International Immunology - recent issues

IN THIS ISSUE

2008-04-24

Modulation of T cell homeostasis and alloreactivity under continuous FTY720 exposure

2008-04-24

The immunomodulator FTY720 inhibits lymph node (LN) and thymic egress, thereby constraining T cell circulation and reducing peripheral T cell numbers. Here, we analyzed in mouse models the as yet scarcely characterized impact of long-term (up to 6 months) FTY720 exposure on T cell homeostasis and possible consequences for alloreactivity. In green fluorescent protein (GFP) hemopoietic chimeras, the turnover of (initially GFP^–) peripheral T cell pools was markedly delayed under FTY720, while normal homeostatic differences between CD4 and CD8 T cell sub-populations were retained or amplified further. Homeostatic proliferation was enhanced, and within shrinking T cell pools, the proportions of effector memory phenotype CD4 T cells (CD4T_PEM) increased in spleens and LNs and of central memory phenotype CD8 T cells (CD8T_PCM) in LNs. By contrast, the fractions of CD8T_PEM and CD4T_PCM remained stably small under FTY720. The enrichment for CD4T_PEM and CD8T_PCM correlated with larger proportions of IFN-producing T cells upon nonspecific but not allospecific stimulation. Splenic CD4 T cells from FTY720-treated mice proliferated more strongly upon transfer to semi-allogeneic hosts. However, heart allograft survival was not compromised in FTY720 pre-treated recipients. It correlated with reduced intra-graft CD8 T cells, and the longest surviving transplants contained the highest numbers of CD4 T cells. Thus, continuous FTY720 exposure reveals differential homeostatic responses by memory phenotype CD4 and CD8 T cell sub-populations, and it may enhance alloreactive CD4 T cell proliferation and tissue infiltration without accelerating allograft rejection.

Requirements for the natural killer cell-mediated induction of IgG1 and IgG2a expression in B lymphocytes

Gao, N., Jennings, P., Yuan, D. — 2008-04-24

Upon interaction with resting B lymphocytes, IL-2-propagated NK cells can initiate the process of Ig constant region switch recombination (CSR) by inducing germ line transcripts for 2a (I2a) as well as increased levels of mRNA for activation-induced cytidine deaminase enzyme. Whereas both these processes are necessary for CSR, they are not sufficient because the cells do not proceed to the expression of mature mRNA for 2a (VDJC2a). In addition, NK cells can also upregulate mRNA for the T-box transcription factor (T-bet) in B cells without being able to induce further differentiation. Using transgenic B cells with B cell receptor specificity for nitrophenol (NP), we have now shown that NP–Ficoll-stimulated B cells can be induced by NK cells to express IgG2a as well as IgG1 presumably due to the completion of the process of switch recombination. The inductive ability of NK cells does not require IFN- but does require signals transmitted via CD48 by direct cell contact. In addition, NP–Ficoll on its own can induce proliferation of antigen-specific B cells as well as germ line transcripts of 1; however, expression of VDJC1 mRNA also requires NK cell interaction with B lymphocytes. Therefore, in the presence of antigen, NK cells can provide a necessary signal that substitutes for cytokines in the induction of IgG2a as well as IgG1 expression. This in vitro analysis provides a mechanistic basis for understanding the documented NK cell effects on T-independent B cell responses in vivo.

Gr1+IL-4-producing innate cells are induced in response to Th2 stimuli and suppress Th1-dependent antibody responses

McKee, A. S., MacLeod, M., White, J., Crawford, F., Kappler, J. W., Marrack, P. — 2008-04-24

Alum is used as a vaccine adjuvant and induces T_h2 responses and T_h2-driven antibody isotype production against co-injected antigens. Alum also promotes the appearance in the spleen of Gr1+IL-4+ innate cells that, via IL-4 production, induce MHC II-mediated signaling in B cells. To investigate whether these Gr1+ cells accumulate in the spleen in response to other T_h2-inducing stimuli and to understand some of their functions, the effects of injection of alum and eggs from the helminth, Schistosoma mansoni, were compared. Like alum, schistosome eggs induced the appearance of Gr1+IL-4+ cells in spleen and promoted MHC II-mediated signaling in B cells. Unlike alum, however, schistosome eggs did not promote CD4 T cell responses against co-injected antigens, suggesting that the effects of alum or schistosome eggs on splenic B cells cannot by themselves explain the T cell adjuvant properties of alum. Accordingly, depletion of IL-4 or Gr1+ cells in alum-injected mice had no effect on the ability of alum to improve expansion of primary CD4 T cells. However, Gr1+ cells and IL-4 played some role in the effects of alum, since depletion of either resulted in antibody responses to antigen that included not only the normal T_h2-driven isotypes, like IgG1, but also a T_h1-driven isotype, IgG2c. These data suggest that alum affects the immune response in at least two ways: one, independent of Gr1+ cells and IL-4, that promotes CD4 T cell proliferation and another, via Gr1+IL-4+ cells, that participates in the polarization of the response.

KLF4 is a FOXO target gene that suppresses B cell proliferation

2008-04-24

Lymphocytes circulate in a quiescent (G₀) state until they encounter specific antigens. In T cells, quiescence is programed by transcription factors of the forkhead box O (FOXO) and Krüppel-like factor (KLF) families. However, the transcription factors that regulate B cell quiescence are not known. KLF4 is a candidate tumor suppressor gene in B lymphocytes, and thus a likely candidate for regulating B cell homeostasis. Here, we show that RNA and protein expression of murine KLF4 decreases following B cell activation. Forced expression of KLF4 in proliferating B cell blasts causes a G₁ cell cycle arrest. This effect requires the DNA binding and transactivation domains of KLF4 and correlates with changes in the expression of known KLF target genes. We present evidence that Klf4 is a target gene for FOXO transcription factors, which also suppress B cell proliferation. To determine the effect of KLF4 loss-of-function, we generated mice with B cell-specific deletion of the Klf4 gene. These mice exhibited normal B cell development and function with no evidence of a lowered activation threshold. Collectively, our findings indicate that KLF4 has growth-suppressive properties in B cells but might be redundant with other members of the KLF family in maintaining B cell quiescence.

Improved methods for detecting selection by mutation analysis of Ig V region sequences

Hershberg, U., Uduman, M., Shlomchik, M. J., Kleinstein, S. H. — 2008-04-24

Statistical methods based on the relative frequency of replacement mutations in B lymphocyte Ig V region sequences have been widely used to detect the forces of selection that shape the B cell repertoire. However, current methods produce an unexpectedly high frequency of false positives and are sensitive to intrinsic biases of somatic hypermutation that can give the appearance of selection. The new statistical test proposed here provides a better trade-off between sensitivity and specificity compared with previous approaches. The low specificity of existing methods was shown in silico to result from an interaction between the effects of positive and negative selection. False detection of positive selection was confirmed in vivo through a re-analysis of published sequence data from diffuse large B cell lymphomas, highlighting the need for re-analysis of some existing studies. The sensitivity of the proposed method to detect selection was validated using new Ig transgenic mouse models in which positive selection was expected to be a significant force, as well as with a simulation-based approach. Previous concerns that intrinsic biases of somatic hypermutation could give the appearance of selection were addressed by extending the current mutation models to more fully account for the impact of microsequence on relative mutability and to include transition bias. High specificity was confirmed using a large set of non-productively rearranged Ig sequences. These results show that selection can be detected in vivo with high specificity using the new method proposed here, allowing greater insight into the existence and direction of antigen-driven selection.

