cells because both CD25(+) and CD25(-) CD4(+) DO11.10 T cells from the CLN could suppress delayed type hypersensitivity responses in adoptive transfer. These findings demonstrate that although striking similarities exist between the differentiation of T(R) and effector T cells, this does not include their function. We are the first to demonstrate that functional T(R) cells, which reside within both CD25(+) and CD25(-) subsets, can be isolated from CLN as early as 3 days after nasal OVA application.     

CD18 is required for intestinal T cell responses at multiple immune checkpoints

The intestinal immune response to oral Ags involves a complex multistep process. The requirements for optimal intestinal T cell responses in this process are unclear. LFA-1 plays a critical role in peripheral T cell trafficking and activation, however, its role in intestinal immune responses has not been precisely defined. To dissect the role of LFA-1 in intestinal immune responses, we used a system that allows for segregation of T cell migration and activation through the adoptive transfer of LFA-1-deficient (CD18(-/-)) CD4(+) T cells from DO11.10 TCR transgenic mice into wild-type BALB/c mice. We find that wild-type mice adoptively transferred with CD18(-/-) DO11.10 CD4(+) T cells demonstrate decreases in the numbers of Ag-specific T cells in the intestinal lamina propria after oral Ag administration. We also find that in addition to its role in trafficking to intestinal secondary lymphoid organs, LFA-1 is required for optimal CD4(+) T cell proliferation in vivo upon oral Ag immunization. Furthermore, CD18(-/-) DO11.10 CD4(+) T cells primed in the intestinal secondary lymphoid organs demonstrate defects in up-regulation of the intestinal-specific trafficking molecules, alpha(4)beta(7) and CCR9. Interestingly, the defect in trafficking of CD18(-/-) DO11.10 CD4(+) T cells to the intestinal lamina propria persists even under conditions of equivalent activation and intestinal-tropic differentiation, implicating a role for CD18 in the trafficking of activated T cells into intestinal tissues independent of the earlier defects in the intestinal immune response. This argues for a complex role for CD18 in the early priming checkpoints and ultimately in the trafficking of T cells to the intestinal tissues during an intestinal immune response.     

Expression of dual TCR on DO11.10 T cells allows for ovalbumin-induced oral tolerance to prevent T cell-mediated colitis directed against unrelated enteric bacterial antigens

The triggering Ag for inflammatory bowel disease and animal models of colitis is not known, but may include gut flora. Feeding OVA to DO11.10 mice with OVA-specific transgenic (Tg) TCR generates Ag-specific immunoregulatory CD4(+) T cells (Treg) cells. We examined the ability of oral Ag-induced Treg cells to suppress T cell-mediated colitis in mice. SCID-bg mice given DO11.10 CD4(+)CD45RB(high) T cells developed colitis, and cotransferring DO11.10 CD45RB(low)CD4(+) T cells prevented CD4(+)CD45RB(high) T cell-induced colitis in the absence of OVA. The induction and prevention of disease by DO11.10 CD4(+) T cell subsets were associated with an increase in endogenous TCRalpha chain expression on Tg T cells. Feeding OVA to SCID-bg mice reconstituted with DO11.10 CD4(+)CD45RB(high) attenuated the colitis in association with increased TGF-beta and IL-10 secretion, and decreased proliferative responses to both OVA and cecal bacteria Ag. OVA feeding also attenuated colitis in SCID-bg mice reconstituted with a mix of BALB/c and DO11.10 CD45RB(high) T cells, suggesting that OVA-induced Treg cells suppressed BALB/c effector cells. The expression of endogenous non-Tg TCR allowed for DO11.10-derived T cells to respond to enteric flora Ag. Furthermore, feeding OVA-induced Treg cells prevented colitis by inducing tolerance in both OVA-reactive and non-OVA-reactive T cells and by inducing Ag-nonspecific Treg cells. Such a mechanism might allow for Ag-nonspecific modulation of intestinal inflammation in inflammatory bowel disease.     

CD8 blockade promotes antigen responsiveness to nontolerizing antigen in tolerant mice by inhibiting apoptosis of CD4+ T cells

Using the DO11.10 CD4+ TCR-transgenic mouse system, we have recently shown that CD8 blockade promotes the expansion of Ag-specific regulatory CD4+ T cells in mice made tolerant to OVA with anti-CD4 mAb. We now show that CD8 blockade is also critical to promoting responses to nontolerizing Ag in anti-CD4 mAb-treated tolerant mice. Previously published work shows that treatment with anti-CD4 mAb without CD8 blockade induces Ag-specific tolerance. We now show that, in addition to inducing tolerance, anti-CD4 mAb treatment also significantly reduces responsiveness to irrelevant, nontolerizing Ag, and this unresponsiveness is associated with significant apoptosis of the CD4+ T cells. Anti-CD4 mAb-induced apoptosis is inhibited by cotreatment with anti-CD8 mAb and responsiveness to irrelevant Ag is restored, while Ag-specific tolerance is maintained. These data suggest that CD8 blockade promotes responsiveness to nontolerizing Ags in tolerant mice by inhibiting CD4+ T cell apoptosis.     

