PspK of Streptococcus pneumoniae increases adherence to epithelial cells and enhances nasopharyngeal colonization.

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and can cause invasive disease aided by the pneumococcal capsule. Group II nontypeable S. pneumoniae (NTSp) lacks a polysaccharide capsule, and a subgroup of NTSp carriage isolates has been found to have a novel gene, pneumococcal surface protein K (pspK), which replaces the capsule locus. A recent rise in the number of NTSp isolates colonizing the human nasopharynx has been observed, but the colonization factors of NTSp have not been well studied. PspK has been shown to play a role in mouse colonization. We therefore examined PspK-mediated immune evasion along with adherence to host cells and colonization. PspK bound human secretory immunoglobulin A (sIgA) but not the complement regulator factor H and did not decrease C3b deposition on the pneumococcal surface. PspK increased binding of pneumococci to epithelial cells and enhanced pneumococcal colonization independently of the genetic background. Understanding how NTSp colonizes and survives within the nasopharynx is important due to the increase in NTSp carriage. Our data suggest that PspK may aid in the persistence of NTSp within the nasopharynx but is not involved in invasion.

Evolution of the capsular gene locus of Streptococcus pneumoniae serogroup 6.

Streptococcus pneumoniae expressing serogroup 6 capsules frequently causes pneumococcal infections and the evolutionary origins of the serogroup 6 strains have been extensively studied. However, these studies were performed when serogroup 6 had only two known members (serotypes 6A and 6B) and before the two new members (serotypes 6C and 6D) expressing wciN(ß) were found. We have therefore reinvestigated the evolutionary origins of serogroup 6 by examining the profiles of the capsule gene loci and the multilocus sequence types (MLSTs) of many serogroup 6 isolates from several continents. We confirmed that there are two classes of cps locus sequences for serogroup 6 isolates. In our study, class 2 cps sequences were limited to a few serotype 6B isolates. Neighbour-joining analysis of cps sequence profiles showed a distinct clade for 6C and moderately distinct clades for class 1 6A and 6B sequences. The serotype 6D cps profile was found within the class 1 6B clade, suggesting that it was created by recombination between 6C and 6B cps loci. Interestingly, all 6C isolates also had a unique wzy allele with a 6 bp deletion. This suggests that serotype switching to 6C involves the transfer of a large (>4 kb) gene segment that includes both the wciN(ß) allele and the 'short' wzy allele. The MLST studies of serotype 6C isolates suggest that the 6C cps locus is incorporated into many different pneumococcal genomic backgrounds but that, interestingly, 6C cps may have preferentially entered strains of the same genomic backgrounds as those of serotype 6A.

Unbalanced X chromosome mosaicism in B cells of mice with X-linked immunodeficiency.

The immunodeficiency in CBA/N mice is reflected by abnormal development of a subset of B lymphocytes. However, it is not clear how xid, the mutant gene in CBA/N mice, affects the development of this subset. Specifically, it is not known if the xid gene influences the development of the B cell subset directly or indirectly by providing the improper developmental milieu through effects on other cells. We investigated this question using female mice heterozygous for two x chromosomal genes, xid and Pgk-1 (phosphoglycerate kinase-1). Since females are mosaic because of x chromosome inactivation, their lymphocytes can be studied for the choice of the x chromosome, using the two PGK-1 isoenzymes as the cytological marker. We find that B lymphocytes in the spleen prefer the x chromosome without xid while the remaining splenocytes and cells from other tissues do not. This suggests that xid affects B lymphocytes directly and not through their developmental milieu. Furthermore, our data suggest that the precursors for IgG1- and IgG3-producing cells may be both few and different.

Subclass restriction of murine antibodies. II. The IgG plaque-forming cell response to thymus-independent type 1 and type 2 antigens in normal mice and mice expressing an X-linked immunodeficiency.

