Deletion of galectin-3 in the host attenuates metastasis of murine melanoma by modulating tumor adhesion and NK cell activity

Galectin-3, a β galactoside–binding lectin, plays an important role in the processes relevant to tumorigenesis such as malignant cell transformation, invasion and metastasis. We have investigated whether deletion of Galectin-3 in the host affects the metastasis of B16F1 malignant melanoma. Galectin-3-deficient (Gal-3^−/−) mice are more resistant to metastatic malignant melanoma as evaluated by number and size of metastatic colonies in the lung. In vitro assays showed lower number of attached malignant cells in the tissue section derived from Gal-3^−/− mice. Furthermore, lack of Galectin-3 correlates with higher serum levels of IFN-γ and IL-17 in tumor bearing hosts. Interestingly, spleens of Gal-3^−/− mice have lower number of Foxp3⁺ T cells after injection of B16F1 melanoma cells. Finally, we found that while CD8⁺ T cell and adherent cell cytotoxicity were similar, there was greater cytotoxic activity of splenic NK cells of Gal-3^−/− mice compared with “wild-type” (Gal-3 ^+/+ ) mice. Despite the reduction in total number of CD3ε⁻NK1.1⁺, Gal-3^−/− mice constitutively have a significantly higher percentage of effective cytotoxic CD27^highCD11b^high NK cells as well as the percentage of immature CD27^highCD11b^low NK cells. In contrast, CD27^lowCD11b^high less functionally exhausted NK cells and NK cells bearing inhibitory KLRG1 receptor were more numerous in Gal-3 ^+/+ mice. It appears that lack of Galectin-3 affects tumor metastasis by at least two independent mechanisms: by a decrease in binding of melanoma cells onto target tissue and by enhanced NK-mediated anti-tumor response suggesting that Galectin-3 may be considered as therapeutic target.     

NKG2D in NK and T Cell-Mediated Immunity

One of the best characterized NK cell receptors is NKG2D, a highly conserved C-type lectin-like membrane glycoprotein expressed on essentially all NK cells, as well as on γδ-TcR⁺ T cells and αβ-TcR⁺ CD8⁺ T cells, in humans and mice. Here we review recent studies implicating NKG2D in T cell and NK cell-mediated immunity to viruses and tumors, and its potential role in autoimmune diseases and allogeneic bone marrow transplantation.     

Potentiation of antitumor effect of NKT cell ligand, α-galactosylceramide by combination with IL-12 on lung metastasis of malignant melanoma cells1

The combined therapeutic effect of natural killer T (NKT) cell ligand α-galactosylceramide (α-GalCer) and IL-12 against highly metastatic B16-BL6-HM melanoma cells was investigated. In comparison with a single administration of α-GalCer or IL-12, the combined treatment of tumor-bearing mice with α-GalCer plus IL-12 caused a super-induction of serum IFN-γ levels, though α-GalCer-induced IL-4 production was rather inhibited. In parallel with the augmented IFN-γ production, the natural killing activity against YAC-1 cells and syngeneic B16- BL6-HM melanoma was greatly augmented by the combined therapy. The major effector cells responsible for natural killing activity induced by α-GalCer plus IL- 12 were enriched in both NK1.1⁺TCRαβ⁺ NKT cells and NK1.1⁺TCRαβ⁻ NK cells. The preventing effect of α-GalCer or IL-12 alone against lung metastasis of B16-BL6-HM was also enhanced by the combination therapy. The antitumor activity of α-GalCer was totally abolished in NKT-deficient mice. However, IL- 12-induced antitumor activity was not eliminated in NKT-deficient mice though it was inhibited by anti-asialo GM1 Ab treatment. These findings suggested that α-GalCer synergistically act with IL-12 to activate both NKT cells and NK cells, which may play a critical role in the strong prevention of distant tumor metastasis at early stages of tumor-bearing. These data will provide a novel tool for the prevention of tumor metastasis using NKT-specific ligands α-GalCer and IL-12. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of α7- and β4-Containing Nicotinic Acetylcholine Receptors in the Affective and Somatic Aspects of Nicotine Withdrawal: Studies in Knockout Mice

