Pre-T Cell Receptors (Pre-TCRs) Leverage Vß Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands.

The pre-T cell receptor (pre-TCR) is a pTα-ß heterodimer functioning in early αß T cell development. Although once thought to be ligand-autonomous, recent studies show that pre-TCRs participate in thymic repertoire formation through recognition of peptides bound to major histocompatibility molecules (pMHC). Using optical tweezers, we probe pre-TCR bonding with pMHC at the single molecule level. Like the αßTCR, the pre-TCR is a mechanosensor undergoing force-based structural transitions that dynamically enhance bond lifetimes and exploiting allosteric control regulated via the Cß FG loop region. The pre-TCR structural transitions exhibit greater reversibility than TCRαß and ordered force-bond lifetime curves. Higher piconewton force requires binding through both complementarity determining region loops and hydrophobic Vß patch apposition. This patch functions in the pre-TCR as a surrogate Vα domain, fostering ligand promiscuity to favor development of ß chains with self-reactivity but is occluded by α subunit replacement of pTα upon αßTCR formation. At the double negative 3 thymocyte stage where the pre-TCR is first expressed, pre-TCR interaction with self-pMHC ligands imparts growth and survival advantages as revealed in thymic stromal cultures, imprinting fundamental self-reactivity in the T cell repertoire. Collectively, our data imply the existence of sequential mechanosensor αßTCR repertoire tuning via the pre-TCR.

Unraveling graft-versus-host disease and graft-versus-leukemia responses using TCR Vß spectratype analysis in a murine bone marrow transplantation model.

The optimum use of allogeneic blood and marrow transplantation (BMT) as a curative therapy for hematological malignancies lies in the successful separation of mature donor T cells that are host reactive and induce graft-versus-host disease (GVHD) from those that are tumor reactive and mediate graft-versus-leukemia (GVL) effects. To study whether this separation was possible in an MHC-matched murine BMT model (B10.BR→CBA) with a CBA-derived myeloid leukemia line, MMC6, we used TCR Vß CDR3-size spectratype analysis to first show that the Vß13 family was highly skewed in the B10.BR anti-MMC6 CD8(+) T cell response but not in the alloresponse against recipient cells alone. Transplantation of CD8(+)Vß13(+) T cells at the dose equivalent of their constituency in 1 × 10(7) CD8(+) T cells, a dose that had been shown to mediate lethal GVHD in recipient mice, induced a slight GVL response with no concomitant GVHD. Increasing doses of CD8(+)Vß13(+) T cells led to more significant GVL responses but also increased GVHD symptoms and associated mortality. Subsequent spectratype analysis of GVHD target tissues revealed involvement of gut-infiltrating CD8(+)Vß13(+) T cells accounting for the observed in vivo effects. When BMT recipients were given MMC6-presensitized CD8(+)Vß13(+) T cells, they displayed a significant GVL response with minimal GVHD. Spectratype analysis of tumor-presensitized, gut-infiltrating CD8(+)Vß13(+) T cells showed preferential usage of tumor-reactive CDR3-size lengths, and these cells expressed increased effector memory phenotype (CD44(+)CD62L(-/lo)). Thus, Vß spectratyping can identify T cells involved in antihost and antitumor reactivity and tumor presensitization can aid in the separation of GVHD and GVL responses.

Antibody humanization by redesign of complementarity-determining region residues proximate to the acceptor framework.

Antibodies are key components of the adaptive immune system and are well-established protein therapeutic agents. Typically high-affinity antibodies are obtained by immunization of rodent species that need to be humanized to reduce their immunogenicity. The complementarity-determining regions (CDRs) contain the residues in a defined loop structure that confer antigen binding, which must be retained in the humanized antibody. To design a humanized antibody, we graft the mature murine CDRs onto a germline human acceptor framework. Structural defects due to mismatches at the graft interface can be fixed by mutating some framework residues to murine, or by mutating some residues on the CDRs' backside to human or to a de novo designed sequence. The first approach, framework redesign, can yield an antibody with binding better than the CDR graft and one equivalent to the mature murine, and reduced immunogenicity. The second approach, CDR redesign, is presented here as a new approach, yielding an antibody with binding better than the CDR graft, and immunogenicity potentially less than that from framework redesign. Application of both approaches to the humanization of anti-α4 integrin antibody HP1/2 is presented and the concept of the hybrid humanization approach that retains "difficult to match" murine framework amino acids and uses de novo CDR design to minimize murine amino acid content and reduce cell-mediated cytotoxicity liabilities is discussed.

