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BackgroundChimeric antigen receptor T (CART) cell therapy targeting the B cell specific differentiation antigen CD19 has shown clinical efficacy in a subset of relapsed/refractory (r/r) diffuse large B cell lymphoma (DLBCL) patients. Despite this heterogeneous response, blood pre-infusion biomarkers predicting responsiveness to CART cell therapy are currently understudied.MethodsBlood cell and serum markers, along with clinical data of DLBCL patients who were scheduled for CART cell therapy were evaluated to search for biomarkers predicting CART cell responsiveness.FindingsCompared to healthy controls (n=24), DLBCL patients (n=33) showed significant lymphopenia, due to low CD3+CD4+ T helper and CD3-CD56+ NK cell counts, while cytotoxic CD3+CD8+ T cell counts were similar. Although lymphopenic, DLBCL patients had significantly more activated HLA-DR+ (P=0.005) blood T cells and a higher frequency of differentiated CD3+CD27-CD28- (28.7 ± 19.0% versus 6.6 ± 5.8%; P+CD27-CD28- T cells (23.3 ± 19.3% versus 35.1 ± 18.0%) were independently associated with OR. This association was even more pronounced when patients were stratified for complete remission (CR versus non-CR: 13.7 ± 11.7% versus 37.7 ± 17.4%, P=0.001). A cut-off value of ≤ 18% of CD3+CD27-CD28- T cells predicted CR at 12 months with high accuracy (PIn vitro, CD3+CD8+CD27-CD28- compared to CD3+CD8+CD27+CD28+ CART cells displayed similar CD19+ target cell-specific cytotoxicity, but were hypoproliferative and produced less cytotoxic cytokines (IFN-γ and TNF-α). CD3+CD8+ T cells outperformed CD3+CD4+ T cells 3- to 6-fold in terms of their ability to kill CD19+ target cells.InterpretationLow frequency of differentiated CD3+CD27-CD28- T cells at leukapheresis represents a novel pre-infusion blood biomarker predicting a favorable response to CART cell treatment in r/r DLBCL patients.

Background The determinants of successful humoral immune response to the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) are of critical importance for the design of effective vaccines and the evaluation of the degree of protective immunity conferred by exposure to the virus. As novel variants emerge, understanding their likelihood of suppression by population antibody repertoires has become increasingly important. Methods In this study, we analyzed the SARS‐CoV‐2 polyclonal antibody response in a large population of clinically well‐characterized patients after mild and severe COVID‐19 using a panel of microarrayed structurally folded and unfolded SARS‐CoV‐2 proteins, as well as sequential peptides, spanning the surface spike protein (S) and the receptor‐binding domain (RBD) of the virus. Results S‐ and RBD‐specific antibody responses were dominated by immunoglobulin G (IgG), mainly IgG1, and directed against structurally folded S and RBD and three distinct peptide epitopes in S2. The virus neutralization activity of patients´ sera was highly correlated with IgG antibodies specific for conformational but not sequential RBD epitopes and their ability to prevent RBD binding to its human receptor angiotensin‐converting enzyme 2 (ACE2). Twenty percent of patients selectively lacked RBD‐specific IgG. Only immunization with folded, but not with unfolded RBD, induced antibodies against conformational epitopes with high virus‐neutralizing activity. Conformational RBD epitopes required for protection do not seem to be altered in the currently emerging virus variants. Conclusion These results are fundamental for estimating the protective activity of antibody responses after natural infection or vaccination and for the design of vaccines, which can induce high levels of SARS‐CoV‐2–neutralizing antibodies conferring sterilizing immunity., IgG response in convalescent COVID‐19 patients is directed to folded but not to unfolded RBD or RBD peptides. IgGs to folded RBD are required for virus neutralization. Twenty percent of convalescent COVID‐19 patients selectively lack RBD‐specific IgG. Only immunization with folded, but not with unfolded RBD, induces antibodies with virus‐neutralizing activity.

