Andreas Sputtek

Antigen-Specific Tolerance by Autologous Myelin Peptide–Coupled Cells: A Phase 1 Trial in Multiple Sclerosis

Antigen-coupled cells result in antigen-specific tolerization for treatment of multiple sclerosis. Multiple Sclerosis Therapy Attached at the Hip In multiple sclerosis (MS), a patient’s own immune cells are thought to attack antigens in the brain and spinal cord. One approach to prevent this attack is through tolerization: harnessing one way the body itself attempts to prevent autoimmunity. Ideally, this would happen in an antigen-specific way so that autoimmunity is blocked, while the protective functions of the immune system remain intact. There has been considerable success inducing antigen-specific tolerance in mouse models of MS by chemically coupling the antigen of choice to carrier cells. Now, Lutterotti et al. take this approach into human patients. The authors coupled peripheral blood mononuclear cells from MS patients with seven different myelin peptides thought to be potentially antigenic in MS. Patients who had T cell responses restricted to at least one of the peptides tested were selected. Indeed, patients who received the highest doses of antigen-coupled cells demonstrated decreases in antigen-specific T cell responses after therapy. Although the patient numbers are small in this first-in-human study, the safety, feasibility, and early results suggest that this approach may provide a promising avenue for future trials. Multiple sclerosis (MS) is a devastating inflammatory disease of the brain and spinal cord that is thought to result from an autoimmune attack directed against antigens in the central nervous system. The aim of this first-in-man trial was to assess the feasibility, safety, and tolerability of a tolerization regimen in MS patients that uses a single infusion of autologous peripheral blood mononuclear cells chemically coupled with seven myelin peptides (MOG1–20, MOG35–55, MBP13–32, MBP83–99, MBP111–129, MBP146–170, and PLP139–154). An open-label, single-center, dose-escalation study was performed in seven relapsing-remitting and two secondary progressive MS patients who were off-treatment for standard therapies. All patients had to show T cell reactivity against at least one of the myelin peptides used in the trial. Neurological, magnetic resonance imaging, laboratory, and immunological examinations were performed to assess the safety, tolerability, and in vivo mechanisms of action of this regimen. Administration of antigen-coupled cells was feasible, had a favorable safety profile, and was well tolerated in MS patients. Patients receiving the higher doses (>1 × 109) of peptide-coupled cells had a decrease in antigen-specific T cell responses after peptide-coupled cell therapy. In summary, this first-in-man clinical trial of autologous peptide-coupled cells in MS patients establishes the feasibility and indicates good tolerability and safety of this therapeutic approach.

The glass transition temperature of mixtures of trehalose and hydroxyethyl starch

Although mixtures of HES and sugars are used to preserve cells during freezing or drying, little is known about the glass transition of HES, or how mixtures of HES and sugars vitrify. These difficulties may be due to the polydispersity between HES samples or differences in preparation techniques, as well as problems in measuring the glass transition temperature (T(g)) using differential scanning calorimetry (DSC). In this report, we examine the T(g) of mixtures of HES and trehalose sugar with <1% moisture content using DSC measurements. By extrapolating these measurements to pure HES using the Gordon-Taylor and Fox equations, we were able to estimate the T(g) of our HES sample at 44 degrees C. These results were additionally confirmed by using mixtures of glucose-HES which yielded a similar extrapolated T(g) value. Our approach to estimating the glass transition temperature of HES may be useful in other cases where glass transitions are not easily identified.

CD34+-Selected Stem Cell Boost without Further Conditioning for Poor Graft Function after Allogeneic Stem Cell Transplantation in Patients with Hematological Malignancies

We retrospectively analyzed outcomes of a CD34(+)-selected stem cell boost (SCB) without prior conditioning in 32 patients (male/22; median age of 54 years; range, 20 to 69) with poor graft function, defined as neutrophils ≤1.5 x 10(9)/L, and/or platelets ≤30 x 10(9)/L, and/or hemoglobin ≤8.5 g/dL). The median interval between stem cell transplantation and SCB was 5 months (range, 2 to 228). The median number of CD34(+) and CD3(+) cells were 3.4 x 10(6)/kg (.96 to 8.30) and 9 x 10(3)/kg body weight (range, 2 to 70), respectively. Hematological improvement was observed in 81% of patients and noted after a median of 30 days (range, 14 to 120) after SCB. The recipients of related grafts responded faster than recipients of unrelated grafts (20 versus 30 days, P = .04). The cumulative incidence of acute (grade II to IV) and chronic graft-versus-host disease (GVHD) after SCB was 17% and 26%, respectively. Patients with acute GVHD received a higher median CD3(+) cell dose. The 2-year probability of overall survival was 45%. We suggest that SCB represents an effective approach to improve poor graft function post transplantation, but optimal timing of SCB administration, anti-infective, and GVHD prophylaxis needs further evaluation.