Shaping of human germline IgH repertoires revealed by deep sequencing.

To understand better how selection processes balance the benefits of Ig repertoire diversity with the risks of autoreactivity and nonfunctionality of highly variable IgH CDR3s, we collected millions of rearranged germline IgH CDR3 sequences by deep sequencing of DNA from mature human naive B cells purified from four individuals and analyzed the data with computational methods. Long HCDR3 regions, often components of HIV-neutralizing Abs, appear to derive not only from incorporation of long D genes and insertion of large N regions but also by usage of multiple D gene segments in tandem. However, comparison of productive and out-of-frame IgH rearrangements revealed a selection bias against long HCDR3 loops, suggesting these may be disproportionately either poorly functional or autoreactive. Our data suggest that developmental selection removes HCDR3 loops containing patches of hydrophobicity, which are commonly found in some auto-antibodies, and at least 69% of the initial productive IgH rearrangements are removed from the repertoire during B cell development. Additionally, we have demonstrated the potential utility of this new technology for vaccine development with the identification in all four individuals of related candidate germline IgH precursors of the HIV-neutralizing Ab 4E10.

Limiting CDR-H3 diversity abrogates the antibody response to the bacterial polysaccharide α 1→3 dextran.

Anti-polysaccharide Ab responses in mice are often oligoclonal, and the mechanisms involved in Ag-specific clone production and selection remain poorly understood. We evaluated the relative contribution of D(H) germline content versus N nucleotide addition in a classic oligoclonal, T-independent Ab response (α 1→3 dextran [DEX]) by challenging adult TdT-sufficient (TdT(+/+)) and TdT-deficient (TdT(-/-)) gene-targeted mice, limited to the use of a single D(H) gene segment (D-limited mice), with Enterobacter cloacae. D-limited mice achieved anti-DEX-specific levels of Abs that were broadly comparable to those of wild-type (WT) BALB/c mice. Sequence analysis of the third CDR of the H chain intervals obtained by PCR amplification of V(H) domain DNA from DEX-specific plasmablasts revealed the near universal presence of an aspartic acid residue (D99) at the V-D junction, irrespective of the composition of the D(H) locus. Although WT mice were able to use germline D(H) (DQ52, DSP, or DST) gene segment sequence, TdT activity, or both to produce D99, all three D-limited mouse strains relied exclusively on N addition. Additionally, in the absence of TdT, D-limited mice failed to produce a DEX response. Coupled with previous studies demonstrating a reduced response to DEX in TdT(-/-) mice with a WT D(H) locus, we concluded that in the case of the anti-DEX repertoire, which uses a short third CDR of the H chain, the anti-DEX response relies more intensely on sequences created by postnatal N nucleotide addition than on the germline sequence of the D(H).

TCR bias of in vivo expanded T cells in pancreatic islets and spleen at the onset in human type 1 diabetes.

Autoreactive T cells, responsible for the destruction of pancreatic ß cells in type 1 diabetes, are known to have a skewed TCR repertoire in the NOD mouse. To define the autoreactive T cell repertoire in human diabetes, we searched for intraislet monoclonal expansions from a recent onset in human pancreas to then trace them down to the patient's peripheral blood and spleen. Islet infiltration was diverse, but five monoclonal TCR ß-chain variable expansions were detected for Vß1, Vß7, Vß11, Vß17, and Vß22 families. To identify any sequence bias in the TCRs from intrapancreatic T cells, we analyzed 139 different CDR3 sequences. We observed amino acid preferences in the NDN region that suggested a skewed TCR repertoire within infiltrating T cells. The monoclonal expanded TCR sequences contained amino acid combinations that fit the observed bias. Using these CDR3 sequences as a marker, we traced some of these expansions in the spleen. There, we identified a Vß22 monoclonal expansion with identical CDR3 sequence to that found in the islets within a polyclonal TCR ß-chain variable repertoire. The same Vß22 TCR was detected in the patient's PBMCs, making a cross talk between the pancreas and spleen that was reflected in peripheral blood evident. No other pancreatic monoclonal expansions were found in peripheral blood or the spleen, suggesting that the Vß22 clone may have expanded or accumulated in situ by an autoantigen present in both the spleen and pancreas. Thus, the patient's spleen might be contributing to disease perpetuation by expanding or retaining some autoreactive T cells.

