CDR3 Region Research Articles

Oligoclonality and innate-like features in the TCR repertoire of type II NKT cells reactive to a β-linked self-glycolipid (143.16)

Abstract CD1d-restricted type II NKT cells are distinct from the invariant type I NKT cells and are poorly characterized. A major subset recognizes the β-linked self-glycolipid sulfatide, playing a role in autoimmunity and anti-tumor immunity. Here, we analyzed the TCR repertoire of this type II NKT cell subset in mouse, using flowcytometry, TCR gene sequencing and spectratyping analysis of sorted sulfatide/CD1d-tetramer+ cells. For the TCR β-chain, we found predominant usage of the Vβ8.1/Vβ3.1 and Jβ2.7 gene segments with few dominant clonotypes of restricted CDR3 lengths and conserved acidic residues. The TCR α-chain was predominantly encoded by Vα1/Vα3 and Jα7/Jα9 gene segments and showed defined CDR3α regions of 8 or 9 a.a. lengths comprising conserved basic residues. CDR3 regions of both TCR α- and β-chains were encoded by germline sequences or N-additions. Though potential CD1d-binding tyrosine residues in the CDR2β region are conserved between most type I and type II NKT TCRs, CDR1α and 3α regions differ significantly between the two subsets. Collectively, these findings emphasize innate-like features of sulfatide-reactive type II NKT cells as well as oligoclonal TCR Vα-Jα/Vβ-Jβ gene usage characteristic of the antigen-specific responses of conventional MHC-restricted T cells. These data provide important clues to the recognition of β-linked glycolipids by CD1d-restricted T cells in general. (Supported by grants from NIH, JDRF, MSNRC and DNRG (to VK) and DFG (to PA).)

Sequential monitoring of minimal residual disease in acute lymphoblastic leukemia: 7-year experience in a pediatric hematology/oncology unit

We evaluated minimal residual disease (MRD) in 91 children with acute lymphoblastic leukemia (ALL) by PCR amplification of clonal rearrangements, immunoglobulin (IgH; VDJ rearrangement, CDR3 region) and T-cell receptor (TCRδ). Sequential monitoring of MRD was performed at different time points during and after chemotherapy and was correlated to patient outcome. In total, 792 bone marrow samples were assessed for MRD at the end of induction, and during and after treatment completion. MRD positivity at the end of induction was detected in 12% of patients and was associated with high incidence of relapse, 54.55% (p = 0.0002), at 5 years. On the other hand, 88% of patients were MRD-negative at the end of induction and the relapse rate at 5 years was very low, 5%. The frequency of MRD decreased to 16% in the first 6 months of chemotherapy; however, the incidence of relapse in MRD-positive patients remained high, 42.8%. After treatment completion (24–36 months from diagnosis), 32% patients were MRD-positive and the relapse rate was 36.5% (p = 0.0009). Our results indicated that monitoring of MRD constituted an essential prognostic marker, and detection of MRD particularly at the end of induction and after treatment completion was strongly predictive for patient outcome.

A Humanized Anti-VEGF Rabbit Monoclonal Antibody Inhibits Angiogenesis and Blocks Tumor Growth in Xenograft Models

Rabbit antibodies have been widely used in research and diagnostics due to their high antigen specificity and affinity. Though these properties are also highly desirable for therapeutic applications, rabbit antibodies have remained untapped for human disease therapy. To evaluate the therapeutic potential of rabbit monoclonal antibodies (RabMAbs), we generated a panel of neutralizing RabMAbs against human vascular endothelial growth factor-A (VEGF). These neutralizing RabMAbs are specific to VEGF and do not cross-react to other members of the VEGF protein family. Guided by sequence and lineage analysis of a panel of neutralizing RabMAbs, we humanized the lead candidate by substituting non-critical residues with human residues within both the frameworks and the CDR regions. We showed that the humanized RabMAb retained its parental biological properties and showed potent inhibition of the growth of H460 lung carcinoma and A673 rhabdomyosarcoma xenografts in mice. These studies provide proof of principle for the feasibility of developing humanized RabMAbs as therapeutics.