Antibody-based tests are available for measuring SARS-CoV-2-specific immune responses but fast T-cell assays remain scarce. Robust T cell-based tests are needed to differentiate specific cellular immune responses after infection from those after vaccination.One hundred seventeen individuals (COVID-19 convalescent patients: n = 40; SARS-CoV-2 vaccinees: n = 41; healthy controls: n = 36) were evaluated for SARS-CoV-2-specific cellular immune responses (proliferation, Th1, Th2, Th17, and inflammatory cytokines, activation-induced marker [AIM] expression) by incubating purified peripheral blood mononuclear cells (PBMC) or whole blood (WB) with SARS-CoV-2 peptides (S, N, or M), vaccine antigens (tetanus toxoid, tick borne encephalitis virus) or polyclonal stimuli (Staphylococcal enterotoxin, phytohemagglutinin).N-peptide mix stimulation of WB identified the combination of IL-2 and IL-13 secretion as superior to IFN-γ secretion to discriminate between COVID-19-convalescent patients and healthy controls (p .0001). Comparable results were obtained with M- or S-peptides, the latter almost comparably recalled IL-2, IFN-γ, and IL-13 responses in WB of vaccinees. Analysis 10 months as opposed to 10 weeks after COVID-19, but not allergic disease status, positively correlated with IL-13 recall responses. WB cytokine responses correlated with cytokine and proliferation responses of PBMC. Antigen-induced neo-expression of the C-type lectin CD69 on CD4Along with N- and S-peptide-induced IL-2 and CD69 neo-expression, we suggest to include the type 2 cytokine IL-13 as T-cellular recall marker for SARS-CoV-2 specific T-cellular immune responses after infection and vaccination.

Background: Prophylactic vaccination against infectious diseases may induce a state of long-term protection in the otherwise healthy host. However, the situation is less predictable in immunocompromised patients and may require adjustment of vaccination schedules and/or basic therapy. Methods: A patient in full remission of multiple myeloma since the last three years and on long-term maintenance therapy with pomalidomide, a drug inhibiting angiogenesis and myeloma cell growth, was vaccinated twice with Comirnaty followed by two vaccinations with Vaxzevria. Seroconversion and SARS-CoV-2-specific cellular responses were monitored. Results: No signs of seroconversion or T cellular memory were observed after the first “full immunization” with Comirnaty. Consequently, long-term-maintenance therapy with Pomalidomide was stopped and two additional shots of Vaxzevria were administered after which the patient seroconverted with Spike(S)-protein specific antibody levels reaching 49 BAU/mL, mild S-peptide pool-specific T cell proliferation, effector cytokine production (IL-2, IL-13), and T cellular activation with increased numbers of CD3+CD4+CD25+ T cells as compared to vaccinated and non-vaccinated control subjects. However, despite suspension of immunosuppression and administration of in total four consecutive heterologous SARS-CoV-2 vaccine shots, the patient did not develop neutralizing RBD-specific antibodies. Conclusions: Despite immunomonitoring-based adjustment of vaccination and/or therapy schedules vaccination success, with clear correlates of protection, the development of RBD-specific antibodies could not be achieved in the immunocompromised patient with current SARS-CoV-2 vaccines. Thus, our report emphasizes the need for improved active and passive immunization strategies for SARS-CoV-2 infections.