To assess which nicotinic acetylcholine receptors (nAChRs) are involved in the aversive aspects of nicotine withdrawal, brain reward function and the somatic signs of nicotine withdrawal were assessed in mice that lack α7 and β4 nAChR subunits. Brain reward function was assessed with the intracranial self-stimulation (ICSS) procedure, in which elevations in ICSS thresholds reflect an anhedonic mood state. At 3–6 h of spontaneous nicotine/saline withdrawal, thresholds were elevated in nicotine-withdrawing α7^+/+ and β4^+/+, but not α7^−/− or β4^−/−, mice compared with saline-withdrawing mice, indicating a delay in the onset of withdrawal in the knockout mice. From 8 to 100 h of withdrawal, thresholds in α7^+/+ and α7^−/− mice were equally elevated, whereas thresholds in β4^+/+ and β4^−/− mice returned to baseline levels. Somatic signs were attenuated in nicotine-withdrawing β4^−/−, but not α7^−/−, mice. Administration of a low dose of the nAChR antagonist mecamylamine induced threshold elevations in α7^−/−, but not α7^+/+, mice, whereas the highest dose tested only elevated thresholds in α7^+/+ mice. Mecamylamine-induced threshold elevations were similar in β4^−/− and β4^+/+ mice. In conclusion, null mutation of the α7 and β4 nAChR subunits resulted in a delayed onset of the anhedonic aspects of the spontaneous nicotine withdrawal syndrome. Previous findings of attenuated somatic signs of nicotine withdrawal in β4^−/−, but not α7^−/−, mice were confirmed in the present study, indicating an important role for β4-containing nAChRs in the somatic signs of nicotine withdrawal. The mecamylamine-precipitated withdrawal data suggest that compensatory adaptations may occur in constitutive α7^−/− mice or that mecamylamine may interact with other receptors besides nAChRs in these mice. In summary, the present results indicate an important role for α7 and β4-containing nAChRs in the anhedonic or somatic signs of nicotine withdrawal.     

Identification of an HLA-DQ6-derived peptide recognized by mouse MHC class I H-2Db-restricted CD8+ T cells in HLA-DQ6 transgenic mice

Summary CD8⁺ T cells from C57BL/6(B6) mice show cytotoxicity to B cell blasts prepared from syngeneic transgenic mice expressing HLA-DQ6 molecules in a mouse MHC class I H-2D^b restricted manner. Although these results suggest that CD8⁺ T cells recognize peptides derived from DQ6 molecule bound to H-2D^b on target cells, no direct evidence so far has been obtained. To clarify this, we synthesized 23 peptides corresponding to DQ6α orβ chain and carrying the motifs of D^b-binding peptides, and examined their capacity to induce cytotoxicity in the CD8⁺ T cell line. We show here that DQA1-2, one of these peptides, induced cytotoxicity of the CD8⁺ T cells when this peptide was pulsed to H-2D^b expressing target cells, as efficiently as HLA-DQ6 expressing target cells did. Thus, our results suggest that DQA1-2 can be naturally processed from DQ6 molecules and recognized by the CD8⁺ T cells in the context of H-2D^b molecules. These results suggest that allogeneic HLA class II molecules are involved in the rejection not only as the ligand for T cell receptor of alloreactive CD4⁺ T cells but also as self-peptides bound to HLA class I molecules recognized by CD8⁺ T cells.     

Identification of CD4+ T-cell epitopes on iron-regulated surface determinant B of Staphylococcus aureus

