Enhanced mutational activity of Vkappa gene rearrangements in systemic lupus erythematosus.

To determine the differential impact of somatic hypermutation and selective influences on the light chain repertoire in systemic lupus erythematosus (SLE), the frequency and pattern of somatic hypermutations were compared between the productive and nonproductive Vkappa gene repertoire manifested by individual CD19(+) B cells in a patient with SLE. The mutational frequency of nonproductive rearrangements in the SLE patient was significantly (P < 0.001) increased (3.7 x 10(-2)) compared to normals (4.8 x 10(-3)). Similarly, the mutational frequency of the productive Vkappa rearrangements was also significantly increased in the SLE patient (2.8 x 10(-2) vs 1.1 x 10(-2)) (P < 0.001). There were no differences in the R/S ratios of mutations in productive and nonproductive Vkappa rearrangements. Moreover, a variety of mutational "hot spots" were noted, but, unexpectedly, in the FRs. As in normals, mutations were found most frequently in RGYW/WRCY sequences accounting for 39.3% (nonproductive) and 40.1% (productive) of all mutations. Of note, nonproductive Vkappa rearrangements harbored significantly more mutations than productive rearrangements (P < 0.05) indicating that there was overall selection against mutations in the expressed repertoire. This was most apparent in the CDR3. These data are most consistent with the conclusion that, in this SLE patient, the mutational machinery was markedly enhanced compared to normals, but with no subsequent positive selection of mutations. The enhanced mutational activity may play a role in the emergence of autoreactivity in this SLE patient.

Ig lambda and heavy chain gene usage in early untreated systemic lupus erythematosus suggests intensive B cell stimulation.

To determine the distribution of Vlambda and Jlambda as well as VH and JH gene usage in a patient with systemic lupus erythematosus (SLE), productive and nonproductive VJ and V(D)J rearrangements were amplified from individual peripheral CD19+ B cells and were analyzed. No differences in the Vlambda and Jlambda or the VH and JH gene usage in the nonproductive gene repertoire of this SLE patient were found compared with the distribution of genes found in normal adults, whereas marked skewing of both Vlambda and VH was noted among the productive rearrangements. The distribution of productive Vlambda rearrangements was skewed, with significantly greater representation of the Jlambda distal cluster C Vlambda genes and the Vlambda distal Jlambda7 element, consistent with the possibility that there was receptor editing of the Vlambda locus in this patient. Significant bias in VH gene usage was also noted with VH3 family members dominating the peripheral B cell repertoire of the SLE patient (83%) compared with that found in normal subjects (55%; p < 0.001). Notably, a clone of B cells employing the VH3-11 gene for the heavy chain and the Vlambda1G segment for the light chain was detected. These data are most consistent with the conclusion that extreme B cell overactivity drives the initial stages of SLE leading to remarkable changes in the peripheral V gene usage that may underlie on fail to prevent the emergence of autoimmunity.

Molecular mechanisms and selection influence the generation of the human V lambda J lambda repertoire.

To define the lambda light chain repertoire in humans, a single-cell PCR technique using genomic DNA obtained from individual peripheral B cells was employed. Of the 30 known functional V lambda genes, 23 were detected in either the nonproductive or productive repertoires. Specific V lambda genes, including 2A2, 2B2, 1G, and 4B, were overexpressed in the nonproductive repertoire, whereas some V lambda genes, such as 3R, 2A2, 2B2, 1C, 1G, and 1B, were overexpressed in the productive repertoire. Comparison of the nonproductive and productive repertoires indicated that no V lambda genes were positively selected, whereas a number of V lambda genes, including 4C, 1G, 5B, and 4B, were negatively regulated. All four of the functional J lambda segments were found in both repertoires, with J lambda 7 observed most often. Evidence of terminal deoxynucleotidyltransferase activity was noted in nearly 80% of nonproductive V lambda J lambda rearrangements, and exonuclease activity was apparent in the majority. Despite this, the mean CDR3 length was 30 base pairs in both productive and nonproductive repertoires, suggesting that it was tightly regulated at the molecular level. These results have provided new insights into the dimensions of the human V lambda repertoire and the influences that shape it.

Somatic hypermutation of human immunoglobulin heavy chain genes: targeting of RGYW motifs on both DNA strands.

