Humoral Immune Responses against the Immature Laminin Receptor Protein Show Prognostic Significance in Patients with Chronic Lymphocytic Leukemia

Patients

Sera and PBMC were obtained from a total of 67 patients with untreated CLL, 11 patients with CLL who had undergone allogeneic stem cell transplantation, and 50 healthy donors serving as negative controls. Diagnosis of CLL was based on morphologic and immunophenotypic features according to the classification of the World Health Organization (WHO) (26). Sample collection took place from 2003 to 2006. Written informed consent was provided according to the Declaration of Helsinki. The study was approved by the review board of the local ethics committee.

The prognostic parameters were performed at time of diagnosis or shortly thereafter. If analysis of somatic hypermutations or fluorescence in situ hybridization (FISH) had not been performed at diagnosis, they were performed at the same time of the immune studies. For all patients except four, information on IgV_H gene mutational status and FISH analysis for genetic abnormalities was available. Patient characteristics are summarized in Table I⇓. Healthy donor samples were obtained from blood donors of the institutional department of transfusion medicine (n = 50, male/female ratio 3:1, mean age 55 years).

CLL cells and nonmalignant subsets

Peripheral blood was drawn from healthy donors or CLL patients, and PBMC were obtained by Ficoll density gradient centrifugation and cryopreserved in liquid nitrogen. Isolation of CD19⁺ B cells, as well as CD4⁺, CD8⁺, CD14⁺, CD56⁺ and CD34⁺ nonmalignant subsets from healthy donors, was conducted using MACS technology (Miltenyi Biotec) following the manufacturer’s instructions. FACS analysis of the different cell population revealed cell purity of >90%. Sera were stored at −20°C until use.

OFA Ab and peptides

Mouse anti-OFA/iLR mAb was used as previously described (21). A set of 42 synthetic peptides (OFA_1–42) with a length of 17–20 aa overlapping by 5 aa and representing the entire sequence of OFA/iLR were obtained from Peptides & Elephants. Peptides were provided with >70% purity.

Flow cytometry for CD38, ZAP70, and OFA/iLR expression

Staining of OFA/iLR on CLL cells and nonmalignant subsets (n = 3 for each subset) was performed as previously described (21). FITC-labeled goat anti-mouse Ig (Dako) was used as a secondary reagent. Assessment of intracellular ZAP70 and cell-surface expression of CD38 was conducted as described earlier using the following Ab conjugates: anti-ZAP70 (clone 1E7.2, Caltag Laboratories), anti-CD19-PerCP/Cy5.5 (clone SJ25C1, Beckman Coulter/Immunotech), and anti-CD38-PE (clone HB-7, Caltag Laboratories), or respective isotype control (7, 27). At least 10,000 cells were acquired for all FACS analysis in the CellQuest program on a BD FACScan flow cytometer (BD Biosciences).

Cytogenetic analysis

Based on conventional cytogenetic studies and data from comparative genomic hybridization, a comprehensive set of FISH DNA probes (Abbott Diagnostics) was developed to diagnose the most frequent genomic aberrations in CLL. Our probe set allowed screening for the following partial deletions, partial trisomies, and translocations: del(6q23), del(11q22), tri12, del(13q14), and del(17p13), as previously described (27).

IgV_H mutational analysis

The sequences were compared with published germline V_H, D, and J_H genes using DNAPLOT software and IMGT database (IMGT, the international ImMunoGeneTics database, http://imgt.cines.fr). Mutational status was calculated as percentage deviation from the closest matching germline V_H segment. As previously decided, a cutoff of ≥98% germline homology defined the group with unmutated IgV_H (28).

Western blot analysis for OFA/iLR expression and for Abs to recombinant OFA/iLR protein

Standard Western blot analysis for OFA/iLR expression was performed as described previously using the monoclonal anti-OFA/iLR Ab at a concentration of 0.75 μg/ml (21).

ELISA for Abs to recombinant OFA/iLR protein

For the analysis of specific anti-OFA/iLRP Abs, a standard ELISA was performed as described elsewhere (29). Purified human OFA/iLR (6 μg/ml) and HIV p24 (1 μg/ml) (Aalto Bio Reagents) were bound to ELISA plates (Greiner Bio-One). Sera were used in a dilution of 1/50. OD was read at 450 nm. Responses against HIV p24 (all patients negative) were used as a negative control and nonspecific reactivity was subtracted from each OFA/iLR OD value. Epitope mapping was performed using 42 overlapping OFA/iLR peptides. For IgG subtype analysis peroxidase-conjugated mouse anti-human IgG1, IgG2, IgG3, or IgG4 were used (all from Zymed Laboratories). All analyses were performed in duplicate.