Bimodal regulation of T cell-mediated immune responses by TIM-4

2008-04-24

T cell Ig and mucin domain (TIM)-4 is preferentially expressed on antigen-presenting cells, and its counter-ligand, TIM-1, is thought to deliver co-stimulating signals to T cells. However, the physiological functions of TIM-4 remain unclear. Here, we demonstrate that TIM-4 inhibits naive T cell activation through a ligand other than TIM-1. The inhibitory effect of TIM-4 was specific to naive T cells which do not express TIM-1, and the effect disappeared in pre-activated T cells. Conversely, antibody-mediated blockade of TIM-4 in vivo substantially suppressed T cell-mediated inflammatory responses despite enhanced generation of antigen-specific T cells. Furthermore, treatment with anti-TIM-4 reduced the inflammatory responses developed in mice that were adoptively transferred with antigen-primed T cells. These results suggest that TIM-4 exerts bimodal functions depending on the activation status of T cells.

Cross-talk among Toll-like receptors and their ligands

2008-04-24

Toll-like receptors (TLRs) 4, 5, 7 and 9 belong to a family of proteins that recognize mainly conserved microbial motifs. Though each TLR has a highly specific ability to recognize a particular microbial pattern, recent papers suggest that some ligands are able to affect the expression of different TLRs. In this paper, we have investigated TLR4, 5, 7 and 9 expression, both at mRNA and protein level, following treatment of different intestinal epithelial cell lines with LPS, flagellin, loxiribine, CpG-oligodeoxynucleotide and peptidoglycan, to assess if the different TLR ligands may modulate the expression of the respective TLR and of the unrelated ones. Our results show that a cross-talk exists between TLRs and various ligands, indicating a cross-regulation among these pattern recognition receptors. In particular, TLR4 was generally down-regulated by treatment with ligands other than LPS, while flagellin and unrelated microbial-associated molecular patterns exerted a general stimulatory activity as regards TLR5 expression. Concerning TLR7 and 9, we have observed a more variable behaviour of the various cell lines with the different ligands. Together, our results demonstrate that the expression of TLRs in intestinal cells is highly dynamic and tightly regulated in response to encountered microbial stimuli.

Sulfates are main targets of immune responses to cruzipain and are involved in heart damage in BALB/c immunized mice

2008-03-21

Trypanosoma cruzi, the agent of Chagas disease contains a major cysteine proteinase, cruzipain (Cz), with an unusual carboxyl-terminal extension (C-T). We have previously reported the presence of sulfate groups in the N-linked oligosaccharide chains of this domain. In order to evaluate the immune responses to sulfated moieties on Cz, BALB/c mice were immunized with purified Cz and C-T prior and after desulfation treatment. The humoral immune response to sulfates on Cz or C-T was mainly IgG2b. Interestingly, the abolishment of IgG2b reactivity when desulfated antigens were used as immunogens demonstrates that esterified sulfate groups are absolutely required for eliciting IgG2b response to Cz. Sera from chronically T. cruzi-infected subjects with mild disease displayed higher levels of total IgG and IgG2 antibodies specific for sulfated epitopes compared with those in more severe forms of the disease. A significant reduction of C-T-specific delayed-type hypersensitivity reaction in C-T-immunized mice was observed when desulfated C-T was challenged, suggesting the involvement of sulfate groups in the generation of memory T-cell responses. Moreover, immunization with C-T in the absence of infection elicited ultrastructural abnormalities in heart tissue. Surprisingly, hearts from sulfate-depleted C-T-immunized mice did not present pathological alterations. This is the first report showing that sulfate-bearing glycoproteins from trypanosomatids are able to elicit specific humoral and cellular immune responses and appeared to be involved in the generation of heart tissue damage. These results represent a further step in the understanding of the role of Cz in the course of T. cruzi infection.

CTLA-4{middle dot}Ig converts naive CD4+CD25- T cells into CD4+CD25+ regulatory T cells

Razmara, M., Hilliard, B., Ziarani, A. K., Chen, Y. H., Tykocinski, M. L. — 2008-03-21

CTLA-4·Ig was originally designed as an immunosuppressive agent capable of interfering with the co-stimulation of T cells. In the present study, we demonstrate that CTLA-4·Ig, in combination with TCR ligation, has the additional capacity to convert naive CD4⁺CD25^– T cells into Foxp3⁺ regulatory T (T_reg) cells, as well as to expand their numbers. The CD4⁺CD25⁺Foxp3⁺ T_reg generated by CTLA-4·Ig treatment in vitro potently suppress effector T cells. Extending this in vivo, we show that systemic administration of CTLA-4·Ig increases the percentage of CD4⁺CD25^hiFoxp3⁺ cells within mixed lymphocyte reaction-induced murine lymph nodes. Significantly, the in vitro conversion of naive CD4⁺CD25^– T cells into T_reg cells is antigen-presenting cell (APC) dependent. This finding, together with the further observation that this conversion can also be driven in vitro by an antibody that engages B7-2 ligand, suggests that CTLA-4·Ig-driven T_reg induction may be predicated upon active CTLA-4·Ig to B7-2 signaling within APC, which elicits from them T_reg-inducing potential. These findings extend CTLA-4·Ig's functional repertoire, and at the same time, reinforce the concept that T cell anergy and active suppression are not entirely distinct processes and may be linked by some common molecular triggers.

Dual role of Cbl links critical events in BCR endocytosis

Jacob, M., Todd, L., Sampson, M. F., Pure, E. — 2008-03-21

Receptor endocytosis down-regulates ligand-induced signaling in a timely manner and depends on cytoskeletal remodeling. In B lymphocytes, internalization of B cell receptors (BCRs) is also critical to antigen presentation. However, the mechanisms underlying BCR endocytosis are not fully understood. Similarly, the molecular mechanisms linking endocytosis to cytoskeletal remodeling remain poorly defined. We used flow cytometry, pull-down assays, immunochemistry and fluorescence microscopy to investigate BCR internalization in the DT40 B cell line. We demonstrate that ablation of Cbl impacts BCR endocytosis and the underlying cytoskeletal dynamics. Specifically, we demonstrate that ligand-induced endocytosis is impaired in Cbl^–/– cells and that the ubiquitin ligase activity is required for Cbl to promote endocytosis. We also show that phosphorylation of CrkII, activation of Rac downstream of CrkII and BCR capping require Cbl. Furthermore, we show that the association of Cbl and CrkII requires phosphorylation of Cbl, but not its ubiquitin ligase activity. Our data indicate that Cbl promotes BCR endocytosis and attenuates ligand-induced signaling by virtue of its ability to orchestrate receptor ubiquitylation and cytoskeletal dynamics.