Single Domain Antibodies Are Specially Suited for Quantitative Determination of Gliadins under Denaturing Conditions

Food intended for celiac patients' consumption must be analyzed for the presence of toxic prolamins using high detectability tests. Though 60% ethanol is the most commonly used solvent for prolamins extraction, 2-mercaptoethanol (2-ME) and guanidinium chloride (GuHCl) can be added to increase protein recovery. However, ethanol and denaturing agents interfere with antigen recognition when conventional antibodies are used. In the present work, a new method for gliadins quantification is shown. The method is based on the selection of llama single domain antibody fragments able to operate under denaturing conditions. Six out of 28 VHH-phages obtained retained their binding capacity in 15% ethanol. Selected clones presented a long CDR3 region containing two additional cysteines that could be responsible for the higher stability. One of the clones (named VHH26) was fully operative in the presence of 15% ethanol, 0.5% 2-ME, and 0.5 M GuHCl. Capture ELISA using VHH26 was able to detect gliadins in samples shown as negatives by conventional ELISA. Therefore, this new strategy appears as an excellent platform for quantitative determination of proteins or any other immunogenic compound, in the presence of denaturing agents, when specific recognition units with high stability are required.

Optimization of the HA-1-Specific T Cell Receptor for Gene Therapy of Hematological Malignancies.

Abstract 4093Poster Board III-1028Relapsed hematological malignancies after HLA-matched allogeneic stem cell transplantation (allo-SCT) are treated by donor lymphocyte infusion (DLI), inducing long-lasting complete remissions. However, treatment of relapsed hematological malignancies after allo-SCT with DLI is associated with induction of graft versus host disease (GvHD). It has been demonstrated that T cells recognizing minor histocompatibility antigens (mHags) selectively expressed on hematopoietic cells mediate anti-leukemic reactivity after allo-SCT without causing GvHD. mHags are derived from genetically polymorphic proteins that can be differentially expressed between donor and recipient. The mHag HA-1 is presented in the context of HLA-A2. The HA-1 tissue distribution is restricted to hematopoietic cells and carcinomas, making it an attractive target antigen to treat hematological malignancies relapsing after allo-SCT when the patient is HA-1+ and the donor is HA-1-. Therefore, adoptive transfer of HA-1-TCR gene modified T cells might be an attractive strategy to separate GvHD from graft versus leukemia effect (GvL). For optimal anti-leukemic reactivity, high expression of introduced TCRs and persistence of the gene modified T cells is important. Based on the previously reported low HA-1-TCR expression on HA-1-TCR modified T cells, optimization of the strategy is required. Several strategies to improve expression of the introduced TCR have been described. Protein expression of the TCR chains can be enhanced by codon optimization. In addition, preferential pairing facilitated by introduction of an extra disulfide bond in the constant regions of the TCR chains can increase the cell surface expression of the transferred TCR. Another strategy based on the fact that TCRs differ in their capacity to compete for cell surface expression, is to select recipient T cells with weak competitor phenotypes. In this study, we investigated the cause of low HA-1-TCR expression after gene transfer, and used the different strategies to increase HA-1-TCR expresssion. To study whether low HA-1-TCR expression was due to inefficiency of the TCRα and β chains to pair, TCR-deficient jurkat cells were transduced with the individual TCRα and TCRβ chains in combination with 17 different TCRα chains and TCRβ chains. Results indicated that low HA-1-TCR expression was not due to inefficient pairing of the HA-1-TCR chains, but caused by low HA-1-TCRβ chain expression on the cell surface. To investigate whether low cell surface expression of the HA-1-BV6S4 chain was due to intrinsic properties, the CDR1 and CDR3 region of the HA-1-TCR BV6S4 chain were exchanged with the CDR1 and CDR3 region of the HA-2-TCRβ (BV6S2) chain. We demonstrated that exchange of the HA-1-TCRβ CDR1 region with the HA-2-TCRβ CDR1 region resulted in improved TCR-expression, however, the HA-1-specificity was completely abolished, indicating that the HA-1-TCRβ CDR1 region is crucial for HA-1-specificity. Furthermore, since there is exclusive TCRBV6S4 chain usage of HA-1-specific T cells, we were unable to select for another HA-1-TCR for clinical use, and were pressed to optimize the HA-1-TCRβ chain. Both codon optimization of the HA-1-TCR chains aiming at improving protein expression and inclusion of cysteine residues in the HA-1-TCR chains aiming at inducing preferential pairing resulted in a significant increase in HA-1-TCR expression. Combining the two strategies increased the HA-1-TCR expression even more, resulting in 70% and 35% of tetramer positive HA-1-TCR transferred weak competitor and strong competitor T cells, respectively. In addition, the HA-1-TCR engineered T cells were able to efficiently recognize target cells that endogenously process and present HA-1, independent of whether the recipient T cells were strong or weak competitor T cells. These results illustrate that engineering of the HA-1-TCR by codon optimization and introduction of an extra cysteine bond resulted in high numbers of high-avidity HA-1-TCR in any T cell of choice irrespective of the properties of the endogenous TCR. Disclosures:No relevant conflicts of interest to declare.

