Affinity-matured antibody with a disulfide bond in H-CDR3 loop.

Affinity maturation increases antigen-binding affinity and specificity of antibodies by somatic hypermutation. Various monoclonal antibodies against (4-hydroxy-3-nitrophenyl)acetyl (NP) were obtained during affinity maturation. Among them, highly matured anti-NP antibodies, such as E11 and E3, possess Cys96H and Cys100H in the complementarity-determining region 3 of the heavy chain, which would form a disulfide bond. In this study, we evaluated the effects of disulfide bonds on antigen binding by generating single-chain Fv (scFv) antibodies of E11 and its mutants, E11_C96KH/C100EH and E11_C96KH/C100QH, and determined their antigen-binding thermodynamics and kinetics. The binding affinities of the Cys mutants were lower than that of E11 scFv, indicating that the disulfide bond contributed to antigen binding, especially for stable complex formation. This was also supported by the decreased affinity of E11 scFv in the presence of a reducing agent. The crystal structures of NP-free and NP-bound E11 scFvs were determined at high resolution, showing the existence of a disulfide bond between Cys96H and Cys100H, and the antigen recognition mechanism, which could be compared with those of other anti-NP antibodies, such as germline-type N1G9 and matured-type C6, as reported previously. These structures could explain the molecular basis of changes in antigen-binding affinity and thermal stability in the absence or presence of antigens. Small-angle X-ray scattering further showed a local conformational change in E11 scFv upon antigen binding in solution.

An alpaca single-domain antibody (VHH) phage display library constructed by CDR shuffling provided high-affinity VHHs against desired protein antigens.

Antigen-combining sites of the camelid heavy-chain antibody variable domain (VHH) are constructed by three complementarity-determining regions (CDR1, CDR2 and CDR3). We prepared cDNA using mRNA extracted from peripheral lymphocytes of alpacas that had been non-immunized or immunized with human serum albumin (HSA). The VHH gene fragments encoding the amino-terminal half-containing CDR1 as well as CDR2 and the carboxy-terminal half-containing CDR3 were amplified independently by PCR, and then full-length VHH gene fragments were generated by overlap extension PCR and cloned into the phagemid vector. This protocol, referred to as CDR shuffling, allowed us to construct an alpaca VHH phage display library possessing repertoires different from those naturally occurring in animals. We asked, first, whether this library was able to provide the functional VHH fragments against HSA, an immunized antigen, and obtained 29 anti-HSA VHH clones, 41% possessed KD values of lower than 10-8 M, 5 of which had KD values of 10-10 M. We also obtained VHH clones against non-immunized protein antigens such as cardiac troponin T and I, Ebola virus glycoprotein 1 and human immunoglobulin G by biopanning. We compared the amino acid sequences and affinities and found that 43% of VHHs had KD values of less than 10-8 M, although those having KD values of 10-10 M were unavailable. These results suggested that the CDR-shuffled VHH phage display library could potentially provide VHHs against non-immunized protein antigens with similar levels of affinities to those against immunized antigens.

A Trade-off Between Thermostability and Binding Affinity of Anti-(4-hydroxy-3-nitrophenyl)Acetyl Antibodies During the Course of Affinity Maturation.

Somatic hypermutation (SHM) is one of the driving forces that increases antibody (Ab) affinity. We studied the effects of SHM on thermostability and affinity using three single-chain Fv fragments (scFvs) of anti-(4-hydroxy-3-nitrophenyl)acetyl Abs, namely 9TG, 9T7, and E11. 9TG has a germline structure that lacks SHM and is an ancestor of 9T7 with 11 mutations. E11, which has 21 mutations, is a mature Ab and has its own ancestor. The thermostabilities and antigen-Ab interactions were analyzed by circular dichroism (CD), differential scanning calorimetry (DSC), and isothermal titration calorimetry (ITC). Far-UV CD spectra showed that all scFvs were folded into a structure referred to as immunoglobulin-fold and were unfolded by heating at different melting temperatures. Comparison of thermodynamic parameters obtained from DSC and ITC revealed that the magnitude of stabilization free energy at 37 °C was in the order, 9TG > 9T7 > E11, while that of the free energy of interaction with antigen was 9TG < 9T7 < E11, suggesting that Abs make a trade-off between stability and affinity during affinity maturation.