PI3K is a negative regulator of IgE production

Doi, T., Obayashi, K., Kadowaki, T., Fujii, H., Koyasu, S. — 2008-03-21

The production of IgE, a main player in allergic disorders such as asthma and atopic dermatitis, is strictly regulated and the serum concentrations of IgE are normally kept at a much lower level than other isotypes. We found that mice deficient for the p85 regulatory subunit of class IA phosphoinositide 3-kinase (PI3K) produced increasing amounts of serum IgE. Purified p85^–/– B cells produced more IgE than wild-type B cells in vitro in response to anti-CD40 mAb and IL-4. PI3K inhibitors wortmannin and IC87114 enhanced IgE production by wild-type B cells stimulated with anti-CD40 mAb and IL-4. Under the same condition, antigen receptor cross-linking induced the expression of inhibitor of differentiation-2 and suppressed the expression of activation-induced cytidine deaminase and class switch recombination (CSR) in a PI3K-dependent manner. IgE production was also suppressed in a concentrated cell culture condition, which was completely reversed by PI3K inhibition. The selective suppression of IgE production by PI3K was also observed at a protein level after CSR. Our results indicate that PI3K negatively regulates IgE production at both CSR and protein levels.

Implication for the CD94/NKG2A-Qa-1 system in the generation and function of ocular-induced splenic CD8+ regulatory T cells

Chattopadhyay, S., O'Rourke, J., Cone, R. E. — 2008-03-21

The injection of antigen into the anterior chamber (AC) induces the production of antigen-specific splenic CD8⁺ regulatory T cells (Tregs) /suppressor T cells that perform the local suppression of delayed-type hypersensitivity (DTH) responses. Because CD94/NKG2A-Qa-1-dependent interactions have been implicated in CD8⁺ Treg-mediated immune suppression and DBA/2J mice are deficient in CD94/NKG2R, we have utilized these mice to test the hypothesis that the CD94/NKG2A-Qa-1 system is essential to the induction and immunosuppressive function of CD8⁺ Tregs in anterior chamber-associated immune deviation (ACAID). We show that: (i) neither ACAID-mediated suppression of DTH to ovalbumin nor splenic Tregs/suppressor T cells was induced in DBA/2J mice that received an injection of antigen into the AC; (ii) splenic CD8⁺ Tregs from ACAID-induced DBA/2NCr mice suppressed the initiation of DTH when transferred to DBA/2J mice; (iii) following injection of antigen into the AC, intravenous administration of splenocytes or Peripheral Blood Mononuclear Cells (PBMC) isolated from DBA/2NCr but not from DBA/2J mice transferred suppression of DTH to DBA/2NCr mice; (iv) antibodies to CD94/NKG2A reduced the ACAID CD8⁺ T cell-mediated suppression of DTH and (v) The deficiency of such immune regulation in DBA/2J mice also correlated with a decreased number of Qa-1^b+ B cells, F4/80⁺ cells, a deficient number of CD94/NKG2AR and Qa-1 tetramer binding by CD8⁺ T cells. These results demonstrate that defective ACAID in DBA/2J mice involves multiple regulatory lesions resulting in a lack of induction of a CD8⁺ Treg response and possibly defective CD94/NKG2A-dependent suppression of peripheral cell-mediated immunity.

Pervasive and stochastic changes in the TCR repertoire of regulatory T-cell-deficient mice

2008-03-21

We hypothesize that regulatory T-cell (Treg)-deficient strains have an altered TCR repertoire in part due to the expansion of autoimmune repertoire by self-antigen. We compared the Vβ family expression profile between B6 and Treg-lacking B6.Cg-Foxp3^sf^/Y (B6.sf) mice using fluorescent anti-Vβ mAbs and observed no changes. However, while the spectratypes of 20 Vβ families among B6 mice were highly similar, the Vβ family spectratypes of B6.sf mice were remarkably different from B6 mice and from each other. Significant spectratype changes in many Vβ families were also observed in Treg-deficient IL-2 knockout (KO) and IL-2R KO mice. Such changes were not observed with anti-CD3 mAb-treated B6 mice or B6 CD4⁺CD25^– T cells. TCR transgenic (OT-II.sf) mice displayed dramatic reduction of clonotypic TCR with concomitant increase in T cells bearing non-transgenic Vβ and V families, including T cells with dual receptors expressing reduced levels of transgenic V and endogenous V. Collectively, the data demonstrate that Treg deficiency allows polyclonal expansion of T cells in a stochastic manner, resulting in widespread changes in the TCR repertoire.

Generation of tumour-rejecting anti-carbohydrate monoclonal antibodies using melanoma modified with Fas ligand

2008-03-21

Carbohydrate antigens such as glycolipids and glycoproteins are over-expressed in a variety of cancers and have therefore been identified as ideal candidates for tumour vaccines. Detection of anti-carbohydrate antibodies is associated with a good prognosis in cancer patients. However, generation of an efficient adaptive immune response has been hampered by the low immunogenicity of carbohydrates due to tolerance. Here, we describe a method by which tumour-rejecting antibodies directed against carbohydrates can be elicited in two different melanoma mouse models. Thus, using the murine melanoma B16F10 over-expressing Fas ligand (FasL), we have generated mAbs against cancer carbohydrate antigens expressed by the melanoma. Importantly, passive transfer of mAbs resulted in rejection of melanoma in vivo. Their protective effect in vivo was dependent on FcR and in vitro antibody-dependent cellular phagocytosis. They were also able to delay tumour growth when injected after the tumour was established. FasL-expressing tumours as an adjuvant are a novel way to generate anti-carbohydrate antibodies able to reject tumours in vivo.

Circulating neutrophils of septic patients constitutively express IL-10R1 and are promptly responsive to IL-10

2008-03-21

Previous studies have demonstrated that neutrophils isolated from the blood of healthy donors do not respond to IL-10 in terms of either activation of signal transducer and activator of transcription-3 (STAT3) tyrosine phosphorylation or induction of suppressor of cytokine signalling (SOCS)-3 protein, unlike autologous mononuclear cells. This was explained by the fact that circulating neutrophils of healthy donors express only IL-10R2, but not IL-10R1, the latter IL-10R chain being essential for mediating IL-10 responsiveness. In this study, we report that peripheral blood neutrophils of septic patients constitutively display, besides IL-10R2, also abundant levels of surface IL-10R1. Consequently, septic neutrophils are promptly responsive to IL-10 in vitro, as revealed by a direct IL-10-mediated induction of STAT3 tyrosine phosphorylation and SOCS-3 gene transcription, mRNA and protein expression. Consistent with the presence of a fully functional IL-10R, modulation of LPS-induced CXCL8, CCL4, tumour necrosis factor- and IL-1ra gene expression was also rapidly induced by IL-10 in septic, but not normal, neutrophils. Collectively, these data uncover that neutrophils of septic patients are predisposed to be promptly responsive to IL-10, presumably to help limiting their pro-inflammatory state. They also fully validate our previous observations, herein in the context of a human disease, that responsiveness of human neutrophils to IL-10 is strictly dependent upon the modulation of IL-10R1 expression.

