Abstract:TCRs of invariant NKT (iNKT) cells bind α-galactosylceramide (αGC) loaded CD1d in a highly conserved fashion and show a characteristic TCR gene usage: An "invariant" α chain with a canonical AV14/AJ18 rearrangement in mice (AV24/AJ18 in humans) is paired with β chains containing characteristic Vβ segments. In the rat, a multimember AV14 gene family increases the variability within this system. This study characterizes CD1d binding of rat AV14 gene segments in TCR transductants as well as CD1d binding and iNKT … Show more
“…In some rearrangements glycine 93 or valine 92 were substituted by valine or alanine, respectively. This is analogous to substitutions that, in rat and mouse, respectively, were found to affect ligand binding . Furthermore, the cytokine production of splenocytes in response to typical iNKT cell antigens such as α GC and PBS57 indicates the existence of a typical iNKT cell population in this species.…”
Section: Discussionsupporting
confidence: 68%
“…a). The CDR1–3 region and the hypervariable region 4 (HV4) are based on published data and indicated in the alignment . The number of identical amino acids/nucleotides was divided by the total length of the alignment, so taking gaps into account, and the results were expressed as a percentage.…”
Section: Resultsmentioning
confidence: 99%
“…This is analogous to substitutions that, in rat and mouse, respectively, were found to affect ligand binding. 28,39 Furthermore, the cytokine production of splenocytes in response to typical iNKT cell antigens such as aGC and PBS57 indicates the existence of a typical iNKT cell population in this species. The levels of IFN-c were about 100-fold higher and for IL-4 twice as high as levels in F344 rats.…”
Section: Discussionmentioning
confidence: 98%
“…Fifty microlitres per well was used to coat wells of U‐bottom 96‐well suspension culture plates and plates were incubated at 4° overnight and afterwards washed three times with PBS. Then, 5 × 10 4 rat iNKT TCR‐expressing mouse T‐cell hybridoma cells, BW r/m CD28 EGN rAV14 S6 93A S65T CDR2+4 L14V, were added in RPMI‐1640 medium [Gibco, Grand Island, NY; supplemented with 10% FBS, 1 m m sodium pyruvate, 0·05 m m glutamine, 0·1 m m non‐essential amino acids, 5 m m β ‐mercaptoethanol, penicillin (100 U/ml), streptomycin (100 μg/ml)], and cultured for 22 hr at 37° in 5% CO 2 . Supernatants were analysed with a mouse interleukin‐2 (IL‐2) sandwich ELISA (BD OptEIA™ mouse IL‐2 ELISA Kit).…”
Section: Methodsmentioning
confidence: 99%
“…The CDR1-3 region and the hypervariable region 4 (HV4) are based on published data and indicated in the alignment. 28 The number of identical amino acids/nucleotides was divided by the total length of the alignment, so taking gaps into account, and the results were expressed as a percentage. Comparable to the sequence analysis of CD1d, the cotton rat AV14/AJ18 rearrangement shared an amino acid (nucleotide) homology of 80% (86%) with the predicted hamster rearrangement, 75% (83%) with rat, 74% (82%) with mouse and only 66% (75%) with the homologous human rearrangement (Fig.…”
Section: Canonical Av14/aj18 Rearrangements In the Cotton Ratmentioning
SummaryThe cotton rat (Sigmodon hispidus) belongs to the rodent family of Cricetidae and provides a powerful model to study the pathogenesis of human respiratory viruses and measles virus. Recent studies in other rodent models have suggested a role for invariant natural killer T (iNKT) cells in antiviral immunity and vaccination against respiratory virus infections. Using new experimental tools, we provide the first evidence for a functional CD1d cell molecule (crCD1d) and iNKT T-cell receptor in cotton rats. The crCD1d cDNA sequence was identified and crCD1d transductants showed that monoclonal antibody WTH-2 stains crCD1d as efficiently as mouse or rat CD1d. The expression of crCD1d was clearly weaker for thymocytes and B cells, and higher for T cells, which is different to what is found in murine species. The antigen-presenting capacity of crCD1d was demonstrated with crCD1d-immunoglobulin dimers loaded with the glycolipid PBS57, which bound iNKT T-cell receptors. Evidence for functional cotton rat iNKT cells was provided by detection of interferon-c and interleukin-4 in cultures of splenocytes stimulated with PBS57 and a-galactosylceramide and by specific staining of about 0Á2% of splenocytes with PBS57-loaded crCD1d dimers. Canonical AV14/ AJ18 rearrangements were identified and found to contain multiple members of the AV14 (AV11) family. One of them was expressed and found to bind CD1d dimers. In summary, these data provide the first evidence for functional CD1d molecules and iNKT T-cell receptors in cotton rats and provide the tools to analyse them both in the cotton rat model of infectious diseases.