ATP Acts as Danger Associated Molecular Pattern and Enhances Acute Graft-Versus-Host Disease Severity Via P2×7-R.

Abstract 1336Poster Board I-358Conditioning prior allogeneic hematopoietic cell transplantation causes local tissue injury. Cellular molecules that are released upon cell stress, injury or cell death can act as potent danger associated molecular patterns (DAMP). Nucleotides like ATP are ideal candidates to serve as such indicators of cell damage, since they are present in the extracellular environment in the nanomolar range under physiological conditions but are released upon unphysiological cell death which causes activation of purinergic receptors. The role of purinergic signaling in alloantigen driven immune responses such as graft-versus-host disease (GvHD) is unclear. We found that ATP levels in the peritoneal fluid increased when GvHD was present in human and mice. Detection of extracellular ATP with a luciferase based in vivo detection system demonstrated early ATP release from the gastrointestinal tract. Blockade of multiple purinergic receptors on T cells or ATP neutralization during GvHD development reduced disease severity and proinflammatory cytokine production significantly. Gene expression analysis of different P2R in GvHD target organs identified the P2X7(R)receptor to be increasingly expressed. P2X7R−/− recipients developed less severe GvHD as compared to wildtype mice. This was paralleled by the induction of STAT5 signaling with increased numbers of Foxp3 positive regulatory T cells while STAT1 phosphorylation and IFN-γ production was reduced. Despite P2X7R blocking, T cells were still able to reconstitute the lymphoid compartment with a polyclonal intact TCR repertoire analyzed by spectratyping DNA sequence analyses of the CDR3 region and to reject established B-cell lymphoma. Therefore, DAMPs such as endogenous nucleotides may not be required for anti-tumor activity but dominantly trigger acute GvHD. Blockade or genetic deficiency of P2X7R in BMT recipients conferred GvHD protection without loss of GvL effects, which has important clinical implications given the availability of P2X7R inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Graft-Versus-Leukemia Antigen CML66 Elicits Coordinated B and T Cell Immunity After Donor Lymphocyte Infusion.