Evaluation of the effect of human {beta}-defensins on neutrophil apoptosis

Nagaoka, I., Niyonsaba, F., Tsutsumi-Ishii, Y., Tamura, H., Hirata, M. — 2008-03-21

Peptide antibiotics possess the potent antimicrobial activities against invading microorganisms and contribute to the innate host defense. Antimicrobial human β-defensins (hBDs) not only exhibit potent bactericidal activities against Gram-negative and Gram-positive bacteria but also function as immunomodulatory molecules by inducing cytokine and chemokine production and inflammatory and immune cell activation. Neutrophil is a critical effector cell in host defense against microbial infection, and its lifespan is regulated by various pathogen- and host-derived substances. Here, to further evaluate the role of hBDs in innate immunity, we investigated the action of hBD-1 to -4 on neutrophil apoptosis. Neutrophil apoptosis was assessed using human blood neutrophils based on the morphological changes. Of note, hBD-3 most potently suppressed neutrophil apoptosis among hBD-1 to -4, accompanied with the down-regulation of truncated Bid (a pro-apoptotic protein), up-regulation of Bcl-x_L (an anti-apoptotic protein) and inhibition of mitochondrial membrane potential change and caspase 3 activity. Furthermore, we revealed that neutrophils expressed CC chemokine receptor (CCR) 6, and the action of hBD-3 was completely abrogated by a neutralizing anti-CCR6 mAb. Collectively, these observations suggest that hBDs, especially hBD-3, can not only kill bacteria but also modulate (suppress) neutrophil apoptosis via the action on CCR6. Suppression of neutrophil apoptosis results in the prolongation of their lifespan and may be advantageous for the host defense against bacterial invasion.

KIR2DS1-mediated activation overrides NKG2A-mediated inhibition in HLA-C C2-negative individuals

Foley, B., De Santis, D., Lathbury, L., Christiansen, F., Witt, C. — 2008-03-21

NK cell cytotoxicity is controlled through a balance of both activating and inhibitory signals. The HLA specificity of alloreactive NK cells has been previously shown to be controlled by inhibitory killer immunoglobulin-like receptors (KIRs). Alloreactive NK cells lyse targets that lack the HLA ligand for their inhibitory KIR. We have characterized in detail an alloreactive NK clone in which the specificity is controlled by an activating receptor, KIR2DS1. Only target cells expressing the HLA-C group 2 (C2) epitope were lysed by this clone and homozygous C2 targets were lysed more strongly than heterozygous C1/C2 targets. Anti-CD158a (KIR2DS1) blocked lysis of targets confirming KIR2DS1 was responsible. Although this NK clone expressed NKG2A, an inhibitory receptor whose ligand is HLA-E, targets with ligands for both KIR2DS1 and NKG2A were lysed by this clone indicating that the KIR2DS1-mediated activation signal overrides the NKG2A-mediated inhibitory signal. KIR2DS1 activated NK clones in polyclonally expanded NK cultures from a donor that lacked the C2 epitope accounted for ~1% of all NK cells. This study highlights a potential role for NK cells controlled by activating KIR in mediating NK alloreactivity.

Methylprednisolone induces preferential and rapid differentiation of CD34+ cord blood precursors toward NK cells

Vitale, C., Cottalasso, F., Montaldo, E., Moretta, L., Mingari, M. C. — 2008-03-21

Previous studies showed that methylprednisolone (MePDN) down-regulates the surface expression of activating NK receptors and sharply inhibits the NK cytotoxicity both in vitro and in vivo. Since MePDN is administered to patients undergoing hemopoietic stem cell transplant to treat acute graft versus host disease (GvHD), we analyzed whether it could also inhibit the NK cell differentiation from CD34⁺ hemopoietic cell precursors, thus interfering with the development of effector cells with anti-leukemic potential. We show that MePDN promotes the in vitro differentiation of CD161⁺CD56^+/– immature NK cells by inducing a rapid expression of NKp46, NKG2D, DNAX-accessory molecule 1 (DNAM-1), leukocyte function-associated antigen-1 and NKG2A and an efficient cytolytic activity. This phenotypic and functional NK cell maturation occurred more rapidly than in parallel control cultures performed in the absence of MePDN. In addition, MePDN induced CD33⁺CD161^–CD56^– myeloid precursors to switch toward NK cells. It is also of note that immature NK cells when cultured in the absence (but not in the presence) of MePDN produced high amounts of IL-8. These data indicate that MePDN can accelerate the in vitro NK cell differentiation, thus revealing a dichotomous effect on immature versus mature NK cells; in addition, interference with the in vitro development of myeloid cells occurred. These effects should be further investigated in hemopoietic stem cell transplanted patients receiving steroids to treat GvHD.

CD4+ICOS+ T lymphocytes inhibit T cell activation 'in vitro' and attenuate autoimmune encephalitis 'in vivo'

2008-03-21

The inducible co-stimulator (ICOS, CD278) is essential to the efficient development of normal and pathological immune reactions. Since ICOS-deficient mice have enhanced susceptibility to experimental allergic encephalomyelitis (EAE), we have functionally analyzed a CD4⁺ICOS⁺ population comprising 6–15% of all CD4⁺ T cells in secondary lymphoid organs of unmanipulated wild-type mice and checked for their ability to suppress EAE. In C57BL/6 mice, CD4⁺ICOS⁺ cells were a major source of cytokines including IFN-, IL-2, IL-4, IL-10 or IL-17A. Upon activation, these cells showed preferentially enhanced production of IL-4 or IL-10 but inhibited IFN- production. In contrast, CD4⁺ICOS^– cells mainly produced IFN-. Interestingly, CD4⁺ICOS⁺ cells partially suppressed the proliferation of CD4⁺ICOS^– or CD4⁺CD25^– lymphocytes ‘in vitro’ by an IL-10-dependent mechanism. Furthermore, CD4⁺ICOS⁺ activated and expanded under appropriate conditions yielded a population enriched in cells producing IL-10 and T_h2 cytokines that also suppressed the proliferation of CD4⁺CD25^– lymphocytes. CD4⁺ICOS⁺ cells, before or after expansion in vitro, reduced the severity of EAE when transferred to ICOS-deficient mice. In the same EAE model, lymph node cells from ICOS-deficient mice receiving ICOS⁺ cells showed reduced IL-17A production and enhanced IL-10 secretion upon antigen activation in vitro. Thus, naturally occurring CD4⁺ICOS⁺ cells, expanded or not in vitro, are functionally relevant cells able of protecting ICOS-deficient mice from severe EAE.

Differential influence of the tumour-specific non-human sialic acid containing GM3 ganglioside on CD4+CD25- effector and naturally occurring CD4+CD25+ regulatory T cells function

2008-03-21

Increasing evidences suggest that the aberrant expression of certain gangliosides on malignant cells could affect host's anti-tumour-specific immune responses. We have recently documented the relevance of the N-glycolylated variant of GM3 ganglioside (NGcGM3), a tumour-specific non-human sialic acid containing ganglioside, for tumour progression. However, evidences about the implication of host's immunity in NGcGM3-promoted cancer progression had not been obtained previously. In this work, we compared tumour growth of X63 myeloma cells pre-treated or not with an inhibitor of the glucosylceramide synthase enzyme, in wild or CD4⁺ T cell-depleted BALB/c mice. Results clearly showed a relationship between the agonistic effect of NGcGM3 in tumour growth and the presence of CD4⁺ T lymphocytes. For the first time, a description of a ganglioside-differential effect over purified CD4⁺CD25^– and naturally occurring regulatory CD4⁺CD25⁺ T cells is provided. While NGcGM3 similarly down-modulated the CD4 expression in both cell populations, the inhibitory capacity of the CD4⁺CD25⁺ lymphocytes and their proliferation, induced by an anti-CD3 mAb and IL2, were not modified. In a different fashion, a reduction in proliferative capacity and a noteworthy secretion of anti-inflammatory cytokines were detected when CD4⁺CD25^– T cells were cultured in the presence of NGcGM3. Considering the relevance of dendritic cells (DC) on primary activation of T cells, the effect of NGcGM3 over DC differentiation and TLR4-mediated maturation was also assessed. Our results indicate that NGcGM3 contributes to cancer progression mainly by influencing DC and CD4⁺CD25^– T lymphocyte functions, rather than increasing the inhibitory capacity of naturally occurring regulatory T cells.

