Emergence in Cx knockout mice of a diverse cytotoxic T lymphocyte repertoire that recognizes a single peptide from the immunoglobulin constant x light chain region

Allotype- or idiotype-specific CD4⁺ T cells have been reported to recognize immunoglobulin (Ig) peptides presented by class II molecules. In contrast, few data are available concerning the generation of Ig peptide-specific CD8⁺ T cells. We have therefore investigated whether T-depleted spleen cells from Ig x light chain-expressing 129/Sv mice (129x^+/+) could induce, in Cx knockout mice (129 x^?/?), the generation of Ig constant x light chain region (Cx)-specific cytotoxic T lymphocytes (CTL). The determination of TCRβ chain expressed by nine CTL clones, together with the use of a library of overlapping peptides spanning the whole Cx sequence, show that the B cells from x^+/+ mice are able to elicit in Cx knockout mice, the emergence of a diverse CTL repertoire that recognizes one single Cx peptide presented by the H-2K^b class I molecule. In addition, these data support the notion that B cells are able to process and present on their class I molecules, peptides generated from their own x light chains.     

Enrichment of the antibodies against the C-terminus of Taiwan cobra cobrotoxin using dimeric glutaraldehyde-modified toxin as an immunogen.

The repertoire of antibodies producing by immunizing rabbits with cobrotoxin and dimeric glutaraldehyde-modified cobrotoxin (dGA-cobrotoxin) was analyzed by studying the immunoreactivity of the two antibody preparations toward cobrotoxin, GA-cobrotoxin and recombinant cobrotoxin. The results of enzyme-linked immunoassay revealed that the two antibody preparations exhibited a higher reactivity against their cognate antigen. Moreover, different behavior was observed for the reactivity of the two antibody preparations against GA-cobrotoxin and recombinant cobrotoxin. Notably, distortion of disulfide linkages at the C-terminus resulted in a reduced decrease in the antigenic activity of recombinant cobrotoxin toward anti-cobrotoxin antibodies compared to anti-dGA-cobrotoxin antibodies. Affinity purification of the antibodies against the C-terminus of cobrotoxin revealed that its amount represented 77% and 35.5% of the total anti-dGA-cobrotoxin antibodies and the total anti-cobrotoxin antibodies, respectively. These findings suggest that the antibody preparation elicited by dGA-cobrotoxin enriches the content of antibodies recognizes the C-terminal region of native cobrotoxin.     

Skewing of the B cell receptor repertoire in myalgic encephalomyelitis/chronic fatigue syndrome

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition characterized by fatigue and post-exertional malaise, accompanied by various signs of neurological and autonomic dysfunction. ME/CFS is often triggered by an infectious episode and associated with an aberrant immune system. Here we report that ME/CFS is a disorder characterized by skewed B cell receptor gene usage. By applying a next-generation sequencing to determine the clone-based IGHV/IGHD/IGHJ repertoires, we revealed a biased usage of several IGHV genes in peripheral blood B cells from ME/CFS patients. Results of receiver operating characteristic (ROC) analysis further indicated a possibility of distinguishing patients from healthy controls, based on the skewed B cell repertoire. Meanwhile, B cell clones using IGHV3-30 and IGHV3-30-3 genes were more frequent in patients with an obvious infection-related episode at onset, and correlated to expression levels of interferon response genes in plasmablasts. Collectively, these results imply that B cell responses in ME/CFS are directed against an infectious agents or priming antigens induced before disease onset.     

Dual reporter system to dissect cis- and trans-effects influencing the mutation rate in a hypermutating cell line

Activation induced cytidine deaminase (AID) plays a key role in the induction of somatic hypermutation and class switching in the immunoglobulin genes of B-lymphocytes. AID expression by itself is sufficient to induce a GC-basepair biased mutator phenotype in lymphoid and non-lymphoid cell lines. Nevertheless a network of cis-regulatory elements and additional trans-factor proteins seems to govern the molecular mechanism of somatic hypermutation. To address the nature of mutation rate changes observed in the hypermutating pre-B cell line 18–81, we extended our previously described green fluorescent protein (GFP) reversion-system. Introducing an additional mutation reporter transgene enables us to discriminate between cis- and trans-factor caused alterations in the mutator phenotype. We show here that in cell line 18–81 the mutation rate declines upon prolonged periods of cell culture. The gradual loss of the mutator phenotype in cell line 18–81 is due to the downregulation of endogenous AID expression and can be reconstituted by overexpression of human AID protein. A correlation between AID mRNA levels and mutation rates is evident and even small changes in AID expression levels cause a significant effect on the mutability of the reporter transgenes.     