Abstract 2449Poster Board II-426Donor lymphocyte infusion (DLI) is a highly effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic hematopoietic stem cell transplantation (HSCT), in which 70-80% patients achieve curative responses. Despite its efficacy and established clinical use, the mechanism by which DLI achieves anti-tumor immunity remains incompletely understood. CML66 was discovered as an overexpressed nonpolymorphic CML-associated antigen that elicited a high-titer (>1:50,000) antibody response in a patient who received CD4+ DLI for relapsed CML. CML66 is preferentially expressed on myeloid leukemia progenitor cells and the development of CML66 antibodies temporally correlated with attainment of molecular remission. Since CD4+ T cell responses are involved in generating both antigen-specific B cell and CD8+T cell responses, we queried whether development of potent B cell immunity was also associated with the development of cytolytic T cell immunity against CML66. To detect specific T cell responses, post-DLI PBMC from this patient were screened for reactivity against pools of overlapping 15- to 18-mer peptides encoding the entire protein sequence of CML66. Patient CD8+ T cells were found to be reactive against a single CML66-derived peptide pool, and specifically an 8 amino acid peptide (HDVDALLW), beginning at residue 459. This T cell epitope is positioned in a different region of the protein compared to the B cell epitope (at residues 198-217: GFYVSLEWVTISKKNQDNK). A peptide-specific T cell clone was isolated, whose MHC class I molecule binding was restricted to HLA B*4403. The CML66-reactive T cell clone recognized the HLA-B4403-expressing CD34+ AML cell line MUTZ-3, providing evidence that this epitope is naturally processed and presented by myeloid leukemia cells. Functional cytotoxic T cell responses to CML66 appeared to precede the appearance of antigen-specific antibody responses. While CML66-specific plasma antibody was only detectable starting 2-3 months after DLI, reactivity to the HLA-B4403-restricted epitope by peripheral blood-derived CD8+ T cells was detected by IFNγ ELISpot as early as one month following DLI. To sensitively detect CD8+ CML66-specific T cells in vivo, we sequenced the CDR3 region specific to the CML66-specific clone, and designed clone-specific quantitative PCR primers. Nested PCR of RNA directly extracted from patient PBMC revealed only minimal detection of the CML66-specific T cell clone prior to DLI, but then expansion between 1 to 6 months after DLI. Similar analysis of marrow-derived RNA also revealed the presence of CML66-specific T cell clone in patient marrow following DLI. Moreover, this clone was already detected in patient marrow prior to DLI, suggesting that marrow is a reservoir of leukemia-specific T cells following hematopoietic stem cell transplantation. Our studies provide the first description of coordinated adaptive immunity developing against a nonpolymorphic leukemia-associated antigen, in close temporal correlation with the attainment of long-lasting immune-mediated clinical remission after allogeneic HSCT. The detected cytolytic T cell response was associated with the secretion of high-titer IgG antibody specific for a distinct epitope in the same target protein. In this setting, our results indicate that infusion of CD4+ donor cells resulted in the rapid activation and expansion of pre-existing marrow-resident leukemia-specific CD8+ T cells, followed by a cascade of antigen-specific immune responses detectable in the periphery. Disclosures:No relevant conflicts of interest to declare.

Clonal Expansions of Cytotoxic T Cells in the Blood of Patients with Waldenstrom's Macroglobulinaemia Are Anergic and Disappear After Nucleoside Analogue Therapy.

Abstract 1820Poster Board I-846T cells contribute to the immunomodulatory control of the tumor in patients with monoclonal gammopathies. We previously found that CD3+CD8+CD57+TCRVβ+restricted cytotoxic T cell expansions were present in 48% of patients with multiple myeloma (n=221) and conferred a significant favorable prognosis. We now report the presence of these expansions in 70% of patients with Waldenstrom's Macroglobulinaemia (WM) (n=20) with a wide spectrum of the TCRVβ repertoire represented. Previous nucleoside analogue (NA) therapy, known to be associated with an increased incidence of transformation to aggressive lymphoma, significantly influenced the presence of TCRVβ expansions (χ2=11.6; p<0.001) as 5 of the 6 patients without and only 1 of the 14 patients with TCRVβ expansions had received NA. Clonality of CD3+CD8+CD57+TCRVβ-restricted cytotoxic T cells was confirmed by determining the size of the TCRVβ chain CDR3 region and by direct sequencing. We identified differential gene expression by using microarray analysis (Affymetrix GeneChip Human Genome U133 plus 2.0 Arrays) and confirmed the expression of selected genes by real-time qPCR and flow cytometry. Data was analysed by paired moderated t-test and then Gene Set Enrichment Analysis (GSEA) for pathway analysis. Clonal T cells not only had upregulated genes from cytotoxic pathways (granzyme B, perforin and FGFBP2) but also genes which suppress apoptosis, proliferation, cell cycle G1/S transition arrest and T cell activation (RAS, CSK and TOB pathways) indicating anergy which was validated by CFSE tracking of proliferation after antiCD3+antiCD28 stimulation. The current studies provide further evidence for the persistence and relevance of clonal T cells in monoclonal gammopathies. DisclosuresNo relevant conflicts of interest to declare.