Three-dimensional structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody possessing a glycine residue at position 95 of the heavy chain.

Antibodies possessing high affinity and specificity are desired as therapeutic reagents and biosensor materials. Such antibodies are often obtained from immunized animals through the process referred to as affinity maturation where antibody affinity increases with time after immunization. Somatic hypermutation (SHM) was shown to be involved in this process; however, structural basis of affinity maturation has not well been understood yet. We analyzed the crystal structure of a high affinity anti-(4-hydroxy-3-nitrophenyl)acetyl antibody, C6, possessing Gly at position 95 of heavy chain and 17 amino acid replacements by SHM. Here, we discuss how the amino acid residues at position 95, introduced at a junction of VH and DH gene segments during gene-recombination, as well as those replaced by SHM contribute to increasing the affinity by comparing the C6 structure with that of a germline low affinity antibody, N1G9, possessing Tyr at position 95.

High-affinity IgM+ memory B cells are defective in differentiation into IgM antibody-secreting cells by re-stimulation with a T cell-dependent antigen.

IgM antibodies (Abs) are thought to play a major role in humoral immunity but only at the early stage of the primary immune response. However, two subsets of IgM+ memory B cells (MBCs), one with high affinity gained by means of multiple somatic hypermutation (SHM) and the other with low affinity and no SHMs, are generated through the germinal center (GC)-dependent and GC-independent (non-GC) pathway, respectively, after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP)-chicken γ-globulin. Surprisingly, an analysis of antibody-secreting cells reveals that a large amount of anti-NP IgM Ab with few SHMs is secreted during the recall response, indicating that only non-GC MBCs have terminal differentiation potential. Since secondary IgM Abs are capable of binding to dinitrophenyl ligands, they likely provide broad cross-reactivity in defense against microbial infection.

Pronounced effect of hapten binding on thermal stability of an anti-(4-hydroxy-3-nitrophenyl)acetyl antibody possessing a glycine residue at position 95 of the heavy chain.

Immune response to T-cell-dependent antigens is highly dynamic; several B-cell clones responsible for antibody production appear alternately during immunization. It was previously shown that at least two-types of antibodies are secreted after immunization with (4-hydroxy-3-nitrophenyl)acetyl (NP); one has Tyr and another has Gly at position 95 of the heavy chain (referred to as Tyr95- and Gly95-type). The former appeared at an early stage, while the latter appeared at a late stage, i.e., after secondary immunization, although Fv domains of these antibodies were encoded by same genes of variable heavy and light chains. We examined whether any biophysical properties of antigen-combing sites relate to this shift in B-cell clones by preparing single-chain Fv (scFv). Thermodynamic and kinetic parameters of the interaction of scFv with various haptens are in accordance with those of intact antibodies, indicating that scFvs are appropriate models for the study on structure and function of antibodies. Next, we measured thermal stability of scFvs using differential scanning calorimetry and found that the apparent melting temperature of free Tyr95-type was 64-66°C,while that of Gly95-type was 47-48°C, indicating that the latter was highly unstable. However, Gly95-type greatly gained thermal stability because of hapten binding. We discussed the relationship between thermal stability resulted by hapten binding and dynamism of antibody response during immunization.

Somatic hypermutation of immunoglobulin genes in Rad18 knockout mice.

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is triggered by the activity of activation-induced cytidine deaminase (AID). AID induces DNA lesions in variable regions of Ig genes, and error-prone DNA repair mechanisms initiated in response to these lesions introduce the mutations that characterize SHM. Error-prone DNA repair in SHM is proposed to be mediated by low-fidelity DNA polymerases such as those that mediate trans-lesion synthesis (TLS); however, the mechanism by which these enzymes are recruited to AID-induced lesions remains unclear. Proliferating cell nuclear antigen (PCNA), the sliding clamp for multiple DNA polymerases, undergoes Rad6/Rad18-dependent ubiquitination in response to DNA damage. Ubiquitinated PCNA promotes the replacement of the replicative DNA polymerase stalled at the site of a DNA lesion with a TLS polymerase. To examine the potential role of Rad18-dependent PCNA ubiquitination in SHM, we analyzed Ig gene mutations in Rad18 knockout (KO) mice immunized with T cell-dependent antigens. We found that SHM in Rad18 KO mice was similar to wild-type mice, suggesting that Rad18 is dispensable for SHM. However, residual levels of ubiquitinated PCNA were observed in Rad18 KO cells, indicating that Rad18-independent PCNA ubiquitination might play a role in SHM.