Iron-regulated surface determinant B (IsdB) of Staphylococcus aureus (S. aureus) is a highly conserved surface protein that can induce protective CD4⁺ T-cell immune response. A pivotal role of CD4⁺ T-cells in effective immunity against S. aureus infection has been proved, but CD4⁺ T-cell epitopes on the S. aureus IsdB have not been well identified. In this study, MHC binding assay was firstly used to predict CD4⁺ T-cell epitopes on S. aureus IsdB protein, and six peptides were synthesized to validate the probable epitopes. Two novel IsdB CD4⁺ T-cell epitopes, P1 (residues 159–178) and P4 (residues 287–306), were for the first time identified using CD4⁺ T-cells obtained from IsdB-immunized C57BL/6 (H-2^b) and BALB/c (H-2^d) mice spleen based on cell proliferation and cytokines response. The results showed that P1 and P4 emulsified in Freund's adjuvant (FA) induced much higher cell proliferation compared with PBS emulsified in FA. CD4⁺ T-cells stimulated with peptides P1 and P4 secreted significantly higher levels of IFN-γ and IL-17A. However, the level of the cytokine IL-4 almost remained unchanged, suggesting that P1 and P4 preferentially elicited polarized Th1-type responses. In addition, BALB/c mice just respond to P4 not P1, while C57BL/6 mice respond to P1 not P4, implying that epitope P1 and P4 were determined as H-2^b and H-2^d restricted epitope, respectively. Taken together, our data may provide an explanation of the IsdB-induced protection against S. aureus and highlight the possibility of developing the epitope-based vaccine against the S. aureus.     

IFN-γ is essential for the inhibition of B16BL6 melanoma lung metastasis in chronic alcohol drinking mice

We previously found that chronic alcohol consumption (20% w/v in drinking water) that models the level consumed by human alcoholics, when administered to female C57BL/6 mice inhibits B16BL6 melanoma metastasis to the lung; however, the mechanism is not known. Chronic alcohol consumption increases IFN-γ producing NK, NKT, CD4⁺, and CD8⁺ T cells. To examine the impact of IFN-γ on metastasis, we inoculated B16BL6 melanoma cells i.v. into control and chronic alcohol drinking IFN-γ knockout (KO) mice. Knockout of the ifn-γ gene abrogated the anti-metastatic effects associated with alcohol consumption. We examined metastasis in common gamma-chain (γC) KO mice, which are deficient in NK, NKT and CD8⁺ T cells, and in Vα14Jα281^−/− KO mice, which are deficient in invariant NKT (iNKT) cells, in order to assess the importance of specific IFN-γ producing cell types to this effect. We found that the antimetastatic effect of alcohol was still present in γC KO mice and also in γC KO mice depleted of Gr-1⁺ cells. Knockout of iNKT cells reduced the degree but not the antimetastatic effect associated with alcohol. These results indicate that the antimetastatic effect induced by chronic alcohol consumption is IFN-γ dependent and that multiple IFN-γ producing cell types contribute to this effect.     

Responses against complex antigens in various models of CD4 T-cell deficiency

The most common models of CD4 T-cell deficiency are mice exogenously injected with anti-CD4 antibody (Ab), CD4 knockout (CD4^−/−) and major histocompatibility complex (MHC) class II knockout (class II^−/−) mice. We recently described the anti-CD4 Ab transgenic mouse (GK) as an improved CD4 cell-deficient model. This review compares this new GK mouse model with the widely available class II^−/− and CD4^−/− mice, when exposed to complex antigens (foreign grafts and during bacterial or viral infection). We highlight here the cytometric and functional differences (including Ab isotype, viral or bacterial clearance, and graft survival) among these CD4 cell-deficient models. For example, whereas grafts are generally rejected in class II^−/− and CD4^−/− mice as quickly as in wild-type mice, they survive longer in GK mice. Also, CD4^−/− mice produce IgG against both simple model and complex antigens, but class II^−/− and GK mice produce small amounts of IgG2a against complex antigens but not simple model antigens. These differences harbinger the caveats in the use of these various mice.     

Absence of cytotoxic molecules in CD8- and/or CD56-positive adult T-cell leukaemia/lymphoma