Anti-HLA-DR-triggered monocytes mediate in vitro T cell anergy

2008-03-21

Monomorphic MHC class II determinants are attractive targets for immunomodulation. HLA-DR ligation on antigen-presenting cells (APCs) can dramatically alter their function or induce cell death. In monocytes, HLA-DR triggering diminishes their capacity to stimulate T cell proliferation. To further investigate this monocyte-dependent T cell inhibition, we activated human T cells ± HLA-DR triggering on APCs and tested whether this can induce T cell anergy. Only anti-HLA-DR, but not anti-proliferative control agent anti-CD45, could modulate monocytes in primary cultures with stimulated T cells, so that T cells were hyporesponsive during re-stimulation. Cell separation studies demonstrated that HLA-DR ligation on monocytes is sufficient for mediating T cell anergy. Secretion of monokines was severely reduced after primary culture. Monocytes anergized independently of soluble factors. Extracellular signal-regulated kinase (ERK) phosphorylation occurred early with anti-HLA-DR, but late with anti-CD45 antibody. However, ERK inhibition did not reverse the T cell-anergizing potential of HLA-DR-ligated monocytes implicating other signaling pathways involved in tolerance induction. When analyzing the anergized T cells, they were refractory to exogenous IL-2 and characterized by defective secretion of various cytokines. Expression of CD25, CD28, intracellular CD3 and CTLA-4 was reduced. The hyporesponsive T cells up-regulated cell-cycle inhibitors p27^kip1 and p21^cip1 in correlation with human T cell anergy. In contrast, caspase-3 and -8, known to contribute to T cell proliferation, were equally decreased in anti-HLA-DR- and anti-CD45-inhibited cultures. In summary, anti-HLA-DR treatment can generate tolerogenic monocytes transmitting T cell anergy that may be exploited for future immunomodulatory strategies to treat immune-mediated disease states.

Mechanisms of organogenesis of primary lymphoid follicles

Beyer, T., Meyer-Hermann, M. — 2008-03-21

Primary lymphoid follicles (PLFs) in secondary lymphoid tissue (SLT) of mammals are the backbone for the formation of follicular dendritic cell (FDC) networks. These are important for germinal center reactions during which affinity maturation creates optimized antibodies in adaptive immune responses. In the context of organogenesis, molecular requirements for the formation of follicles have been identified. The present study complements these findings with a simulation of the dynamics of the PLF formation, and a critical analysis of the relevant molecular interactions. In contrast to other problems of pattern formation, the homeostasis of cell populations in SLTs is not governed by a growth-death balance but by a flow equilibrium of migrating cells. The influx of cells into these tissues has been extensively studied. However, less information is available about the efflux of lymphocytes from SLTs. This study formulates the minimal requirements for cell efflux that guarantee a flow equilibrium and, thus, a stable PLF. The model predicts that in addition to already identified regulatory mechanisms, a negative regulation of the generation of FDCs is required. Furthermore, a comparison with data concerning the microanatomy of SLTs yields the conclusion that dynamical changes of the lymphatic endothelium during the formation of FDC networks are necessary to understand the genesis and maintenance of follicles.

Application of CD27 as a marker for distinguishing human NK cell subsets

Silva, A., Andrews, D. M., Brooks, A. G., Smyth, M. J., Hayakawa, Y. — 2008-03-21

It has long been recognized that human NK cells can be divided into two phenotypically and functionally distinct subsets, based on their levels of expression of CD56. We recently found that CD27 distinguishes subsets of mature mouse NK cells. Here we report that CD27 can be used as a marker to discriminate human NK cell subsets. The majority of peripheral blood human NK cells were CD27^lo/CD56^dim NK cells, whereas the minor CD27^hi NK cell population correspondingly displayed a CD56^bright phenotype. Distinctions between CD27^lo and CD27^hi NK cells in their receptor expression and typical NK cell functions such as cytotoxicity and cytokine production can be easily delineated. Therefore, we propose the dual use of CD27 and CD56 as maturation/subset markers for human NK cells. The identification of CD27 subsets in both mice and humans will allow more accurate projections of the role of NK cell subsets in murine models of human pathologies where NK cells are involved.

NK cells and NKT cells collaborate in host protection from methylcholanthrene-induced fibrosarcoma

Smyth, M. J. — 2008-03-21

Recombinant antibodies for delivery of antigen: a single loop between {beta}-strands in the constant region can accommodate long, complex and tandem T cell epitopes

2008-02-26

Recombinant antibodies are increasingly used for efficient delivery of T cell epitopes to antigen-presenting cells (APCs), both for vaccination purposes and for immune modulation. We have previously shown that recombinant antibodies can accommodate single T cell epitopes inserted into loops between β-strands in constant (C) domains. Such recombinant antibodies have in addition been equipped with variable regions that target APCs for increased delivery of C region T cell epitopes. We here show that loop 6 (loop FG) in C_H1 of human 3 can be exchanged with (i) long T cell epitopes up to 37 amino acids, (ii) epitopes with complex secondary structure such as gluten epitopes with a type II polyproline helical confirmation and (iii) two tandemly linked T cell epitopes. T cell responses increased with T cell epitope elongation, presumably due to a positive influence of flanking residues. Recombinant antibodies targeted to either CD14 on monocytes or HLA-DP on monocytes and dendritic cells gave similar results and were 2–4 logs more efficient at stimulating human T cells than were non-targeted controls. Thus, single loops in C regions of recombinant antibodies seem versatile and may be used for delivery of lengthy, complex and multiple T cell epitopes to human APCs.

CD4+CD25+ regulatory T cells in the small intestinal lamina propria show an effector/memory phenotype

2008-02-26

CD4⁺CD25⁺ regulatory T cells (Tregs) have been implicated in the suppression of pathogenic responses to both self- and non-self-antigens in the intestine. However, their precise properties and functions in the gut, as well as the molecular basis of their recruitment to the gut, are poorly understood. Here, we found that most of the CD4⁺CD25⁺ T cells in the small intestinal lamina propria (LP) express Foxp3 and exhibit an ‘effector/memory’ phenotype, CD44^hiCD45RB^loCD62L^–, whereas only a minority of the Foxp3⁺CD4⁺CD25⁺ T cells in the spleen and mesenteric lymph nodes showed this phenotype. The Tregs in the small intestinal LP (LP-Tregs) expressed higher levels of CCR4 and CCR9 and a substantially lower level of CCR7 than the Tregs in the spleen. In vitro, the LP-Tregs showed chemotaxis to CCL25/thymus-expressed chemokine. In addition, they showed efficient chemotaxis to the CCR4 ligands, CCL17/thymus and activation-regulated chemokine and CCL22/macrophage-derived chemokine, which are abundantly expressed by dendritic cells (DCs) in the small intestinal LP. In vivo, ~50% of the LP-Tregs were closely associated or in direct contact with LP-DCs. These findings demonstrate that LP-Tregs are phenotypically and functionally unique and raise the possibility that they are retained in the small intestinal LP through the action of CCL17 and CCL22, which are locally produced by LP-DCs.

