A public T cell receptor recognized by a monoclonal antibody specific for the D-J junction of the ß-chain.

It is becoming increasingly clear that T cell responses against many antigens are dominated by public α/ß T cell receptors (TCRs) with restricted heterogeneity. Because expression of public TCRs may be related to resistance, or predisposition to diseases, it is relevant to measure their frequencies. Although staining with tetrameric peptide/major histocompatibility complex (pMHC) molecules gives information about specificity, it does not give information about the TCR composition of the individual T cells that stain. Moreover, next-generation sequencing of TCR does not yield information on pairing of α- and ß-chains in single T cells. In an effort to overcome these limitations, we have here investigated the possibility of raising a monoclonal antibody (moAb) that recognizes a public TCR. As a model system, we have used T cells responding to the 91-101 CDR3 peptide of an Ig L-chain (λ2³¹5), presented by the MHC class II molecule I-E(d). The CD4⁺ T cell responses against this pMHC are dominated by a receptor composed of Vα3Jα1;Vß6DßJß1.1. Even the V(D)J junctions are to a large extent shared between T cell clones derived from different BALB/c mice. We here describe a murine moAb (AB10) of B10.D2 origin that recognizes this public TCR, while binding to peripheral T cells is negligible. Binding of the moAb is abrogated by introduction of two Gly residues in the D-J junction of the CDR3 of the ß-chain. A model for the public TCR determinant is presented.

3-O sulfation of syndecan-1 mediated by the sulfotransferase HS3ST3a1 enhances myeloma aggressiveness.

Multiple myeloma is a hematological neoplasm derived from plasma cells invariably developing in the bone marrow (BM). The persisting clinical challenge in MM resides in its high ability to resist drugs as shown by the frequent relapses observed in patients regardless of the treatment applied. In a mouse model of MM, we identified a subpopulation of cells harboring increased resistance to current MM drugs. These cells bound a proliferation inducing ligand (APRIL), a key MM promoting/survival factor. APRIL binding involved the heparan sulfate (HS) chain present on syndecan-1 (SDC-1), and correlated with reactivity to the anti-HS antibody 10e4. 10e4+cells had a high proliferation activity, and were able to form colonies in 3-D cultures. 10e4+ cells were the only cells able to develop in BM after intravenous injection. They also resisted drugs in vivo, since their number increased after treatment in BM. Notably, 10e4+ cells differentiated into 10e4- cells upon in vitro and in vivo expansion. Expression of one sulfotransferase, HS3ST3a1, allowed modification of syndecan-1 to confer reactivity to 10e4 and binding to APRIL. HS3ST3a1 deletion inhibited tumorigenesis in BM. Notably, the two populations coexisted at a variable frequency in the BM of MM patients at diagnosis. In total, our results indicate that 3-O-sulfation on SDC-1 carried out by HS3ST3a1 defines aggressive MM cells, and that targeting of this enzyme could possibly be used to better control drug resistance.

Generation of antibody-producing hybridomas following one single immunization with a targeted DNA vaccine.

The standard protocol for generating antibody (Ab)-producing hybridomas is based on fusion of plasmacytoma cells with Ab-producing B cells harvested from immunized mice. To increase the yield of hybridomas, it is important to use immunization protocols that induce a high frequency of B cells producing specific Abs. Our laboratory has developed a vaccine format, denoted vaccibody that promotes the immune responses towards the delivered antigen. The vaccine format targets antigens in a bivalent form to surface receptors on antigen-presenting cells (APCs). Here, we used the fluorescent protein (FP) mCherry as antigen and targeted it to APCs by use of either the natural ligand CCL3/MIP-1α or single-chain variable fragment specific for major histocompatibility complex class II. The vaccine format was delivered to mouse muscle as DNA combined with electroporation. By this procedure, we developed two monoclonal Abs that can be utilized to detect the FC mCherry in various applications. The data suggest that the targeted DNA vaccine format can be utilized to enhance the number of Ab-producing hybridomas and thereby be a tool to improve the B cell hybridoma technology.

