Differential IL-12 responsiveness of T cells but not of NK cells from tumor-bearing mice in IL-12-responsive versus -unresponsive tumor models.
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While IL-12 administration induces tumor regression through stimulating T cells in tumor-bearing mice, this IL-12 effect is observed in some but not all tumor models. The present study aimed to compare IL-12 responsiveness of T cells from tumor-bearing mice in IL-12-responsive (CSA1M and OV-HM) and -unresponsive (Meth A) tumor models. Tumor regression in IL-12-responsive tumor models required the participation of T cells, but not of NK1.1(+) cells. Because a NK1.1(+) cell population was the major producer of IFN-gamma, comparable levels of IFN-gamma production were induced in IL-12-responsive and -unresponsive tumor-bearing mice. This indicates that the amount of IFN-gamma produced in tumor-bearing individuals does not correlate with the anti-tumor efficacy of IL-12. In contrast, IL-12 responsiveness of T cells differed between the responsive and unresponsive models: purified T cells from CSA1M/OV-HM-bearing or Meth A-bearing mice exhibited high or low IL-12 responsiveness respectively, when evaluated by the amounts of IFN-gamma produced in response to IL-12. T cells from CSA1M- or OV-HM-bearing but not from Meth A-bearing mice exhibited enhanced levels of mRNA for the IL-12 receptor (IL-12R). These results indicate that a fundamental difference exists in IL-12 responsiveness of T cells between IL-12-responsive and -unresponsive tumor models, and that such a difference is associated with the expression of IL-12R on T cells. (+info)
The mouse tumor cell lines EL4 and RMA display mosaic expression of NK-related and certain other surface molecules and appear to have a common origin.
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As a potential means for facilitating studies of NK cell-related molecules, we examined the expression of these molecules on a range of mouse tumor cell lines. Of the lines we initially examined, only EL4 and RMA expressed such molecules, both lines expressing several members of the Ly49 and NKRP1 families. Unexpectedly, several of the NK-related molecules, together with certain other molecules including CD2, CD3, CD4, CD32, and CD44, were often expressed in a mosaic manner, even on freshly derived clones, indicating frequent switching in expression. In each case examined, switching was controlled at the mRNA level, with expression of CD3zeta determining expression of the entire CD3-TCR complex. Each of the variable molecules was expressed independently, with the exception that CD3 was restricted to cells that also expressed CD2. Treatment with drugs that affect DNA methylation and histone acetylation could augment the expression of at least some of the variable molecules. The striking phenotypic similarity between EL4 and RMA led us to examine the state of their TCRbeta genes. Both lines had identical rearrangements on both chromosomes, indicating that RMA is in fact a subline of EL4. Overall, these findings suggest that EL4 is an NK-T cell tumor that may have retained a genetic mechanism that permits the variable expression of a restricted group of molecules involved in recognition and signaling. (+info)
Characterization of Ly-6M, a novel member of the Ly-6 family of hematopoietic proteins.
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The Ly-6 family includes a number of highly homologous, low molecular weight glycophosphatidylinositol-linked proteins expressed on hematopoietic and lymphoid cells. The best characterized family member is Sca-1 (Ly-6A/E), an antigen commonly used for purification of murine pluripotent hematopoietic cells. We sought to characterize the genomic locus surrounding the Sca-1 gene. We identified several overlapping P1 artificial chromosomes containing the Sca-1 gene and mapped one of these to mouse chromosome 15D3.1-3.3, the region previously shown to contain members of the murine Ly-6 gene family. We then mapped this clone and found that the Sca-2 gene lies 35.4 kilobase (kb) downstream of Sca-1 in the opposite transcriptional orientation. This is the first direct demonstration of physical linkage of Ly-6 genes. A novel gene, highly homologous to Sca-1 was identified and localized 13.4 kb downstream of Sca-1. This gene, which we designated Ly-6M, shares several structural features conserved among members of the Ly-6 family. Ly-6M messenger RNA (mRNA) is easily detectable in hematopoietic tissue (bone marrow, spleen, thymus, peritoneal macrophages) as well as kidney and lung. No mRNA expression was detected in heart, stomach, liver, small intestine, brain, or skin. Ly-6M protein is detectable on 10% to 15% of peripheral blood leukocytes, including monocytes and a subpopulation of B220(+) cells. Ly-6M is broadly distributed in the bone marrow, with prominent expression on monocytes and myeloid precursors. The identification and characterization of Ly-6M adds a new member to a complex family of homologous, tightly linked genes that have proven extremely useful reagents for defining populations within the hematopoietic system. (+info)
Mice with a selective deletion of the CC chemokine receptors 5 or 2 are protected from dextran sodium sulfate-mediated colitis: lack of CC chemokine receptor 5 expression results in a NK1.1+ lymphocyte-associated Th2-type immune response in the intestine.