Tissue-specific expression of B-cell translocation gene 2 (BTG2) and its function in T-cell immune responses in a transgenic mouse model

Terra, R., Luo, H., Qiao, X., Wu, J. — 2008-02-26

B-cell translocation gene 2 (BTG2) belongs to the anti-proliferative gene family. According to previous in vitro studies, BTG2 overexpression leads to delayed cell cycling. We investigated BTG2 expression during mouse ontogeny and its immune and circadian functions in this study. In situ hybridization showed that BTG2 was expressed at high levels in the central nervous system, liver, stomach, thymus, spleen, skin, adrenal gland, pituitary gland and salivary glands during embryonic days (E10–E17), postnatal days (P1 and P10) and adult stages. Expression was observed in organs and tissues from adult mice with and without a robust proliferation program. Thus, the gene might have important functions that are both related and unrelated to proliferation. BTG2 expression was induced after in vitro T-cell receptor stimulation in T cells using anti-CD3 antibodies. However, transgenic (Tg) mice with actin promoter-driven expression of BTG2 showed normal in vitro and in vivo T-cell responses, such as thymus development, T-cell activation marker expression, T-cell proliferation and migration, as well as in vivo delayed-type hypersensitivity reactions. Although BTG2 was expressed in the suprachiasmatic nucleus and pineal gland in the brain, BTG2 Tg mice had no abnormal circadian behavior. Our data on BTG2 expression during ontogeny provide useful clues for the further investigation of BTG2 function. Additional studies are warranted to examine its role in immune and other systems.

Prolactin-induced production of cytokines in macrophages in vitro involves JAK/STAT and JNK MAPK pathways

Tripathi, A., Sodhi, A. — 2008-02-26

Macrophages play a crucial role in host immunosurveillance against pathogens and malignancies. The enhanced productions of pro-inflammatory cytokines are central to the regulatory role of macrophages and induction of robust immune response. The excessive inflammatory response of macrophages can result into pathological conditions in host. We have previously reported that prolactin (PRL) induces the production of nitric oxide (NO) and tumor necrosis factor (TNF)- in murine peritoneal macrophages. It was suggested that protein tyrosine kinases (PTKs), mitogen-activated protein kinases (MAPKs) and Ca⁺⁺ signaling were involved in the NO production by macrophages on PRL treatment. In this manuscript, we investigated the role of PTKs [Janus kinase (JAK) 2 and phosphoinositide 3-kinase (PI3K)] and c-Jun N-terminal kinase (JNK) MAPK in PRL-induced activation of murine peritoneal macrophages. It is reported that PRL-induced activation of macrophages in vitro is dependent on JAK/signal transducers and activators of transcription (STAT) and JNK MAPK-signaling pathways. It is observed that pre-treatment of macrophages with JNK inhibitor, SP600125; tyrosine kinase inhibitor, genistein; PI3K inhibitor, Wortmannin and JAK2 inhibitor, AG490 inhibited the phosphorylation of JNK MAPK. Further, pre-treatment of macrophages with SP600125 inhibited the PRL-induced production of IFN- and TNF-. AG490, inhibitor of JAK2, down-regulated transcription factors c-jun and STAT1 and inhibited the PRL-induced IFN-, TNF-, IL-1β and IL-12p40 production in macrophages.

Critical role of dendritic cells in determining the Th1/Th2 balance upon Leishmania major infection

Suzue, K., Kobayashi, S., Takeuchi, T., Suzuki, M., Koyasu, S. — 2008-02-26

The onset of T_h1 immunity is in part regulated by genetic background. To elucidate the cell type carrying critical factors determining the T_h1 response, we employed Rag-2^–/– mice on Leishmania major-susceptible BALB/c and -resistant B10.D2 backgrounds. By using bone marrow (BM) chimeras generated by the transplantation of B10.D2 BM cells into BALB/c-Rag-2^–/– mice, and vice versa, it was shown that hematopoietic cells carry factors determining the disease outcome and T_h1 response against L. major infection. B10.D2-Rag-2^–/– mice reconstituted with BALB/c CD4⁺ T cells exhibited a T_h1 response and controlled L. major infection. Wild-type BALB/c mice inoculated with L. major-parasitized B10.D2-Rag-2^–/– splenocytes also exhibited a T_h1 response and a mild disease outcome, whereas such a T_h1 response was not induced when CD11c⁺ dendritic cells (DCs) were depleted from parasitized B10.D2-Rag-2^–/– splenocytes. T_h1 response was reconstituted by the addition of L. major-parasitized B10.D2 DCs but not L. major-parasitized BALB/c DCs to DC-depleted parasitized B10.D2-Rag-2^–/– splenocytes. These results indicate that DCs determine the outcome of the disease upon L. major infection.

Distinct regulatory functions of SLP-76 and MIST in NK cell cytotoxicity and IFN-{gamma} production

2008-02-26

Activation of NK cells is triggered by multiple receptors. We demonstrate here that SLP-76 is required for CD16- and NKG2D-mediated NK cell cytotoxicity, while MIST negatively regulates these responses in an SLP-76-dependent manner. Exceptionally, MIST acts as a positive regulator of cytotoxicity against YAC-1 cells, although SLP-76 plays a more key role. SLP-76 acts as a dominant positive regulator for both NKG2D-mediated and YAC-1 cell-triggered IFN- production. Although NKG2D-mediated IFN- production depends on phospholipase C (PLC) 2, YAC-1 cell-triggered IFN- production is PLC2- and Syk/ZAP-70 independent and nuclear factor-kappa B mediated. SLP-76 is required for this process in the presence of MIST but is dispensable in the absence of MIST. Thus, YAC-1 cell-triggered NKG2D-independent IFN- production appears to be regulated by SLP-76-dependent and -independent pathways, in which the latter is negatively regulated by MIST. Taken together, these results suggest that SLP-76 and MIST distinctly but interactively regulate NK cell cytotoxicity and IFN- production.

FAK-mediated activation of ERK for eosinophil migration: a novel mechanism for infection-induced allergic inflammation

Cheung, P. F.-Y., Wong, C.-K., Ip, W.-K., Lam, C. W.-K. — 2008-02-26

Bacterial and viral infections often induce the exacerbation of allergic diseases. In this study, we investigated the activation of human eosinophils by different microbial products via Toll-like receptors (TLRs). The underlying intracellular mechanism involving activation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK), an integrin-associated focal adhesion molecule, was also examined. Seven TLR ligands were studied for their abilities in promoting survival, modulating the expression of adhesion molecules and facilitating chemotactic migration of eosinophils. While peptidoglycan (PGN) (TLR2 ligand) showed the most prominent effects, flagellin (TLR5 ligand) and imiquimod R837 (TLR7 ligand) were also effective in activating eosinophils. However, little or no effect was observed for double-stranded polyinosinic–polycytidylic acid (TLR3 ligand), ultra-purified LPS (TLR4 ligand), single-stranded RNA (ssRNA) (TLR8 ligand) and CpG-DNA (TLR9 ligand). Further investigation confirmed that PGN, flagellin and R837 commonly transmitted signals through ERK activation that required prior phosphorylation of tyrosine 925, but not tyrosine 577, on FAK. Moreover, the inhibition of ERK activation by selective inhibitor PD98059 and FAK expression by FAK-specific RNA interference could significantly abolish the stimulatory effects induced by PGN, flagellin and R837. Taken together, our findings indicate the involvement of FAK-dependent activation of ERK1 in TLR-mediated eosinophil stimulation. A potential role of eosinophils was also suggested in exacerbating allergic inflammation in response to microbial infections.

