A Prospective Trial of Extracorporeal Photopheresis for Chronic Graft-versus-Host Disease Reveals Significant Disease Response and No Association with Frequency of Regulatory T Cells.

Extracorporeal photopheresis (ECP) is an accepted treatment for chronic graft-versus-host disease (cGVHD); however, the mechanism of action is unclear. We conducted a prospective multicenter clinical trial to assess ECP response rates using the 2005 National Institutes of Health (NIH) consensus criteria and to assess the relationship between regulatory T cells (Tregs) and treatment response (NCT01324908). Eighty-three patients with any NIH subtype of cGVHD were enrolled, irrespective of number of prior lines of treatment, and 6 were subsequently excluded because of the absence of follow-up from cancer relapse, infection, or study withdrawal. Study outcomes were provider-assessed response and formal response by 2005 NIH criteria. Peripheral blood samples were collected at prespecified study visits and were analyzed by flow cytometry for Tregs. In a heavily pretreated cohort of patients, with a median of 2 prior lines of therapy, 62.3% of patients had a provider-assessed response to ECP and 43.5% had response by NIH criteria. These assessments showed only a slight agreement (kappa statistic, .09). In a logistic regression model that included previously identified risk factors such as bilirubin, platelet count, and time from transplant to study entry, no clinical factors were associated with the provider's response assessment. Furthermore, there was no significant difference in percentage of Tregs in blood leukocytes at study entry and completion or in overall change in Treg frequency between ECP responders and nonresponders. ECP was associated with a clinically significant decrease in median prednisone dose (.36 to .14 mg/kg, P < .001) from study entry to last visit and a significant decrease in global severity of cGVHD and total body surface area with erythematous rash. Overall, ECP was able to deliver response using NIH response criteria in a highly pretreated cohort with moderate and severe cGVHD independent of most previous risk factors for adverse outcomes of cGVHD.

Dynamical System Modeling of Immune Reconstitution after Allogeneic Stem Cell Transplantation Identifies Patients at Risk for Adverse Outcomes.

Systems that evolve over time and follow mathematical laws as they evolve are called dynamical systems. Lymphocyte recovery and clinical outcomes in 41 allograft recipients conditioned using antithymocyte globulin (ATG) and 4.5-Gy total body irradiation were studied to determine if immune reconstitution could be described as a dynamical system. Survival, relapse, and graft-versus-host disease (GVHD) were not significantly different in 2 cohorts of patients receiving different doses of ATG. However, donor-derived CD3(+) cell reconstitution was superior in the lower ATG dose cohort, and there were fewer instances of donor lymphocyte infusion (DLI). Lymphoid recovery was plotted in each individual over time and demonstrated 1 of 3 sigmoid growth patterns: Pattern A (n = 15) had rapid growth with high lymphocyte counts, pattern B (n = 14) had slower growth with intermediate recovery, and pattern C (n = 10) had poor lymphocyte reconstitution. There was a significant association between lymphocyte recovery patterns and both the rate of change of donor-derived CD3(+) at day 30 after stem cell transplantation (SCT) and clinical outcomes. GVHD was observed more frequently with pattern A, relapse and DLI more so with pattern C, with a consequent survival advantage in patients with patterns A and B. We conclude that evaluating immune reconstitution after SCT as a dynamical system may differentiate patients at risk of adverse outcomes and allow early intervention to modulate that risk.

Genetic variation in recipient B-cell activating factor modulates phenotype of GVHD.

B-cell activating factor (BAFF) single nucleotide polymorphisms (SNPs) are associated with autoimmune diseases. Because patients with classic and overlap chronic GVHD (cGVHD) have features of autoimmune diseases, we studied the association of recipient and/or donor BAFF SNPs with the phenotype of GVHD after allogeneic stem cell transplantation. Twenty tagSNPs of the BAFF gene were genotyped in 164 recipient/donor pairs. GVHD after day 100 occurred in 124 (76%) patients: acute GVHD (aGVHD) subtypes (n = 23), overlap GVHD (n = 29), and classic cGVHD (n = 72). In SNP analyses, 9 of the 20 tag SNPs were significant comparing classic/overlap cGVHD versus aGVHD subtypes/no GVHD. In multivariate analyses, 4 recipient BAFF SNPs (rs16972217 [odds ratio = 2.72, P = .004], rs7993590 [odds ratio = 2.35, P = .011], rs12428930 [odds ratio2.53, P = .008], and rs2893321 [odds ratio = 2.48, P = .009]) were independent predictors of GVHD subtypes, adjusted for conventional predictors of cGVHD. This study shows that genetic variation of BAFF modulates GVHD phenotype after allogeneic stem cell transplantation.