Clonal expansions and loss of receptor diversity in the naive CD8 T cell repertoire of aged mice.

There are well-characterized age-related changes in the peripheral repertoire of CD8 T cells characterized by reductions in the ratio of naive:memory T cells and the development of large clonal expansions in the memory pool. In addition, the TCR repertoire of naive T cells is reduced with aging. Because a diverse repertoire of naive T cells is essential for a vigorous response to new infections and vaccinations, there is much interest in understanding the mechanisms responsible for declining repertoire diversity. It has been proposed that one reason for declining repertoire diversity in the naive T cell pool is an increasing dependence on homeostatic proliferation in the absence of new thymic emigrants for maintenance of the naive peripheral pool. In this study, we have analyzed the naive CD8 T cell repertoire in young and aged mice by DNA spectratype and sequence analysis. Our data show that naive T cells from aged mice have perturbed spectratype profiles compared with the normally Gaussian spectratype profiles characteristic of naive CD8 T cells from young mice. In addition, DNA sequence analysis formally demonstrated a loss of diversity associated with skewed spectratype profiles. Unexpectedly, we found multiple repeats of the same sequence in naive T cells from aged but not young mice, consistent with clonal expansions previously described only in the memory T cell pool. Clonal expansions among naive T cells suggests dysregulation in the normal homeostatic proliferative mechanisms that operate in young mice to maintain diversity in the naive T cell repertoire.

The heavy chain variable segment gene repertoire in chronic Chagas' heart disease.

Patients chronically infected with Trypanosoma cruzi develop chronic Chagas' heart disease (cChHD). Their Ab response is suspected to be involved in the cardiac pathogenesis. Reactivity of serum Abs from these patients has been extensively studied but little is known about the diversity of the in vivo IgG repertoire. We analyzed 125 variable H chain (VH) genes and compared it to repertoires from healthy individuals, and patients with autoimmune processes and other infections. VH were from plasma cells isolated from heart tissue of three cChHD patients and from a Fab combinatorial library derived from bone marrow of another cChHD patient. The role of the parasite in shaping the Ab repertoire was assessed analyzing VH genes before and after panning against T. cruzi Ag. Among recovered VH genes, a significantly increased representation of VH4 was observed. Plasma cells at the site of cardiac infiltration showed an increased VH1 usage. CDR3 lengths were similar to the ones found in the healthy repertoire and significantly shorter than in other infections. VH derived from anti-T. cruzi Fab and plasma cells showed a higher proportion of hypermutated genes, 46.9% and 43.75%, respectively, vs 30.9% of the cChHD patient repertoire, pointing to the role of parasite Ags in the shaping of the humoral response in Chagas' disease. No histological evidence of germinal center-like structures was observed in heart tissue. In accordance, VH analysis of heart plasmocytes revealed no evidence of clonal B cell expansion, suggesting that they migrated into heart tissue from secondary lymphoid organs.

Two distinct populations of H chain-edited B cells show differential surrogate L chain dependence.

Developing autoreactive B cells may edit (change) their specificity by secondary H or L chain gene rearrangement. Recently, using mice hemizygous for a site-directed VDJH and VJkappa transgene (tg) encoding an autoreactive Ab, we reported ongoing L chain editing not only in bone marrow cells with a pre-B/immature B cell phenotype but also in immature/transitional splenic B cells. Using the same transgenic model, we report here that editing at the H chain locus appears to occur exclusively in bone marrow cells with a pro-B phenotype. H chain editing is shown to involve VH replacement at the tg allele or VH rearrangement at the wild-type (wt) allele when the tg is inactivated by nonproductive VH replacement. VH replacement/rearrangement at the tg/wt alleles was found to entail diverse usage of VH genes. Whereas the development of edited B cells expressing the wt allele was dependent on the lambda5 component of the surrogate L chain, the development of B cells expressing the tg allele, including those with VH replacement, appeared to be lambda5 independent. We suggest that the unique CDR3 region of the tg-encoded muH chain is responsible for the lambda5 independence of tg-expressing B cells.