The impact of the somatic hypermutational machinery was examined by analyzing the frequency and distribution of mutations in nonproductive V(H)DJ(H) rearrangements obtained from individual human peripheral B cells. A strong bias toward nucleotide substitutions within the quadruplet motif RGYW was observed. In addition, there was a comparably increased frequency of mutations of the inverse repeat of RGYW, WRCY. Together, mutations of RGYW/WRCY accounted for 37% of all nucleotide substitutions. No significant strand polarity of the distribution of mutations was evident when nucleotide substitutions of highly mutated quartets and triplets as well as of their inverse repeats were analyzed. Furthermore, detailed analysis of mutations of specific triplets, such as AGC and TAC provided evidence that they were mutated more frequently when they were included within RGYW and WRCY, respectively. Despite being a target of the mutational machinery, neither RGYW nor WRCY was mutated in the absence of a large number of substitutions of other nucleotides in the same sequence. These results indicate that the mutational machinery targets RGYW sequences for mutations on either DNA strand and do not support the contention that the mutational machinery exhibits DNA strand polarity.

Immunoglobulin kappa chain receptor editing in systemic lupus erythematosus.

To determine whether receptor editing of Vkappa genes was involved in the pathogenesis of systemic lupus erythematosus (SLE), the usage of Vkappa and Jkappa gene elements from individual peripheral CD19(+) B cells obtained from a patient with untreated SLE was examined. No differences in the Vkappa and Jkappa gene usage in the nonproductive gene repertoire of this SLE patient were noted compared with the distribution of genes found in normal adults. However, an increased usage of Jkappa5 segments, and a significant overrepresentation of the Vkappa1 and Vkappa4 families, especially the L15, O14/O4, and B3 genes characterized the productive Vkappa gene repertoire of the SLE patient. Furthermore, Jkappa5-containing Vkappa gene rearrangements in the productive but not the nonproductive repertoire manifested significantly fewer mutations compared with Vkappa genes recombined with Jkappa1-4. These data are consistent with the conclusion that receptor editing of Vkappa is much more apparent in this SLE patient than in normals and suggest that a deficiency in this means to counteract the emergence of autoimmunity is not an essential feature of SLE.

Differential quantitative effects of interleukin (IL)-2 and IL-15 on cytotoxic activity and proliferation by lymphocytes from patients receiving in vivo IL-2 therapy.

Lymphocytes from patients receiving in vivo interleukin (IL)-2 therapy possess enhanced in vitro proliferative and cytotoxic responses to IL-2. The cells from these patients that respond to exogenous IL-2 are CD56+ natural killer cells expressing intermediate-affinity IL-2 receptor betagamma(c) complexes. Because IL-15 activates cells via these same betagamma(c) receptors, we hypothesized that IL-15 would also activate lymphocytes from patients treated with in vivo IL-2 therapy and therefore that IL-15 might potentially be useful as an immunotherapeutic agent alone or in combination with IL-2. We report here that peripheral blood mononuclear cells (PBMCs) from patients receiving in vivo IL-2 therapy do proliferate in response to IL-15. However, a greater dose of IL-15 is needed to reach the same level of proliferation stimulated by IL-2. The EC50 for IL-2 is 0.21 +/- 0.04 nM (mean +/- SE; n = 18), whereas the EC50 for IL-15-stimulated proliferation is 1.16 +/- 0.16 nM (n = 18). In contrast to the proliferative response, equivalent doses of IL-2 and IL-15 stimulate patient PBMCs to mediate similar levels of cytotoxicity against Daudi, K562, and LA-N-5 tumor targets. Notably, low concentrations of IL-15 that do not stimulate a substantial proliferative response (e.g., 1.0 ng/ml) do boost PBMCs to mediate cytotoxicity against these tumor targets. These distinct dose-response curves for proliferation compared to cytotoxicity suggest that IL-15 should be evaluated for its potential as an immunotherapeutic agent to treat cancer, particularly in regimens providing doses that might minimize the proliferative response (associated with cytokine release and toxic side effects) while maintaining the cytolytic antitumor response.

Comparable impact of mutational and selective influences in shaping the expressed repertoire of peripheral IgM+/CD5- and IgM+/CD5+ B cells.

Somatic hypermutation and subsequent selection play a significant role in shaping the peripheral B cell repertoire. This repertoire is composed of CD5+ (5%) and CD5- B cells (95%) which are known to traffic through different lymphoid compartments. Previous studies have shown that V(H) gene usage by CD5+ and CD5- B cells is similar, although mutations are more frequent in the latter. However, the effect of mutation and subsequent selection on the expressed V(H) repertoire of CD5+ and CD5- B cells has not been delineated in detail. This study, therefore, analyzed the mutational pattern of individual IgM+/CD5+ and IgM+/CD5- B cells. In both populations, mutations can occur without heavy chain isotype switching. Despite the differences in mutational frequency, the patterns of mutation and subsequent selection were comparable in CD5+ and CD5- B cells. These results imply that although mutations are more frequent in CD5- B cells, the overall mechanisms governing somatic hypermutation and subsequent positive and negative selection are similar in CD5+ and CD5- B cells.