Cryopreservation of Erythrocytes, Thrombocytes, and Lymphocytes

The cryopreservation of blood cells can be regarded as a classical field of development and application of low temperature biology. Cryopreservation methods have been developed for erythrocytes, which are commonly frozen with glycerol as the cryoprotective additive although hydroxyethyl starch (HES) shows considerable promise. Cryopreserved erythrocytes for transfusion are of advantage in the case of patients with rare blood groups, adverse antibody problems, autologous use and civil as well as military disasters. Additionally they can be used for blood typing, antibody screening and compatibility testing. Cryopreservation methods for thrombocytes, lymphocytes and hematopoietic stem cells usually involve dimethyl sulfoxide (DMSO) as the cryoprotective additive. Low temperature preservation of thrombocytes offers the possibility of making HPA- and/or HLA-typed platelet concentrates available in blood banks at any time. The use of cryopreserved lymphocytes is well established and a routine procedure for clinical laboratory testing. Recently there is a growing clinical interest in cryopreserved lymphocytes in addition to hematopoietic progenitor cells for the supplemental treatment of patients after blood stem cell transplantation. Despite occasional reports, it is our opinion that no clinically suitable method for the preservation of human granulocytes has been developed so far.

Purification of CD4+ T Cells for Adoptive Immunotherapy after Allogeneic Hematopoietic Stem Cell Transplantation

Donor lymphocyte infusions (DLIs) are used for adoptive immunotherapy to prevent or treat relapse and infectious complications after allogeneic hematopoietic stem cell transplantation (HSCT). Unmanipulated DLIs are associated with a risk of graft-versus-host disease (GVHD), probably related to CD8(+) T cell activity. We investigated an automated clinical-scale human-CD4(+)-cell purification method to deplete CD8(+) cells. Twenty-four stem cell recipients received a total of 24 leukapheresis products being enriched for CD4(+) cells using magnetic associated cell sorting (MACS) with an automated device (CliniMACS(®)) before DLIs. MACS resulted in a mean CD4(+) cell count of 16 × 10(6)/kg bw corresponding to 3.4-fold CD4(+) cell enrichment. Mean yield and purity of CD45(+)CD3(+)CD4(+)CD14(-)7AAD(-) were 74% ± 23% and 82% ± 11%, respectively. Median initial dose of DLIs was 1.1 × 10(6) CD4(+)/kg. During a median follow-up of 25 months, 7 (30%) patients experienced GVHD (acute II-IV: n = 4, 17%; acute III-IV: n = 2, 8%; chronic limited: n = 2, 8%; chronic extensive: n = 1, 4%). Thirteen of 21 further evaluable patients (62%) showed measurable clinical response, 2 patients with therapy refractory infectious complications (HSV) showed remarkable immunologic improvement. Automated enrichment of CD4(+) by magnetic cell sorting provides an efficient and rapid method for processing donor lymphocytes. Additional studies should further investigate this approach in terms of efficacy and the risk of GVHD.

No proinflammatory signature in CD34+ hematopoietic progenitor cells in multiple sclerosis patients.

Autologous hematopoietic stem cell transplantation (aHSCT) has been used as a therapeutic approach in multiple sclerosis (MS). However, it is still unclear if the immune system that emerges from autologous CD34+ hematopoietic progenitor cells (HPC) of MS patients is pre-conditioned to re-develop the proinflammatory phenotype. The objective of this article is to compare the whole genome gene and microRNA expression signature in CD34+ HPC of MS patients and healthy donors (HD). CD34+ HPC were isolated from peripheral blood of eight MS patients and five HD and analyzed by whole genome gene expression and microRNA expression microarray. Among the differentially expressed genes (DEGs) only TNNT1 reached statistical significance (logFC=3.1, p<0.01). The microRNA expression was not significantly different between MS patients and HD. We did not find significant alterations of gene expression or microRNA profiles in CD34+ HPCs of MS patients. Our results support the use of aHSCT for treatment of MS.

CD34+-selected stem cell boost for delayed or insufficient engraftment after allogeneic stem cell transplantation

BACKGROUND Poor graft function without rejection may occur after stem cell transplantation (SCT). CD34(+) stem cell boost (SCB) can restore marrow function but may induce or exacerbate GvHD. We therefore investigated the feasibility and efficacy of CD34(+)-selected SCB in some patients with poor graft function. We present the results for eight patients (median age 46 years) transplanted initially for myelofibrosis, acute leukemia, myeloma and NHL. Six patients had received HLA-matched and two mismatched grafts (PB, BM; n=5, 3). After a median of 128 days post-transplant, the median leukocyte and platelet counts were, respectively, 2.05/nL and 18/nL. None had achieved platelet counts >50/nL even though donor chimerism was >95% in seven recipients. METHODS Positive selection of CD34(+) stem cells was performed on a CliniMACS device, observing GMP and achieving a median of 98.5% purity. The patients received a median of 1.7 x 10(6)/kg CD34(+) cells and 2.5 x 10(3)/kg CD3(+) T lymphocytes. RESULTS Hemograms at days +30, +60 and +90, respectively, showed steadily increasing median leukocyte (2.55, 3.15 and 4.20/nL) and platelet (29, 39 and 95/nL) counts. After a median follow-up of 144 days, five patients remained alive. No patient had developed acute or chronic GvHD. One patient died of leukemic relapse and two others of systemic mycosis. DISCUSSION These preliminary results point to the possibility of safely improving graft function using CD34(+) positively selected stem cells without necessarily increasing the incidence of GvHD in patients with poor graft function post-SCT. Experience with more patients and longer follow-up should clarify the optimal role for this procedure.