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The chemokine receptors CCR2 and CCR5 and their respective ligands regulate leukocyte chemotaxis and activation. To determine the role of these chemokine receptors in the regulation of the intestinal immune response, we induced colitis in CCR2- and CCR5-deficient mice by continuous oral administration of dextran sodium sulfate (DSS). Both CCR2- and CCR5-deficient mice were susceptible to DSS-induced intestinal inflammation. The lack of CCR2 or CCR5 did not reduce the DSS-induced migration of macrophages into the colonic lamina propria. However, both CCR5-deficient mice and, to a lesser degree, CCR2-deficient mice were protected from DSS-induced intestinal adhesions and mucosal ulcerations. CCR5-deficient mice were characterized by a greater relative infiltration of CD4+ and NK1.1+ lymphocyte in the colonic lamina propria when compared to wild-type and CCR2-deficient mice. In CCR5-deficient mice, mucosal mRNA expression of IL-4, IL-5, and IL-10 was increased, whereas that of IFN-gamma was decreased, corresponding to a Th2 pattern of T cell activation. In CCR2-deficient mice, the infiltration of Th2-type T cells in the lamina propria was absent, but increased levels of IL-10 and decreased levels of IFN-gamma may have down regulated mucosal inflammation. Our data indicate that CCR5 may be critical for the promotion of intestinal Th1-type immune responses in mice. (+info)
Synergistic effects of in vivo depletion of Ly-49A and Ly-49G2 natural killer cell subsets in the rejection of H2(b) bone marrow cell allografts.
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Subsets of murine natural killer (NK) cells exist that express the Ly-49 family of molecules that recognize different major histocompatibility complex (MHC) determinants. Bone marrow transplantation studies were performed to examine the in vivo functions of 2 of these subsets. Subsets of Ly-49A and Ly-49G2 NK share specificity for the same MHC class 1 ligand, D(d), binding of which results in an inhibitory signal to the NK cell but allows them to lyse H2(b) targets in vitro. We therefore examined the ability of these subsets to reject H2(b) bone marrow cell allografts in lethally irradiated mice. Surprisingly, depletion of Ly-49A(+) NK cells in BALB/c or B10.D2 mice (both H2(d)) had no effect on the rejection of H2(b) BMC. However, Ly-49A depletion did partially abrogate the ability of B10.BR (H2(k)) mice to reject H2(b) allografts. Although depletion of either Ly-49A(+) or Ly-49G2(+) NK cells alone had no effect on the ability of B10.D2 mice to reject H2(b) BMC, depletion of both subsets dramatically and synergistically abrogated rejection. Studies with various B10 congenic mice and their F(1) hybrids indicate that this synergy between Ly49A and Ly4G2 depletion occurs in every instance. Thus, Ly-49A(+) NK cells appear to play a role in the rejection H2(b) bone marrow allografts, but, in most strains of mice studied, Ly-49G2(+) NK cells must also be eliminated. The putative roles of these NK cell subsets in clinical transplantation remains to be elucidated. (Blood. 2000;95:3840-3844) (+info)
Peptide specificity of RT1-A1(c), an inhibitory rat major histocompatibility complex class I natural killer cell ligand.
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The rat major histocompatibility complex class Ia allelomorph RT1-A1(c) is a potent ligand for the recently identified inhibitory rLy-49 receptor, STOK-2. With the ultimate objective of studying the interactions of these molecules using structural and functional methods, we undertook a detailed study of its peptide specificity. The study revealed that designing an "ideal peptide" by choosing the most abundant residues in the "binding motif" obtained by pool sequencing does not necessarily yield an optimal binding peptide. For RT1-A1(c), as many as four positions, P2, P4, P5, and P9, were detected as putative anchors. Since this molecule displays a preference for highly hydrophobic peptides, we tested binding of peptides derived from the known leader peptide sequences of other rat histocompatibility complex class I molecules. One such peptide, found to bind well, requiring 1.6 microm peptide to achieve 50% stabilization, was searched for in vivo. Natural RT1-A1(c) binding peptides were purified from rat splenocytes and characterized by mass spectrometry using a combined matrix-assisted laser desorption ionization/time-of-flight and quadrupole time-of-flight approach. Results showed that the signal sequence-derived peptide was not detectable in the purified peptide pool, which was composed of a complex spectrum of peptides. Seven of these self-peptides were successfully sequenced. (+info)
Positive impact of inhibitory Ly49 receptor-MHC class I interaction on NK cell development.
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NK cells can kill MHC-different or MHC-deficient but not syngeneic MHC-expressing target cells. This MHC class I-specific tolerance is acquired during NK cell development. MHC recognition by murine NK cells largely depends on clonally distributed Ly49 family receptors, which inhibit NK cell function upon ligand engagement. We investigated whether these receptors play a role for the development of NK cells and provide evidence that the expression of a Ly49 receptor transgene on developing NK cells endowed these cells with a significant developmental advantage over NK cells lacking such a receptor, but only if the relevant MHC ligand was present in the environment. The data suggest that the transgenic Ly49 receptor accelerates and/or rescues the development of NK cells which would otherwise fail to acquire sufficient numbers of self-MHC-specific receptors. Interestingly, the positive effect on NK cell development is most prominent when the MHC ligand is simultaneously present on both hemopoietic and nonhemopoietic cells. These findings correlate with functional data showing that MHC class I ligand on all cells is required to generate functionally mature NK cells capable of reacting to cells lacking the respective MHC ligand. We conclude that the engagement of inhibitory MHC receptors during NK cell development provides signals that are important for further NK cell differentiation and/or maturation. (+info)
CD1d-specific NK1.1+ T cells with a transgenic variant TCR.
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The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Valpha14-Jalpha281 rearrangement and a limited set of TCR Vbeta segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Valpha3. 2/Vbeta9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-gamma, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the "non-Valpha 14" diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics. (+info)