Ballistic strength training in adults with cerebral palsy may increase rate of force development in plantar flexors, but transition to walking remains unclear: a case series.

BACKGROUND: Persons with cerebral palsy (CP) walk with reduced ankle plantar flexor power compared to typically developing. In this study, we investigated whether a ballistic strength-training programme targeting ankle plantar flexors could improve muscle strength, muscle architecture and walking function in adults with CP. METHODS: Eight adults (mildly affected CP) underwent eight weeks of ballistic strength training, with two sessions per week. Before and after the intervention preferred walking speed, ankle plantar flexion rate of force development (RFD), maximal voluntary contraction (MVC), muscle thickness, pennation angle and fascicle length were measured. Data are presented for individuals, as well as for groups. Group changes were analysed using the Wilcoxon signed-rank test. RESULTS: Data were analysed for eight participants (five women, mean age 37.9 years; six GMFCS I and two GMFCS II). Two participants increased their walking speed, but there were no significant group changes. In terms of muscle strength, there were significant group changes for RFD at 100 ms and MVC. In the case of muscle architecture, there were no group changes. CONCLUSION: In this study, we found that eight weeks of ballistic strength training improved ankle plantar flexor muscle strength but walking function and muscle architecture were unchanged. Larger studies will be needed to obtain conclusive evidence of the efficacy of this training method.

Is secretion of tumour-specific antigen important for cancer eradication by CD4(+) T cells?--Implications for cancer immunotherapy by adoptive T cell transfer.

The potential for cancer immunotherapy by adoptive transfer of CD4(+) T cells is gaining increased attention. Most cancer cells lack major histocompatibility complex (MHC) class II molecules and cannot present tumour-specific antigens (TSA) directly to CD4(+) T cells. We have reported that tumour-specific CD4(+) T cells collaborate with macrophages and dendritic cells. These professional antigen-presenting cells endocytose and process TSA to display antigenic peptides on their MHC class II molecules for indirect cancer cell recognition by CD4(+) T cells. We hypothesized that this critical step may depend on secretion of TSA by cancer cells. This was investigated in a mouse model for myeloma immunosurveillance mediated by CD4(+) T cells. From this study, several conclusions could be drawn. First, TSA secretion facilitates cancer immunosurveillance. Second, TSA secretion results in stronger activation of naïve tumour-specific CD4(+) T cells in lymph nodes. Third, TSA concentration within the tumour extracellular matrix must reach a certain threshold to allow successful cancer immunosurveillance. Fourth, treatment by local injection of purified TSA enhances immunity against cancer cells that do not secrete TSA. Fifth, secretion of TSA by at least some cancer cells within a tumour favours antitumour immunity. Therefore, we propose that CD4(+) T cells that recognize secreted TSA may be superior for immunotherapy by T cell transfer, because the local extracellular antigen concentration will be higher for secreted TSA. Thus, it is anticipated that secreted TSA will be more readily detected in vivo by transferred CD4(+) T cells, resulting in more efficient tumour eradication.

T helper cells recognize an idiotope located on peptide 88-114/117 of the light chain variable domain of an isologous myeloma protein (315).

Isolated variable region light chain 315 (VL-315), the VL domain of a myeloma protein of BALB/c origin, induces T cells of BALB/c (H-2d) mice that help the adoptive secondary anti-4-hydroxy-3-iodo-5-nitrophenylacetyl (NIP) antibody response to NIP-Fab315. The location of the epitope recognized by helper cells was examined with two fragments of VL-315, obtained by cleavage with cyanogen bromide at Met 87. Both N-terminal fragment 1-86 and C-terminal fragment 88-114/117 elicited BALB/c antibodies that bound to the respective fragments and to VL-315. By contrast, only fragment 88-114/117, which consists of the third hypervariable region, J region, and 5-7 amino acids of the C region, induced helper cells that augmented the anti-NIP response to NIP-Fab 315.

The inhibitor of death receptor signaling, FLICE-inhibitory protein defines a new class of tumor progression factors.