Structural dynamics of a single-chain Fv antibody against (4-hydroxy-3-nitrophenyl)acetyl.

Protein structure dynamics are critical for understanding structure-function relationships. An antibody can recognize its antigen, and can evolve toward the immunogen to increase binding strength, in a process referred to as affinity maturation. In this study, a single-chain Fv (scFv) antibody against (4-hydroxy-3-nitrophenyl)acetyl, derived from affinity matured type, C6, was designed to comprise the variable regions of light and heavy chains connected by a (GGGGS)3 linker peptide. This scFv was expressed in Escherichia coli in the insoluble fraction, solubilized in the presence of urea, and refolded by stepwise dialysis. The correctly refolded scFv was purified, and its structural, physical, and functional properties were analyzed using analytical ultracentrifugation, circular dichroism spectrometry, differential scanning calorimetry, and surface plasmon resonance biosensor. Thermal stability of C6 scFv increased greatly upon antigen binding, due to favorable enthalpic contributions. Antigen binding kinetics were comparable to those of the intact C6 antibody. Structural dynamics were analyzed using the diffracted X-ray tracking method, showing that fluctuations were suppressed upon antigen binding. The antigen binding energy determined from the angular diffusion coefficients was in good agreement with that calculated from the kinetics analysis, indicating that the fluctuations detected at single-molecule level are well reflected by antigen binding events.

Affinity maturation of anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies accompanies a modulation of antigen specificity.

Anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies bearing λ1 chains are known to possess fine specificity, referred to as heterocliticity, which causes these antibodies to bind to hapten analogues such as (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and (4-hydroxy-3,5-dinitrophenyl)acetyl (NNP) with higher affinity than to the autologous hapten, NP. They also show preferential binding to the phenolate form of hapten than to the phenolic form. We address here the question of whether affinity maturation accompanies in the fine specificity of these antibodies by analyzing the interaction between NP1-, NIP1-, or NNP1-hen egg lysozyme and anti-NP antibodies that possess different association constants to NP using a surface plasmon resonance biosensor. We measured interactions at various pH values and found that heterocliticity as well as preferential binding to the phenolate form of hapten were most prominent in a germline antibody having immature affinity and that fine specificity becomes less evident, i.e., anti-NP antibodies become more specific to the immunizing antigen, NP during the process of affinity maturation.

High affinity IgM(+) memory B cells are generated through a germinal center-dependent pathway.

During a T cell-dependent immune response, B cells undergo clonal expansion and selection and the induction of isotype switching and somatic hypermutation (SHM). Although somatically mutated IgM(+) memory B cells have been reported, it has not been established whether they are really high affinity B cells. We tracked (4-hydroxy-3-nitrophenyl) acetyl hapten-specific GC B cells from normal immunized mice based on affinity of their B cell receptor (BCR) and performed BCR sequence analysis. SHM was evident by day 7 postimmunization and increased with time, such that high affinity IgM(+) as well as IgG(+) memory B cells continued to be generated up to day 42. In contrast, class-switch recombination (CSR) was almost completed by day 7 and then the ratio of IgG1(+)/IgM(+) GC B cells remained unchanged. Together these findings suggest that IgM(+) B cells undergo SHM in the GC to generate high affinity IgM(+) memory cells and that this process continues even after CSR is accomplished.

An asymmetric antibody repertoire is shaped between plasmablasts and plasma cells after secondary immunization with (4-hydroxy-3-nitrophenyl)acetyl chicken γ-globulin.

