David Senitzer

Population coverage by HLA class-I restricted cytotoxic T-lymphocyte epitopes.

Abstract. Vaccination using cytotoxic T-lymphocyte (CTL) epitopes has become a widely used immunization strategy, especially because their structure makes them an attractive alternative to the delivery of whole proteins as immunogens. Nonetheless, their use is limited, in particular because of their specificity, being recognized only by cognate HLA alleles. The potential for immunizing a substantial portion of an ethnically diverse population using a modest number of peptides has been aided by the identification of HLA supertypes. However, the derivation of epitopes is often guided by methods that do not guarantee cross-reactivity, so we consider the feasibility of providing vaccine coverage to a multi-ethnic population under different assumptions. In particular, two large datasets are used to estimate the number of peptides needed to provide ≥90% group-specific coverage of a multi-ethnic population, when specificity is assumed to be either to a single serologic or molecular type. These assumptions are evaluated utilizing a clinically important epitope repertoire derived from two human cytomegalovirus proteins, and data on the in vitro memory response elicited by these peptides is presented. In summary, our combined theoretical and empiric studies suggest that 90% coverage of some ethnic groups is attainable with 11 uniquely defined HLA-restricted CTL epitopes. The derivation of four or more additional CTL epitopes is needed to attain 90% coverage of Blacks or Asians, the minimally covered groups. Ninety percent coverage of all major ethnic groups in a multi-ethnic population appears feasible without relying on cross-reactivity, but may require two to three times more CTL epitopes than estimated for serologic data, homogenous populations, or HLA alleles grouped as supertypes.

Hematopoietic Stem-Cell Contribution to Ectopic Skeletogenesis

BACKGROUND Fibrodysplasia ossificans progressiva is a rare genetic disorder of ectopic skeletogenesis associated with dysregulation of bone morphogenetic protein (BMP) signaling. Hematopoietic cells have been implicated in the ectopic skeletogenesis of fibrodysplasia ossificans progressiva, and their replacement has been postulated as a possible cure. However, the definitive contribution of hematopoietic cells to the pathogenesis of ectopic skeletogenesis remains obscure. METHODS We employed both careful clinical observation and in vivo murine transplantation studies to more precisely determine the contribution of hematopoietic cells to ectopic skeletogenesis. We identified a patient with fibrodysplasia ossificans progressiva who had undergone bone marrow transplantation for the treatment of intercurrent aplastic anemia twenty-five years earlier and investigated whether the clinical course of the fibrodysplasia ossificans progressiva had been influenced by bone marrow replacement or immunosuppression, or both. In complementary studies, we transplanted hematopoietic stem cells from constitutively expressing LacZ transgenic mice to identify the contribution of hematopoietic cells to BMP4-induced heterotopic ossification, a histopathologic model of fibrodysplasia ossificans progressiva. RESULTS We found that replacement of hematopoietic cells was not sufficient to prevent ectopic skeletogenesis in the patient with fibrodysplasia ossificans progressiva but pharmacologic suppression of the apparently normal donor immune system following transplantation in the new host modulated the activity of the fibrodysplasia ossificans progressiva and diminished the expression of skeletal ectopia. In complementary murine transplantation studies, we found that cells of hematopoietic origin contributed to the early inflammatory and late marrow-repopulating stages of BMP4-induced heterotopic ossification but were not represented in the fibroproliferative, chondrogenic, or osteogenic stages of heterotopic ossification. Interestingly, both recombinant human BMP4 induction in an animal model and the dysregulated BMP signaling pathway in a patient with fibrodysplasia ossificans progressiva were sufficient to recruit at least two populations of cells, one of hematopoietic origin and at least one of non-hematopoietic origin, that contribute to the formation of an ectopic skeleton. CONCLUSIONS Taken together, these findings demonstrate that bone marrow transplantation did not cure fibrodysplasia ossificans progressiva in the patient in this study, most likely because the hematopoietic cell population is not the site, or at least not the dominant site, of the intrinsic dysregulation of the BMP signaling pathway in fibrodysplasia ossificans progressiva. However, following transplantation of bone marrow from a presumably normal donor, immunosuppression of the immune system appeared to ameliorate activation of ectopic skeletogenesis in a genetically susceptible host. Thus, cells of hematopoietic origin may contribute to the formation of an ectopic skeleton, although they are not sufficient to initiate the process alone.