Background SARS‐CoV‐2 has triggered a pandemic that is now claiming many lives. Several studies have investigated cellular immune responses in COVID‐19‐infected patients during disease but little is known regarding a possible protracted impact of COVID‐19 on the adaptive and innate immune system in COVID‐19 convalescent patients. Methods We used multiparametric flow cytometry to analyze whole peripheral blood samples and determined SARS‐CoV‐2‐specific antibody levels against the S‐protein, its RBD‐subunit, and viral nucleocapsid in a cohort of COVID‐19 convalescent patients who had mild disease ~10 weeks after infection (n = 109) and healthy control subjects (n = 98). Furthermore, we correlated immunological changes with clinical and demographic parameters. Results Even ten weeks after disease COVID‐19 convalescent patients had fewer neutrophils, while their cytotoxic CD8+ T cells were activated, reflected as higher HLA‐DR and CD38 expression. Multiparametric regression analyses showed that in COVID‐19‐infected patients both CD3+CD4+ and CD3+CD8+ effector memory cells were higher, while CD25+Foxp3+ T regulatory cells were lower. In addition, both transitional B cell and plasmablast levels were significantly elevated in COVID‐19‐infected patients. Fever (duration, level) correlated with numbers of central memory CD4+ T cells and anti‐S and anti‐RBD, but not anti‐NC antibody levels. Moreover, a “young immunological age” as determined by numbers of CD3+CD45RA+CD62L+CD31+ recent thymic emigrants was associated with a loss of sense of taste and/or smell. Conclusion Acute SARS‐CoV‐2 infection leaves protracted beneficial (ie, activation of T cells) and potentially harmful (ie, reduction of neutrophils) imprints in the cellular immune system in addition to induction of specific antibody responses., Ten weeks after disease, COVID‐19 patients had fewer neutrophils compared to subjects without COVID‐19, while their cytotoxic CD8+ T cells were still activated. In COVID‐19 patients both CD3+CD4+ and CD3+CD8+ effector memory cells, transitional B cells and plasmablast levels were higher, while CD25+Foxp3+ T regulatory cells were lower than in subjects without COVID‐19. Fever duration correlated with higher numbers of central memory CD4+ T cells, anti‐S and anti‐RBD antibody levels, while loss of taste/smell was associated with higher levels of recent thymic emigrants. Abbreviations: COVID‐19, coronavirus disease 2019; ELISA, enzyme‐linked immunosorbent assay; KRECs, kappa‐deleting recombination excision circles; qPCR, quantitative PCR; RBD, receptor‐binding domain of SARS‐CoV2 spike protein; TRECs, T‐cell receptor excision circles; S, spike protein of SARS‐Cov2; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Recent data revealed the importance of immune reconstitution (IR) for the evaluation of possible biomarkers in National Institutes of Health (NIH)–defined chronic graft-vs.-host disease (cGVHD) and its clinical aspects. In this large pediatric study (n = 146), we have analyzed whether cellular and humoral parameters of IR in the long-term follow-up (FU) with a special emphasis on B-cell reconstitution correlate with NIH-defined cGVHD criteria. HYPOTHESIS: we were especially interested in whether meaningful cGVHD biomarkers could be defined in a large pediatric cohort. We here demonstrate for the first time in a highly homogenous pediatric patient cohort that both cGVHD (n = 38) and its activity were associated with the perturbation of the B-cell compartment, including low frequencies of CD19+CD27+ memory B-cells and increased frequencies of circulating CD19+CD21low B-cells, a well-known hyperactivated B-cell subset frequently found elevated in chronic infection and autoimmunity. Notably, resolution of cGVHD correlated with expansion of CD19+CD27+ memory B-cells and normalization of CD19+CD21low B-cell frequencies. Moreover, we found that the severity of cGVHD had an impact on parameters of IR and that severe cGVHD was associated with increased CD19+CD21low B-cell frequencies. When comparing the clinical characteristics of the active and non-active cGVHD patients (in detail at time of analyses), we found a correlation between activity and a higher overall severity of cGVHD, which means that in the active cGVHD patient group were more patients with a higher disease burden of cGVHD—despite similar risk profiles for cGVHD. Our data also provide solid evidence that the time point of analysis regarding both hematopoietic stem cell transplantation (HSCT) FU and cGVHD disease activity may be of critical importance for the detailed investigation of pediatric cohorts. Finally, we have proven that the differences in risk factors and patterns of IR, with cGVHD as its main confounding factor, between malignant and non-malignant diseases, are important to be considered in future studies aiming at identification of novel biomarkers for cGVHD.

Background: Primary immunodeficiencies represent the ‘molecular Achilles’ heels’ of human immunity. Detailed analyses of primary immunodeficiencies extend our knowledge of pivotal immunological processes, lead to novel diagnostic algorithms and shorten the time to diagnosis. Methods: Clinical/immunological phenotypes of 2 unrelated patients from Austria with combined immunodeficiency were determined. Leukocyte subpopulations of these patients, their parents and healthy controls were analyzed by flow cytometry. Patient-derived Epstein-Barr virus (EBV)-transformed B cell lines were established and complemented by candidate cDNAs. Suspected mutations were confirmed by DNA sequencing. Results: Phenotyping revealed a lack of constitutive human leukocyte antigen (HLA) class II expression on antigen-presenting cells of both patients, compatible with MHC class II deficiency. Rapid vector-based complementation analysis of the patients’ B cells identified HLA class II transactivator (CIITA) deficiency in patient VIP1 and regulatory factor X (RFX)AP deficiency in patient VIP2. CIITA deficiency was caused by a homozygous p.Glu381X mutation. RFXAP deficiency resulted from a homozygous p.Ser123ThrfsX15 mutation, not described in the Middle European population so far. Of note, HLA class II-associated invariant chain (Ii) expression levels were significantly reduced in VIP1 and 3 additional EBV-transformed B cell lines of CIITA-deficient patients but normal in EBV-transformed B cells from VIP2. In addition, peripheral blood B cells from the parents of VIP1 showed significantly reduced HLA-DR and -DP expression levels compared to healthy controls. Conclusions: Analysis of patients’ intracellular Ii and their parents’ surface HLA class II expression levels might help to identify CIITA-deficient patients already during initial phenotyping.

