Poonam Dharmani-Khan

Immune Cell Subset Counts Associated with Graft-versus-Host Disease

Graft-versus-host disease (GVHD) is a major transplantation complication. The purpose of this study was to measure immune cell subsets by flow cytometry early after transplantation (before median day of GVHD onset) to identify subsets that may play a role in GVHD pathogenesis. We also measured the subsets later after transplantation to determine which subsets may be influenced by GVHD or its treatment. We studied 219 patients. We found that acute GVHD (aGVHD) was preceded by high counts of CD4 T cells and CD8 T cells. It was followed by low counts of total and naive B cells, total and cytolytic NK cells, and myeloid and plasmacytoid dendritic cells. Chronic GVHD (cGVHD) was preceded by low counts of memory B cells. In conclusion, both CD4 and CD8 T cells appear to play a role in the pathogenesis of aGVHD. Generation of B cells, NK cells, and dendritic cells may be hampered by aGVHD and/or its treatment. Memory B cells may inhibit the development of cGVHD.

High Serum Level of Antithymocyte Globulin Immediately before Graft Infusion Is Associated with a Low Likelihood of Chronic, But Not Acute, Graft-versus-Host Disease

Rabbit antithymocyte globulin (ATG) is administered during transplant conditioning to decrease the risk of both acute graft-versus-host disease (aGVHD) and chronic graft-versus-host disease (cGVHD). Here we evaluated the relationship between the serum concentration of ATG (capable of binding to lymphocytes) immediately before graft infusion (day 0) or on dayxa0+7 orxa0+28 post-transplantation and the development of aGVHD or cGVHD. We studied 180 patients whose conditioning included 4.5 mg/kg antithymocyte globulin (ATG; Thymoglobulin). For aGVHD, we found no association with ATG levels on day 0. Nevertheless, high dayxa0+7 andxa0+28 ATG levels were associated with a low likelihood of aGVHD. For cGVHD, high ATG levels at all 3 time points (days 0,xa0+7, andxa0+28) were associated with a low likelihood of cGVHD. In conclusion, high-dose ATG administration at the time of graft infusion appears to inhibit the development of cGVHD, but not aGVHD; however, higher ATG levels on daysxa0+7 andxa0+28 are associated with lower rates of both aGVHD and cGVHD.

Antithymocyte Globulin at Clinically Relevant Concentrations Kills Leukemic Blasts

In contrast to cyclosporine or methotrexate, rabbit antithymocyte globulin (ATG) used for graft-versus-host disease (GVHD) prophylaxis with myeloablative conditioning does not increase the risk of relapse after hematopoietic cell transplantation. The reason for this is unknown. We hypothesized that ATG at concentrations achieved with our standard ATG dose of 4.5xa0mg/kg exerts antileukemic activity. We measured ATG-induced killing of leukemic blasts via complement-dependent cytotoxicity (CDC) and via complement-independent cytotoxicity (CIC) in marrow or blood from 36 patients with newly diagnosed acute leukemia. The median percentage of blasts killed by CDC was 0.3% at 1xa0mg/L ATG, 2.8% at 10xa0mg/L ATG, 12.6% at 25xa0mg/L ATG, and 42.2% at 50xa0mg/L ATG. The median percentage of blasts killed by CIC after a 4-hour incubation with ATG was 1.9% at 1xa0mg/L ATG, 7.15% at 10xa0mg/L ATG, 12.1% at 25xa0mg/L ATG, and 13.9% at 50xa0mg/L ATG. CIC appeared to represent a direct induction of apoptosis by ATG. There was a high variability in the sensitivity of the blasts to ATG; at 50xa0mg/L, the percentage of blasts killed ranged from 2.6% to 97.2% via CDC and from 1.4% to 69.9% via CIC. In conclusion, ATG at clinically relevant concentrations kills leukemic blasts inxa0vitro. Some acute leukemias are highly sensitive to ATG, whereas others are relatively resistant. This finding could lead to personalized administration of ATG.