Death receptor-mediated apoptosis can be modulated by several antiapoptotic proteins, such as the FLICE (FADD [Fas-associated death domain]-like IL-1beta-converting enzyme)-inhibitory proteins (FLIPs). The FLIP family includes both cellular and viral members. The Kaposi's sarcoma-associated herpesvirus protein (KSHV)-FLIP is expressed by human herpesvirus 8 (HHV-8), which is associated with malignancies such as Kaposi's sarcoma and certain lymphomas. In this paper, we demonstrate that KSHV-FLIP protects cells from Fas-mediated apoptosis by inhibiting caspase activation and permits clonal growth in the presence of death stimuli in vitro. Furthermore, we show that KSHV-FLIP can act as a tumor progression factor by promoting tumor establishment and growth in vivo. When injected into immunocompetent recipient mouse strains, murine B lymphoma cells (A20) transduced with KSHV-FLIP rapidly develop into aggressive tumors showing a high rate of survival and growth. The tumor-progressive activity of KSHV-FLIP is mediated by prevention of death receptor-induced apoptosis triggered by conventional T cells. Consequently, inhibitors of death receptor signaling can be regarded as a new class of tumor progression factors, and HHV-8-associated tumors may represent naturally occurring examples of the tumorigenic effect of such inhibitors.

T cell recognition of the dominant I-A(k)-restricted hen egg lysozyme epitope: critical role for asparagine deamidation.

Type-B T cells raised against the immunodominant peptide in hen egg lysozyme (HEL(48-62)) do not respond to whole lysozyme, and this has been thought to indicate that peptide can bind to l-A(k) in different conformations. Here we demonstrate that such T cells recognize a deamidated form of the HEL peptide and not the native peptide. The sequence of the HEL epitope facilitates rapid and spontaneous deamidation when present as a free peptide or within a flexible domain. However, this deamidated epitope is not created within intact lysozyme, most likely because it resides in a highly structured part of the protein. These findings argue against the existence of multiple conformations of the same peptide-MHC complex and have important implications for the design of peptide-based vaccines. Furthermore, as the type-B T cells are known to selectively evade induction of tolerance when HEL is expressed as a transgene, these results suggest that recognition of posttranslationally modified self-antigen may play a role in autoimmunity.

Muscle Strength as a Predictor of Gait Variability after Two Years in Community-Living Older Adults.

BACKGROUND: Stride-to-stride fluctuations, or gait variability, can be captured easily using body worn inertial sensors. Previously, sensor-measured gait variability has been found to be associated with fall risk and central nervous changes. However, further research is needed to clarify the clinical relevance of this method. OBJECTIVES: In this study, we look at how gait variability is associated with muscle strength, measured two years earlier. DESIGN, SETTING AND PARTICIPANTS: This is study of longitudinal associations. Participants were community-dwelling volunteers between 70-81 years. MEASUREMENTS: Participants were tested while walking with a single sensor at their lower back, and they walked back and forth over a distance of 6.5 meters under four conditions: at preferred speed, at fast speed, with an added cognitive task, and while walking across an uneven surface. Gait variability in the anteroposterior (AP), mediolateral (ML) and vertical (V) directions was identified. A muscle strength score was composed by transforming hand grip strength, isometric knee extension strength and the 30 second chair rise-test to z-scores and adding them. RESULTS: 56 individuals were analysed (mean age at baseline 75.8 (SD 3.43), 60 percent women). In a backwards regression method using age, gender and baseline walking speed as covariates, muscle strength predicted gait variability after two years for AP variability during preferred speed (Beta= .314, p=.025) and uneven surface walking (Beta=.326, p=.018). Further, muscle strength was associated with ML variability during preferred speed (Beta=.364, p=.048) and fast speed (Beta=.419, p=.042), and V variability during preferred speed (Beta=.402, p=.002), fast speed (Beta=.394, p=.004) and uneven surface walking (Beta=.369, p=.004). CONCLUSIONS: Sensor-measured gait variability tended to be associated with muscle strength measured two years earlier. This finding could emphasize the relevance of this relatively novel measure of gait in older adults for both research and clinical practice.