Antigens have been classified previously into three categories, thymus-dependent (TD), thymus-independent type (TI) 1, and TI-2, based upon thymic dependence and ability to stimulate an immunodeficient strain of mouse, CBA/N. Here we demonstrate that the different antigen classes elicit IgG antibodies of different subclasses. TD antigens stimulate predominantly IgG1 antibodies, with smaller amounts of IgG2 and IgG3 being expressed. TI-1 antigens stimulate almost no IgG1 antibodies and equal amounts of IgG2 and IgG3. TI-2 antigens elicit predominantly IgG3 antibodies. Mice expressing the CBA/N phenotype are known to be nonresponsive to TI-2 antigens. This was confirmed in this study. In addition, we demonstrate that the IgG3 component of the response to TI-1 antigens is virtually absent in mice expressing the CBA/N phenotype, which supports our previous finding that the CBA/N defect may be restricted to a B-lymphocyte subpopulation containing most of the precursors of IgG3-secreting cells.

Antiphosphocholine antibodies found in normal mouse serum are protective against intravenous infection with type 3 streptococcus pneumoniae.

The antiphosphocholine (PC) antibody in normal mouse sera (NMS) provides protection against intravenous infection with encapsulated strain WU2 of type 3 Streptococcus pneumoniae. Mice unable to make anti-PC antibody, as a result of suppression with anti-T-15 idiotype or inheritance of the xid gene of CAB/N mice, are highly susceptible to infection with strain WU2. Mice inheriting the xid gene can be protected with NMS from immunologically normal mice or with IgM hybridoma anti-PC antibody. The protective effect of NMS can be removed with PC-containing immunoabsorbents.

Immunoglobulin subclass-specific immunodeficiency in mice with an X-linked B-lymphocyte defect.

CBA/N mice express an X-linked deficiency in their antibody response to many bacterial carbohydrates; we have shown recently that these antigens normally elicit antibody responses predominantly of the IgM and IgG3 isotypes. Here we demonstrate that mice, with the CBA/N phenotype have perferential deficiencies of IgM and IgG3 immunoglobulin expression, both when measured in serum and in cells secreting these isotypes, and that this deficiency is only partially corrected by polyclonal activation of B cells. This suggests that CBA/N mice may lack a subpopulation of B cells that contain most of the IgG3 precursors.

Multiple VH gene segments encode murine antistreptococcal antibodies.

Most mouse strains are able to mount a diverse antibody response against group A streptococcal carbohydrate (GAC). We have previously reported that murine anti-GAC antibodies are for the most part restricted to IgM and IgG3 subclasses. In addition, despite extensive heterogeneity in their isoelectric focusing patterns, greater than 50% of A/J anti-GAC antibodies share a common light chain defined by spectrotypic and idiotypic (VK1GAC) criteria. We have used protein and DNA sequencing strategies to examine the genetic basis of diversity in murine anti-GAC antibodies. In particular, we report that, (a) multiple, closely homologous VH gene segments contribute to the generation of anti-GAC antibodies, (b) a common framework sequence, related to the VK27 subgroup, probably defines VK1GAC, and (c) the A/J anti-GAC VH regions and BALB/c anti-inulin VH sequences are 95% homologous at the protein level and are likely encoded by overlapping VH gene families. Lastly, we discuss the genetic mechanisms that might permit the evolution of multiple, closely homologous germline VH gene segments in the context of highly divergent flanking region sequences.

Distinct roles of lymphotoxin alpha and the type I tumor necrosis factor (TNF) receptor in the establishment of follicular dendritic cells from non-bone marrow-derived cells.

In mice deficient in either lymphotoxin alpha (LT-alpha) or type I tumor necrosis factor receptor (TNFR-I), organized clusters of follicular dendritic cells (FDC) and germinal centers (GC) are absent from the spleen. We investigated the role of LT-alpha and TNFR-I in the establishment of spleen FDC and GC structure by using reciprocal bone marrow (BM) transfer. When LT-alpha-deficient mice were reconstituted with wild-type BM, FDC organization and the ability to form GC were restored, indicating that the LT-alpha-expressing cells required to establish organized FDC are derived from BM. The role of LT-alpha in establishing organized FDC structure was further investigated by the transfer of complement receptor 1 and 2 (CR1/2)-deficient BM cells into LT-alpha-deficient mice. Organized FDC were identified with both the FDC-M1 and anti-CR1 monoclonal antibodies in these BM-chimeric mice, indicating that these cells were derived from the LT-alpha-deficient recipient. Thus, expression of LT-alpha in the BM-derived cells, but not in the non-BM-derived cells, is required for the maturation of FDC from non-BM precursor cells. In contrast, when TNFR-I-deficient mice were reconstituted with wild-type BM, they showed no detectable FDC clusters or GC formation. This indicates that TNFR-I expression on non-BM-derived cellular components is necessary for the establishment of these lymphoid structures. TNFR-I-deficient BM was able to restore FDC organization and GC formation in LT-alpha-deficient mice, indicating that formation of these structures does not require TNFR-I expression on BM-derived cells. The data in this study demonstrate that FDC organization and GC formation are controlled by both LT-alpha-expressing BM-derived cells and by TNFR-I-expressing non-BM-derived cells.

Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers.

In mice deficient in either lymphotoxin-alpha (LT-alpha) or the type I tumor necrosis factor (TNF) receptor, but not the type II TNF receptor, germinal centers failed to develop in peripheral lymphoid organs. Germinal center formation was restored in LT-alpha-deficient mice by transplantation of normal bone marrow, indicating that the LT-alpha-expressing cells required to establish this lymphoid structure are derived from bone marrow.

A rapid pneumococcal serotyping system based on monoclonal antibodies and PCR.

Streptococcus pneumoniae expresses at least 91 different polysaccharide (PS) capsules and the currently available serotyping methods are tedious to perform. We have been developing a rapid pneumococcal serotyping assay (named the 'multibead assay') based on the capacity of pneumococcal lysates to inhibit the ability of 24 different anti-capsule antibodies to bind to latex beads coated with 24 different PSs (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9N, 9V, 14, 18C, 19A, 19F, 23F, 2, 8, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F). Because the polyclonal antibodies used for 10 serotypes (2, 8, 10A, 11A, 12F, 15B, 17F, 20, 22F and 33F) had limited serotype specificity, we replaced them with monoclonal antibodies for the 10 serotypes. To extend the serotype coverage beyond the 24 serotypes, we have adapted multiplexed PCR for five additional serotypes (15A, 15C, 16F, 35B and 38) to be useful with the pneumococcal lysates prepared for the multibead assay. We then validated the combined assay with 157 clinical isolates from the Centers for Disease Control and Prevention and found that the new combined assay produced results that are concordant with the quellung reaction. The combined assay is robust and could be used to rapidly identify the serotypes of the majority of pneumococci ( approximately 90 %). In addition, the assay validation study suggests the presence of serological subtypes within serotype 11A.

An idiotypic marker associated with a germ-line encoded kappa light chain variable region that predominates the vaccine-induced human antibody response to the Haemophilus influenzae b polysaccharide.

Human antibodies specific for the Haemophilus influenzae b polysaccharide (Hib PS) frequently express a cross-reactive idiotype (CRI), and commonly utilize a VL region that is the product of the V kappa II gene A2. To examine further anti-Hib PS V region expression and to determine whether CRI expression is correlated with the V kappa IIA2 chain, we isolated a monoclonal antibody (MAb) reactive with an idiotypic determinant of anti-Hib PS antibodies. This MAb inhibited Hib PS binding but did not react with Ig isotypic determinants. The CRI recognized by this MAb, designated HibId-1, was associated with the Hib PS-combining site since the reactivity of the MAb with anti-Hib PS antibodies could be inhibited by Hib PS. HibId-1 was expressed by 17 of 17 clonally purified and sequence-defined anti-Hib PS antibodies having V kappa IIA2 L chains. In contrast, 0 of 10 anti-Hib PS antibodies having either V lambda, V kappa I, or V kappa III chains expressed HibId-1. Western blot analysis showed that the MAb anti-CRI reacted with isolated anti-Hib PS V kappa IIA2 L chains but not with H chains or other L chains, indicating that the HibId-1 determinant is localized to the V kappa IIA2 chain, and does not require pairing with H chain for expression. Anti-Hib PS antibodies bearing HibId-1 were present in at least 85% of subjects immunized with either free Hib PS or Hib PS coupled to diphtheria toxoid (Hib PS-DT), and comprised on the average 60% of the total vaccine-induced serum anti-Hib PS. HibId-1 expression was not related to age at vaccination inasmuch as infants, children, and adults had similar distributions of HibId-1-positive anti-Hib PS after vaccination with Hib PS-DT. HibId-1 was expressed at a lower frequency and comprised a smaller fraction of the total anti-Hib PS antibody in adult preimmunization sera as compared to post-Hib PS immunization sera, suggesting that immunization preferentially stimulates HibId-1-positive B cells. These data demonstrate that antibodies bearing HibId-1/V kappa IIA2 comprise a predominant component of the anti-Hib PS response induced by immunization, and that this pattern of VL expression is established early in ontogeny.