Genetic variation in donor CTLA-4 regulatory region is a strong predictor of outcome after allogeneic hematopoietic cell transplantation for hematologic malignancies.

Relapse remains a major cause of death after allogeneic hematopoietic cell transplantation (allo-HCT). Graft-versus-tumor effect is primarily mediated by donor T cells. Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a critical inhibitor of T cell proliferation. Single nucleotide polymorphisms (SNPs) in CTLA-4 may affect immune responses. We hypothesized that CTLA-4 SNPs will be associated with disease control after allo-HCT. One hundred sixty-four adult patients with the availability of pretransplantation recipient and donor DNA samples were included in this analysis. Ten tagSNPs of the CTLA-4 gene were identified. Donor CTLA-4 SNP rs4553808 was associated with decreased relapse-free survival (RFS) (P = .019) and overall survival (OS) (P = .033). In multivariable analysis of an additive genetic model, genotype of CTLA-4 SNP rs4553808 was an independent risk factor for inferior RFS (hazard ratio [HR] = 1.73, 95% confidence interval [CI] 1.10-2.71, P = .017) and OS (HR = 1.84, 95% CI 1.13-3.0, P = .015). CTLA-4 SNPs can be used to identify high-risk patient subsets that may benefit from preemptive immunomodulation to decrease relapse rates and improve survival.

Favorable outcomes in patients with high donor-derived T cell count after in vivo T cell-depleted reduced-intensity allogeneic stem cell transplantation.

Patients with hematologic malignancies were conditioned using a rabbit antithymocyte globulin-based reduced-intensity conditioning regimen for allogeneic stem cell transplantation. Donor-derived CD3(+) cell count (ddCD3), a product of CD3(+) cell chimerism and absolute CD3(+) cell count, when <110/µL at 8 weeks post-stem cell transplantation predicted a high risk of sustained mixed chimerism and relapse. Alternatively, patients with a higher ddCD3 developed graft-versus-host disease more frequently, and when partially chimeric, had higher rates of conversion to full donor chimerism after withdrawal of immunosuppression. Early data from our small cohort of patients indicate that ddCD3 at 8 weeks may be used to guide decisions regarding withdrawal of immunosuppression and administration of donor lymphocyte infusion in partially T cell-depleted reduced-intensity regimens.

Epigenetic induction of adaptive immune response in multiple myeloma: sequential azacitidine and lenalidomide generate cancer testis antigen-specific cellular immunity.

Patients with multiple myeloma (MM) undergoing high dose therapy and autologous stem cell transplantation (SCT) remain at risk for disease progression. Induction of the expression of highly immunogenic cancer testis antigens (CTA) in malignant plasma cells in MM patients may trigger a protective immune response following SCT. We initiated a phase II clinical trial of the DNA hypomethylating agent, azacitidine (Aza) administered sequentially with lenalidomide (Rev) in patients with MM. Three cycles of Aza and Rev were administered and autologous lymphocytes were collected following the 2nd and 3rd cycles of Aza-Rev and cryopreserved. Subsequent stem cell mobilization was followed by high-dose melphalan and SCT. Autologous lymphocyte infusion (ALI) was performed in the second month following transplantation. Fourteen patients have completed the investigational therapy; autologous lymphocytes were collected from all of the patients. Thirteen patients have successfully completed SCT and 11 have undergone ALI. Six patients tested have demonstrated CTA up-regulation in either unfractionated bone marrow (n = 4) or CD138+ cells (n = 2). CTA (CTAG1B)-specific T cell response has been observed in all three patients tested and persists following SCT. Epigenetic induction of an adaptive immune response to cancer testis antigens is safe and feasible in MM patients undergoing SCT.

Persistent anemia after implantation of the total artificial heart.