Delineation of selective influences shaping the mutated expressed human Ig heavy chain repertoire.

After Ag exposure, somatic hypermutation and subsequent selection play significant roles in shaping the peripheral B cell repertoire. However, the disparate impact of each process has not been completely delineated. To address this, the mutational patterns of a large panel of productive V(H)DJ(H) rearrangements of individual human B cells were analyzed and compared with those of a previously reported panel of nonproductive V(H)DJ(H) rearrangements. The productive V(H) rearrangements exhibited a significantly lower mutational frequency and a significantly smaller number of replacement mutations than the nonproductively rearranged genes, suggesting that structural constraints of the Ig molecule and selective influences both impacted the repertoire, militating against replacement mutations. Positive selection favored a mean of four to six replacements in complementarity-determining region 1 (CDR1) and CDR2, and less than two replacements in the framework regions (FRs). In contrast, the negative impact of replacement mutations generated an increased number of silent mutations within both the CDRs and FRs of the productive repertoire accompanied by a net increase in the ratio of replacement to silent mutations in the CDRs compared with that in the FRs. Moreover, there was a negative influence on the distribution of amino acid changes resulting from mutations of highly mutable codons, such as AGY, TAY, GTA, and GCT, preferentially leading to conservative changes in the expressed Ig repertoire. The results are consistent with the conclusion that the expressed repertoire is limited, compared with the potential generated by the mutational machinery, by the dual requirements of avoiding autoreactivity and satisfying structural constraints of an intact Ig molecule.

Distinctions in lymphocyte responses to IL-2 and IL-15 reflect differential ligand binding interactions with the IL-2Rbeta chain and suggest differential roles for the IL-2Ralpha and IL-15Ralpha subunits.

More interleukin 15 (IL-15) than IL-2 was needed to generate comparable proliferative responses by phytohaemagglutinin (PHA) blasts and Tf-1beta cells expressing high affinity and intermediate affinity IL-2 receptor (IL-2R) complexes, respectively. The focus of these experiments was to determine the contribution of the shared IL-2 and IL-15 receptor components to these dose-response differences. Some of this difference can be attributed to the role of the IL-2Rbeta chain, in that HuMikbeta1, a monoclonal antibody recognizing the IL-2Rbeta chain, blocks 92.2+/-2.5% (mean+/-SE) of the IL-2 proliferative response by Tf-1beta cells but only inhibits 57.9+/-3.7% of the IL-15 response, indicating that IL-2 and IL-15 may physically utilize the IL-2Rbeta chain differently. Monoclonal antibody 341, which recognizes IL-2Rbeta but does not inhibit IL-2 binding to the IL-2Rbeta chain, blocks 35.4+/-2.3% of IL-15-stimulated proliferation of PHA blasts, while not affecting the IL-2-stimulated proliferation. Finally, although HuMikbeta1 does not inhibit IL-2 responses by PHA blasts bearing high affinity IL-2 receptors, HuMikbeta1 does block IL-15-stimulated proliferation by these same cells bearing high affinity IL-15 receptors (88.5+/-1.6% inhibition). This indicates that the role of IL-15Ralpha in the high affinity IL-15R complex is distinct from that of IL-2Ralpha in the high affinity IL-2R complex. Overall, these studies show that the physical interactions of the IL-2Rbetagammac complex with IL-2 are different than the interactions with IL-15.

Alteration of the CD34+ Tf-1 beta cell line profile in response to long-term exposure to IL-15.

Interleukin 15 (IL-15) is a cytokine with many functional characteristics that are similar to IL-2. Most of the functional activities that IL-2 and IL-15 support have been evaluated in short-term assays. It was our intention, then, to determine the long-term effects of IL-15 in comparison to IL-2. These studies were performed using the growth factor-dependent myelomonocytic cell line, Tf-1, which has been well characterized with regard to morphology, CD marker expression, responses to certain growth factors and cytokines (GM-CSF, IL-4, erythropoietin), and can differentiate through the myeloid and erythroid lineages. In order to study IL-2 and IL-15 responses, Tf-1 cells were retrovirally infected with the IL-2R beta chain gene as a means to confer IL-2 responsiveness to this cell type. The results of this study demonstrate that retroviral infection of Tf-1 successfully generated a stable IL-2 responsive cell line, Tf-1 beta, without interfering with the original characteristics of the Tf-1 cell. Tf-1 beta cells respond functionally to both IL-2 and IL-15. When Tf-1 beta cells are grown for 8 weeks in IL-2 (Tf-1 beta 2), rather than GM-CSF, the original morphology, CD marker expression, esterase activity and proliferative response is unaltered in comparison to that of the original Tf-1 beta line maintained in GM-CSF. However, long-term growth of Tf-1 beta in IL-15 (Tf-1 beta 15) results in morphological alterations, downregulation of CD33, CD38, and HLA-DR, and a decreased response to IL-15 in comparison to Tf-1 beta 2. These studies support the concept that retroviral infection, even when it confers new functions upon a cell, does not necessarily alter all other functions, as assessed by evaluation of its phenotypic profile. Furthermore, the production of the Tf-1 beta 2 and Tf-1 beta 15 sublines demonstrates that IL-2 and IL-15 can support long-term cell growth. However, this long-term growth in IL-15 leads to subtle alterations in the cell profile that are not seen with IL-2, suggesting that distinctions in IL-2 and IL-15 function do exist. Further study of the Tf-1 beta 15 cell line will be useful to clarify these functional distinctions between IL-2 and IL-15.