The Effect of Single and Combined Activating Killer Immunoglobulin-like Receptor Genotypes on Cytomegalovirus Infection and Immunity after Hematopoietic Cell Transplantation

It has been shown that activating killer Ig-like receptor (aKIR) genes are important for control of cytomegalovirus (CMV) reactivation after hematopoietic cell transplantation (HCT). To date, using the broad classification of KIR haplotypes A and B, the precise role of individual KIR genes in the control of infection cannot be discerned. To address this, a consecutive case series of 211 non-T cell-depleted HCT patients all at risk for CMV were monitored biweekly for CMV DNA in plasma by quantitative polymerase chain reaction (Q-PCR) and at intervals for CMV-specific T cell immunity. Comparing patients with CMV reactivation (n = 152) to those with no reactivation (n = 59), the presence of specific aKIR haplotypes in the donor, but not in the recipient, were associated with protection from CMV reactivation and control of peak plasma CMV DNA (P < .001). A donor aKIR profile, predictive for low risk of CMV reactivation, contained either aKIR2DS2 and aKIR2DS4 or had >/=5 aKIR genes. Neither donor nor recipient inhibitory KIR (iKIR) played a role in a protective effect. CD4(+)- and CD8(+)-specific CMV immunity did not explain reduced CMV infection. The initial control of CMV infection after HCT is managed by aKIR functions, and donor aKIR haplotypes deserve further evaluation in donor selection for optimized HCT outcome.

Reduced-intensity conditioning followed by peripheral blood stem cell transplantation for adult patients with high-risk acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) with high-risk features has a poor prognosis in adults despite aggressive chemotherapy. Reduced-intensity conditioning (RIC) is a lower toxicity alternative for high-risk patients requiring hematopoietic cell transplantation (HCT); however, it has not been widely used for ALL. We conducted a retrospective study of 24 high-risk adult ALL patients who received an RIC regimen of fludarabine (Flu)/melphalan (Mel) prior to allogeneic peripheral blood stem cell transplantation (PBSCT) between 6/14/02 and 6/15/07 at the City of Hope. Indications for the RIC regimen were: (1) aged 50 years or older (42%), (2) compromised organ function (54%), or (3) recipient of a previous HCT (37.5%). Patients had a median age of 47.5 years and the median follow-up was 28.5 months for living patients. Both overall survival (OS) and disease-free survival (DFS) at 2 years was 61.5%. Relapse incidence was 21.1% and nonrelapse mortality (NRM) was 21.5% at 2 years. Chronic graft-versus-host (cGVHD) developed in 86% of evaluable patients. In this series, no significant correlations were made between outcomes and patient age, presence of Philadelphia chromosome, relatedness of donor source, or prior HCT. These high survival rates for high-risk ALL patients following RIC HCT may offer a promising option for patients not eligible for a standard myeloablative transplant.

Killer immunoglobulin-like receptor ligand HLA-Bw4 protects against multiple sclerosis†

Multiple sclerosis (MS) is a chronic inflammatory disease affecting the central nervous system. A human leukocyte antigen (HLA) class II association is well established (DRB1*1501‐DQB1*0602), but more recently HLA class II–independent associations with HLA class I variants have also been reported. The HLA class I (HLA‐A, ‐B, ‐C) molecules serve as ligands for both T‐cell receptors and killer immunoglobulin‐like receptors (KIRs). We investigated the HLA class I alleles defined by their KIR binding motifs and the KIR genes to evaluate whether these genes could influence MS susceptibility or severity, alone or in combination.

Genetic susceptibility to anthracycline-related congestive heart failure in survivors of haematopoietic cell transplantation.