“…In some rearrangements glycine 93 or valine 92 were substituted by valine or alanine, respectively. This is analogous to substitutions that, in rat and mouse, respectively, were found to affect ligand binding . Furthermore, the cytokine production of splenocytes in response to typical iNKT cell antigens such as α GC and PBS57 indicates the existence of a typical iNKT cell population in this species.…”
Section: Discussionsupporting
confidence: 68%
“…a). The CDR1–3 region and the hypervariable region 4 (HV4) are based on published data and indicated in the alignment . The number of identical amino acids/nucleotides was divided by the total length of the alignment, so taking gaps into account, and the results were expressed as a percentage.…”
Section: Resultsmentioning
confidence: 99%
“…This is analogous to substitutions that, in rat and mouse, respectively, were found to affect ligand binding. 28,39 Furthermore, the cytokine production of splenocytes in response to typical iNKT cell antigens such as aGC and PBS57 indicates the existence of a typical iNKT cell population in this species. The levels of IFN-c were about 100-fold higher and for IL-4 twice as high as levels in F344 rats.…”
Section: Discussionmentioning
confidence: 98%
“…Fifty microlitres per well was used to coat wells of U‐bottom 96‐well suspension culture plates and plates were incubated at 4° overnight and afterwards washed three times with PBS. Then, 5 × 10 4 rat iNKT TCR‐expressing mouse T‐cell hybridoma cells, BW r/m CD28 EGN rAV14 S6 93A S65T CDR2+4 L14V, were added in RPMI‐1640 medium [Gibco, Grand Island, NY; supplemented with 10% FBS, 1 m m sodium pyruvate, 0·05 m m glutamine, 0·1 m m non‐essential amino acids, 5 m m β ‐mercaptoethanol, penicillin (100 U/ml), streptomycin (100 μg/ml)], and cultured for 22 hr at 37° in 5% CO 2 . Supernatants were analysed with a mouse interleukin‐2 (IL‐2) sandwich ELISA (BD OptEIA™ mouse IL‐2 ELISA Kit).…”
Section: Methodsmentioning
confidence: 99%
“…The CDR1-3 region and the hypervariable region 4 (HV4) are based on published data and indicated in the alignment. 28 The number of identical amino acids/nucleotides was divided by the total length of the alignment, so taking gaps into account, and the results were expressed as a percentage. Comparable to the sequence analysis of CD1d, the cotton rat AV14/AJ18 rearrangement shared an amino acid (nucleotide) homology of 80% (86%) with the predicted hamster rearrangement, 75% (83%) with rat, 74% (82%) with mouse and only 66% (75%) with the homologous human rearrangement (Fig.…”
Section: Canonical Av14/aj18 Rearrangements In the Cotton Ratmentioning
SummaryThe cotton rat (Sigmodon hispidus) belongs to the rodent family of Cricetidae and provides a powerful model to study the pathogenesis of human respiratory viruses and measles virus. Recent studies in other rodent models have suggested a role for invariant natural killer T (iNKT) cells in antiviral immunity and vaccination against respiratory virus infections. Using new experimental tools, we provide the first evidence for a functional CD1d cell molecule (crCD1d) and iNKT T-cell receptor in cotton rats. The crCD1d cDNA sequence was identified and crCD1d transductants showed that monoclonal antibody WTH-2 stains crCD1d as efficiently as mouse or rat CD1d. The expression of crCD1d was clearly weaker for thymocytes and B cells, and higher for T cells, which is different to what is found in murine species. The antigen-presenting capacity of crCD1d was demonstrated with crCD1d-immunoglobulin dimers loaded with the glycolipid PBS57, which bound iNKT T-cell receptors. Evidence for functional cotton rat iNKT cells was provided by detection of interferon-c and interleukin-4 in cultures of splenocytes stimulated with PBS57 and a-galactosylceramide and by specific staining of about 0Á2% of splenocytes with PBS57-loaded crCD1d dimers. Canonical AV14/ AJ18 rearrangements were identified and found to contain multiple members of the AV14 (AV11) family. One of them was expressed and found to bind CD1d dimers. In summary, these data provide the first evidence for functional CD1d molecules and iNKT T-cell receptors in cotton rats and provide the tools to analyse them both in the cotton rat model of infectious diseases.
Nonclonal innate immune responses mediated by germ line–encoded receptors, such as Toll-like receptors or natural killer receptors, are commonly contrasted with diverse, clonotypic adaptive responses of lymphocyte antigen receptors generated by somatic recombination. However, the Variable (V) regions of antigen receptors include germ line–encoded motifs unaltered by somatic recombination, and theoretically available to mediate nonclonal, innate responses, that are independent of or largely override clonotypic responses. Recent evidence demonstrates that such responses exist, underpinning the associations of particular γδ T cell receptors (TCRs) with specific anatomical sites. Thus, TCRγδ can make innate and adaptive responses with distinct functional outcomes. Given that αβ T cells and B cells can also make nonclonal responses, we consider that innate responses of antigen receptor V-regions may be more widespread, for example, inducing states of preparedness from which adaptive clones are better selected. We likewise consider that potent, nonclonal T cell responses to microbial superantigens may reflect subversion of physiologic innate responses of TCRα/β chains.
CD1d molecules are MHC class I-like molecules that present glycolipids to iNKT cells. The highly conserved interaction between CD1d:α-Galactosylceramide (αGC) complexes and the iNKT TCR not only defines this population of αβ T cells but can also be used for its direct identification. Therefore, CD1d oligomers are a widely used tool for iNKT cell related investigations. To this end, the lipid chains of the antigen have to be inserted into the hydrophobic pockets of the CD1d binding cleft, often with help of surfactants. In this study, we investigated the influence of different surfactants (Triton X-100, Tween 20, Tyloxapol) on in vitro loading of CD1d molecules derived from four different species (human, mouse, rat and cotton rat) with αGC and derivatives carrying modifications of the acyl-chain (DB01-1, PBS44) and a 6-acetamido-6-deoxy-addition at the galactosyl head group (PBS57). We also compared rat CD1d dimers with tetramers and staining of an iNKT TCR transductant was used as readout for loading efficacy. The results underlined the importance of CD1d loading efficacy for proper analysis of iNKT TCR binding and demonstrated the necessity to adjust loading conditions for each oligomer/glycolipid combination. The efficient usage of surfactants as a tool for CD1d loading was revealed to be species-specific and depending on the origin of the CD1d producing cells. Additional variation of surfactant-dependent loading efficacy between tested glycolipids was influenced by the acyl-chain length and the modification of the galactosyl head group with PBS57 showing the least dependence on surfactants and the lowest degree of species-dependent differences.
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