BACKGROUND: The total artificial heart (TAH) replaces the heart with 2 pneumatic pumps and 4 tilting disk mechanical valves. It was hypothesized that patients receiving TAH support have persistent hemolysis that resolves after heart transplantation (HT). METHODS AND RESULTS: Hematocrit (HCT) was compared in patients on TAH to left ventricular assist device (LVAD) support for bridge to HT. Data were compared with t tests. The TAH (n = 36; mean age 47 ± 13 years) and LVAD patients (n = 14; mean age 53 ± 12 years) were supported for a median of 83 (interquartile range [IQR] 43-115) and 106 days (IQR 84-134), respectively. Hematocrit was similar between the TAH and LVAD patients (34 ± 6% vs 37 ± 5%; P = .07) at baseline. After placement, TAH patients had lower HCT at 2 (20 ± 2% vs 24 ± 3%), 4 (22 ± 3% vs 26 ± 3%), 6 (22 ± 4% vs 30 ± 4%), and 8 weeks (23 ± 4% vs 33 ± 5%; P < .001 for all). There were no differences in HCT at 1 (30 ± 4% vs 29 ± 7%; P = .42) and 3 months (35 ± 7% vs 35 ± 4%; P = .98) after removal of the devices for HT. TAH patients had undetectable haptoglobin in 96% of assessments, increased lactate dehydrogenase (1,128 ± 384 units/L), and detectable plasma free hemoglobin in 40% of measurements (21 ± 15 mg/dL). High sensitivity C-reactive protein (52 ± 50 mg/dL) was elevated, and reticulocyte production index was decreased (1.6 ± 0.6). CONCLUSIONS: Patients implanted with a TAH have persistent anemia that resolves only after HT. The association of hemolysis, ineffective erythropoiesis, and inflammation with the TAH warrants further study.

The influence of lymphoid reconstitution kinetics on clinical outcomes in allogeneic stem cell transplantation.

Lymphoid recovery following myeloablative stem cell transplantation (SCT) displays a logistic pattern of exponential growth followed by a plateau. Within this logistic framework, lymphoid recovery is characterized by the parameters R (slope of ascent), a (time of maximal rate of ascent) and K (plateau), the 'steady-state' lymphocyte count. A retrospective analysis of allogeneic SCT performed from 2008 to 2013 was undertaken to compare lymphoid recovery and clinical outcomes in 131 patients with acute myelogenous leukemia, acute lymphocytic leukemia, and myelodysplastic syndromes. Using Prism software, a logistic curve was successfully fit to the absolute lymphocyte count recovery in all patients. Patients were classified according to the magnitude and rate of lymphoid recovery; pattern A achieved an absolute lymphocyte counts (ALC) of >1000/µL by day 45, pattern B an ALC 500 < x < 1000/µL, and pattern C an ALC <500/µL. Pattern A was characterized by a higher mean K (p < .0001) compared with patterns B and C. Patients with patterns B and C were more likely to have mixed T cell chimerism at 90 d following SCT (p = .01). There was a trend towards improved survival (and relapse-free survival) in those with pattern A and B at 1 year compared to pattern C (p = .073). There was no difference in cGVHD (p = .42) or relapse (p = .45) between pattern types. Cytomegalovirus (CMV), aGVHD, and all relapse were heralded by deviation from logistic behavior. Pattern C patients were more likely to require donor lymphocyte infusion (DLI) (p = .017). Weaning of tacrolimus post-transplant was associated with a second, separate logistic expansion in some patients. This study demonstrated that lymphoid reconstitution follows a prototypical logistic recovery and that pattern observed correlates with T cell chimerism and need for DLI, and may influence survival.

Stem cell transplantation as a dynamical system: are clinical outcomes deterministic?

Outcomes in stem cell transplantation (SCT) are modeled using probability theory. However, the clinical course following SCT appears to demonstrate many characteristics of dynamical systems, especially when outcomes are considered in the context of immune reconstitution. Dynamical systems tend to evolve over time according to mathematically determined rules. Characteristically, the future states of the system are predicated on the states preceding them, and there is sensitivity to initial conditions. In SCT, the interaction between donor T cells and the recipient may be considered as such a system in which, graft source, conditioning, and early immunosuppression profoundly influence immune reconstitution over time. This eventually determines clinical outcomes, either the emergence of tolerance or the development of graft versus host disease. In this paper, parallels between SCT and dynamical systems are explored and a conceptual framework for developing mathematical models to understand disparate transplant outcomes is proposed.

Specific Antibodies and Their Potential Role in Periodontal Diseases.

Periodontal diseases are thought to result from inflammatory responses to bacterial challenges in the gingival crevicular area. Antibodies are a major host-protective mechanism in many bacterial infections. Consequently, the antibody responses to suspected periodontal pathogenic bacteria have been extensively measured as to their relationship to diseases and specificity for suspected pathogens associated with progressing disease sites. Recently, studies on the bacterial immunogen characterization, antibodysubclass identification, and antibody biological capabilities have been reported. Although increased antibody levels to certain suspected periodontal pathogens were associated with periodontal diseases in humans, little evidence exists as to the role of these antibodies in the infectious process. In vivo experiments in animals indicated that specific antibodies against certain suspected periodontal pathogens were associated with suppression of bacterial colonization, limiting the spread of infection, and a decrease in alveolar bone loss. However, in vitro as well as in vivo experiments suggested that phagocytic cells are required for efficient bactericidal activity of antibodies and that the presence of other sensitized immune cells may either have inhibited or enhanced the infectivity of certain periodontal pathogens. Possible explanations for the observed inconsistencies are presented and the potential for utilization of specific anti-periodontal pathogen responses in the understanding and prevention of diseases is discussed. J Periodontol 1993; 64:807- 818.