Overlap between in vitro donor antihost and in vivo posttransplantation TCR Vbeta use: a new paradigm for designer allogeneic blood and marrow transplantation.

Following allogeneic blood and marrow transplantation (BMT), mature donor T cells can enhance engraftment, counteract opportunistic infections, and mount graft-versus-tumor (GVT) responses, but at the risk of developing graft-versus-host disease (GVHD). With the aim of separating the beneficial effects of donor T cells from GVHD, one approach would be to selectively deplete subsets of alloreactive T cells in the hematopoietic cell inoculum. In this regard, TCR Vbeta repertoire analysis by CDR3-size spectratyping can be a powerful tool for the characterization of alloreactive T-cell responses. We investigated the potential of this spectratype approach by comparing the donor T-cell alloresponses generated in vitro against patient peripheral blood lymphocytes (PBLs) with those detected in vivo posttransplantation. The results indicated that for most Vbeta families that exhibited alloreactive CDR3-size skewing, there was a robust overlap between the in vitro antipatient and in vivo spectratype histograms. Thus, in vitro spectratype analysis may be useful for determining the alloreactive T-cell response involved in GVHD development and, thereby, could serve to guide select Vbeta family depletion for designer transplants to improve outcomes.

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting.

Humanization of nonhuman antibodies (Abs) has been carried out mainly for Abs which bind to antigen without catalytic activity. Here we report humanization of mouse-originated 3D8 (m3D8) mAbs (scFv, VH, and VL) with DNA hydrolyzing catalytic activity by grafting the complementarity determining regions (CDRs) into the corresponding regions of a fixed human framework scaffold, generating humanized 3D8 (h3D8) Abs in the respective format of scFv, VH, and VL. h3D8 Abs retained comparable DNA binding and hydrolyzing activities to those of the corresponding m3D8 Abs. Our results suggest that CDRs of anti-DNA hydrolyzing Abs might possess the intrinsic properties of DNA binding and hydrolyzing activities.

ALTHEA Gold Libraries™: antibody libraries for therapeutic antibody discovery.

We describe here the design, construction and validation of ALTHEA Gold Libraries™. These single-chain variable fragment (scFv), semisynthetic libraries are built on synthetic human well-known IGHV and IGKV germline genes combined with natural human complementarity-determining region (CDR)-H3/JH (H3J) fragments. One IGHV gene provided a universal VH scaffold and was paired with two IGKV scaffolds to furnish different topographies for binding distinct epitopes. The scaffolds were diversified at positions identified as in contact with antigens in the known antigen-antibody complex structures. The diversification regime consisted of high-usage amino acids found at those positions in human antibody sequences. Functionality, stability and diversity of the libraries were improved throughout a three-step construction process. In a first step, fully synthetic primary libraries were generated by combining the diversified scaffolds with a set of synthetic neutral H3J germline gene fragments. The second step consisted of selecting the primary libraries for enhanced thermostability based on the natural capacity of Protein A to bind the universal VH scaffold. In the third and final step, the resultant stable synthetic antibody fragments were combined with natural H3J fragments obtained from peripheral blood mononuclear cells of a large pool of 200 donors. Validation of ALTHEA Gold Libraries™ with seven targets yielded specific antibodies in all the cases. Further characterization of the isolated antibodies indicated KD values as human IgG1 molecules in the single-digit and sub-nM range. The thermal stability (Tm) of all the antigen-binding fragments was 75°C-80°C, demonstrating that ALTHEA Gold Libraries™ are a valuable source of specific, high affinity and highly stable antibodies.

Unmutated and mutated chronic lymphocytic leukemias derive from self-reactive B cell precursors despite expressing different antibody reactivity.