MHC Drives TCR Repertoire Shaping, but not Maturation, in Recent Thymic Emigrants

After developing in the thymus, recent thymic emigrants (RTEs) enter the lymphoid periphery and undergo a maturation process as they transition into the mature naive (MN) T cell compartment. This maturation presumably shapes RTEs into a pool of T cells best fit to function robustly in the periphery without causing autoimmunity; however, the mechanism and consequences of this maturation process remain unknown. Using a transgenic mouse system that specifically labels RTEs, we tested the influence of MHC molecules, key drivers of intrathymic T cell selection and naive peripheral T cell homeostasis, in shaping the RTE pool in the lymphoid periphery. We found that the TCRs expressed by RTEs are skewed to longer CDR3 regions compared with those of MN T cells, suggesting that MHC does streamline the TCR repertoire of T cells as they transition from the RTE to the MN T cell stage. This conclusion is borne out in studies in which the representation of individual TCRs was followed as a function of time since thymic egress. Surprisingly, we found that MHC is dispensable for the phenotypic and functional maturation of RTEs.

Control of CD56 expression and tumor cell cytotoxicity in human Vγ2Vδ2 T cells

BackgroundIn lymphocyte subsets, expression of CD56 (neural cell adhesion molecule-1) correlates with cytotoxic effector activity. For cells bearing the Vγ2Vδ2 T cell receptor, isoprenoid pyrophosphate stimulation leads to uniform activation and proliferation, but only a fraction of cells express CD56 and display potent cytotoxic activity against tumor cells. Our goal was to show whether CD56 expression was regulated stochastically, similar to conventional activation antigens, or whether CD56 defined a lineage of cells committed to the cytotoxic phenotype.ResultsTracking individual cell clones defined by their Vγ2 chain CDR3 region sequences, we found that CD56 was expressed on precursor cytotoxic T cells already present in the population irrespective of their capacity to proliferate after antigen stimulation. Public T cell receptor sequences found in the CD56+ subset from one individual might appear in the CD56- subset of another donor. The commitment of individual clones to CD56+ or CD56- lineages was stable for each donor over a 1 year interval.ConclusionThe ability to express CD56 was not predicted by TCR sequence or by the strength of signal received by the TCR. For γδ T cells, cytotoxic effector function is acquired when cytotoxic precursors within the population are stimulated to proliferate and express CD56. Expression of CD56 defines a committed lineage to the cytotoxic phenotype.

HIV-1 gp41-specific monoclonal mucosal IgAs derived from highly exposed but IgG-seronegative individuals block HIV-1 epithelial transcytosis and neutralize CD4+ cell infection: an IgA gene and functional analysis