Complement-mediated cytotoxicity and Ab-dependent cellular cytotoxicity (ADCC)

Specific cell lysis with Ab and complement was measured using a 2-h ⁵¹Cr-release assay as previously described by Bellucci et al. (30). For complement-dependent cytotoxicity, a 1/4 dilution of rabbit complement (Calbiochem) was chosen. For ADCC, PBMC from healthy donors were used as effector cells at an E:T ratio of 10:1. OFA/iLR-expressing CLL cells (autologous or allogeneic), cells from patients with CLL in Richter transformation that were negative for OFA/iLR expression, and CD19⁺ purified B cells from healthy donors were used as target cells and incubated with different dilutions of patient serum. All CLL samples used as targets were analyzed by FACS for CD19⁺ surface expression and contained >90% CD19⁺ cells, either for high leukemic burden or after CD19⁺ isolation using MACS as described above.

Statistical methods

To establish cut-off points for OFA/iLR OD values that distinguished between negative and positive cases, we used different approaches including the mean plus two SDs of the healthy donor samples and the receiver operator characteristic curve (31, 32). Using these approaches, the suggested cut-off points lie between 0.06 and 0.13 OD. As for the exploratory nature of the study and to avoid false-positives, an OD of 0.11 for IgG and 0.13 for IgM were chosen as initial cut-off values for determination of positive Ab reactivity for OFA/iLR and were subsequently applied to healthy donors and CLL patients. Student’s t test was used to determine differences in anti-OFA/iLR Abs between healthy donors and CLL patients. Following Dohner et al.’s hierarchical classification, high-risk cytogenetics was defined as the presence of del17p and/or del11q (3). As six of seven patients with tri12 concomitantly had a del11q, the remaining patient with isolated tri12 was also included in the group of high-risk cytogenetics. Survival data were obtained from clinical records. Progression-free survival (PFS) was defined as the time from diagnosis to either disease progression or death from any cause (events) or last follow-up (censored) and was estimated using the method of Kaplan and Meier. In a univariate analysis we compared the Kaplan-Meier curves by the log-rank test and additionally by the Breslow test, which gives more emphasis to early events and is preferred in situations when the proportional hazard assumption may be violated by the data (33). Since CLL is an incurable disease, all patients will eventually experience disease progression, and early differences in events are important. Multivariate Cox regression models, adjusted for established risk factors, were fitted to evaluate the impact of the humoral immune response as a novel prognostic factor on PFS. Relative risks and the corresponding 95% CIs were calculated. p values ≤0.05 were considered to be significant. All statistical analyses were performed using SPSS/PC+ software (version 12.0) for Windows.

Expression of OFA/iLR in patients with CLL and normal B cells

Expression of OFA/iLR protein on CLL cells and B lymphocytes purified from the blood of healthy individuals was determined by flow cytometry and Western blot analysis using the previously described monoclonal OFA/iLR-specific Ab (21). OFA/iLR expression was detectable in all of the 67 samples of CLL patients, although the range of OFA/iLR expression levels was relatively broad (10–98%, mean 63.3%). As described earlier, no OFA/iLR expression was found by FACS on blood cells, including both CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T lymphocytes, CD 14⁺ monocytes, CD19⁺ B cells, CD34⁺ progenitor cells, and CD56⁺/CD16⁺ NK cells (21). Interestingly, cells from patients with Richter’s syndrome (n = 5), a secondary high-grade lymphoma developing in patients with CLL, did not show OFA/iLR expression. Fig. 1⇓A shows flow cytometric analyses of purified CD19⁺ B lymphocytes from one healthy donor and the expression profile of OFA/iLR on malignant B cells of four CLL patients. OFA/iLR expression seen by flow cytometry was confirmed by Western blot analysis for healthy donors and CLL patients (Fig. 1⇓B).

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FIGURE 1.