Low Counts of B Cells, Natural Killer Cells, Monocytes, Dendritic Cells, Basophils, and Eosinophils are Associated with Postengraftment Infections after Allogeneic Hematopoietic Cell Transplantation

Hematopoietic cell transplant (HCT) recipients are immunocompromised and thus predisposed to infections. We set out to determine the deficiency of which immune cell subset(s) may predispose to postengraftment infections. We determined day 28, 56, 84, and 180 blood counts of multiple immune cell subsets in 219 allogeneic transplant recipients conditioned with busulfan, fludarabine, and Thymoglobulin. Deficiency of a subset was considered to be associated with infections if the low subset count was significantly associated with subsequent high infection rate per multivariate analysis in both discovery and validation cohorts. Low counts of monocytes (total and inflammatory) and basophils, and low IgA levels were associated with viral infections. Low plasmacytoid dendritic cell (PDC) counts were associated with bacterial infections. Low inflammatory monocyte counts were associated with fungal infections. Low counts of total and naive Bxa0cells, total and CD56(high) natural killer (NK) cells, total and inflammatory monocytes, myeloid dendritic cells (MDCs), PDCs, basophils and eosinophils, and low levels of IgA were associated with any infections (due to any pathogen or presumed). In conclusion, deficiencies of Bxa0cells, NK cells, monocytes, MDCs, PDCs, basophils, eosinophils, and/or IgA plasma cells appear to predispose to postengraftment infections.

Donor-Recipient Matching for KIR Genotypes Reduces Chronic GVHD and Missing Inhibitory KIR Ligands Protect against Relapse after Myeloablative, HLA Matched Hematopoietic Cell Transplantation

Background Allogeneic hematopoietic cell transplantation (HCT) can be curative for many hematologic diseases. However, complications such as graft-versus-host disease (GVHD) and relapse of primary malignancy remain significant and are the leading causes of morbidity and mortality. Effects of killer Ig-like receptors (KIR)-influenced NK cells on HCT outcomes have been extensively pursued over the last decade. However, the relevance of the reported algorithms on HLA matched myeloablative HCT with rabbit antithymocyte globulin (ATG) is used for GVHD prophylaxis remains elusive. Here we examined the role of KIR and KIR-ligands of donor-recipient pairs in modifying the outcomes of ATG conditioned HLA matched sibling and unrelated donor HCT Methods and Findings The study cohort consisted of 281 HLA matched sibling and unrelated donor-recipient pairs of first allogeneic marrow or blood stem cell transplantation allocated into ‘discovery’ (135 pairs) and ‘validation’ (146 pairs) cohorts. High resolution HLA typing was obtained from the medical charts and KIR gene repertoires were obtained by a Luminex® based SSO method. All surviving patients were followed-up for a minimum of two years. KIR and HLA class I distributions of HCT pairs were stratified as per applicable definitions and were tested for their association with cause specific outcomes [acute GVHD grade II-IV (aGVHD), chronic GVHD needing systemic therapy (cGVHD) and relapse] using a multivariate competing risks regression model as well as with survival outcomes [relapse-free survival (RFS), cGVHD & relapse free survival (cGRFS) and overall survival (OS)] by multivariate Cox proportional hazards regression model. A significant association between KIR genotype mismatching (KIR-B/x donor into KIR-AA recipient or vice versa) and cGVHD was found in both discovery (p = 0.001; SHR = 2.78; 95%CI: 1.50–5.17) and validation cohorts (p = 0.005; SHR = 2.61; 95%CI: 1.33–5.11). High incidence of cGVHD associated with KIR genotype mismatching was applicable to both sibling and unrelated donors and was specific to recipients who had one or two C1 bearing HLA-C epitopes (HLA-C1/x, p = 0.001; SHR = 2.40; 95%CI: 1.42–4.06). When compared with KIR genotype mismatched transplants, HLA-C1/x patients receiving grafts from KIR genotype matched donors had a significantly improved cGRFS (p = 0.013; HR = 1.62; 95%CI: 1.11–2.39). Although there was no effect of KIR genotype matching on survival outcomes, a significantly reduced incidence of relapse (p = 0.001; SHR = 0.22; 95%CI: 0.10–0.54) and improved relapse-free survival (p = 0.038; HR = 0.40; 95%CI: 0.17–0.95) was observed with one or more missing ligands for donor inhibitory KIR among the recipients of unrelated donor transplants. Conclusions The present study for the first time presents the beneficial effects of KIR genotype matching in reducing cGVHD in myeloablative transplant setting using HLA matched (sibling and unrelated) donors. The findings offer a clinically applicable donor selection strategy that can help control cGVHD without affecting the risk of relapse and/or identify patients at a high risk of developing cGVHD as potential candidates for preemptive therapy. The findings also affirm the beneficial effect of one or more missing inhibitory KIR ligands in the recipient in reducing relapse and improving a relapse free survival in unrelated donor transplants.