Antigen-dependent monophasic or recurrent autoimmune uveitis in rats

Diedrichs-Mohring, M., Hoffmann, C., Wildner, G. — 2008-02-26

Experimental autoimmune uveitis (EAU) in the Lewis rat has been regarded as an acute and monophasic disease. Uveitis can be induced by immunization with retinal soluble antigen (S-Ag), interphotoreceptor retinoid-binding protein (IRBP) or their peptide derivatives (PDSAg from S-Ag and R14 from IRBP) in CFA as well as by the transfer of activated, antigen-specific T cells. Previously, it has been shown that adoptively transferred, IRBP peptide-specific, but not S-Ag peptide-specific T cells can induce relapsing uveitis in rats. We observed spontaneous recurrences of intra-ocular inflammation even after immunization with R14 in CFA and were able to experimentally re-induce uveitis in rats previously immunized with autoantigen peptide in CFA. The efficiency of re-induction was dependent on the mode of pre-treatment [immunization or adoptive transfer (AT)] as well as on the antigen itself. Primary PDSAg-responses prevented subsequent re-induction of disease much more efficiently than primary R14-mediated EAU. In our model, the suppressive effect of CFA did not play a key role in preventing re-induction or spontaneous relapses. Furthermore, epitope spreading could not be demonstrated as a cause for recurrent inflammation. These data suggest that autoimmune responses with different antigen specificities could underlie similar clinical pictures while being differently regulated, which may help explain the variations in the disease courses in patients and the differential responses to therapeutic modalities.

Accelerated age-dependent transition of human regulatory T cells to effector memory phenotype

2008-02-26

We and others recently described a method for isolating viable forkhead boxp3 (FoxP3⁺) T regulatory cells (Tregs) by means of the surface phenotype CD4⁺CD127^loCD25⁺. In this study, we used the new strategy to measure Treg numbers, phenotype and function at different ages. Mean percentages of CD4⁺CD127^loCD25⁺ Tregs increased only slightly throughout life, from 6.10% in cord blood to 7.22% in PBMC from adults between 20 and 25 years and 7.50% in PBMC from adults over the age of 60. In all age groups, a higher proportion of Tregs had acquired a CD45RA^– memory phenotype compared with CD4⁺Foxp3^– conventional T cells. This increase was entirely attributable to increased Tregs with an effector memory phenotype, whereas central memory phenotype cells were comparably represented within the Treg and conventional CD4⁺ T-cell populations. Expression of CD95 also differed between Tregs and conventional CD4⁺ T cells at all ages. However there was no difference in the suppressive capacity of the different naive and memory Treg subsets. These results suggest that, compared with their conventional CD4⁺ T-cell counterparts, Tregs undergo preferential differentiation from a naive to an effector memory phenotype, driven by their specificity for self- rather than foreign antigen. However, number and function are remarkably stable throughout life.

Predominant donor CD103+CD8+ T cell infiltration into the gut epithelium during acute GvHD: a role of gut lymph nodes

Zhou, S., Ueta, H., Xu, X.-D., Shi, C., Matsuno, K. — 2008-02-26

The existence of donor effector cell subsets responsible for either gut or skin graft-versus-host disease (GvHD) is still undetermined. We examined the trafficking and role of donor CD8⁺ intra-epithelial lymphocytes (IELs) in the gut and skin epithelia concerning Eβ7 integrin (CD103) expression, using a rat acute lethal GvHD model. Most CD103⁺ donor cells were CD8⁺ and showed a proliferative activity in the target epithelia. On the other hand, activated donor T cells in the host lymphoid tissues did not express CD103, indicating the presence of CD8⁺ IEL precursors in the lymphoid tissues that may up-regulate CD103 only after migrating to the target organs. At the late stage of GvHD, while >80% of the donor CD8⁺ IELs were CD103⁺ in the gut epithelium, both CD4⁺ and CD103⁺CD8⁺ T cells evenly accumulated in the skin epidermis. The CD103 expression by donor CD8⁺ IELs especially in the gut was also correlated with the clinical GvHD manifestations. Furthermore, the selective removal of gut lymph nodes (LNs) but not skin LNs suppressed the infiltration of CD103⁺ donor IELs in the gut and alleviated intestinal GvHD. In conclusion, CD103⁺CD8⁺ donor T cells predominantly infiltrate into the gut epithelium and are responsible for the manifestations of intestinal GvHD. This pathology is at least partly dependent on the gut LNs.

BART is essential for nuclear retention of STAT3

2008-02-26

Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation and survival in immune responses, hematopoiesis, neurogenesis and other biological processes. STAT3, for example, is involved in the epithelial–mesenchymal transition during gastrulation, organogenesis, wound healing and cancer progression. STAT activity is regulated by a variety of mechanisms, including nuclear translocation. To clarify the molecular mechanisms underlying the regulation of STAT activity, we performed yeast two-hybrid screening. Here, we identified binder of ADP-ribosylation factor-like two (BART) as a novel STAT-binding partner. Importantly, we showed that BART is essential for the transcriptional activity and nuclear retention of STAT3. Furthermore, an effector of BART, ADP-ribosylation factor-like 2 (ARL2) was also involved in nuclear retention of STAT3. These results indicate that BART plays an essential role in the nuclear retention of STAT3 through interaction with ARL2.

Role of human non-invariant NKT lymphocytes in the maintenance of type 2 T helper environment during pregnancy

2008-02-26

The molecular and cellular mechanisms that generate the T_h2 cytokine environment necessary for the maintenance of pregnancy are still not fully understood. We herein show that the human decidua is highly enriched for TCRβ⁺CD161⁺ NKT cells. They express non-invariant antigen receptors encoded by diverse TCRV- and Vβ-chain gene segments, thereby referred to as non-invariant NKT (non-iNKT) cells. In spite of their diverse TCR expression, they do not recognize fetal allo-antigens but specifically responded to CD1d-transfected cell lines. In contrast to the peripheral blood non-iNKT cells, the decidua-residing non-iNKT cells had a marked T_h2 bias. In addition, they suppress the mixed leukocyte reaction directed against the paternal antigens. The T_h2 cytokines have been known to stimulate trophoblast outgrowth and invasion. Thereby, the non-iNKT cells residing in the decidual tissue may have a functionally important interaction with the villous and extravillous trophoblast cells expressing CD1d and may therefore play a pivotal role in successful pregnancy by inhibiting fetal rejection and enhancing placental growth. These findings may reflect one mechanism that is an essential component for the T_h2 environment necessary for the maintenance of pregnancy.