Expression and antigenicity of OFA/iLR in CLL patients and healthy donors. A, One representative experiment of FACScan analysis of CD19⁺ lymphocytes. The filled profile indicates isotype control; nonfilled gray profile, healthy donor; nonfilled dotted profiles, CLL patient nos. 1–4. B, Detection of OFA/iLR using Western blot analysis. Total protein from a healthy donor (lane 1) and different CLL patients (lanes 2–7) were separated by SDS-PAGE and blotted to nitrocellulose filter membranes. Monoclonal anti-OFA/iLR Ab was used as a primary and goat anti-mouse as a secondary Ab. C, Dot blot showing specific recognition of OFA/iLR protein by anti-OFA/iLR Abs. Recombinant His-tagged OFA/iLR protein and recombinant non-His-tagged surviving protein were blotted on a nitrocellulose membrane. Truncated, non-His-tagged HIV protein was used as a negative control. Proteins were incubated with anti-OFA/iLR mAb, anti-survivin polyclonal Ab, and anti-His-tagged Ab.

OFA/iLR Abs are present in sera from untreated patients with CLL as well as in patient sera after allogeneic stem cell transplantation

First, specificity of the monoclonal anti-OFA/iLR Ab used for all subsequent ELISA analysis was confirmed by dot blot shown in Fig. 1⇑C. Anti-OFA/iLR Abs specifically detected the recombinant protein but not the nonrelated protein survivin and, conversely, OFA/iLR protein was not recognized by the nonrelated anti-survivin Ab. Given the frequent expression of OFA/iLR on the surface of CLL cells, we next analyzed whether patients developed spontaneous humoral immune responses toward this Ag. The presence of anti-OFA/iLR IgG and IgM Abs was evaluated by means of an ELISA assay using the recombinant OFA/iLR protein as a coating agent. Sixty-seven untreated patients with CLL and 50 healthy volunteers were analyzed. In 22 of 67 patients (32.8%), IgG OFA/iLR Abs were detected (Fig. 2⇓A). In 12 of 67 patients (17.9%), IgM OFA/iLR Abs were identified (Fig. 2⇓B), whereas 5 (7.4%) patients had both IgG and IgM Abs detected. Four of 50 healthy donors (8%) had IgG or IgM OD levels above the cut-off point. Untreated patients with advanced stage of disease (Binet C) did not show any detectable levels of IgG or IgM Abs against OFA/iLR (n = 10, data not shown). CLL patients had statistically significant higher levels of anti-OFA/iLR-IgG and -IgM (p = 0.012 and p = 0.049, respectively) while there was no statistically significant difference between the humoral response of mutated and unmutated patients (Fig. 2⇓, A and B).

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FIGURE 2.

Titers of IgG and IgM anti-OFA/iLR Abs in untreated CLL patients, healthy donors, and CLL patients after allogeneic transplantation. A and B, Results from anti-OFA/iLR ELISA are shown for healthy donors (HD) (▪), untreated CLL patients (CLL) (□), mutated CLL patients (▴), and unmutated CLL patients (○) for (A) IgG and (B) IgM, respectively. Black horizontal bars indicate cut-off values for IgG (0, 11) and IgM (0, 12). Ab response is displayed as OD. C, OD levels from an ELISA analysis for four CLL patients after allogeneic transplantation. Time point −1 represents pretransplant donor sera.

We also looked for anti-OFA/iLR Ab levels in 11 patients with CLL who had undergone allogeneic stem cell transplantation (allo-SCT). The time from allo-SCT until first analysis of sera varied from 4 to 44 mo (median 14.8 mo). Eight of 11 sera showed detectable IgM and/or IgG anti-OFA/iLR Abs after allo-SCT. Interestingly, six of eight patients who achieved a complete remission after transplantation with complete donor chimerism also showed significant anti-OFA/iLR responses. Characteristics of allotransplanted patients and Ab responses are shown in Table II⇓. For four patients, donor sera were available, which all tested negative for anti-OFA/iLR Abs. Immune responses against OFA/iLR developed after allo-SCT sustained for several months, but decreased over time without signs of relapse or loss of full donor chimerism (Fig. 2⇑C).