Haematopoietic cell transplantation (HCT) survivors are at increased risk for developing congestive heart failure (CHF), primarily due to pre‐HCT exposure to anthracyclines. We examined the association between the development of CHF after HCT and polymorphisms in 16 candidate genes involved in anthracycline metabolism, iron homeostasis, anti‐oxidant defence, and myocardial remodelling. A nested case‐control study design was used. Cases (post‐HCT CHF) were identified from 2950 patients who underwent HCT between 1988 and 2007 at City of Hope and had survived ≥1 year. This cohort formed the sampling frame for selecting controls (without CHF) matched on: age, race/ethnicity, cumulative anthracycline exposure, stem cell source (allogeneic, autologous), and length of follow‐up. Seventy‐seven cases with pre‐HCT germline DNA and 178 controls were genotyped. Multivariate analysis revealed that the odds of CHF was higher in females [Odds Ratio (OR) = 2·9, P < 0·01], individuals with pre‐HCT chest radiation (OR = 4·7, P = 0·05), hypertension (OR = 2·9, P = 0·01), and with variants of genes coding for the NAD(P)H‐oxidase subunit RAC2 (rs13058338, 7508T→A; OR = 2·8, P < 0·01), HFE (rs1799945, 63C→G; OR = 2·5, P = 0·05) or the doxorubicin efflux transporter ABCC2 (rs8187710, 1515G→A; OR = 4·3, P < 0·01). A combined (clinical and genetic) CHF predictive model performed better [area under the curve (AUC), 0·79] than the genetic (AUC = 0·67) or the clinical (AUC = 0·69) models alone.

Impact of Graft Cell Dose on Transplant Outcomes following Unrelated Donor Allogeneic Peripheral Blood Stem Cell Transplantation: Higher CD34+ Cell Doses Are Associated with Decreased Relapse Rates

Peripheral blood stem cells (PBSC) have been increasingly used in the matched unrelated donor (MUD) transplant setting, but the impact of CD34(+) cell dose on outcomes in this setting have not been well characterized. We analyzed 181 consecutive patients who underwent MUD-PBSC transplantation at the City of Hope between August 2000 to December 2004. Patients were conditioned with either full-intensity regimen or reduced-intensity regimen. There was a significant inverse relationship between higher CD34(+) cell dose and faster neutrophil engraftment (r = -0.16, P = .035). By univariate analysis, a CD34(+) cell dose > or =4.2 x 10(6)/kg (above the lowest quartile) was associated with significantly lower relapse risk (hazard ratio [HR] = 0.67, P = .0126), with a trend for corresponding improvement for disease-free survival (HR = 0.84, P = .12) but not overall survival (HR = 0.91, P = .46). The impact of the CD34(+) cell dose remained significant in multivariate analysis. The higher CD34(+) cell dose was significantly associated with faster recovery of absolute lymphocyte counts on day +30 posttransplant. Subset analysis demonstrated that the higher CD34(+) cell dose was associated with (1) greater reduction in relapse in myeloid malignancies than that in lymphoid malignancies, (2) greater reduction in reduced-intensity conditioning than in full-intensity conditioning, (3) greater reduction in relapse when there is a inhibitory killer-cell immunoglobulin-like receptor ligand (iKIRL)-mismatch in the gravft-versus-host (GVH) direction, and (4) greater reduction in relapse when there is a lack of iKIRL, suggesting that the protective effect of CD34(+) cell dose against relapse may be immune-mediated, possibly through NK cell recovery.

One-Antigen Mismatched Related versus HLA-Matched Unrelated Donor Hematopoietic Stem Cell Transplantation in Adults with Acute Leukemia: Center for International Blood and Marrow Transplant Research Results in the Era of Molecular HLA Typing