Assignment of Opsonic Values to Pneumococcal Reference Serum 007sp for Use in Opsonophagocytic Assays for 13 Serotypes.

Opsonophagocytic assays (OPAs) are routinely used for assessing the immunogenicity of pneumococcal vaccines, with OPA data often being utilized for licensure of new vaccine formulations. However, no reference serum for pneumococcal OPAs is available, making evaluation of data among different laboratories difficult. This international collaboration was initiated to (i) assign consensus opsonic indexes (OIs) to FDA pneumococcal reference serum lot 007sp (here referred to as 007sp) and a panel of serum samples used for calibration of the OPA and (ii) determine if the normalization of the OPA results obtained with test samples to those obtained with 007sp decreases the variability in OPA results among laboratories. To meet these goals, six participating laboratories tested a panel of serum samples in five runs for 13 serotypes. For each serum sample, consensus OIs were obtained using a mixed-effects analysis of variance model. For the calibration serum samples, normalized consensus values were also determined on the basis of the results obtained with 007sp. For each serotype, the overall reduction in interlaboratory variability was calculated by comparing the coefficients of variation of the unadjusted and the normalized values. Normalization of the results substantially reduced the interlaboratory variability, ranging from a 15% reduction in variability for serotype 9V to a 64% reduction for serotype 7F. Normalization also increased the proportion of data within 2-fold of the consensus value from approximately 70% (average for all serotypes) to >90%. On the basis of the data obtained in this study, pneumococcal reference standard lot 007sp will likely be a useful reagent for the normalization of pneumococcal OPA results from different laboratories. The data also support the use of the 16 FDA serum samples used for calibration of the OPA as part of the initial evaluation of new assays or periodic assessment of established assays.

Physiological performance of beech (Fagus sylvatica L.) at its southeastern distribution limit in Europe: seasonal changes in nitrogen, carbon and water balance.

To assess the physiological performance of drought-sensitive European beech ( Fagus sylvatica L.) under the dry Mediterranean climate prevailing at its southeastern distribution limit in Europe, we analyzed seasonal changes in carbon, nitrogen and water balance of naturally grown adult trees. We determined the foliar C and N contents, delta13C and delta18O signatures, total soluble non-protein nitrogen compounds (TSNN) in xylem, leaves, and phloem, as well as leaf water potential and photosynthetic quantum yield in northern Greece during 2003. Tissue sampling was performed in May, July, and September, while field measurements were conducted regularly. Climatic conditions for the 2003 growing season fall within the typical range of the studied area. The N- and C-related parameters displayed distinct seasonal courses. TSNN was highest in May in all tissues, and asparagine (Asn) was then the most abundant compound. Thereafter, TSNN decreased significantly in all tissues and both its concentration and composition remained constant in July and September. In both months, glutamate (Glu) prevailed in leaves, gamma-aminobutyric acid (GABA) in phloem exudates from twigs and trunks, and arginine (Arg) in the xylem sap, where loading with amino acids was rather low during that period, amounting to only 0.8 micromol N ml-1 in September. Highest total foliar N and C contents were detected in May, and the elevated abundance of nutrients as well as an increased foliar delta13C signature at the beginning of the growing season is attributed to remobilization processes. The signatures of delta18O, quantum yield and leaf water potentials varied only slightly throughout the growing season. Although summer precipitation at the study site was considerably lower compared to what is usual for typical central European beech forests, no intensive drought responses of the physiological apparatus were detected in the studied beech trees. This suggests efficient internal regulation mechanisms, constantly ensuring a favourable physiological status under the relatively dry Mediterranean climate.

Human IgG subclass assays using a novel assay method.