Studies on the structural basis of antibody affinity maturation have been carried out by measuring the affinity of secreted antibodies, and information on structures has often been obtained from nucleotide sequences of BCRs of memory B cells. We considered it important to establish whether the repertoire of secreted antibodies from plasma cells is really in accord with that of BCRs on memory B cells at the same time points post-immunization. We isolated plasma cells secreting antibodies specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten by affinity matrix technology using biotin-anti-CD138 and streptavidin-NP-allophycocyanin, to which anti-NP antibodies secreted by autologous plasma cells bound preferentially. We found that plasmablasts occupied >90% of the antibody-secreting cell compartment in the primary response and that they secreted antibodies whose VH regions were encoded by V186.2(+)Tyr95(+) sequences, which provided an increase in the medium level of affinity by somatic hypermutation (SHM) of heavy chains at position 33. After secondary immunization, a further increase in antibody affinity was observed, which was explained by the appearance of a number of plasma cells secreting V186.2(+)Gly95(+) antibodies that acquired high affinity by multiple SHMs as well as plasmablasts secreting V186.2(+)Tyr95(+) antibodies. However, we did not detect any plasmablasts secreting V186.2(+)Gly95(+) antibodies, showing that plasmablasts and plasma cells have a different antibody repertoire, i.e. their respective repertoires are asymmetric. On the basis of these findings, we discussed the relationship between the BCR affinity of memory B cells and plasmablasts as well as plasma cells as pertaining to their ontogeny.

Detection and isolation of anti-hapten antibody-secreting cells by cellular affinity matrix technology.

We developed a method to detect and isolate plasma cells that produce antigen-specific antibodies. An affinity matrix of hapten was constructed on a cell surface, and subsequent incubation allowed cells to secrete antibodies. Anti-hapten antibodies preferentially bound to the affinity matrix on the cells from which they were secreted. We showed that the combination of surface biotinylation and streptavidin which was conjugated with a high valence of hapten was suitable for sensitive detection of antibody binding. Using this protocol, anti-hapten plasma cells from immunized mouse spleen were detected and enriched by flow cytometry. This method allows for isolation of intact plasma cells according to the antibody specificity and may be useful for highly efficient and precise analysis of an antibody repertoire.

Generation of a Tlx1(CreER-Venus) knock-in mouse strain for the study of spleen development.

The spleen is a lymphoid organ that serves as a unique niche for immune reactions, extramedullary hematopoiesis, and the removal of aged erythrocytes from the circulation. While much is known about the immunological functions of the spleen, the mechanisms governing the development and organization of its stromal microenvironment remain poorly understood. Here we report the generation and analysis of a Tlx1(Cre) (ER) (-Venus) knock-in mouse strain engineered to simultaneously express tamoxifen-inducible CreER(T2) and Venus fluorescent protein under the control of regulatory elements of the Tlx1 gene, which encodes a transcription factor essential for spleen development. We demonstrated that Venus as well as CreER expression recapitulates endogenous Tlx1 transcription within the spleen microenvironment. When Tlx1(Cre) (ER) (-Venus) mice were crossed with the Cre-inducible reporter strain, Tlx1-expressing cells as well as their descendants were specifically labeled following tamoxifen administration. We also showed by cell lineage tracing that asplenia caused by Tlx1 deficiency is attributable to altered contribution of mesenchymal cells in the spleen anlage to the pancreatic mesenchyme. Thus, Tlx1(Cre) (ER) (-Venus) mice represent a new tool for lineage tracing and conditional gene manipulation of spleen mesenchymal cells, essential approaches for understanding the molecular mechanisms of spleen development.

Low-affinity IgM antibodies lacking somatic hypermutations are produced in the secondary response of C57BL/6 mice to (4-hydroxy-3-nitrophenyl)acetyl hapten.