B cell chronic lymphocytic leukemia (CLL) is a disease of expanding monoclonal B cells whose B cell receptor (BCR) mutational status defines 2 subgroups; patients with mutated BCRs have a more favorable prognosis than those with unmutated BCRs. CLL B cells express a restricted BCR repertoire including antibodies with quasi-identical complementarity-determining region 3 (CDR3), which suggests specific antigen recognition. The antigens recognized by CLL antibodies may include autoantigens since about half of CLL B cells produce autoreactive antibodies. However, the distribution of autoreactive antibodies between Ig heavy-chain variable-unmutated (IgV-unmutated) CLL (UM-CLL) and IgV-mutated CLL (M-CLL) is unknown. To determine the role of antibody reactivity and the impact of somatic hypermutation (SHM) on CLL antibody specificity, we cloned and expressed in vitro recombinant antibodies from M- and UM-CLL B cells and tested their reactivity by ELISA. We found that UM-CLL B cells expressed highly polyreactive antibodies whereas most M-CLL B cells did not. When mutated nonautoreactive CLL antibody sequences were reverted in vitro to their germline counterparts, they encoded polyreactive and autoreactive antibodies. We concluded that both UM-CLLs and M-CLLs originate from self-reactive B cell precursors and that SHM plays an important role in the development of the disease by altering original BCR autoreactivity.

Statistical analysis of CDR3 length distributions for the assessment of T and B cell repertoire biases.

Complementarity-determining region 3 (CDR3) length distribution analysis explores the diversity of the T cell receptor (TCR) and immunoglobulin (Ig) repertoire at the transcriptome level. Studies of the CDR3, the most hypervariable part of these molecules, have been frequently used to identify recruitment of T and B cell clones involved in immunological responses. CDR3 length distribution analysis gives a clear perception of repertoire variations between individuals and over time. However, the complexity of CDR3 length distribution patterns and the high number of possible repertoire alterations per individual called for the development of robust data analysis methods. The goal of these methods is to identify, quantify and statistically assess differences between repertoires so as to offer a better diagnostic or predictive tool for pathologies involving the immune system. In this review we will explain the benefit of analyzing CDR3 length distribution for the study of immune cell diversity. We will start by describing this technology and its associated data processing, and will subsequently focus on the statistical methods used to compare CDR3 length distribution patterns. Finally, we will address the various methods for assessing CDR3 length distribution gene signatures in pathological states.

Single amino acid substitution in LC-CDR1 induces Russell body phenotype that attenuates cellular protein synthesis through eIF2α phosphorylation and thereby downregulates IgG secretion despite operational secretory pathway traffic.

Amino acid sequence differences in the variable region of immunoglobulin (Ig) cause wide variations in secretion outputs. To address how a primary sequence difference comes to modulate Ig secretion, we investigated the biosynthetic process of 2 human IgG2κ monoclonal antibodies (mAbs) that differ only by one amino acid in the light chain complementarity-determining region 1 while showing ∼20-fold variance in secretion titer. Although poorly secreted, the lower-secreting mAb of the 2 was by no means defective in terms of its folding stability, antigen binding, and in vitro biologic activity. However, upon overexpression in HEK293 cells, the low-secreting mAb revealed a high propensity to aggregate into enlarged globular structures called Russell bodies (RBs) in the endoplasmic reticulum. While Golgi morphology was affected by the formation of RBs, secretory pathway membrane traffic remained operational in those cells. Importantly, cellular protein synthesis was severely suppressed in RB-positive cells through the phosphorylation of eIF2α. PERK-dependent signaling was implicated in this event, given the upregulation and nuclear accumulation of downstream effectors such as ATF4 and CHOP. These findings illustrated that the underlining process of poor Ig secretion in RB-positive cells was due to downregulation of Ig synthesis instead of a disruption or blockade of secretory pathway trafficking. Therefore, RB formation signifies an end of active Ig production at the protein translation level. Consequently, depending on how soon and how severely an antibody-expressing cell develops the RB phenotype, the productive window of Ig secretion can vary widely among the cells expressing different mAbs.

[Immunoglobulin expression in colon cancer cell line HT-29 and its biological activities].