Chronic graft-versus-host disease (cGVHD) is a serious and frequent complication of allogeneic hematopoietic stem cell transplantation (HCT). Currently, no biomarkers for prediction and diagnosis of cGVHD are available. We performed a large prospective study focusing on noninvasive biomarkers for National Institutes of Health-defined cGVHD patients (n = 163) in comparison to time-matched HCT recipients who never experienced cGVHD (n = 64), analyzed from day 100 after HCT. In logistic regression analysis, CD19(+)CD21(low) B cells (P = .002; hazard ratio [HR], 3.31; 95% confidence interval [CI], 1.53 to 7.17) and CD4(+)CD45RA(+)CD31(+) T cells (P.001; HR, 3.88; 95% CI, 1.88 to 7.99) assessed on day 100 after HCT were significantly associated with subsequent development of cGVHD, independent of clinical parameters. A significant association with diagnosis of cGVHD was only observed for CD19(+)CD21(low) B cells (P = .008; HR, 3.00; 95% CI, 1.33 to 6.75) and CD4(+)CD45RA(+)CD31(+) T cells (P = .017; HR, 2.80; 95% CI, 1.19 to 6.55). CD19(+)CD21(low) B cells were found to have the highest discriminatory value with an area under the receiver operating curve of .77 (95% CI, .64 to .90). Our results demonstrate that CD19(+)CD21(low) B cells and CD4(+)CD45RA(+)CD31(+) T cells are significantly elevated in patients with newly diagnosed cGVHD.

Bronchiolitis obliterans syndrome (BOS), pathognomonic for chronic graft-versus-host disease (cGVHD) of the lung, is a progressive and often fatal complication after allogeneic hematopoietic cell transplantation (HCT). Biomarkers for the prediction and diagnosis of BOS are urgently needed to improve patients' prognosis. We prospectively evaluated B-cell subpopulations and B-cell activating factor (BAFF) in 136 patients (46 BOS, 41 no cGVHD, 49 cutaneous cGVHD) to define novel biomarkers for early diagnosis of National Institutes of Health-defined BOS diagnosed a median of 11 mo after HCT. Patients with newly diagnosed BOS had significantly higher percentages of CD19(+)CD21(low) B cells (25.5 versus 6.6%, P.0001), BAFF (7.3 versus 3.5 ng/mL, P = .02), and BAFF/CD19(+) ratio (0.18 versus 0.02 ng/10(3) CD19(+) B cells, P 5 .007) compared with patients without cGVHD. The area under the receiver operating curve for CD19(+)CD21(low) B cells was 0.97 (95% confidence interval, 0.94-0.99) and a cutoff point9% was optimal for diagnosing BOS in patients with first drop of pulmonary function tests with a sensitivity of 96% and a negative predictive value of 94%. Thus, elevated levels of CD19(+)CD21(low) B cells are a potential novel biomarker for HCT patients at risk for developing BOS at an early stage and could allow improvement of patient outcome.