Review: to what extent are T cells tolerant to immunoglobulin variable regions?

During the last 25 years it has become increasingly clear that short peptides derived from Ig V-regions are displayed on MHC class II molecules. Recognition of such idiotypic(Id)-peptide/MHC class II complexes by Id-specific CD4(+) T cells plays a role in (1) Id-driven T-B collaboration, (2) immunosurveillance of B cell cancers and (3) Id-vaccination. A crucial question is then: to what extent are T cells tolerized to Ig V-region sequences? Or rephrased: how large is the T-cell repertoire for Ig V-region sequences presented by MHC class II molecules? We argue that T cells are to a large extent tolerant to germline-encoded V-region sequences but that there is a T-cell repertoire for rare Id-sequences that arise as a consequence of somatic hyper mutation or N-region diversity. Moreover, when otherwise rare Id-sequences increase in concentration, T-cell tolerance is induced (Fig. 1). For these reasons, T cells that recognize rare Id-peptides, arising as a consequence of somatic genetic events unique to each B cell, may play a special importance in Id-driven T-B collaboration, immunosurveillance of B-cell malignancies, and Id-vaccination.

Thymic skewing of the CD4/CD8 ratio maps with the T-cell receptor alpha-chain locus.

The thymic preference for CD4+ T cells over CD8+ T cells is often attributed to a default pathway favouring CD4+ T cells or to homeostatic mechanisms. It is also clear, however, that T-cell receptor (TCR) preferences for major histocompatibility complex (MHC) class I versus class II binding will strongly influence an individual clone's skewing to the CD4 or CD8 subset. The variable region of each TCR alpha chain (V alpha) studied to date is found to be overrepresented in either CD4+ or CD8+ cells, suggesting that each V alpha element can interact more favourably with either MHC class I or class II molecules. Indeed, TCRs appear to have an intrinsic ability to interact with MHC molecules, and single amino acid residues present in germline-encoded complementarity determining region 1 (CDR1) and CDR2 of the V alpha element can be responsible for determining MHC specificity. Interestingly, the degree of CD4/CD8 skewing is variable among different mouse strains and in human populations. Here, we have shown that polymorphism in CD4/CD8 skewing between B6 and BALB/c mice is determined by the stem cell genotype and not by environmental effects, and that it maps in or near the TCR alpha-chain complex, Tcra. This was confirmed by comparing Tcra(b) with Tcra(a) or Tcra(c) haplotypes in congenic mice. We propose that the array of V alpha genes in various Tcra haplotypes exerts influence over the proportion of CD4 and CD8 subsets generated and may account in part for the observed thymic skewing. Thus, while it has been suggested that the TCR genes have been selected by evolution for MHC binding, our results further indicate selection for class II MHC preference.

Functional phage display of two murine alpha/beta T-cell receptors is strongly dependent on fusion format, mode and periplasmic folding assistance.

Phage display has been instrumental for the success of antibody (Ab) technology. The aim of the present study was to explore phage display of soluble T-cell receptors (TCRs). A library platform that supports engineering and selection of improved TCRs to be used as detection reagents for specific antigen presentation will be very useful. In such applications, high, equal and clone independent display levels are a prerequisite for 'fair' selection. Therefore, we explored how different pIII fusion formats and modes affected the display levels of two murine alpha/beta TCRs. Both are derived from T-cell clones associated with the MOPC315 myeloma model. The results show that the design of the pIII fusion particle significantly affects the subsequent display levels. Furthermore, successful display may be obtained both in phagemid and phage versions. Importantly, improvement of poor display can be achieved by over-expressing the periplasmic chaperone FkpA.

Antibodies engineered with IgD specificity efficiently deliver integrated T-cell epitopes for antigen presentation by B cells.