Adult T-cell leukaemia/lymphoma (ATLL) cells usually exhibit a CD4⁺ (helper/inducer) phenotype (CD4⁺/8^–/56^–), and only a minority of tumours express the CD8 (cytotoxic/suppressor) or CD56 (natural killer [NK]-associated) antigens. TIA-1 is a cytotoxic granule-associated protein expressed in NK cells and cytotoxic T lymphocytes (CTLs). Granzyme B, perforin and Fas ligand (FasL) are also expressed in activated CTLs and NK cells. To clarify the cytotoxic potential of ATLL cells, immunohistochemistry was performed in CD8⁺ and/or CD56⁺ ATLL cells, using anti-TIA-1, anti-granzyme B, anti-perforin and anti-FasL antibodies. We studied nine cases of CD8⁺ and/or CD56+ ATLL, all of which exhibited monoclonal integration of human T-cell leukaemia virus type 1 (HTLV-1) proviral DNA. Four cases exhibited a CD8⁺/CD56^– phenotype, four others had a CD8^–/CD56⁺ phenotype, and one was CD8⁺/CD56⁺. All but one case also expressed the surface antigens CD3, TCR αβ, and CD4. Expression of granzyme B and TIA-1 were demonstrated in three and two cases, respectively, but none expressed perforin or FasL. In the control study, 10 cases with typical CD3⁺/4⁺/8^–/56^– ATLL demonstrated no expression of those cytotoxic-associated proteins. Our findings suggest that CD8 and/or CD56 positivity probably confer(s) no cytotoxic function on ATLL cells, and it is possible that CD8 and CD56 may be simply aberrant surface markers in ATLL. Received: 20 January 1999 / Accepted: 13 April 1999     

Mice deficient in Vβ8<Superscript>+</Superscript>NKT cells are resistant to experimental hepatitis but are partially susceptible to generalised Shwartzman reaction

Abstract NKT cells are responsible for hepatitis induced either by concanavalin A (Con-A) or α-galactosylceramide (α-GalCer), and they are also profoundly involved in the generalised Shwartzman reaction (GSR) induced by consecutive injections of interleukin (IL)-12 and lipopolysaccharide (LPS). In the present study, using NC/Nga (NC) mice and SJL mice lacking the Vβ⁺8 gene, we examined the role of Vβ⁺8+NKT cells in hepatitis models and in the GSR. The absence of Vβ⁺8+NKT cells in the liver mononuclear cells (MNC) was confirmed by the α-GalCer/CD1d/Ig dimer. Unexpectedly, other dimer+NKT cells including Vβ7⁺NKT cells in these mice were found to decrease in comparison to that of C57BL/6 mice. No significant hepatocyte injury was observed after α-GalCer or Con-A administration in either mice. The serum interferon (IFN)-γ, IL-4 and tumour necrosis factor (TNF) levels did not increase in these mice after α-GalCer injection, however these cytokines substantially increased after Con-A administration, thus suggesting that the roles of NKT cells differ between the two hepatitis models. However, in GSR, although neither mice showed lower IFN-γ levels after a priming IL-12 injection, they showed TNF levels comparable to those in normal mice after LPS injection, and thus resulted in a decreased but substantial mortality. Although liver MNC from IL-12-injected SJL mice showed an impaired antitumour cytotoxicity, liver MNC of NC mice exhibited a greater antitumour cytotoxicity than that of C57BL/6 mice because liver NK cells proportionally increased in NC mice. These results confirm the critical role that Vβ8⁺NKT cells play in both liver and multi-organ injury.     

Positive and Negative Selection in Transgenic Mice Expressing a T-Cell Receptor Specific for Influenza Nucleoprotein and Endogenous Superantigen

A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8⁺ and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC D^b (Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2^b, IE^-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2^k and BALB/c H-2^d) and the endogenous Mtv ligands. Positive selection of CD8⁺ T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2^b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of ^{in vitro} culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8⁺ peripheral T-cells in these (H-2^k or H-2^d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2^d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired.     

Evidence for the causal role of endogenous interferon-alpha/beta in the regulation of angiogenesis,tumorigenicity, and metastasis of cutaneous neoplasms