AIDS is mainly a sexually transmitted disease, and accordingly, mucosal tissues are the primary sites of natural human immunodeficiency virus type-1 (HIV-1) transmission. Mucosal immunoglobulin A (IgA) antibody specific for HIV-1 envelope gp41 subunit is one correlate of protection in individuals who are highly sexually exposed to HIV-1 but remain persistently IgG seronegative (HEPS). Understanding these peculiar IgAs at the gene and functional level is possible only with monoclonal IgAs. We have constructed a mucosal Fab IgA library from HEPS and have characterized a series of HIV-1 IgAs specific for gp41 that, in vitro, are transcytosis-blocking and infection-neutralizing. Characterization of their IgA genes shows that Fab specific for the gp41 membrane-proximal region harbors a long heavy-chain CDR3 loop (CDRH3) similar to the two broadly neutralizing IgG monoclonal antibodies, 2F5 and 4E10. Furthermore, the selected Fab IgA shows extensive somatic mutations that cluster in the CDR regions, indicating that affinity maturation due to an antigen-driven process had occurred in HEPS individuals, presumably upon multiple exposures to HIV. This analysis of HEPS monoclonal IgA gives a unique opportunity to correlate an antibody function (resistance to a pathogen in vivo) with an antibody gene. Such neutralizing monoclonal IgAs could be used in microbicide formulation.

Non-equilibrium and differential function between intraepithelial and lamina propria virus-specific TCRαβ+ CD8αβ+ T cells in the small intestinal mucosa

The gastrointestinal mucosa regularly encounters commensal and pathogenic microbiota. Gut mucosal lymphocytes consist of two phenotypically different populations residing in the intestinal intraepithelial (IEL) compartment and lamina propria (LP). Little is known about compositional and functional differences of antigen-specific T cells from these mucosal compartments after mucosal infection, or the degree of trafficking between them. We here studied the B8R20−27-specific CD8 T-cell response in LP and IEL compartments after intrarectal immunization with modified vaccinia virus Ankara (MVA). CD8+ T cells in the IEL compartment had much lower avidity than in the LP or spleen during acute and memory phases. Surprisingly, the TCR Vβ-chain distribution of antigen-specific T cells and the length of the CDR3 region of the dominant Vβ genes showed substantial dissimilarities between IEL and LP antigen-specific CD8αβ T cells in individual mice, increasing with time. We show functional and compositional differences between these mucosal compartments during the effector and memory phases of the immune response, indicating limited crosstalk and microenvironmental differences between the IEL, LP, and spleen. The restricted migration of cells from each of these mucosal compartments could partly account for a founder effect we observed in the IEL TCRαβ CD8αβ epitope-specific repertoire that might impact protective efficacy.

Characterization of the photodegradation of a human IgG1 monoclonal antibody formulated as a high-concentration liquid dosage form

The photodegradation of a human IgG1 monoclonal antibody has been examined in a high concentration (100 mg/mL) liquid formulation. It was observed that a yellowish color is generated when the formulation is exposed to intense and prolonged light exposure, and this discoloration occurs along with a loss in bioactivity. Extensive analytical characterization was performed to determine light induced degradation pathways that occur during exposure to intense light of ICH photodegradation conditions. It has been shown that the monoclonal antibody undergoes a combination of physical and chemical reactions under these conditions, including covalent aggregate formation, fragmentation at the hinge region, oxidation of Trp, His, and Met residues, and deamidation of Asn residues. Oxidation of Trp 94 and deamidation of Asn 93, located in the light chain CDR region, correlates with loss of bioactivity under these conditions. A series of formulation experiments were performed to elucidate the impact of the extent of light exposure, oxygen, protein concentration, and solution pH on the photostability of the formulation. Results demonstrated that photodegradation of the IgG, after intensive light exposure, can be prevented by proper secondary packaging. In addition, it is also shown that a high concentration, liquid dosage form of a human monoclonal antibody is stable upon exposure to the ambient light conditions encountered during routine manufacturing, long-term storage, and administration with proper design of formulation conditions, the primary container as well as the secondary package. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3117–3130, 2009

Mutagenicity and Potential Carcinogenicity of Thiopurine Treatment in Patients with Inflammatory Bowel Disease