Approximately 13% of patients lacking an HLA-identical sibling have a one-antigen-mismatched related donor (MMRD). Historically, outcomes from the use of a one-antigen MMRD were considered equivalent to those from the use of a matched unrelated donor (UD). Recent improvements in UD stem cell transplantation (SCT) resulting from better molecular HLA matching justifies investigating whether UD should be preferred over MMRD in adult patients with acute leukemia. Here, we compared the outcomes of MMRD (n = 89) and HLA-A, -B, -C, and -DRB1 allele-matched UD (n = 700) SCT reported to the Center for International Blood and Marrow Transplant Research between 1995 and 2005. The patients underwent transplantation for acute myelogenous leukemia or acute lymphoblastic leukemia in first or second complete remission. Donor type was not associated with hematologic recovery. Univariate and multivariate comparisons of MMRD versus HLA-matched UD transplants showed no statistically significant differences in overall survival, disease-free survival, treatment-related mortality, relapse, or 100-day grade III-IV acute graft-versus-host disease (GVHD). MMRD SCT was associated with a lower rate of chronic GVHD at 1 year (35% vs 47%; P = .03), which was confirmed by multivariate analysis (relative risk, 0.58; 95% confidence interval, 0.39-0.85; P < .01). According to our data, HLA-matched UD and MMRD SCT are associated with comparable survival. Given that less chronic GVHD was observed in the MMRD transplantations, this option, when available, remains the first choice in patients with acute leukemia without an HLA-identical sibling in need of allogeneic SCT.

Killer Ig-like receptor (KIR) compatibility plays a role in the prevalence of acute GVHD in unrelated hematopoietic cell transplants for AML

Summary:Killer Ig-like receptor (KIR) is a major cluster of the natural killer cell receptors and may play a role in the outcome of hematopoietic cell transplants. A total of 65 AML cases transplanted with T-replete hematopoietic cells from unrelated donors were retrospectively KIR-genotyped by a multiplex PCR method of our own design. The KIR gene frequency and genotype patterns in these 130 samples were consistent with the data in the literature. Based upon overall inhibitory and activating KIR genes in both donors and patients, we developed an algorithm to calculate a compatibility score for each transplant case as plus, zero or minus. Significantly higher incidence (18/20, 90%) of acute (a) GVHD (grade II–IV) was found in the transplant cases with plus scores than that (25/45, 56%) in the cases with zero or minus scores (P<0.01). When the scores are sorted in the opposite way, fewer cases (13/26, 50%) of aGVHD were found in the transplants with minus scores than that (30/39, 77%) in the transplants with zero or plus scores (P<0.05). The difference of aGVHD prevalence between the plus score and minus score groups is highly significant (P<0.01). KIR genotype compatibility calculated by this algorithm may predict aGVHD incidence and be helpful in choosing donors.

Does the KIR2DS5 Gene Protect from Some Human Diseases

Background KIR2DS5 gene encodes an activating natural killer cell receptor whose ligand is not known. It was recently reported to affect the outcome of hematopoietic stem cell transplantation. Methodology/Principal Findings In our studies on KIR2DS5 gene associations with human diseases, we compared the frequencies of this gene in patients and relevant controls. Typing for KIR2DS5 gene was performed by either individual or multiplex polymerase chain reactions which, when compared in the same samples, gave concordant results. We noted an apparently protective effect of KIR2DS5 gene presence in several clinical conditions, but not in others. Namely, this effect was observed in ankylosing spondylitis (p = 0.003, odds ratio [OR] = 0.47, confidence interval [CI] = 0.28–0.79), endometriosis (p = 0.03, OR = 0.25, CI = 0.07–0.82) and acute rejection of kidney graft (p = 0.0056, OR = 0.44, CI = 0.24–0.80), but not in non-small-cell lung carcinoma, rheumatoid arthritis, spontaneous abortion, or leukemia (all p>0.05). In addition, the simultaneous presence of KIR2DS5 gene and HLA-C C1 allotype exhibited an even stronger protective effect on ankylosing spondylitis (p = 0.0003, OR = 0.35, CI = 0.19–0.65), whereas a lack of KIR2DS5 and the presence of the HLA-C C2 allotype was associated with ankylosing spondylitis (p = 0.0017, OR = 1.92, CI = 1.28–2.89), whereas a lack of KIR2DS5 and presence of C1 allotype was associated with rheumatoid arthritis (p = 0.005, OR = 1.47, CI = 1.13–1.92). The presence of both KIR2DS5 and C1 seemed to protect from acute kidney graft rejection (p = 0.017, OR = 0.47, CI = 0.25–0.89), whereas lack of KIR2DS5 and presence of C2 seemed to favor rejection (p = 0.0015, OR = 2.13, CI = 1.34–3.37). Conclusions/Significance Our results suggest that KIR2DS5 may protect from endometriosis, ankylosing spondylitis, and acute rejection of kidney graft.