Primary tumor growth and metastasis depend on angiogenesis, which is determined by the balance between proangiogenic and antiangiogenic molecules. Interferon (IFN)-α and -β inhibit angiogenesis through downregulation of interleukin-8, matrix metalloproteinase-9, and basic fibroblast growth factor. To provide evidence for the causal role of IFN-α/β in the induction of neoplasms, their angiogenesis, and hence, progressive growth, we carried out experiments using 129S6 IFN-α/β receptor −/− mice back-crossed to BALB/c nude mice. Subcutaneous angiogenesis was determined following implantation of gelfoam sponges containing 0.4% agarose and several proangiogenic molecules. Tumorigenicity and production of lung metastasis were determined subsequent to subcutaneous and intravenous injections, respectively, of highly metastatic A375SM human melanoma cells. Carcinogenesis was induced by chronic exposure of mice to UVB radiation (5 kJ/m², 3 times/week). Angiogenesis, tumorigenicity, and production of metastasis, as well as development of autochthonous skin tumors, were all accelerated in IFN-α/β receptor −/− mice as compared to control mice. Collectively, the data show that inability to respond to endogenous IFN-α/β (through a mutation in the IFN-α/β receptor) leads to increased susceptibility to carcinogenesis, enhanced angiogenesis, tumorigenicity, and metastasis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nod2 Mutation Enhances NF-kappaB Activity and Bacterial Killing Activity of Macrophages

NOD2, an intracellular sensor of bacteria, are linked to increased susceptibility to bacteria in Crohn’s disease (CD). The NOD2 protein is expressed mainly by macrophages and dendritic cells. This study is to examine the role of NOD2 in the innate response of macrophages to bacterial challenge. First, peritoneal macrophages and alveolar macrophages were harvested from WT, Nod2^2939iC, as well as TLR4^−/− mice and incubated with E. coli or P. aeruginosa. Bacterial killing activity; IL-1β and TLR4 protein expression; NF-κB DNA binding activity assay; as well as IL-1β, TNFα, TLR2, TLR4 and TLR9 mRNA expression of macrophages were examined. We found that alveolar macrophages and peritoneal macrophages of Nod2^2939iC mice but not WT mice or TLR4^−/− mice demonstrated a significant increase of E. coli killing activity. Bacterial challenge also induced a significant increase of pro-IL-1β protein expression; NF-κB DNA binding activity; as well as IL-1β and TNFα mRNA expression of the peritoneal macrophages in Nod2^2939iC mice. Collectively, the increase of bacterial killing activity, IL-1β expression, and NF-κB DNA binding activity of macrophages in Nod2^2939iC mice suggests that NOD2 is a positive regulator of NF-κB/IL-1β-mediated innate response to bacteria challenge in Crohn’s disease.     

Association of Graves’ Disease and Prevalence of Circulating IFN-γ-producing CD28<Superscript>−</Superscript> T Cells

Background  Peripheral blood CD4⁺ and CD8⁺ T-cell subsets lacking surface CD28 have been suggested to predispose patients to immune-mediated disorders. Materials and Methods  To determine the role of CD28⁻ T-cell subset in Graves’ disease (GD), we characterized peripheral blood CD4⁺CD28⁻ and CD8⁺CD28⁻ T cell from early onset GD patients. Results and Discussion  GD patients had significantly higher percentages of CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells than did healthy donors. Both CD28⁻ T cells expressed mostly CD45RO, suggesting that they are activated and/or are memory T cells. GD patient-derived CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells produced more intracellular IFN-γ than their counterparts from healthy donors. Furthermore, CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells from GD patients with Graves’ ophthalmopathy (GO) secreted higher level of intracellular IFN-γ than those CD28⁻ T cells from GD patients without GO. Retrospective analysis showed that the increased levels of CD4⁺CD28⁻ T cells and their IFN-γ-producing subgroups were positively correlated to the serum anti-thyrotropin receptor (TSHR) autoantibodies (TRAb). Our observations suggest that increased IFN-γ-producing CD28⁻ T cells in GD patients may play an important role in the pathogenesis of GD. Zhiping Sun and Weixue Zhong contributed equally to this paper.     