OBJECTIVE: To determine the expression of immunoglobulins in HT-29 cells (an established colon cancer cell line, and explore their effect on the biological activities of the cancer cells.) METHODS: The transcripts of variable regions of immunoglobulin heavy chains in HT-29 cells were detected by RT-PCR. Antisense CDR3 (specific to HT-29)-pIRES 1 neo vector was constructed, then transfected into HT-29 cells by electroporation. Programmed cell death and growth inhibition of HT-29 cells were detected by FCM and MTT, respectively. RESULTS: The transcripts of Ig heavy chain (V(H) CDR3 region) were expressed in HT-29 cells. Moreover, they showed a monoclonal characteristic after being sequenced. After transfection of the antisense vector of CDR3 (specific to HT-29)-pIRES 1 neo, expression level of Ig in HT-29 cells was significantly decreased, and growth inhibition (P < 0.05) and apoptosis (P < 0.01) were induced. CONCLUSION: These results suggest that tumor derived Ig could promote the survival and growth of tumor cells.

Adult lupus-prone MRL/MpJ2+ mice express a primary antibody repertoire that differs in CDR-H3 length distribution and hydrophobicity from that expressed in the C3H parental strain.

Anti-dsDNA antibodies tend to be enriched for heavy chain complementarity determining region 3 (CDR-H3) intervals of above average length that contain an increased frequency of charged amino acids. It is unclear whether these types of CDR-H3s are more common in the primary B-cell repertoire of auto-immune prone strains or whether their increased prevalence in affected individuals reflects positive selection and expansion of atypical CDR-H3s in the pathogenic response to self-antigen. Here, we present evidence that when compared to C3H, a MRL/MpJ(2+) parental strain, CDR-H3 intervals from pre-B cells of adult lupus-prone MRL/MpJ(2+) mice are longer on average and are enriched for charged amino acids. The predicted prevalence of deformed loops per Shirai H3 criteria is also higher. In contrast, the frequency of charge, the distribution of length, and the pattern of predicted deformed loop structures did not differ in sequences obtained from neonates of the same two strains. These observations suggest that the mechanisms that serve to shape the initial CDR-H3 repertoire in adults, but not neonates, are being regulated differently in C3H versus MRL/MpJ(2+). Dysregulation of the adult pre-B CDR-H3 antibody repertoire could be a contributing factor for the development of florid auto-immune disease in MRL/MpJ(2+) mice.

Functional inhibition of ß-catenin-mediated Wnt signaling by intracellular VHH antibodies.

The Wnt signaling pathway is of central importance in embryogenesis, development and adult tissue homeostasis, and dysregulation of this pathway is associated with cancer and other diseases. Despite the developmental and potential therapeutic significance of this pathway, many aspects of Wnt signaling, including the control of the master transcriptional co-activator ß-catenin, remain poorly understood. In order to explore this aspect, a diverse immune llama VHH phagemid library was constructed and panned against ß-catenin. VHH antibody fragments from the library were expressed intracellularly, and a number of antibodies were shown to possess function-modifying intracellular activity in a luciferase-based Wnt signaling HEK293 reporter bioassay. Further characterization of one such VHH (named LL3) confirmed that it bound endogenous ß-catenin, and that it inhibited the Wnt signaling pathway downstream of the destruction complex, while production of a control Ala-substituted complementarity-determining region (CDR)3 mutant demonstrated that the inhibition of ß-catenin activity by the parent intracellular antibody was dependent on the specific CDR sequence of the antibody.

Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application.

Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.

A hierarchy of T cell receptor motifs determines responsiveness to the immunodominant epitope in experimental autoimmune myasthenia gravis.

The predominant murine T lymphocyte population responding to Talpha146-162, the immunodominant epitope in EAMG, expresses the TCRBV 6 gene segment. However, cells expressing other TCRBV gene segments also react with this peptide. In order to more precisely characterize the Talpha146-162-specific TCR repertoire, we isolated CD4high cells from peptide-immunized mice. The majority of CD4high cells utilized an acidic TCR beta chain CDR3 motif regardless of TCRBV gene usage. Analysis of T cell clones demonstrated a fourfold higher avidity of Vbeta6+ than non-Vbeta6 cells for Talpha146-162 indicating that a hierarchy of TCR motifs determines T cell responsiveness in EAMG.