Plexin-A4 negatively regulates T lymphocyte responses

2008-02-26

Semaphorins and their receptors play crucial roles not only in axon guidance during neuronal development but also in the regulation of immune responses. Plexin-A4, a member of the plexin-A subfamily, forms a receptor complex with neuropilins and transduces signals for class III semaphorins in the nervous system. Although plexin-A4 is also expressed in the lymphoid tissues, the involvement of plexin-A4 in immune responses remains unknown. To explore the role of plexin-A4 in the immune system, we analyzed immune responses in plexin-A4-deficient (plexin-A4–/–) mice. Among immune cells, plexin-A4 mRNA was detected in T cells, dendritic cells and macrophages but not in B cells and NK cells. Plexin-A4–/– mice had normal numbers and cell surface markers for each lymphocyte subset, suggesting that plexin-A4 is not essential for lymphocyte development. However, plexin-A4–/– mice exhibited enhanced antigen-specific T cell responses and heightened sensitivity to experimental autoimmune encephalomyelitis. Plexin-A4–/– T cells exhibited hyperproliferative responses to anti-CD3 stimulation and to allogeneic dendritic cells in vitro. Furthermore, this hyperproliferation was also observed in both T cells from neuropilin-1 mutant (npn-1^Sema–) mice, in which the binding site of class III semaphorins is disrupted, and T cells from Sema3A-deficient (Sema-3A–/–) mice. Collectively, these results suggest that plexin-A4, as a component of the receptor complex for class III semaphorins, negatively regulates T cell-mediated immune responses.

STAT5-signaling cytokines regulate the expression of FOXP3 in CD4+CD25+ regulatory T cells and CD4+CD25- effector T cells

2008-02-26

Forkhead box P3 (FOXP3) is considered a specific marker for CD4⁺CD25⁺ regulatory T (Treg) cells, but increasing evidence suggests that human CD4⁺CD25^– effector T (Teff) cells can transiently express FOXP3 upon activation. We demonstrate that the signal transducer and activator of transcription 5 (STAT5)-signaling cytokines, IL-2, IL-15 and to a lesser extent IL-7, induce FOXP3 up-regulation in vitro in activated human Teff cells. In contrast, cytokines which do not activate STAT5, such as IL-4 or transforming growth factor-β alone, do not directly induce FOXP3 expression in activated Teff cells. Moreover, expression of a constitutively active form of STAT5a is sufficient to induce FOXP3 expression in Teff cells. Expression of FOXP3 in activated Teff cells requires both TCR-mediated activation and endogenous IL-2, but is not dependent on cell division and does not induce suppressive function. The presence of STAT5-activating cytokines is also required to maintain high FOXP3 expression and suppressive activity of Treg cells in vitro. These data indicate that activation of STAT5 sustains FOXP3 expression in both Treg and Teff cells and contribute to our understanding of how cytokines affect the expression of FOXP3.

Hsp60-mediated T cell stimulation is independent of TLR4 and IL-12

Osterloh, A., Veit, A., Gessner, A., Fleischer, B., Breloer, M. — 2008-02-26

Heat shock protein (Hsp) 60 is thought to function as endogenous danger signal by activating professional antigen-presenting cells (APC) through toll-like receptor (TLR) 4 and CD14, a mechanism that is also used by bacterial LPS. We recently showed that Hsp60 binds LPS and enhances LPS-induced immune stimulation. On the other hand, we also observed immune stimulation by Hsp60 independent of LPS which was partially mediated by Hsp60-induced IFN. Here, we study the mechanisms involved in immune stimulation mediated by endotoxin-free Hsp60. We show that T cell co-stimulation induced by LPS-free Hsp60 was independent of TLR4 and the TLR-associated myeloide differentiation factor 88-signaling pathway. LPS-free Hsp60 did not induce IL-6, IL-12 or tumor necrosis factor production in APC nor were these cytokines needed for Hsp60-mediated T cell co-stimulation in the absence of LPS. In contrast to endotoxin-free Hsp60, T cell co-stimulation induced by LPS or Hsp60/LPS complexes strictly depended on IL-12 and functional TLR-4. Furthermore, we show that LPS-free Hsp60 enhances IFN expression in APC and that this cytokine represents one important mediator in immune stimulation by Hsp60 in the absence of LPS. Taken together, we provide evidence that endotoxin-free Hsp60 and LPS or Hsp60/LPS complexes employ different signaling mechanisms to transduce co-stimulatory signals.

Comprehensive analysis of antibody responses to streptococcal and tissue antigens in patients with acute rheumatic fever

2008-02-26

Acute rheumatic fever (ARF) is an autoimmune disease occurring in individuals following untreated group A streptococcal infection believed to be triggered by antibodies to bacterial components that cross-react with human tissues. We developed a multiplexed immunoassay for the simultaneous quantitation of antibodies to nine streptococcal-related antigens including streptolysin O (SLO), DNase B, collagen I and IV, fibronectin, myosin, group A carbohydrate, M6 protein and streptococcal C5a peptidase. Utilizing this method, we examined serum from 49 ARF, 58 pharyngitis patients and age- and sex-matched controls in samples collected at initial disease onset, and at 4 weeks, 6 months and 1 year after diagnosis. Antibody responses were significantly higher for SLO, DNase B, M6 protein, group A carbohydrate and the cross-reactive antigens collagen I and myosin in ARF compared with pharyngitis patients (P ≤ 0.05). Moreover, we found significantly elevated antibody responses in the ARF patients with rheumatic heart disease to fibronectin and collagen I compared with ARF patients without heart disease. The major differences between the ARF patients with and without carditis appear to be in the immune response to the putative heart valve components, collagen I and fibronectin.

Regulation of the plasma cell transcription factor Blimp-1 gene by Bach2 and Bcl6

Ochiai, K., Muto, A., Tanaka, H., Takahashi, S., Igarashi, K. — 2008-02-26

B lymphocyte-induced maturation protein 1 (Blimp-1) is a key regulator for plasma cell differentiation. Prior to the terminal differentiation into plasma cells, Blimp-1 expression is suppressed in B cells by transcription repressors BTB and CNC homology 2 (Bach2) and B cell lymphoma 6 (Bcl6). Bach2 binds to the Maf recognition element (MARE) of the promoter upstream region of the Blimp-1 gene (Prdm1) by forming a heterodimer with MafK. Bach2 and Bcl6 were found to interact with each other in B cells. While both Bach2 and Bcl6 possess the BTB domain which mediates protein–protein interactions, they interacted in a BTB-independent manner. Bcl6 is known to repress Prdm1 through a Bcl6 recognition element 1 in the intron 5, in which a putative, evolutionarily conserved MARE was identified. Both repressed the expression of a reporter gene containing the intron 5 region depending on the presence of the respective binding sites in 18-81 pre-B cells. Co-expression of Bach2 and Bcl6 resulted in further repression of the reporter plasmid. Chromatin immunoprecipitation assays showed MafK to bind to the intron MARE in various B cell lines, thus suggesting that it binds as a heterodimer with Bach2. Therefore, the interaction between Bach2 and Bcl6 might be crucial for the proper repression of Prdm1 in B cells.