The thiopurines azathioprine and 6-mercaptopurine (6-MP) are effective immune modulators and cytotoxic agents extensively used in the treatment of autoimmune diseases, graft rejection, and cancer. There is compelling epidemiologic evidence that thiopurine treatment increases the risk for a variety of tumors by mechanisms that are unclear. We investigated the in vivo mutagenicity of long-term thiopurine treatment by determining the frequency and spectra of somatic mutation events at the hypoxanthine phosphoribosyltransferase (HPRT) locus in peripheral T lymphocytes as well as the prevalence of mutant clonal proliferation in a cross-sectional analysis of data from 119 children and adults with inflammatory bowel disease (IBD). ANOVA and regression were performed to assess relationships among the frequency and spectra of HPRT mutations with disease, duration of illness, duration of treatment, and total therapeutic dose of azathioprine and 6-MP. We observed a significant increase in the frequency of somatic mutations in 56 subjects treated with thiopurines for IBD compared with 63 subjects not treated with thiopurines. This increase was related to both total dose (P < 0.001) and duration of treatment (P < 0.001). Comparative mutation spectra analysis of 1,020 mutant isolates revealed a significant increase in the proportion of all transitions (P < 0.001), particularly G:C to A:T transitions (P < 0.001). Combined analyses of two signatures for mutant clonality, HPRT mutation, and T-cell receptor beta CDR3 region unique gene sequence also showed a significant thiopurine-dependent increase in mutant cell clonal proliferation (P < 0.001). These findings provide in vivo evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention.

Molecular hallmarks of anti-chromatin antibodies associated with the lupus susceptibility locus, Sle1

Anti-nuclear antibodies constitute the hallmark of lupus. The NZM2410-derived Sle1 lupus susceptibility interval on murine chromosome 1 breaches tolerance, leading to the emergence of anti-nuclear autoantibodies targeting nucleosomes. However, little is known about the molecular structure of the anti-nucleosome autoantibodies from this genetically simplified mouse model of lupus. In this study, the immunoglobulin heavy chain and light chain sequences of 50 anti-nuclear monoclonal antibodies derived from five B6. Sle1 z mice were compared to non-nuclear antibody controls. Compared to two different sets of non-nuclear antibodies, anti-nucleosome antibodies derived from B6. Sle1 z congenic mice exhibited a high degree of clonal expansion and three distinct sequence motifs in their heavy chains – cationic CDR3 stretches, non-anionic CDR2 regions, and an increased frequency of aspartate residues at H50, which together increased the likelihood of an antibody being chromatin-reactive by ∼4-fold.

Oligoclonal CD4+T Cells Promote Host Memory Immune Responses to Zwitterionic Polysaccharide ofStreptococcus pneumoniae

Zwitterionic polysaccharides of the normal flora bacteria represent a novel class of antigens in that they correct systemic CD4(+) T-cell deficiencies and direct lymphoid organogenesis during colonization of the host. Presentation of these polysaccharides to CD4(+) T cells depends on major histocompatibility complex class II- and DM-dependent retrograde transport from lysosomes to the cell surface. Yet the phenotype and clonality of the immune response to the polysaccharide in the mature host immune system have not been studied. Using the zwitterionic capsular polysaccharide Sp1 of Streptococcus pneumoniae, a transient member of the bacterial flora, in an experimental mouse model of cellular immunity, we demonstrated the accumulation of TH1- and TH17-polarized CD4(+) CD44(high) CD62(low) CD25(-) memory T cells. Subcutaneous immunization with Sp1 resulted in an increase of serum immunoglobulin G (IgG), predominantly of the IgG1 subclass, and suggested the presence of a humoral memory response to the polysaccharide. CD4(+) T cells stimulated with polysaccharide in vitro and in vivo showed a nonrestricted pattern for the T-cell receptor (TCR) beta-chain variable region, as demonstrated by semiquantitative reverse transcription-PCR and flow cytometry. Clonotype mapping of in vivo and in vitro polysaccharide-activated CD4(+) T cells revealed clonotypic TCR transcripts. Taken together, the data show the induction of clonal expansion of CD4(+) T cells by polysaccharides of commensal bacteria. Cellular and humoral memory host responses imply the ability of these polysaccharides to mediate the expansion of T cells via recognition within the CDR3 region of the TCR.