Contrasting impacts of immunosuppressive agents (rapamycin,FK506, cyclosporin A,and dexamethasone) on bidirectional dendritic cell-T cell interaction during antigen presentation

Rapamycin (RAP), tacrolimus (FK506), cyclosporin A, and glucocorticoids represent modern and classic immunosuppressive agents being used clinically. Although these agents have distinct molecular mechanisms of action and exhibit different immunoregulatory profiles, their direct influences on Ag presentation processes remain relatively unknown. Here we report quantitative and qualitative differences among the above four immunosuppressants in their impact on Ag-specific, bidirectional interaction between dendritic cells (DC) and CD4(+) T cells. In the presence of relevant Ag, bone marrow-derived DC delivered activation signals to CD4(+) T cells isolated from the DO11.10 TCR transgenic mice, leading to clonal expansion; secretion of IFN-gamma, IL-2, and IL-4; and surface expression of CD69. Conversely, DO11.10 T cells delivered maturation signals to DC, leading to IL-6 and IL-12 production and CD40 up-regulation. FK506 (10(-10)-10(-8) M) and cyclosporin A (10(-9)-10(-7) M) each blocked efficiently and uniformly all the changes resulting from intercellular signaling in both DC-->T cell and T cell-->DC directions. Dexamethasone (10(-9)-10(-6) M) suppressed all changes, except for CD69 up-regulation, rather incompletely. Remarkably, RAP (10(-10)-10(-8) M) efficiently inhibited DC-induced T cell proliferation and T cell-mediated CD40 up-regulation by DC without abrogating other changes. Interestingly, T cell-independent DC maturation triggered by LPS stimulation was inhibited by dexamethasone, but not by other agents. Our results demonstrate contrasting pharmacological effects of RAP vs calcineurin inhibitors on Ag presentation, thus forming a conceptual framework for rationale-based selection (and combination) of immunosuppressive agents for clinical application.     

Galectin-8 tandem-repeat structure is essential for T-cell proliferation but not for co-stimulation

Gal (galectin)-8 is a tandem-repeat Gal containing N-CRDs (Nterminal carbohydrate-recognition domains) and C-CRDs (C-terminal carbohydrate-recognition domains) with differential glycan-binding specificity fused by a linker peptide. Gal-8 has two distinct effects on CD4 T-cells: at high concentrations it induces antigen-independent proliferation, whereas at low concentrations it co-stimulates antigen-specific responses. Associated Gal-8 structural requirements were dissected in the present study. Recombinant homodimers N-N (two N-terminal CRD chimaera) and C-C (two C-terminal CRD chimaera), but not single C-CRDs or N-CRDs, induced proliferation; however, single domains induced co-stimulation. These results indicate that the tandem-repeat structure was essential only for the proliferative effect, suggesting the involvement of lattice formation, whereas co-stimulation could be mediated by agonistic interactions. In both cases, C-C chimaeras displayed higher activity than Gal-8, indicating that the C-CRD was mainly involved, as was further supported by the strong inhibition of proliferation and co-stimulation in the presence of blood group B antigen, specifically recognized by this domain. Classic Gal inhibitors (lactose and thiodigalactoside) prevented proliferation but not co-stimulatory activity, which was inhibited by 3-O-β-D-galactopyranosyl-D-arabinose. Interestingly, Gal-8 induced proliferation of na?ve human CD4 T-cells, varying from non- to high-responder individuals, whereas it promoted cell death of phytohaemagglutinin or CD3/CD28 pre-activated cells. The findings of the present study delineate the differential molecular requirements for Gal-8 activities on T-cells, and suggest a dual activity relying on activation state.     

Normal development and activation but altered cytokine production of Fyn-deficient CD4+ T cells

The Src family kinase Fyn is expressed in T cells and has been shown to phosphorylate proteins involved in TCR signaling, cytoskeletal reorganization, and IL-4 production. Fyn-deficient mice have greatly decreased numbers of NKT cells and have thymocytes and T cells with compromised responses following Ab crosslinking of their TCRs. Herein we have addressed the role of Fyn in peptide/MHC class II-induced CD4(+) T cell responses. In Fyn-deficient mice, CD4(+) T cells expressing the DO11.10 TCR transgene developed normally, and the number and phenotype of naive and regulatory DO11.10(+)CD4(+) T cells in the periphery were comparable with their wild-type counterparts. Conjugation with chicken OVA peptide 323-339-loaded APCs, and the subsequent proliferation in vitro or in vivo of DO11.10(+) Fyn-deficient CD4(+) T cells, was virtually indistinguishable from the response of DO11.10(+) wild-type CD4(+) T cells. Proliferation of Fyn-deficient T cells was not more dependent on costimulation through CD28. Additionally, we have found that differentiation, in vitro or in vivo, of transgenic CD4(+) Fyn-deficient T cells into IL-4-secreting effector cells was unimpaired, and under certain conditions DO11.10(+) Fyn-deficient CD4(+) T cells were more potent cytokine-producing cells than DO11.10(+) wild-type CD4(+) T cells. These data demonstrate that ablation of Fyn expression does not alter most Ag-driven CD4(+) T cell responses, with the exception of cytokine production, which under some circumstances is enhanced in Fyn-deficient CD4(+) T cells.     