The little brown bat, M. lucifugus, displays a highly diverse VH, DH and JH repertoire but little evidence of somatic hypermutation

Myotis lucifugus populations in Northeastern US are being decimated by a fungal disease. Since almost nothing is known about the immune system of bats, we are characterizing the immunoglobulin genes of bats. We show that M. lucifugus has a diverse V_H gene repertoire comprised of five of the seven human V_H gene families and an estimated 236 V_H3 genes. 95% of these germline VH3 genes differ in FR3. A comparison of 67 expressed V_H3 genes with 75 germline V_H3 genes revealed a mutation frequency similar to fetal piglets never exposed to environmental antigens. Analysis of CDR3 regions identified at least 13 putative J_H segments and a large D_H repertoire. The low mutation frequency, highly diverse V_H, D_H, and J_H germline repertoire suggests that this species may rely more on combinatorial and junctional diversity than on somatic hypermutation raising questions about the ability of M. lucifugus to respond rapidly to emerging pathogens.     

Memory precursor phenotype of CD8+ T cells reflects early antigenic experience rather than memory numbers in a model of localized acute influenza infection

The mechanistic basis of memory T‐cell development is poorly defined. Phenotypic markers that define precursors at effector stages have been characterized for acute systemic infections with high antigen load. We asked whether such markers can identify memory precursors from early effectors (d6) to late memory (>d500) for two immunodominant CD8⁺ responses during the course of a localized low‐load influenza infection in mice. CD8⁺ T cells stained with the D^bNP₃₆₆ and D^bPA₂₂₄ tetramers were characterized as IL‐7Rα^hi, IL‐7Rα^hiCD62L^hi or IL‐7Rα^hiKLRG1^lo. While the D^bNP₃₆₆‐ and D^bPA₂₂₄‐specific responses were comparable in size, decay kinetics and memory precursor frequency, their expansion characteristics differed. This correlated with a divergence in the IL‐7Rα^hi, IL‐7Rα^hiCD62L^hi and IL‐7Rα^hiKLRG1^lo phenotypes on effector, but not naïve, CD8⁺ populations. That effect was abrogated by priming with viruses engineered to present equivalent levels of NP₃₆₆ and PA₂₂₄ peptides, indicating that memory phenotypes reflect early antigenic experience rather than memory potential. Thus, the IL‐7Rα^hiKLRG1^lo phenotype had a poor predictive value in identifying memory precursors in the spleen and at the site of infection. Greater consistency in influenza‐specific IL‐7Rα^hiKLRG1^loCD8⁺ T‐cell numbers was found in draining lymph nodes, suggesting that this may be the preferential site for memory establishment and maintenance following localized virus infections.     

Adult BM generates CD5+ B1 cells containing abundant N‐region additions

The self‐renewing capacity of B1 cells infers homeostatic regulation; however, previous work suggests the low level of N‐region addition characterizing B1 cells early in life increases with age, which implies that the B1‐cell population is not a closed system. To explore this, we evaluated N‐region addition in CD5⁺ B1 cells generated from adult BM. Adult BM cells were marked with GFP introduced by mouse stem cell virus transduction, and were then adoptively transferred into lethally irradiated recipients. Within 2–3 months, we found GFP‐marked CD5⁺ B cells in the peritoneal cavities of recipients, which we demonstrate here meet a variety of criteria for B1‐cell traits including Mac‐1 surface expression; annexin, elfin, and Pax‐5 gene expression; mitogenic responsiveness to phorbol ester; and spontaneous immunoglobulin secretion. Notably, we found by single‐cell PCR that this population of BM‐derived CD5⁺ B1 cells expressed immunoglobulin with abundant N‐region addition (and little V_H11/V_H12 skewing), unlike CD5⁺ B1 cells obtained from unmanipulated animals but reminiscent of B2 cells. Further, we confirmed that native CD5⁺ B1 cells from older mice contain more N‐region additions than native CD5⁺ B1 cells from younger mice. These results suggest that adult BM progenitors contribute to the peritoneal CD5⁺ B1‐cell pool over time.     

Maturational stage-dependent thymocyte responses to TCR engagement

Thymocyte positive and negative selection are dependent on avidity-driven TCR-mediated recognition events in the thymus. High-avidity recognition events result in negative selection, while low-avidity recognition events result in positive selection. However, it has not been established how thymocytes maturation stages affect their responses to TCR signals of different avidities. We gained insight into this question when we reduced thymocyte selection to an in vitro system, in which full maturation of developmentally synchronized immature double-positive thymocytes was induced on a cloned line of thymic epithelial cells. Our analysis of the kinetics of thymocyte development supports a multi-phasic model of thymic selection. In it, thymocyte maturation stages as well as interaction avidity control the outcome TCR stimulation. Positive selection is initiated during a primary recognition event that proceeds independently of the TCR avidity. During a secondary recognition event the final fate of thymocyte, full maturation versus negative selection, is determined by TCR avidity.