We have developed a strategy for improving the stimulation of T cells during immune responses by constructing recombinant antibodies that enhance the delivery of antigen to antigen-presenting cells, such as B cells. These antibodies have variable regions specific for surface molecules on B cells, and a constant region with an inserted antigen. In vitro, such antibodies make B cells approximately 1000-fold more efficient at presenting antigen and stimulating specific T cells. In vivo, the antibodies turn B cells of the spleen into potent stimulators of T cells. This approach may be useful for the generation of new vaccines.

The Association between Daily Walking Behavior and Self-Reported Physical Function in Community-Dwelling Older Adults.

Many older people do not participate in organized exercise, and daily walking may be the most substantial contributor to physical activity. To investigate the association between daily walking behavior and self-reported health-related physical function, older community-dwelling volunteers wore activity-registering sensors for three days. Self-reported health-related physical functioning was measured using the SF36 10-item Physical Function subscale. Forty-six participants wore a sensor (mean age 77.6, SD 3.6, 61 % women). In a multiple regression model, steps per day (B=.005, p≤.001) and walks per day (B=-.174, p=.010) were associated with the SF36-PF subscale. The association between physical functioning and walks per day was negative: Those who took many walks per day may have been walking more indoors. Health professionals are likely justified in advising older people to incorporate walking into daily life for health purposes. The cross-sectional design does not allow for inferences about causality.

Autologous bone marrow Th cells can support multiple myeloma cell proliferation in vitro and in xenografted mice.

Multiple myeloma (MM) is a plasma cell malignancy where MM cell growth is supported by the bone marrow (BM) microenvironment with poorly defined cellular and molecular mechanisms. MM cells express CD40, a receptor known to activate autocrine secretion of cytokines and elicit proliferation. Activated T helper (Th) cells express CD40 ligand (CD40L) and BM Th cells are significantly increased in MM patients. We hypothesized that activated BM Th cells could support MM cell growth. We here found that activated autologous BM Th cells supported MM cell growth in a contact- and CD40L-dependent manner in vitro. MM cells had retained the ability to activate Th cells that reciprocated and stimulated MM cell proliferation. Autologous BM Th cells supported MM cell growth in xenografted mice and were found in close contact with MM cells. MM cells secreted chemokines that attracted Th cells, secretion was augmented by CD40-stimulation. Within 14 days of culture of whole BM aspirates in autologous serum, MM cells and Th cells mutually stimulated each other, and MM cells required Th cells for further expansion in vitro and in mice. The results suggest that Th cells may support the expansion of MM cells in patients.

Identifying Low Muscle Mass in Patients with Hip Fracture: Validation of Biolectrical Impedance Analysis and Anthropometry Compared to Dual Energy X-ray Absorptiometry.

BACKGROUND: Older hip fracture patients often have reduced muscle mass, which is associated with adverse outcomes. Dual energy X-ray absorptiometry (DXA) can determine muscle mass, but is not practical in the acute phase. We investigated bioelectrical impedance analysis (BIA) and anthropometry compared against DXA for detecting low muscle mass in hip fracture patients. METHODS: This was a cross-sectional validation study at two Norwegian hospitals on 162 hip fracture patients aged ≥ 65 years. Appendicular lean mass (ALM) was determined by DXA, BIA and anthropometry 3 months after hip fracture. ALM by BIA was calculated by the Kyle, Janssen, Tengvall and Sergi equations, and ALM by anthropometry by the Heymsfield and Villani equations. The area under the receiver operating characteristic curve (AUC) was used to compare BIA and anthropometry for determining low ALM (≤5.67 kg/m2 for women and ≤7.25kg/m2 for men). RESULTS: Mean age was 79 years (SD 7.9), 74% were female. Mean ALM by DXA was 14.8 kg (SD 2.3) for women and 20.8 kg (SD 4.2) for men and 45% of women and 60% of men had low ALM. BIA (Kyle) in women (AUC 0.81, 95% confidence interval 0.72-0.89) and BIA (Sergi) in men (AUC 0.89, 95% CI 0.80-0.98) were best able to discriminate between low and normal ALM. Anthropometry (Heymsfield) was less accurate than BIA in women (AUC 0.64, 95% CI 0.54-0.75), and equal to BIA in men (AUC 0.72, 95% CI 0.72 0.56-0.87). CONCLUSION: BIA (Sergi, Kyle and Tengvall) and anthropometry (Heymsfield) can identify low muscle mass in hip fracture patients.

