Ramit Mehr

CD1d Endosomal Trafficking Is Independently Regulated by an Intrinsic CD1d-Encoded Tyrosine Motif and by the Invariant Chain

Endosomal trafficking is an essential component of the CD1 pathway of lipid antigen presentation to T cells. We demonstrate that CD1d access to endosomal compartments is under dual regulation by an intrinsic tyrosine-based motif, which governs intense recycling between the plasma membrane and the endosome, and by the invariant chain, with which CD1d associates in the endoplasmic reticulum. Both pathways independently enhance antigen presentation to V(alpha)14(+) NKT cells, the main subset of CD1d-restricted T cells. These results reveal the complexity of CD1d trafficking and suggest that the invariant chain was a component of ancestral antigen presentation pathways prior to the evolution of MHC and CD1.

Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules

The ability of murine NK cells to reject cells lacking self MHC class I expression results from an in vivo education process. To study the impact of individual MHC class I alleles on this process, we generated mice expressing single MHC class I alleles (Kb, Db, Dd, or Ld) or combinations of two or more alleles. All single MHC class I mice rejected MHC class I–deficient cells in an NK cell–dependent way. Expression of Kb or Dd conveyed strong rejection of MHC class I–deficient cells, whereas the expression of Db or Ld resulted in weaker responses. The educating impact of weak ligands (Db and Ld) was further attenuated by the introduction of additional MHC class I alleles, whereas strong ligands (Kb and Dd) maintained their educating impact under such conditions. An analysis of activating and inhibitory receptors in single MHC class I mice suggested that the educating impact of a given MHC class I molecule was controlled both by the number of NK cells affected and by the strength of each MHC class I–Ly49 receptor interaction, indicating that NK cell education may be regulated by a combination of qualitative and quantitative events.

Diversity, cellular origin and autoreactivity of antibody-secreting cell population expansions in acute systemic lupus erythematosus

Acute systemic lupus erythematosus (SLE) courses with surges of antibody-secreting cells (ASCs) whose origin, diversity and contribution to serum autoantibodies remain unknown. Here, deep sequencing, proteomic profiling of autoantibodies and single-cell analysis demonstrated highly diversified ASCs punctuated by clones expressing the variable heavy-chain region VH4-34 that produced dominant serum autoantibodies. A fraction of ASC clones contained autoantibodies without mutation, a finding consistent with differentiation outside the germinal centers. A substantial ASC segment was derived from a distinct subset of newly activated naive cells of considerable clonality that persisted in the circulation for several months. Thus, selection of SLE autoreactivities occurred during polyclonal activation, with prolonged recruitment of recently activated naive B cells. Our findings shed light on the pathogenesis of SLE, help explain the benefit of agents that target B cells and should facilitate the design of future therapies.

Age- and tissue-specific differences in human germinal center B cell selection revealed by analysis of IgVH gene hypermutation and lineage trees.

The elderly produce increased levels of antibodies to autologous antigens and are less able to make high‐affinity antibodies to foreign antigens. Ig gene hypermutation is integral to the affinity maturation process but previous studies of hypermutation with age have yielded conflicting results. The cells studied have represented post‐germinal center (GC) populations and, therefore, the results may be complicated by possible differences in activation history. We studied Ig genes from GC B cells to elucidate which factors in the affinity maturation process change with age. Age‐related changes in the pattern of hypermutation were seen, although the analysis of variable region heavy chain (VH) genes and their lineage trees shows that an alteration in the mechanism of somatic hypermutation is unlikely. The changes are due to founder cell effects and/or the process of selection. Striking tissue‐specific differences were seen. All measurements indicated that selectionof Ig genes may decrease in Peyers patch GC but increase in splenic GC with age. These tissue‐specific differences highlight the importance of considering the activation and effector sites when studying immune senescence.

IgTree © : Creating Immunoglobulin variable region gene lineage trees

Lineage trees describe the microevolution of cells within an organism. They have been useful in the study of B cell affinity maturation, which is based on somatic hypermutation of immunoglobulin genes in germinal centers and selection of the resulting mutants. Our aim was to create and implement an algorithm that can generate lineage trees from immunoglobulin variable region gene sequences. The IgTree program implements the algorithm we developed, and generates lineage trees. Original sequences found in experiments are assigned to either leaves or internal nodes of the tree. Each tree node represents a single mutation separating the sequences. The mutations that separate the sequences from each other can be point mutations, deletions or insertions. The program can deal with gaps and find potential reversion mutations. The program also enumerates mutation frequencies and sequence motifs around each mutation, on a per-tree basis. The algorithm has proven useful in several studies of immunoglobulin variable region gene mutations.

Diversification of memory B cells drives the continuous adaptation of secretory antibodies to gut microbiota

Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn.

