Functional autoantibodies against SSX-2 and NY-ESO-1 in multiple myeloma patients after allogeneic stem cell transplantation

Abstract

Background: Multiple myeloma (MM) is the malignancy with the most frequent expression of the highly immunogenic cancer-testis antigens (CTA), and we have performed the first analysis of longitudinal expression, immunological properties, and fine specificity of CTA-specific antibody responses in MM.

Methods: Frequency and characteristics of antibody responses against cancer-testis antigens MAGE-A3, NY-ESO-1, PRAME, and SSX-2 were analyzed using peripheral blood (N = 1094) and bone marrow (N = 200) plasma samples from 194 MM patients.

Results: We found that antibody responses against CTA were surprisingly rare, only 2.6 and 3.1 % of patients evidenced NY-ESO-1- and SSX-2-specific antibodies, respectively. NY-ESO-1-specific responses were observed during disease progression, while anti-SSX-2 antibodies appeared after allogeneic stem cell transplantation and persisted during clinical remission. We found that NY-ESO-1- and SSX-2-specific antibodies were both capable of activating complement and increasing CTA uptake by antigen-presenting cells. SSX-2-specific antibodies were restricted to IgG3, NY-ESO-1 responses to IgG1 and IgG3. Remarkably, NY-ESO-1-positive sera recognized various non-contiguous regions, while SSX-2-specific responses were directed against a single 6mer epitope, SSX-2(85-90).

Conclusions: We conclude that primary autoantibodies against intracellular MM-specific tumor antigens SSX-2 and NY-ESO-1 are rare but functional. While their contribution to disease control still remains unclear, our data demonstrate their theoretic ability to affect cellular anti-tumor immunity by formation and uptake of mono- and polyvalent immune complexes.

Fig. 1

NY-ESO-1-specific antibodies correlate with tumor load and disease progression while donor-derived anti-SSX-2 antibodies indicate disease remission. a Longitudinal analysis of humoral responses and antigen expression in the BM for individual patients. Black dots indicate reciprocal titers of anti-NY-ESO-1 and anti-SSX-2 antibodies. White dots indicate expression of NY-ESO-1 or SSX-2 RNA, and white triangles demonstrate expression of LAGE and SSX-4 RNA, respectively, in the BM of the patients. Within single graphs, white areas indicate the presence of clinical remission while gray areas indicate recurrence and progression of the disease. The application of alloSCT is indicated by small black arrows. b Comparison of means of reciprocal titer means for each patient during phases of the indicated clinical condition. Paired samples t tests indicate significant differences in antibody titers between clinical conditions for SSX-2 and NY-ESO-1

Fig. 2

Functional characteristics of NY-ESO-1- and SSX-2-specific patient antibodies. a Western blot analysis of the complement-activating capacity of NY-ESO-1- and SSX-2-specific antibodies. Tetanus toxoid (TT) served as positive control for complement activation by TT-specific antibodies. Complement activation was detected through a complement factor C3-specific antibody. Sera from NY-ESO-1 antibody-positive patient UKE-21 and from SSX-2 antibody-positive patient UKE-64 were able to activate complement when bound to the respective CTA. The same phenomenon was seen with the patients’ TT-specific antibodies. When exposed to control protein GST, no complement activation occurred. Results of one representative experiment are shown. b Uptake of FITC-labeled CTA protein after immune complex formation with either SSX-2- or NY-ESO1-specific antibodies from high-titered MM patients. Bars indicate mean logarithmic fold changes over antigen alone for the respective CTA for 6 different healthy blood donors. Error bars indicate standard error of means (SEM). c Partial confocal sections of one representative antigen uptake experiment. Formation of specific immune complexes comprised of FITC-labeled antigen, and the corresponding MM patient-derived antibodies increased antigen uptake in CD64⁺ APCs from one healthy donor. In contrast, the same antibody did not increase uptake in the unspecific context

Fig. 3

Levels of anti-SSX-2 antibodies correlate with the number of SSX-2-specific T cells. a In patient UKE-64, anti-SSX-2 antibody titers were followed after induction by alloSCT and numbers of T cells specific for SSX-2 were determined by IFN-γ ELISPOT. The black line indicates the reciprocal titer of anti-SSX-2 IgG antibodies, and gray bars indicate mean numbers (+SEM) of CD4⁺ T cells specific for the SSX-2^31–50 epitope. Below each bars examples of ELISPOT results for the given timepoints are shown. b Dot plots represent examples of intracellular cytokine staining followed by flow cytometry. CD4⁺ T cells from patient UKE-64 were cultured for 2–3 weeks with a pool of three 20-mer SSX-2 peptides covering amino acid region 31–70. After being re-exposed to the respective SSX-2 peptides, the patient’s CD4⁺ T cells secreted IFN-γ (left box) while exposure toward T-APC pulsed with an NY-ESO-1 control peptide (right box) did not provoke IFN-γ production. c As indicated by this exemplary dot plot, the patient’s CD4⁺ T cells specific for SSX-2 were negative for FOXP3 suggesting that these cells did not represent T regulatory cells

Fig. 4

IgG subtype analysis and epitope mapping of anti-NY-ESO-1 and anti-SSX-2 antibody responses. a Analysis of the distribution of patient-derived NY-ESO-1- and SSX-2-specific antibodies regarding IgG subtypes. Bars indicate the percentage of seropositive patients evidencing the given subtype of CTA-specific IgG. b Target epitopes of the NY-ESO-1 (left) and SSX-2-specific (right) antibody responses of MM patients were identified using overlapping 20-mer peptides spanning the complete sequence of the respective CTA. Bars indicate percentages of seropositive patients recognizing the given epitope. In the case of SSX-2-specific antibodies, an additional series of overlapping 10-mer peptides was used to further define the exact target epitope of the patients’ humoral responses. Results were validated by three independent experiments. c Competition with 6mer peptides covering SSX-2^81–90 at 250 μM for binding of full-length SSX-2. d Dilution series of single-alanine substitution variants of initial 10mer SSX-2^81–90 competing for binding of full-length SSX-2 protein. e Competition with 5mer peptides containing the minimal SSX-2 epitope with and without substitution of proline in position 88 at 250 μM. Competition assays were performed for all SSX-2 antibody-positive patients. Data are shown for one representative patient. f SSX-2-recognizing sera of 6 patients were tested against SSX-1, SSX-2, and SSX-5 full-length proteins in an ELISA assay. Recombinant full-length MAGE-C2 protein was used as a control. Results are shown for one representative patient