Class-switched memory B cells, which are generated through the processes of somatic hypermutation (SHM) and affinity-based selection in germinal centers, contribute to the production of affinity-matured IgG antibodies in the secondary immune response. However, changes in the affinity of IgM antibodies during the immune response have not yet been studied, although IgM(+) memory B cells have been shown to be generated. In order to understand the relationship between IgM affinity and the recall immune response, we prepared hybridomas producing anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) IgM antibodies from C57BL/6 mice and from activation-induced cytidine deaminase (AID)-deficient mice. Binding analysis by ELISA showed that mAbs obtained from the secondary immune response contained IgM mAbs with affinity lower than the affinity of mAbs obtained from the primary response. By analyzing sequences of the IgM genes of hybridomas and plasma cells, we found many unmutated VH genes. VH genes that had neither tyrosine nor glycine at position 95 were frequent. The repertoire change may correlate with the lower affinity of IgM antibodies in the secondary response. The sequence and affinity changes in IgM antibodies were shown to be independent of SHM by analyzing hybridomas from AID-deficient mice. A functional assay revealed a reciprocal relationship between affinity and complement-dependent hemolytic activity toward NP-conjugated sheep RBCs; IgM antibodies with lower affinities had higher hemolytic activity. These findings indicate that lower affinity IgM antibodies with enhanced complement activation function are produced in the secondary immune response.

Multiarray formation of CHO spheroids cocultured with feeder cells for highly efficient protein production in serum-free medium.

Functional proteins like antibody, cytokine and growth factor have been widely used for basic biological research, diagnosis and cancer therapy. Particularly, antibody drugs as attractive biopharmaceuticals will be expected to create an enormous new market. Chinese hamster ovay (CHO) cells are being increasingly used in industry for the production of recombinant therapeutic proteins including antibody drugs. Although three-dimensional culture is preferred to two-dimensional monolayer culture for the efficient large scale culture of CHO cells and subsequent mass production of recombinant proteins, it has the limitation of low protein production. Therefore, a new cell culture em essentially required for an efficient protein production. Here we report on a new three-dimensional cell culture system as a spheroid cell culture on the micropattern array for efficient production of protein in CHO cells. Furthermore, cocultivation of CHO spheroids with feeder cells including bovine aortic endothelial cells (BAEC) and NIH 3T3 was essential to more increase a protein production. The results indicated that CHO heterospheroids cocultured with BAECs were much superior to either CHO monolayers or CHO homospheroids in protein production. Significantly, the above cocultured spheroids in the serum-free medium drastically enhanced protein expression level up to 3-fold compared with CHO spheroids in serum medium, suggesting that a coculture of spheroid system with feeder layer cells is a promising method to enhance protein production under serum-free condition. The spheroid array constructed here is highly usuful as a platform of biopharmaceutical manufacturing as well as tissue and cell based biosensors to detect a wide variety of clinically active compounds through a cellular physiological response.

RBMX: a regulator for maintenance and centromeric protection of sister chromatid cohesion.

Cohesion is essential for the identification of sister chromatids and for the biorientation of chromosomes until their segregation. Here, we have demonstrated that an RNA-binding motif protein encoded on the X chromosome (RBMX) plays an essential role in chromosome morphogenesis through its association with chromatin, but not with RNA. Depletion of RBMX by RNA interference (RNAi) causes the loss of cohesin from the centromeric regions before anaphase, resulting in premature chromatid separation accompanied by delocalization of the shugoshin complex and outer kinetochore proteins. Cohesion defects caused by RBMX depletion can be detected as early as the G2 phase. Moreover, RBMX associates with the cohesin subunits, Scc1 and Smc3, and with the cohesion regulator, Wapl. RBMX is required for cohesion only in the presence of Wapl, suggesting that RBMX is an inhibitor of Wapl. We propose that RBMX is a cohesion regulator that maintains the proper cohesion of sister chromatids.

In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo.

In response to T cell-dependent antigens, B cells proliferate extensively to form germinal centres (GC), and then differentiate into memory B (B(mem)) cells or long-lived plasma cells (LLPCs) by largely unknown mechanisms. Here we show a new culture system in which mouse naïve B cells undergo massive expansion and isotype switching, and generate GC-phenotype B (iGB) cells. The iGB cells expressing IgG1 or IgM/D, but not IgE, differentiate into B(mem) cells in vivo after adoptive transfer and can elicit rapid immune responses with the help of cognate T cells. Secondary culture with IL-21 maintains the proliferation of the iGB cells, while shifting their in vivo developmental fate from B(mem) cells to LLPCs, an outcome that can be reversed by withdrawal of IL-21 in tertiary cultures. Thus, this system enables in vitro manipulation of B-cell fate, into either B(mem) cells or LLPCs, and will facilitate dissection of GC-B cell differentiation programs.