Influence of Chemotherapy on Allergen-Specific IgE

Background: Atopy is defined as excess allergen-specific IgE (A-IgE). IgE is produced by plasma cells that differentiate from allergen-specific B cells. B cells are known to be killed by chemotherapy; however, it is not known whether A-IgE-secreting plasma cells are killed or inhibited by chemotherapy. If yes, serum A-IgE levels would be expected to decrease after chemotherapy. Objectives: We aimed to determine whether A-IgE levels in atopic individuals (serum A-IgE ≥0.35 kUA/L) decrease into the nonatopic range (< 0.35 kUA/L) after chemotherapy. Methods: In 105 patients undergoing chemotherapy for acute leukemia, we measured serum A-IgE before and after chemotherapy. In a subset of these patients, we also measured B cell counts before and after chemotherapy. Results: Of the 105 patients, 36 were atopic. In these patients, median A-IgE level before chemotherapy was 1.6 kUA/L whereas the median level after chemotherapy was 0.6 kUA/L (p < 0.001). In 12/36 (33%) patients, A-IgE levels decreased into the nonatopic range. In nonatopic patients (n = 69), the median A-IgE level also dropped: from 0.04 kUA/L before to 0.03 kUA/L after chemotherapy (p = 0.001). Among the total patients (n = 105), the median pre:post-chemotherapy A-IgE ratio was 1.8 (2.6 in atopic and 1.5 in nonatopic patients). In contrast, the median ratio of pre:post-chemotherapy B cell counts was 87.6. Conclusions: A-IgE levels decrease after chemotherapy but markedly less than B cell counts. Thus, at least some A-IgE plasma cells appear to survive chemotherapy.

Anti-thymocyte globulin’s activity against acute myeloid leukemia stem cells

Rabbit anti-thymocyte globulin (ATG (Thymoglobulin)) kills T cells in vitro and probably also in vivo as it prevents graft-vs-host disease (GvHD) in patients. Recently we demonstrated that ATG at a clinically relevant concentration (10–50u2009mg/L) kills in vitro not only T cells but also leukemic blasts. In the present study, we investigated whether ATG kills not only leukemic blasts but also leukemic stem cells (LSCs). We used a flow cytometric assay of complement-mediated cytotoxicityxa0(CDC). ATG-induced death of acute myeloid leukemia (AML) cells from patients newly diagnosed with AML was measured among blasts as well as LSCs. At 10u2009mg/L ATG, blasts but not LSCs were killed. At 50u2009mg/L ATG, both blasts and LSCs were killed. We also measured ATG-mediated killing of healthy individuals’ hematopoietic stem cells (HSCs). Median 2% HSCs from blood and 15% HSCs from filgrastim-mobilized grafts were killed with 50u2009mg/L ATG, compared to 30% LSCs from the blood of AML patients (pu2009=u20090.001 and 0.022, respectively). In conclusion, LSCs are sensitive to ATG, however, only at a relatively high ATG concentration. At that concentration, LSCs are killed to a higher degree than HSCs.