Thymopoiesis, Regulatory T Cells, and TCRVβ Expression in Thymoma With and Without Myasthenia Gravis, and Modulatory Effects of Steroid Therapy

We analyzed thymocyte and thymic regulatory T cell (CD4SPCD25⁺Foxp3⁺cells, Treg) development in thymoma with and without myasthenia gravis (MG, MG-thymoma, non-MG-thymoma) and in MG-associated non-neoplastic thymus (MG-NNT). An increased number of immature CD4⁺CD8⁻CD3⁻ thymocytes through the CD4⁺CD8⁺ to CD4⁺CD8⁻ transition and an abnormal T cell receptor Vβ (TCRVβ) development through the CD4⁺CD8⁺ to CD4⁻CD8⁺ transition were seen both in MG-and non-MG-thymomas. Terminal thymopoiesis, i.e., CD45RA⁺ cells within the CD4⁺CD8⁻CD3⁺ and CD8⁺CD4⁻CD3⁺ subsets, was skewed towards the CD4⁺ compartment in MG-thymoma and CD8⁺ compartment in non-MG-thymoma, but thymic export was increased only in the latter in keeping with the hypothesis that CD8⁺ lymphocytes may play a role in the initial stages of autosensitization and in disagreement with the relevance of an increased output of CD4⁺ T lymphocytes in paraneoplastic MG. Treg level in normal thymus and MG-NNT and both MG- and non-MG-thymoma was similar, and TCRVβ development in Treg cells was slightly altered in thymoma but irrespective of MG presence. Thus, the relevance of a defective Treg development in MG context remains to be established. Most alterations in thymopoiesis were corrected by therapeutic corticosteroid administration, and the effects of steroid administration may be mediated by thymic microenvironment.     

Clinical, immunohistochemical and phenotypic features of aggressive nodal cytotoxic lymphomas, including α/β, γ/δ T-cell and natural killer cell types

Cytotoxic cells include natural killer (NK) cells and cytotoxic αβ and γδ T lymphocytes (CTLs). These cells express cytotoxic molecules of T-cell restricted intracellular antigen(TIA-1), and activated cytotoxic molecules of perforin, granzyme B, and FasL. Recent studies suggest that most extranodal T-cell lymphomas are derived from CTLs, and that NK cell lymphomas are extranodal. However, only a few nodal NK and cytotoxic lymphomas have been described so far. We present here the clinicopathological features of seven cases of nodal cytotoxic T and NK cell lymphomas. The study excluded anaplastic large-cell lymphomas expressing cytotoxic molecules. The neoplastic cells of all cases contained activated cytotoxic molecules of TIA-1, granzyme B, Fas ligand, and/or perforin. Phenotypically and genotypically, four cases showed αβ T cell type [CD2+, CD3+, T-cell receptor (TCR) δ-1–, βF1+, and TCR gene rearrangement], two cases showed γδ T-cell type [CD2+, CD3+, T-cell receptor (TCR)δ–1+, βF1–, and TCR gene rearrangement], and one case showed NK cell type [CD2+, CD3-, CD56+, T-cell receptor (TCR)δ-1–, βF1–, and TCR gene germline]. Using Southern blot analysis, Epstein-Barr virus (EBV) sequences were detected in six cases, and monoclonal terminal repeat proliferation was confirmed. In addition, in situ hybridization (ISH) studies for EBV showed EBV infection in almost all neoplastic cells. Clinically, all patients presented with peripheral lymphadenopathy in high clinical stages and showed an aggressive course. Hepatosplenomegaly was detected in six cases. During the course of the disease, bone marrow and extranodal invasion were noted in five cases. The nodal type showed an aggressive clinical course in all cases but one, as did the extranodal type. The nodal type varied in phenotype, but was closely associated with EBV infection.     

The T/B cell interaction involved in induction of the mouse IgG2ah suppression is restricted by major histocompatibility complex class I,but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2a^b suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igh^a haplotype, when neonatally transferred into histocompatible Igh^a/b heterozygotes, are able to induce a specific and total suppression of the IgG2a^b allotype. Nevertheless, only transfer of IgG2a^b-primed Igh^a T splenocytes induces this suppression in Igh^b/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8⁺ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4⁺CD8⁺ T cell cooperation and operated via the recognition by the involved TCR of Cγ2a^b-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Igh^b mice genetically deficient for MHC class I (β2-microglobulin^%, or β2m^%) or class II (I-Aβ^%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2a^b T cells transferred into Igh^b H-2^b I-Aβ^% mice carry out the suppression process normally, while in Igh^b H-2^b β2m^% recipients, their suppression induction capacity is significantly inhibited. Moreover, the Cγ2a^b 103–118 peptide, identified as the sole Cγ2a^b-derived peptide able to amplify the anti-IgG2a^b T cell reactivity in Igh^a H-2^b mice, is also able to stabilize the H-2D^b, but not the H-2K^b class I molecules at the surface of RMA-S (TAP2^?, H-2^b) cells. These results indicate that, despite the CD4⁺/CD8⁺ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2a^b+ B cells for suppression establishment.     