To facilitate assays for human IgG subclasses, we have adapted a novel assay method (particle concentration fluorescence immunoassay) (Jolley et al., 1984; MacCrindle et al., 1985) using one polyclonal and several monoclonal antibodies for human IgG subclasses. The advantages of this new assay over previously described methods are sensitivity (0.3-3 micrograms/ml), and automated measurement of multiple samples in a short time (2 h). We found that a monkey antibody for IgG2 and monoclonal antibodies for IgG1, IgG4b epitope, and IgG3 can be adapted to this method. We evaluated 7 different antibodies to IgG4 without finding a suitable monoclonal antibody for this assay method. Several of these IgG4 hybridoma antibodies, however, could be used in a competitive radioimmunoassay using polyvinyl microtiter plates. The usefulness of a monoclonal antibody to the IgG4b epitope was evaluated because no suitable monoclonal antibodies for IgG2 are available. Because IgG4 levels are usually much smaller than IgG2 levels, and the IgG4b epitope is expressed on all IgG2 alleles and only some IgG4 alleles (Kunkel et al., 1970), the antibody for IgG4b is potentially useful to screen a large number of samples for IgG2 deficiency. However, when the monoclonal antibody for IgG4b was compared with an IgG2 specific antibody produced in a monkey, the IgG4b antibody could identify only about half of the patients with known IgG2 deficiency.

Study of the DBA/2Ha immunodeficiency: X-chromosome mosaicism and in vivo immunoresponses.

DBA/2Ha mice have an X-chromosome-linked immunodeficiency and lack the receptor to a TRF (T cell replacing factor) on a subpopulation of B cells. Their immunodeficiency is considered to resemble that of CBA/N, another X-chromosome-linked immunodeficiency. To facilitate direct comparisons of the two immunodeficiencies and to study the in vivo manifestations of DBA/2Ha immunodeficiency, we measured phenotypes and functions of B cells of DBA/2Ha mice. We found that the expression of sIgM among B cells is normal in DBA/2Ha mice, heterozygous females equally express both affected and normal B cell subpopulations, and DBA/2Ha mice respond well to a TI-2 antigen (TNP-Ficoll) and a polyclonal activator (LPS). Unlike CBA/N, DBA/2Ha mice demonstrate very little in vivo immunodeficiencies.

Subpopulations of B lymphocytes in germinal centers, II. A germinal center B cell subpopulation expresses sIgD and CD23.

To understand B cell development in germinal centers, it is important to delineate the expression of surface antigens among germinal center cells. Because it is unclear whether germinal center cells express common antigens such as sIgD and CD23, we studied their expression among tonsillar lymphocytes with flow cytometry, immunohistochemistry, and in vitro stimulation. Upon studying a large number of tonsils with flow cytometry, we found that occasional tonsils have a very large number of sIgD+ cells among their PNA+ cells. Furthermore, the occasional tonsils with a large number of sIgD+ and PNA+ cells also have many CD23+ cells among their PNA+ cells. Tonsil sections stained immunohistochemically revealed germinal centers containing sIgD+ cells. In addition, PNA- and sIgD+ cells can be induced to express PNA binding sites in vitro without losing the expression of sIgD. Taking these findings together, we conclude that a subpopulation of germinal center B cells coexpresses sIgD and CD23.

Cytokine production by T helper cell subpopulations during prolonged in vitro stimulation.

In man, CD4+ T cells can be divided into phenotypically distinguishable subsets with different function whereas CD4+ T cells with the opposite pheno-CD45RO and low levels of CD45RA antigen provide help for mitogen-induced immunoglobulin production whereas CD4+ cells with the opposite phenotype suppress immunoglobulin production. However, studies examining cytokine production by phenotypically defined CD4+ T cell subsets have led to different conclusions. Further, very few studies have examined cytokine production by freshly isolated CD4+ T cell subsets during extended culture periods. Thus, we examined the production of several cytokines (at various time points) by CD4+ T cell subsets that were isolated in several ways, and stimulated with PWM, Con A, and PHA in a well-defined serum-free culture system. We found that CD4+, CD45RA- (or CD45RO+) T cells consistently produced the most IL-2, IFN-gamma, and TNF-alpha after mitogen stimulation for 2 days. PWM induced the largest quantities of each cytokine, although a similar pattern of production was observed in response to Con A and PHA. We were unable to detect IL-4 production by mononuclear cells and CD4+ T cell subsets suggesting that, if it is produced at all, IL-4 is produced in extremely small quantities. When the culture period of initially CD45RO- T cells was extended beyond 2 days, the culture supernatant contained increased quantities of each cytokine and the cells in the culture had an increased number of cells expressing CD45RO antigen. Together, these data indicate that CD4, CD45RA- (or CD45RO+) T cells in peripheral blood are the major producers of IL-2, IFN-gamma, and TNF-alpha following short-term mitogen stimulation, and that phenotypically defined peripheral blood T cell subsets do not maintain a distinct pattern of cytokines during extended culture periods.