Osmotic virial coefficients of hydroxyethyl starch from aqueous hydroxyethyl starch-sodium chloride vapor pressure osmometry.

Hydroxyethyl starch (HES) is an important industrial additive in the paper, textile, food, and cosmetic industries and has been shown to be an effective cryoprotectant for red blood cells; however, little is known about its thermodynamic solution properties. In many applications, in particular those in biology, HES is used in an aqueous solution with sodium chloride (NaCl). The osmotic virial solution thermodynamics approach accurately captures the dependence of osmolality on molality for many types of solutes in aqueous systems, including electrolytes, sugars, alcohols, proteins, and starches. Elliott et al. proposed mixing rules for the osmotic virial equation to be used for osmolality of multisolute aqueous solutions [Elliott, J. A. W.; et al. J. Phys. Chem. B 2007, 111, 1775-1785] and recently applied this approach to the fitting of one set of aqueous HES-NaCl solution data reported by Jochem and Körber [Cryobiology 1987, 24, 513-536], indicating that the HES osmotic virial coefficients are dependent on HES-to-NaCl mass ratios. The current study reports new aqueous HES-NaCl vapor pressure osmometry data which are analyzed using the osmotic virial equation. HES modifications were measured after dialysis (membrane cut off: 10,000 g/mol) and freeze-drying using vapor pressure osmometry at different mass ratios of HES to NaCl for HES up to 50% and NaCl up to 25% with three different HES modifications (weight average molecular weights [g/mol]/degree of substitution: 40,000/0.5; 200,000/0.5; 450,000/0.7). Equations were then fit to the data to provide a model for HES osmotic virial coefficient dependence on mass ratio of HES to NaCl. The osmolality data of the three HES modifications were accurately described over a broad range of HES-to-NaCl mass ratios using only four parameters, illustrating the power of the osmotic virial approach in analyzing complex data sets. As expected, the second osmotic virial coefficients increase with molecular weight of the HES and increase with HES-to-NaCl mass ratio.

Assessment of physiologic natural killer cell cytotoxicity in vitro

Here, we describe an improved (51)chromium release assay (CRA) to compare donor natural killer (NK) cell activity. To validate the assay, we analyzed sample preparation, incubation, and cryopreservation of NK cells. The effector-to-target ratio was corrected for the percentage of NK cells. A logarithmic curve was fitted to the data of the CRA for calculation of the maximum activity. The specific lysis was standardized to a reference sample and normalized to the mean specific lysis of the reference. We found that a longer time span involved with both the addition and the removal of DMSO increased the recovery of NK cell activity. Freezing and thawing reduced the cytotoxicity of NK cells but sustained the relative differences that were seen between freshly prepared NK cells. In contrast, medium incubation of thawed cells markedly increased the cytotoxic potential but also deranged these relative differences. Those were widely equalized when cells were stimulated with IL-2. In conclusion, we established a standardized assay with cryopreserved peripheral blood mononuclear cells as an appropriate tool for investigation of individual physiologic NK cell activity. This assay may help to predict donor NK cell activity in vivo, to reconcile conflicting data about NK cells obtained in transplantation studies.

Looking Back from the Future to the Present: Biopreservation Will Get Us There!

Biopreservation is a rapidly emerging field of specimen procurement, processing, preservation and banking. Such biospecimens can be macromolecules, fungi, viruses, bacteria, cells, tissues and organs. The discipline requires a close collaboration between clinicians, engineers and scientists coming from academia, small private enterprises or huge industrial companies. What they all have in common is the ultimate goal to preserve life, hence the word ‘biopreservation’ (from ‘bios’ (Greek) = life, and ‘preservare’ (Latin) = to keep). But what is the definition of ‘life’? Provided a rather limited biological definition is acceptable here, life can defined as ‘a process requiring energy and mass exchange, growth, reproduction and reactions to changes in the environment, completed by coordination via communication. Some of these criteria can be found in technical, physical and chemical systems, while other criteria can be found in living organisms. However, the minimum quality of all living systems is autopoiesis: the capability of self-preservation and reproduction’ [1].