A Role for Antibiotics in the Treatment of Refractory Periodontitis.

Refractory periodontitis is considered by many investigators to be a separate disease entity that is descriptive of a particular patient who has multiple sites, rather than a few individual sites, that do not respond to conventional periodontal treatment modalities. Such patients continue to demonstrate loss of attachment and alveolar bone despite frequent periodontal treatment which includes surgical intervention, scaling and root planing, and often systemically-administered tetracycline. Controlled clinical studies have demonstrated that both clindamycin-hydrochloride and amoxicillin/clavulanate potassium (Au) are beneficial when used in conjunction with periodontal scaling. Gordon et al. found improvements in attachment levels, inflammation, suppuration, and a decrease in pocket depths for up to 2 years following a 7-day course of Clindamycin given in conjunction with a full-mouth scaling. The incidence of disease activity decreased from an annual rate of 8% of all sites prior to antibiotic treatment to 0.5% after treatment. Magnusson, reporting on a similar group treated with a 14-day course of Au, found an average loss of attachment of 2.2 mm and an increase in pocket depth of 1.5 mm in sites demonstrating disease progression prior to antibiotic treatment. At 3 months post-antibiotic therapy, these sites had regained an average of 2 mm of attachment and pocket depths had decreased an equivalent amount. Both attachment levels and pocket depths remained relatively stable for up to 12 months post-therapy. In an ongoing study, 30 subjects with refractory Periodontitis were treated with either Clindamycin or Au in conjunction with scaling or scaling plus a placebo. Prior to antibiotic treatment, but while being scaled at 3-month intervals, sites with disease activity lost an average 2.4 mm of attachment. At 3 months post-treatment, the clindamycin-treated group showed an average gain of 2.1 mm, the Au-treated group gained 1.9 mm, and the scaling group gained 1.4 mm in attachment. The clindamycin group remained relatively stable for up to 21 months and the Au group remained stable for about 15 months without additional treatment. Five of the 6 subjects treated with scaling alone required additional treatment within 9 months. Preliminary analyses have indicated that at least two patterns or rates of attachment loss may be associated with refractory periodontitis and that each pattern may be indicative of a different microflora. The pattern associated with a relatively rapid loss of attachment was characterized by a Gram-negative flora which contained spirochetes, P. intermedia, and Fusobacterium species. A slow, continuous rate was associated with a predominantly Gram-positive flora containing a high proportion of S. intermedius and/or a S. intermedius-like organism. J Periodontol 1993; 64:772-781.

Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow.

Adult human bone marrow (ABM) is an important source of hematopoietic stem cells for transplantation in the treatment of malignant and nonmalignant diseases. However, in contrast to the recent progress that has been achieved with umbilical cord blood, methods to expand ABM stem cells for therapeutic applications have been disappointing. In this study, we describe a novel culture method that uses human brain endothelial cells (HUBECs) and that supports the quantitative expansion of the most primitive measurable cell within the adult bone marrow compartment, the nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cell (SRC). Coculture of human ABM CD34(+) cells with brain endothelial cells for 7 days supported a 5.4-fold increase in CD34(+) cells, induced more than 95% of the CD34(+)CD38(-) subset to enter cell division, and produced progeny that engrafted NOD/SCID mice at significantly higher rates than fresh ABM CD34(+) cells. Using a limiting dilution analysis, we found the frequency of SRCs within fresh ABM CD34(+) cells to be 1 in 9.9 x 10(5) cells. Following HUBEC culture, the estimated frequency of SRCs increased to 1 in 2.4 x 10(5) cells. All mice that received transplants of HUBEC-cultured cells showed B-lymphoid and myeloid differentiation, indicating that a primitive hematopoietic cell was preserved during culture. Noncontact HUBEC cultures also maintained SRCs at a level comparable to contact HUBEC cultures, suggesting that cell-to-cell contact was not required. These data demonstrate that human brain endothelial cells possess a unique hematopoietic activity that increases the repopulating capacity of adult human bone marrow.