Germinal center T cells are distinct helper-inducer T cells.

Germinal centers (GCs) contain a significant number of CD4+ T cells, but what role these T cells may play in the development of GC B cells has not been determined. To gain insight into their role, we studied the phenotype of GC T cells and the lymphokines secreted by GC T cells isolated from human tonsils obtained after tonsillectomies. In addition to confirming that a large fraction of GC T cells are Leu-7(CD57)+ and Leu-8-, we found that they have no binding sites for peanut agglutinin. Furthermore, we found that they are CD45RA- and CD45R0+, the phenotype of helper-inducer T cells. We also found that Leu-7(CD57)+ cells display CD69, a phenotypic marker of very early cell activation, but do not display three other markers of cell activation: CD25 [interleukin-2 (IL-2) receptor], CD71 (transferrin receptor), and DR. When isolated, Leu-7(CD57)+ cells were stimulated in vitro with a mitogen that can induce peripheral blood T cells with the helper-inducer phenotype to produce various cytokines, Leu-7(CD57)+ cells did not produce IL-2, interleukin-4 (IL-4), interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha) in significant amounts. Taken together, GC T cells from a distinct subpopulation of T cells with helper-inducer phenotype by their histologic location, by their surface phenotype, and by their ability to produce lymphokines. This finding is consistent with the possibility that GC T cells have been selectively recruited to actively help B cells develop in GCs.

Mitogen-induced human IgG subclass expression. II. IgG1 and IgG3 subclasses are preferentially stimulated by a combination of Staphylococcus aureus Cowan I and pokeweed mitogen.

Mitogens generally stimulate human IgG subclass production in amounts proportional to their abundance in serum (IgG1 greater than IgG2 greater than IgG3 greater than IgG4). We report here that a combination of Staphylococcus aureus Cowan strain I and pokeweed mitogen consistently stimulates human peripheral blood lymphocytes in vitro to preferentially produce more IgG1 and IgG3 than IgG2. This preferential stimulation can be measured by increases in the number of immunoblasts (cells with detectable cytoplasmic immunoglobulin) as well as in secreted immunoglobulin. The preferential stimulation pattern is established by the fourth day of culture and is maintained at least until the tenth day. Removal of T cells and subsequent stimulation of B cells with S. aureus Cowan I and interleukin 1 (IL-1) interleukin 2 (IL-2), interleukin 4 (IL-4), or interferon-gamma (IFN-gamma) failed to enhance any IgG subclass production, indicating the requirement for multiple lymphokines in IgG subclass production. The significance of these findings is discussed with respect to B-cell regulatory molecules and the coordinate expression of IgG subclasses.

The human antibody V region repertoire to the type B capsular polysaccharide of Haemophilus influenzae.

The V region repertoire of the human antibody response to the type b capsular polysaccharide of Haemophilus influenzae (Hib-PS) is being defined at the molecular level using antibodies purified from serum of immunized adults. The VH of this response is restricted to the VHIII subgroup while the VL can be divided into two categories. The most common VL, expressed in > 90% of adults and usually constituting the majority of a subjects anti-Hib-PS antibody response, is restricted to the product of a single V kappa II gene known as A2 that probably lacks somatic mutations. The product of the A2 gene is invariably joined to one of several J kappa products by an inserted arginine at the V kappa-J kappa junction. In contrast to the restricted nature of the dominant VL clonotype, the second category of VL constitutes a heterogeneous group of at least seven different VL gene products that often contain somatic mutations and generally exhibit crossreactivity with a related polysaccharide from E. coli. Elucidation of anti-Hib-PS V regions at the molecular level will permit examination of structure-function relationships among these clinically important antibodies and should make the V region repertoire to Hib-PS a useful model for studying human V gene responses.