Evidence of sex-based differences in natural killer cell responses to exercise and carbohydrate intake in children

Distinct natural killer (NK) cell subsets (CD56^bright and CD56^dim) are mobilized with exercise and these cells may serve adaptive functions. We determined the distribution of NK cell subsets in response to exercise and carbohydrate (CHO) intake in young girls and compared these responses with previous findings in young boys of the same age. Twelve girls (12 years old) cycled for 60 min at 70% while drinking 6% CHO or flavoured water. Blood was collected at rest, during (30 and 60 min) and following (30 and 60 min) exercise to identify NK cells as CD3⁻CD56^bright or CD3⁻CD56^dim. CD69 expression on total CD3⁻CD56⁺ cells was also determined. A trend (P = 0.07) was found for a trial × time interaction in CD56^dim cell counts, with values lower with CHO than with water. CHO intake did not influence CD56^bright responses (P ≥ 0.39). The CD56^bright:CD56^dim ratio increased during recovery from exercise (P < 0.001), compared to rest, with no effect of CHO intake (P = 0.48). CD69 expression was not different between exercise or recovery and rest. Like young boys, girls experience an elevated CD56^bright:CD56^dim ratio during recovery from exercise and CHO intake attenuates the exercise-induced CD56^dim but not CD56^bright cell response. Unlike young boys, girls do not experience a CHO-induced increase in the CD56^bright:CD56^dim ratio during recovery and CD69 expression does not increase on CD3⁻CD56⁺ cells during recovery. We conclude that even in young children sex-based differences exist in the NK cell response to exercise and CHO intake.     

Inflammation, T-Cell Phenotype, and Inflammatory Cytokines in Chronic Kidney Disease Patients Under Hemodialysis and its Relationship to Resistance to Recombinant Human Erythropoietin Therapy

Background Resistance to recombinant human erythropoietin (rhEPO) occurs in some chronic kidney disease (CKD) patients, which may be due to enhanced systemic inflammatory response and to the erythropoiesis-suppressing effect of pro-inflammatory cytokines, some of which are produced by T cells. Aim of study The aim of this study was to investigate the relationship between resistance to rhEPO therapy in hemodialysis CKD patients and inflammatory markers [C-reactive protein (CRP), soluble interleukin (IL)-2 receptor (sIL2R), and serum albumin levels], blood cell counts, T-cell phenotype, cytokine production by T cells, and serum cytokine levels. Materials and Methods We studied 50 hemodialysis CKD patients, 25 responders and 25 nonresponders to rhEPO, and compared them to each other and with 25 healthy controls. When compared to controls, CKD patients showed increased serum levels of CRP, IL-6, and sIL2R and a T-cell lymphopenia, due to decreased numbers of both CD4⁺ and CD8⁺ T cells. T cells from CKD patients had an immunophenotype compatible with chronic T-cell stimulation as shown by the increased percentage of CD28⁻, CD57⁺, HLA-DR⁺, CD28⁻HLA-DR⁺, and CD57⁺ HLA-DR⁺ T cells and produce higher levels of IL-2, INF-γ, and TNF-α after short-term in vitro stimulation, although Th1 cytokines were not detectable in serum. Statistically significant differences were found between responders and nonresponders to rhEPO therapy for total lymphocyte and CD4⁺ T-lymphocyte counts, albumin (lower in nonresponders) and CRP (higher in nonresponders) levels. Conclusion CKD patients under hemodialysis present with raised inflammatory markers and decrease of total lymphocyte and CD4⁺ T-lymphocyte counts when compared with controls. Some of those markers are even further enhanced in nonresponders to rhEPO therapy patients, but resistance to this therapy cannot be justified by a Th1 polarized T-cell response.