Manifestations of chronic graft-versus-host disease (cGVHD) can resemble those seen in immunodeficiency states and autoimmune disorders. Reports by us and others suggest an involvement of B cells in the pathogenesis of cGVHD. We investigated B-lymphocyte subpopulations in cGVHD cohorts defined by serum immunoglobulin G (IgG) levels to characterize novel biomarkers for impairment of humoral immunity after allogeneic hematopoietic stem cell transplantation. Seventy-six patients were enrolled a median of 46 months after hematopoietic stem cell transplantation. The hypogammaglobulinemia group had significantly diminished CD19+ B cells (165 vs 454 vs 417 × 106/L) with elevated CD19+CD21low immature (16.5%, 7.7%, and 9.1%) and CD19+CD21int-highCD38highIgMhigh transitional (10.5% vs 4.2% vs 6.3%) B-cell proportions compared with the normogammaglobulinemia and hypergammaglobulinemia groups. CD19+CD10−CD27−CD21high naive B cells were highly elevated in all patients with cGVHD. CD19+CD27+IgD+ non–class-switched (4 vs 12 vs 11 × 106/L) and class-switched (7 vs 35 vs 42 × 106/L) memory B cells were significantly lower in the hypogammaglobulinemia group compared with the others. Besides significantly higher B-cell activation factor/B-cell ratios, significantly more cGVHD patients with hypergammaglobulinemia had autoantibodies compared with the hypogammaglobulinemia subgroup (68% vs 24%, P = .024). In conclusion, B-cell subpopulations can serve as novel cellular biomarkers for immunodeficiency and autoimmunity indicating different pathogenetic mechanisms of cGVHD and encouraging future prospective longitudinal studies.

To the editor: B lymphocytes are increasingly assigned an important role in the pathogenesis of auto-and alloimmune diseases including chronic graft-versus-host disease (cGVHD)[1][1][⇓][2][⇓][3]–[4][4] where abnormalities of B-cell homeostasis in patients with active cGVHD have been observed

Abstract 900 Introduction: Bronchiolitis obliterans syndrome (BOS) characterized by the new development of fixed airflow obstruction after allogeneic hematopoietic cell transplantation (HCT) is an increasingly recognized manifestation of chronic graft-versus-host disease (cGVHD) affecting up to 30% of patients. BOS is associated with increased non-relapse mortality and poor patient outcome after HCT. Its cause is currently unknown but allorecognition of lung antigens is the suspected etiology since BOS has been observed in states of alloimmunity such as cGVHD and after lung transplantation. Currently, the diagnosis of BOS is based on radiologic findings in high resolution expiratory chest CTs (HR-CT) and pulmonary function tests (PFTs). For early diagnosis of BOS biomarkers would be highly desirable allowing efficient treatment of early disease before progression to irreversibility is observed. B-cells are of importance in cGVHD and excess of B-cell activation factor (BAFF), increased CD19+CD21- immature transitional B-cells and dysgammaglobulinemia characterize cGVHD. Objective: We investigated circulating B-cell subpopulations in cGVHD patients to define novel cellular biomarkers for diagnosis of BOS and scoring its severity. Patients and methods: One hundred and thirty-five patients (74 males, 61 females) with a median age of 46 (range, 20–65) years, given myeloablative (n=76) or reduced-intensity (n=59) conditioning for related (n=108) or unrelated donor (n=27) HCT were prospectively evaluated for presence of cGVHD from day 100 after HCT and thereafter serially every 3 months for 2 years. At the same time points PFTs, multicolor flow cytometric analyses of peripheral blood (PB) cells stained for CD19, CD21, CD27, CD10, CD38, IgM, IgD, CD3, CD4, CD8, and CD56, measurements of serum immunoglobulin levels and BAFF were performed. BOS and its severity were defined according to the NIH consensus criteria. All patients with BOS except 2 had HR-CTs of the lung, bronchoalveolar lavage and transbronchial biopsies performed. Results: After a mean follow-up of 250 (range, 100–640) days after HCT 35 of 135 (26%) patients developed BOS. Other manifestations of cGVHD included skin in 72%, eyes in 58%, oral mucosa in 50%, and liver in 65% of patients. Severity of BOS consisted of NIH score I (FEV1=60-79%) in 14 (40%), NIH score II (FEV1=40-59%) in 16 (46%), and NIH score III (FEV Conclusion: Disturbance of B-cell homeostasis is more pronounced in cGVHD of the lung compared to other main organ manifestations. B-cell subpopulation numbers and excess of BAFF can serve as novel cellular biomarkers for BOS. Disclosures: No relevant conflicts of interest to declare.