Specific recognition of OFA/iLR epitopes differs between untreated patients and patients after allogeneic SCT

All patient sera with significant anti-OFA Ab levels (IgG, n = 20; IgM, n = 9; IgG + IgM, n = 5) were included in an epitope mapping assay to confirm epitope specificity. A panel of 42 overlapping peptides representing the entire sequence of the OFA/iLR protein was used in an IgG and IgM ELISA. Twenty-three patients recognized at least one distinct epitope of the OFA/iLR sequence. To exclude nonspecific reactivity, only those patients with specific epitope recognition were considered to have reactive sera. Fifteen patients with reactive sera recognized more than one epitope. Fig. 3⇓A shows the distribution of all recognized peptide epitopes from untreated CLL patients in relation to the localization of functional domains of OFA/iLR. The distribution of specific IgG and IgM responses includes association with intracellular as well as extracellular located epitopes. CLL patients after allogeneic stem cell transplantation who mounted Ab responses against OFA/iLR (n = 8) exclusively recognized peptide epitopes from aa 159–290 located toward the extracellular C-terminal end of the OFA/iLR protein sequence. In contrast to the untreated patient group, intracellular domains were not recognized (Fig. 3⇓B).

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FIGURE 3.

Distribution of recognized epitopes by IgG and IgM anti-OFA/iLR Abs in relation to the OFA/iLR sequence in untreated CLL patients and postallogeneic stem cell transplantation. Schematic view of the OFA/iLR protein and its identified functional domains. OFA/iLR spans the plasma membrane once from aa 86–101 with its C terminus directed toward the extracellular space. Laminin binding sites are identified at aa 161–180 and 205–229. A, Distribution of recognized IgG and IgM OFA/iLR epitopes in relation to the functional OFA/iLR peptide sequence. Results from OFA/iLR ELISA using 42 overlapping peptides. B, Post-allo-SCT sera recognize extracellular located epitopes of the OFA/iLR sequence. OD for IgG and IgM is displayed for each of the 42 epitopes. A and B, Black bars represent IgM, gray bars represent IgG.

Binding of anti-OFA/iLR Abs can be specifically inhibited

To validate the specificity of the detected anti-OFA/iLR Abs, an inhibition assay was performed in the presence of recombinant OFA/iLR protein, which was formerly used as a coating Ag in the ELISA. As demonstrated in Fig. 4⇓A, reactivity to OFA/iLR of respective patient sera was inhibited by addition of the OFA/iLR protein in a dose-dependent manner. No inhibitory effect was observed when HIV p24 protein or β₂-microglobulin was added. Using reactive serum as the source of primary Ab and a FITC-conjugated secondary Ab in a FACS analysis, CD19⁺ purified malignant B lymphocytes (CLL cells) from an OFA/iLR positive patient were recognized but not OFA/iLR-negative CLL cells (Fig. 4⇓B). Serial dilutions of serum at 1/25, 1/50, and 1/100 showed dose-dependent recognition.

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FIGURE 4.

Anti-OFA/iLR Abs are specifically inhibited by addition of OFA/iLR protein and bind to OFA/iLR-expressing cells. A, Reactive sera from two CLL patients were incubated with either recombinant OFA/iLR-protein, HIV p24-truncated protein, or β₂-microglobulin and analyzed in an ELISA. Results are shown as percentage of inhibition in regard to initial OD values for OFA/iLR ELISA. B, Reactive serum recognizes OFA/iLR-positive CLL cells but not OFA/iLR-negative CLL cells. CD19⁺ purified B lymphocytes from two OFA/iLR-positive CLL patients (I and II) and cells from a patient with CLL in Richter transformation (III) were incubated with OFA/iLR-reactive serum in serial dilutions of 1/25, 1/50, and 1/100 as a source of primary Ab and a rabbit anti-human IgG as a secondary Ab.

Predominant subclasses of OFA/iLR Abs are IgG1 and IgG3 and implicate a Th1-biased immune response

For further characterization of OFA/iLR-reactive sera, IgG subclass analysis was performed in an ELISA assay. Sera from patients recognizing at least one specific OFA/iLR epitope were subclassified. Sera from healthy donors were also analyzed for control purposes although they were not reactive in the screening of ELISA for OFA/iLR reactivity. Fig. 5⇓A shows values of OD for IgG1, IgG2, IgG3, and IgG4 Abs. The distribution of subclasses is shown in Fig. 5⇓B, which defines the highest value for each patient as predominant IgG subclass. IgG1 was the the predominant subclass in 14 of 18 patients, while IgG3 anti-OFA/iLR Ab was the major IgG subclass for 8 patients. IgG4 was found to be predominant in serum from only one patient. In five patients, high values for both IgG1 and IgG3 Abs were detectable. No correlation was found between the detection of predominant IgG subclass and the mutational status or OFA/iLR expression of CLL cells (data not shown).

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FIGURE 5.