Somatic hypermutation and antigen-driven selection of B cells are altered in autoimmune diseases

B cells have been found to play a critical role in the pathogenesis of several autoimmune (AI) diseases. A common feature amongst many AI diseases is the formation of ectopic germinal centers (GC) within the afflicted tissue or organ, in which activated B cells expand and undergo somatic hypermutation (SHM) and antigen-driven selection on their immunoglobulin variable region (IgV) genes. However, it is not yet clear whether these processes occurring in ectopic GCs are identical to those in normal GCs. The analysis of IgV mutations has aided in revealing many aspects concerning B cell expansion, mutation and selection in GC reactions. We have applied several mutation analysis methods, based on lineage tree construction, to a large set of data, containing IgV productive and non-productive heavy and light chain sequences from several different tissues, to examine three of the most profoundly studied AI diseases - Rheumatoid Arthritis (RA), Multiple Sclerosis (MS) and Sjögrens Syndrome (SS). We have found that RA and MS sequences exhibited normal mutation spectra and targeting motifs, but a stricter selection compared to normal controls, which was more apparent in RA. SS sequence analysis results deviated from normal controls in both mutation spectra and indications of selection, also showing differences between light and heavy chain IgV and between different tissues. The differences revealed between AI diseases and normal control mutation patterns may result from the different microenvironmental influences to which ectopic GCs are exposed, relative to those in normal secondary lymphoid tissues.

Evidence for large diversity in the human transcriptome created by Alu RNA editing

Adenosine-to-inosine (A-to-I) RNA editing alters the original genomic content of the human transcriptome and is essential for maintenance of normal life in mammals. A-to-I editing in Alu repeats is abundant in the human genome, with many thousands of expressed Alu sequences undergoing editing. Little is known so far about the contribution of Alu editing to transcriptome complexity. Transcripts derived from a single edited Alu sequence can be edited in multiple sites, and thus could theoretically generate a large number of different transcripts. Here we explored whether the combinatorial potential nature of edited Alu sequences is actually fulfilled in the human transcriptome. We analyzed datasets of editing sites and performed an analysis of a detailed transcript set of one edited Alu sequence. We found that editing appears at many more sites than detected by earlier genomic screens. To a large extent, editing of different sites within the same transcript is only weakly correlated. Thus, rather than finding a few versions of each transcript, a large number of edited variants arise, resulting in immense transcript diversity that eclipses alternative splicing as mechanism of transcriptome diversity, although with less impact on the proteome.

Models for antigen receptor gene rearrangement: CDR3 length

Despite the various processing steps involved in V(D)J recombination, which could potentially introduce many biases in the length distribution of complementarity determining region 3 (CDR3) segments, the observed CDR3 length distributions for complete repertoires are very close to a normal‐like distribution. This raises the question of whether this distribution is simply a result of the random steps included in the process of gene rearrangement, or has been optimized during evolution. We have addressed this issue by constructing a simulation of gene rearrangement, which takes into account the DNA modification steps included in the process, namely hairpin opening, nucleotide additions, and nucleotide deletions. We found that the near‐Gaussian‐ shape of CDR3 length distribution can only be obtained under a relatively narrow set of parameter values, and thus our model suggests that specific biases govern the rearrangement process. In both B‐cell receptor (BCR) heavy chain and T‐cell receptor β chain, we obtained a Gaussian distribution using identical parameters, despite the difference in the number and the lengths of the D segments. Hence our results suggest that these parameters most likely reflect the optimal conditions under which the rearrangement process occurs. We have subsequently used the insights gained in this study to estimate the probability of occurrence of two exactly identical BCRs over the course of a human lifetime. Whereas identical rearrangements of the heavy chain are highly unlikely to occur within one human lifetime, for the light chain we found that this probability is not negligible, and hence the light chain CDR3 alone cannot serve as an indicator of B‐cell clonality.

Re-utilization of germinal centers in multiple Peyer's patches results in highly synchronized, oligoclonal, and affinity-matured gut IgA responses

Whereas gut IgA responses to the microbiota may be multi-centered and diverse, little is known about IgA responses to T-cell-dependent antigens following oral immunizations. Using a novel approach, gut IgA responses to oral hapten (4-hydroxy-3-nitrophenyl)acetyl-cholera toxin (NP-CT) conjugates were followed at the cellular and molecular level. Surprisingly, these responses were highly synchronized, strongly oligoclonal, and dominated by affinity matured cells. Extensive lineage trees revealed clonal relationships between NP-specific IgA cells in gut inductive and effector sites, suggesting expansion of the same B-cell clone in multiple Peyers patches (PPs). Adoptive transfer experiments showed that this was achieved through re-utilization of already existing germinal centers (GCs) in multiple PPs by previously activated GC GL7+ B cells, provided oral NP-CT was given before cell transfer. Taken together, these results explain why repeated oral immunizations are mandatory for an effective oral vaccine.