Abstract 2226 Poster Board II-203 Chronic graft-versus-host disease (cGVHD) is a major cause of mortality after allogeneic hematopoietic stem cell transplantation (HCT) and deleteriously affects the quality of life in surviving patients who otherwise have been cured of their underlying disease. Corticosteroids are the mainstay of therapy for cGVHD. Patients with steroid-refractory or steroid-intolerant cGVHD have received numerous immunosuppressive agents with a wide range of responses. Due to possible side effects of long-term immunosuppression including infectious complications and severe organ toxicities, biomarkers for prediction of response would be highly desirable. Recently, we reported that proportions of immature CD19+CD21- B-lymphocytes predict the response to extracorporeal photopheresis (ECP) in patients with cGVHD (Blood 114: 744-746, 2009). Whether these findings were ECP-specific had to remain speculative since no other treatment cohorts had been analyzed and the mechanisms of action of ECP are still being elucidated. Therefore, we investigated B cell subpopulations in peripheral blood (PB) of 74 patients (median age 40 years, range 20-59 years) given different therapies including cyclosporine A (CSA n=24), tacrolimus (n=13), sirolimus (n=18) and ECP (n=19) for moderate (n=29) or severe (n=45) cGVHD according to the NIH Consensus. Duration of cGVHD before assessment of B cell subpopulations was a median of 14 (range, 0.5-120) months. Organ involvement at study entry consisted of skin in 45 patients (61%), oral mucosa in 50 (68%), eyes in 28 (38%), liver in 22 (30%) and lungs in 23 (31%), respectively . Seventy-three percent of patients had more than 2 organs affected by cGVHD. PB leukocytes were analyzed by multiparameter flow cytometry after staining for CD19, CD27, CD21 and surface Ig. Patients were assessed for cGVHD activity and response to therapy according to the NIH Consensus Development Project criteria prior to start of immunosuppressive therapy and every 3 months thereafter. B cell subpopulations of responders and non-responders after 6 months of therapy were compared. Overall, 45 patients (61%) responded to therapy including 20/24 (83%) given CSA, 6/13 (46%) on tacrolimus, 7/18 (39%) on sirolimus, and 12/19 (63%) given ECP, respectively. Among responders 16 (9 on CSA, 7 on ECP) achieved a complete resolution of cGVHD after 6 months of therapy. Prior to start of immunosuppressive therapy non-responders had significantly (p=0.03) higher proportions of immature CD19+CD21- B-lymphocytes with a mean of 19.4% (range, 4.17-45) compared with a mean of 13.3% (range, 1.3-54) in responders to 6 months of therapy. Highest immature CD19+CD21- B-lymphocyte numbers were observed in patients not responding to 6 months of therapy with sirolimus or ECP. Comparisons revealed significantly higher proportions of immature CD19+CD21- B-lymphocytes in non-responders compared to responders to either sirolimus (mean of 23% vs 9.3%, p=0.02), tacrolimus (mean of 17.9% vs 8.6%,p=0.02), or ECP (mean of 22% vs 13.7%, p=0.02), respectively. In the CSA cohort no significant difference in immature CD19+CD21- B-lymphocyte numbers between responders and non-responders was observed. In addition, no significant difference in memory CD27+ B-lymphocytes was observed prior to start of immunosuppressive therapy in any patient cohort between responders and non-responders. After 6 months of immunosuppressive therapy all responding patients had a significant (p=0.01) decrease of percentages of immature CD19+CD21- B-lymphocytes from a mean of 13.3% (range, 1.3-54) prior to therapy to a mean of 8.2% (range, 1.1-30) 6 months later. In addition, in complete responders the immature CD19+CD21- B-lymphocytes significantly (p=0.04) decreased from a mean of 14.06% (range, 1.3-54.6) to a mean of 7% (range, 1.1-36) after 6 months. In all non-responder percentages of immature CD19+CD21- B-lymphocytes either increased or remained unchanged after 6 months of therapy. In conclusion, relative amounts of immature CD19+CD21- B-lymphocytes assessed prior to start of cGVHD therapy may predict response to ECP, tacrolimus, and sirolimus. Increased proportions of CD21- B-lymphocytes could be part of the autoimmune pathogenesis resulting in autoreactive B-cells in cGVHD. The correlation of numbers of B cell subsets and activity of cGVHD as observed in our study supports this hypothesis. Therefore, this novel cellular biomarker should be investigated in larger treatment cohorts of cGVHD patients. Disclosures: Off Label Use: Sirolimus and ECP for therapy of chronic GVHD. Greinix:Therakos: Consultancy, Speakers Bureau. Knobler:Therakos: Consultancy, Speakers Bureau.