IgG1 and IgG3 are the predominant subclasses of OFA/iLR-specific Abs. A, All patients recognizing at least one specific OFA/iLR epitope were analyzed for IgG subclasses. Values of OD for IgG1, IgG2, IgG3, and IgG4 anti-OFA/iLR Abs are shown. B, Summary of predominant IgG subclass of each patient.

Anti-OFA/iLR Abs induce ADCC and complement activation

We next determined the functional properties of the reactive sera. The ability of OFA/iLR Abs to lyse OFA/iLR-expressing CLL cells was determined in an ADCC assay and complement-dependent cytotoxicity (CDC) assay. Sera from patients that showed strong responses in the initial screening ELISA as being responsive to one specific OFA/iLR epitope in the epitope mapping were able to induce lysis of autologous OFA/iLR-positive CLL cells (Fig. 6⇓, A and E) in a dose-dependent manner. Furthermore, reactive sera were able to induce lysis of allogeneic, OFA/iLR-expressing CLL (Fig. 6⇓, B and F) cells but not normal CD19⁺ B cells (Fig. 6⇓, C and G) or CLL cells from patients with Richter transformation, which are negative for OFA/iLR expression (Fig. 6⇓, D and H). In contrast, no lytic effect in ADCC or CDC was observed using healthy donor sera against OFA/iLR expressing CLL cells (Fig. 6⇓, A, B, E, and F). In ADCC, a constant background lysis of 10% of the CLL cells was observed for all serum dilutions owing to spontaneous NK cell-mediated lysis.

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FIGURE 6.

OFA/iLR-reactive patient serum mediates specific CDC and ADCC. A–D, Ab-mediated cellular cytotoxicity; healthy donor PBMC were added in an E:T ratio of 1:10. E–H, Complement-dependent cytotoxicity. A and E, OFA/iLR-reactive patient sera (CLL1 and CLL2) mediate lysis of autologous CLL cells via ADCC (A) and CDC (E). B and F, OFA/iLR-reactive sera from untreated patients (CLL3 and CLL4) and patients after allogeneic SCT (allo1 and allo2) lyse OFA/iLR-expressing cells (CLL1 and CLL2). A, B, E, and F, Anti-OFA/iLR Ab was used as a positive control. Healthy donor sera (HD1 and HD2) were used as negative controls. C and G, Negative control with normal B cells as target cells. D and H, Negative control with OFA/iLR-negative CLL cells in Richter transformation as target cells.

PFS in patients with humoral reactivity to OFA/iLR

To determine whether the presence of specific anti-OFA/iLR Abs influenced PFS, patients were grouped according to the presence/absence of a humoral immune response, which specifically recognized at least one OFA/iLR epitope, and were then analyzed using Kaplan-Meier plots. The median duration of follow-up was 72 mo. In a univariate analysis, the group of patients with reactive sera (n = 23) showed a significantly lower probability of disease progression compared with patients with nonreactive sera (Fig. 7⇓A). Median PFS for patients with reactive sera was 114 mo compared with 66 mo for the nonreactive group (p = 0.029). Patients with unmutated V_H genes had a PFS of 70 mo compared with 96 mo for mutated V_H genes (p = 0.128) (Fig. 7⇓B). A PFS of 36 vs 96 mo was found for patients with presence or absence of high-risk cytogenetics, respectively (p = 0.005) (Fig. 7⇓C). Comparing the reactive and nonreactive groups of patients, the distribution of V_H status did not vary significantly due to the low number of patients. The number of patients with high-risk cytogenetics differed significantly (reactive 8.6% vs nonreactive 40.9%) between the two subgroups. Exact characteristics of the two groups are displayed in Table III⇓. Keeping in mind the possible limitations, we investigated whether the presence of humoral immune responses influenced the time to progression in a multivariate analysis. In the final model with adjustment for V_H mutational status and high-risk cytogenetics, the presence of humoral immune responses against OFA/iLR remained significant and revealed a relative risk of 0.36 (95% CI 0.14–0.94; p = 0.038). Results from the statistical analysis are summarized in Table IV⇓.

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FIGURE 7.

Superior PFS for patients with OFA/iLR-reactive sera. PFS of 67 untreated CLL patients grouped according to (A) their ability to specifically recognize at least one OFA/iLR ELISA epitope, (B) mutated or unmutated status of the V_H genes, and (C) presence or absence of either del17p, tri12, and/or del11q.