Nippostrongylus brasiliensis can induce B7-independent antigen-specific development of IL-4-producing T cells from naive CD4 T cells in vivo

Th2 immune responses to a number of infectious pathogens are dependent on B7-1/B7-2 costimulatory molecule interactions. We have now examined the Th2 immune response to Nippostrongylus brasiliensis (Nb) in B7-1/B7-2(-/-) mice and show that Th2 effector cells develop that can mediate worm expulsion and produce substantial Th2 cytokines comparable with wild-type infected mice; however, in marked contrast, B cell Ag-specific Ab production is abrogated after B7 blockade. To examine the mechanism of T cell activation, OVA-specific DO11.10 T cells were transferred to recipient mice, which were then immunized with a combination of Nb plus OVA or either alone. Only the combination of Nb plus OVA triggered T cell differentiation to OVA-specific Th2 cells, suggesting that Nb acts as an adjuvant to stimulate Ag-specific naive T cells to differentiate to effector Th2 cells. Furthermore, using the DO11.10 TCR-transgenic T cell adoptive transfer model, we show that blocking B7-1/B7-2 interactions does not impair nonparasite Ag-specific DO11.10 Th2 cell differentiation; however, DO11.10 T cell cycle progression and migration to the B cell zone are inhibited.     

Stimulation by soluble CD70 promotes strong primary and secondary CD8+ cytotoxic T cell responses in vivo

Identification of the signals required for optimal differentiation of naive CD8(+) T cells into effector and memory cells is critical for the design of effective vaccines. In this study we demonstrate that CD27 stimulation by soluble CD70 considerably enhances the magnitude and quality of the CD8(+) T cell response. Stimulation with soluble CD70 in the presence of Ag significantly enhanced the proliferation of CD8(+) T cells and their ability to produce IL-2 and IFN-gamma in vitro. Administration of Ag and soluble CD70 resulted in a massive (>300-fold) expansion of Ag-specific CD8(+) T cells in vivo, which was due to the enhanced proliferation and survival of activated T cells. In mice that received Ag and soluble CD70, CD8(+) T cells developed into effectors with direct ex vivo cytotoxicity. Furthermore, unlike peptide immunization, which resulted in a diminished response after rechallenge, CD27 stimulation during the primary challenge evoked a strong secondary response upon rechallenge with the antigenic peptide. Thus, in addition to increasing the frequency of primed Ag-specific T cells, CD27 signaling during the primary response instills a program of differentiation that allows CD8(+) T cells to overcome a state of unresponsiveness. Taken together these results demonstrate that soluble CD70 has potent in vivo adjuvant effects for CD8(+) T cell responses.     

CD8 cell division maintaining cytotoxic memory occurs predominantly in the bone marrow

Long-term persistence of Ag-experienced CD8 cells, a class of T lymphocytes with cytotoxic function, contributes to immunological memory against intracellular pathogens. After Ag clearance, memory CD8 cells are maintained over time by a slow proliferation, primarily cytokine driven. In this article, we show that the bone marrow (BM) is the crucial organ where such basal division of memory CD8 cells occurs. BM memory CD8 cells contain a higher percentage of proliferating cells than their corresponding cells in either spleen or lymph nodes from C57BL/6 mice. This occurs both in the case of memory-phenotype CD44(high) CD8 cells and in the case of Ag-specific memory CD8 cells. Importantly, the absolute number of Ag-specific memory CD8 cells dividing in the BM largely exceeds that in spleen, lymph nodes, liver, and lung taken together. In the BM, Ag-specific memory CD8 cells express lower levels of CD127, i.e., the alpha-chain of IL-7R, than in either spleen or lymph nodes. We interpret these results as indirect evidence that Ag-specific memory CD8 cells receive proliferative signals by IL-7 and/or IL-15 in the BM and propose that the BM acts as a saturable "niche" for the Ag-independent proliferation of memory CD8 cells. Taken together, our novel findings indicate that the BM plays a relevant role in the maintenance of cytotoxic T cell memory, in addition to its previously described involvement in long-term Ab responses.