Interaction of IL-15 with the shared IL-2 receptor beta and gamma c subunits. The IL-15/beta/gamma c receptor-ligand complex is less stable than the IL-2/beta/gamma c receptor-ligand complex.

This study was designed to compare the interactions of IL-2 and IL-15 with the IL-2R beta and IL-2R gamma c subunits, as differences in receptor interactions between IL-2 and IL-15 might contribute to the functional differences between these two cytokines. The results suggest the existence of a human IL-15R alpha subunit, although physical evidence of this molecule was not obtained. Proliferation of anti-CD3 (OKT3)-stimulated human PBL was compared for responsiveness to IL-2, IL-15, and F42K, and IL-2 mutant that does not bind the IL-2R alpha chain. F42K was more potent than IL-15 in activating a dose-dependent response. This fact, along with Scatchard binding analyses of IL-15 on OKT3 blasts and YT cells revealing both high and intermediate affinity receptors, supports the existence of IL-15R alpha on these cells. Additional characterization of the IL-15R utilized covalent cross-linking to affinity label IL-2R and IL-15R on YT cells and OKT3 blasts. Consistent with previously reported functional data, IL-2R alpha was not co-precipitated from the [125I]IL-15 receptor-ligand complex, demonstrating that IL-15 does not interact physically with the IL-2R alpha subunit. While IL-2R alpha did co-precipitate with IL-2R beta and IL-2R gamma c in the presence of IL-2, IL-15R alpha did not co-precipitate with the IL-2R beta/gamma c complex. Finally, YT cells equilibrated with IL-2 and then precipitated through IL-2R beta showed that IL-2R beta and IL-2R gamma c co-precipitate in a 1:1 ratio, while only IL-2R beta was found in the immunoprecipitates of YT cells equilibrated with IL-15. This indicates that IL-15 creates a less stable bridge between the IL-2R beta and IL-2R gamma c chains than does IL-2 on YT cells. This result was identical for both surface-iodinated YT cells and immunoprecipitates that were probed for IL-2R beta and IL-2R gamma c on Western blots.

Distinction between gamma c detection and function in YT lymphoid cells and in the granulocyte-macrophage colony-stimulating factor-responsive human myeloid cell line, Tf-1.

Peripheral blood monocytes respond to interleukin-2 (lL-2) and express the gamma common (gamma c) subunit of the lL-2 receptor (lL-2R) complex. However, the role of lL-2 in myeloid development has recently become of interest for several reasons, including the effect gamma c mutations may or may not have on myeloid development in patients with XSCID. Many studies of lL-2 function in the myeloid cell lineage have been performed on a murine background. To study gamma c expression and function in human myeloid precursors, we introduced the human myelomonocytic cell line, Tf-1, with a retroviral vector containing the human lL-2R beta subunit to create functional human intermediate lL-2R consisting of beta gamma c dimers. We have characterized this transfected variant of Tf-1 (Tf-1 beta) with regard to its response to lL-2. Unlike the parental Tf-1 cell line that is deficient in both lL-2R alpha and lL-2R beta expression, the Tf-1 beta transfectant binds and responds to lL-2 through intermediate-affinity lL-2Rs. Scatchard analyses indicate the number of intermediate-affinity receptors on Tf-1 beta is similar to the number found on the well-characterized YT cell line. However, detection of gamma c on Tf-1 beta cells is dramatically less than on YT cells by Western blot analysis and is undetectable by flow cytometric studies and surface iodinations. The gamma c component on YT cells is readily detected by all three methods. We conclude from these studies that the intermediate-affinity lL-2Rs on the Tf-1 cell line behave differently than those on YT cells with respect to gamma c detection. Either the gamma c molecule itself is different, or the cellular environment in which it functions is altered. Elucidation of gamma c function on this cell line will allow for its use as a model in which other cytokines using gamma c (including lL-2, lL-4, and lL-15) can be studied on the same cellular background.