Specificity of antibody and helper T-cell responses to the isologous myeloma protein W3129 and its subunits.

The specificity of BALB/c antibodies and Th elicited by BALB/c myeloma protein W3129 (alpha, kappa) and its subunits was studied. Antibodies were detected with RIA and ELISA techniques. Th were demonstrated by their ability to augment a secondary anti-NIP antibody response in a Mitchison type assay of adoptive immunity. The major proportion of antibodies elicited by the complete W3129 was directed to an idiotypic determinant(s) that depended on assembled H + L chains. The determinant(s) was probably located in or near the antibody combining site because binding was hapten-inhibitable. A second minor antibody population bound an idiotypic determinant(s) on VW3129H expressed on isolated H-chain as well as on the complete myeloma protein. A third and very weakly reactive set of antibodies was specific for a C alpha antigenic site(s) which was expressed much more efficiently on free than on assembled alpha-chains. The antibody response to free kappa W3129 was directed to idiotypic determinants that were inaccessible in the complete molecule. By contrast, free kappa W3129 elicited Th that responded to an idiotypic determinant(s) on VW3129K; the determinant(s) was expressed on both the isolated chain and the complete W3129, suggesting that Th responded to an idiotope not recognized by B-cells. Priming with free alpha W3129 failed in four out of five experiments to induce Th that responded to the complete W3129, demonstrating that a major difference existed between VH and VL of W3129 regarding their immunogenicity for Th. Nevertheless, free alpha W3129 did elicit antibody responses that displayed high reactivity with the complete molecule, indicating that certain serologically defined antigenic sites on the surface of W3129 are also expressed on isolated alpha W3129. Thus, certain differences were detected as to the specificities of Th and B-cells for W3129 and its subunits since they recognized separate idiotopes located in the VL- or VH-region, respectively. The pattern of Th recognition of W3129 resembled that of another isologous myeloma protein, M315, but was unlike that of a third, J558, previously described from this laboratory.

Immunoglobulin as a vehicle for foreign antigenic peptides immunogenic to T cells.

Antibody (Ab) molecules may serve as targeting vehicles for delivery of foreign antigenic peptides to antigen presenting cells (APC). An attractive strategy is to substitute segments between beta-strands of immunoglobulin (Ig) constant (C)-region domains with antigenic peptides. For this to work, the mutant Ab must maintain its conformation so that it can be secreted from transfected cells. Furthermore, the antigenic peptides must be excised by the processing machinery of APC and loaded onto major histo-compatibility complex (MHC) class II molecules. To test this, we have introduced a peptide of eleven amino acids (a.a.) as either of three different loops in the first C-region domain of the heavy (H) chain (CH1) of human IgG3. When the resulting mutant H chain genes were expressed in a fibroblast cell line equipped with proper class II molecules, the H chains were retained intracellularly, probably due to the light (L) chain deficiency of the fibroblasts. Nevertheless, by the endogenous class II processing pathway, presentation of the epitope to CD4+ cells was observed for all three mutants. The presentation efficiency, however, depended on the position of the peptide in the H chain. This could be due to influence of flanking sequences, which differ in the three loop replacement mutants. When L chain-expressing Chinese hamster ovary (CHO) lambda cells were transfected with the same constructs, two out of the three mutant Ig were secreted. The mutants had the expected antigen specificity and were recognized by anti-IgG Ab. When added exogenously to dendritic cell APC, the mutant IgG3 were processed, and the liberated foreign epitopes presented to T cells. The results suggest that the loops connecting beta-strands in the Ig fold may be replaced by foreign peptides, which upon processing become stimulatory to CD4+ T cells. Combined with the well-known targeting function of antibodies, this principle may be useful for construction of a new generation of vaccines.