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

Patients with myelodysplastic syndromes display several T-cell expansions, which are mostly polyclonal in the CD4 + subset and oligoclonal in the CD8 + subset

Immune dysregulation plays a role in the pathophysiology of myelodysplastic syndromes (MDS), as T-cell clones seem to be involved in the inhibition of hematopoietic precursors. The purpose of this study was to analyze the T-cell receptor (TCR) repertoire of MDS patients, focusing on the third complementarity determining region (CDR3) pattern of their CD4(+) and CD8(+) lymphocyte expansions. The study involved 30 patients and 15 age-matched controls. The beta-variable (betaV) subfamily flow-cytometry analysis was performed on peripheral CD4(+) and CD8(+) T-cells. Spectratyping TCR-CDR3 analysis was carried out on isolated helper and cytotoxic T lymphocytes after immunomagnetic separation and reverse-transcriptase polymerase chain reaction. We first identified by flow cytometry an increased frequency of expanded betaVs in both CD4(+) and CD8(+) T-cells in MDS patients. We then showed, by spectratyping, that the CDR3 profile was mostly Gaussian in their CD4(+) T cells, whereas CD8(+) T cells usually showed skewed or oligoclonal CDR3 regions. When we compared spectratyping and flow-cytometry findings in each patient, we showed that most CD4(+) lymphocyte expansions detected by flow cytometry had Gaussian CDR3 profiles, whereas most CD8(+) expansions were oligoclonal. We confirm that in MDS patients the TCR-betaV repertoire is overall extremely contracted, especially in cytotoxic T cells. This pattern is mainly determined by selective proliferations of both helper and cytotoxic T cells, which are, however, mostly polyclonal in the former and oligoclonal in the latter. Such a difference, possibly related to the different human leukocyte antigen restriction, could reflect the selective involvement of cytotoxic T cells either in the anti-tumor immune surveillance or in an autoreactive aggression toward hematopoietic precursors.

Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA(3-286)) from strain R36A. Abs to PspA(3-286) were encoded by diverse V(H) and V(kappa) families/genes. The H chain CDR3 and L chain CDR3 lengths were 3-13 (7.8 +/- 0.5) and 8-9 (8.7 +/- 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack D(H) gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of D(H) gene-derived amino acids did not prevent anti-PspA(3-286) mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA(3-286) polyclonal Ab response in PspA(3-286)- and R36A-immunized mice. Compared with PspA(3-286)-immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. D(H) genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of D(H)-less Abs.

Diversification of TCR repertoire in melanoma patients treated with chemotherapy before peptides-vaccination (41.19)

Abstract Combining chemotherapy and immunotherapy is an attractive antitumor strategy. Recently we have reported that the administration of dacarbazine (DTIC) one day before peptide-vaccination in disease-free melanoma patients induced an increase of peptide-specific effector-memory CD8+ lymphocytes. To investigate the functional differences between anti-Melan-A CD8+ T cell response elicited by vaccination alone (Arm 1) or by chemotherapy plus vaccination (Arm 2), we generated T cell clones from 5 patients of both Arms, before and after the treatments. A significant enhancement of tumor lytic activity of Melan-A+ clones generated by patients of Arm 2 after the treatment was observed, whereas no significant differences of tumor recognition efficiency were evident among the two arms before treatment. Ninety-six sequences were obtained and 47 clonotypes with different CDR3 regions were observed. A progressive enhancement of TCR repertoire diversity was found only in patients treated with DTIC before vaccination, whereas patients treated with vaccine alone showed a reduction of TCR repertoire diversity accompanied by a reduction of tumor lytic activity in one patient. These results suggest that a diverse Melan-A-specific T cell repertoire occurs in melanoma patients treated with DTIC plus vaccination, which may offer a new clinically advantageous anti-tumor strategy.