Characterization of memory B cells responsible for affinity maturation of anti- (4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies.

We searched for memory B cells responsible for high-affinity anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody production by C57BL/6 mice immunized with NP-chicken γ-globulin (CGG), using flow cytometry. We first prepared transfectants expressing B-cell antigen receptor (BCR) of known affinity as a memory B-cell model as well as NP-allophycocyanin (APC) of different NP valences, NP(lo), NP(med) and NP(hi). We then used the latter as probes capable of distinguishing BCR affinities: NP(lo)-APC bound to BCRs with an affinity higher than 3.4 × 10(6) M(-1), while NP(med)-APC bound to those with a higher than germline affinity. B cells capable of binding to NP(lo)-APC appeared in spleens on day 14 post-immunization, and harbored Tyr95 (Tyr95 type) as well as a mutation from Trp33 to Leu. B cells with BCRs harboring Gly95 (Gly95 type) appeared only in the NP(med)-APC-binding fraction on day 56 and in the NP(lo)-APC-binding fraction on day 77, indicating that this long duration was necessary for Gly95 type B cells to acquire high affinity and to become a member of the group of memory B cells with high affinity. Administration of NP-CGG on day 77 caused little change in the proportion of the Gly95 type in NP(lo)-APC-binding B cells in the following 2 weeks but brought about an increase in the number of high-affinity antibody-secreting cells (ASC), suggesting that the memory B-cell compartment established was maintained at a later stage and supplied high-affinity ASCs. The relationship between these Gly95 type memory B cells and ASCs is discussed.

Conformational analysis of human serum albumin and its non-enzymatic glycation products using monoclonal antibodies.

Monoclonal antibodies (mAbs) were prepared to analyse the conformation of human serum albumin (HSA) and its non-enzymatic glycation (NEG) products. We first determined the epitopes of the mAbs using HSA subdomains expressed on the surface of yeast. Each mAb was classified as belonging to one of two groups; Type I mAbs which recognized a single subdomain structure and Type II mAbs which bound to plural subdomains. We analysed the pH-dependent conformational change in HSA. We found that one Type II mAb, HAy2, detected the normal to base form (N-B) transition while the other did not, suggesting that N-B transition occurred around Domain I accompanied by topological isomerization of subdomains without changing the subdomain structure itself. Next, we analysed the conformations of the NEG products. Since all mAbs reacted with the early NEG products, no structural change was thought to have occurred in the early NEG products. On the other hand, only a Type I mAb, HAy1, had full binding activity with the advanced glycation end products (AGE) while the other mAbs had lost or had diminished activity, suggesting that the over-all tertiary structure of HSA was altered except for a subdomain, sDOM Ia, in AGE.

The amino acid residue at position 95 and the third CDR region in the H chain determine the ceiling affinity and the maturation pathway of an anti-(4-hydroxy-3-nitrophenyl)acetyl antibody.

Two groups of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) Abs each possessing a different amino acid, Tyr or Gly, at position 95, appeared respectively at early and late stages of immunization. The early Abs predominantly harbored Tyr95 and were referred to as the Tyr95 type. These had ∼100-fold lower ceiling affinity than the late Abs harboring Gly95, which were referred to as the Gly95 type. We found that in order to raise affinity, the Tyr95 type utilized a mutation at position 33 in V(H), while the Gly95 type used multiple mutations in both V(H) and V(L), and that the effect of the mutations was reciprocal; the former mutation had a positive effect on Tyr95 type Abs but a negative effect on Gly95 type Abs, and vice versa. The reciprocal effect of these mutations on affinity enabled us to assess the type of Abs prepared by introducing 20 different amino acids at position 95. We found that Abs harboring Lys95, Arg95, Pro95, and Tyr95 belonged to the Tyr95 type and those with Ala95 and Gly95, to the Gly95 type. Since this dependency on the amino acid at position 95 was observed in H chains whose third CDR (CDR 3H) consisted of 9 amino acids and not 11, the CDR 3H region was also considered to play an important role in determining the maturation pathway and the magnitude of the ceiling affinity.