Cryopreservation of peripheral blood mononuclear cells for use in proliferation assays: First step towards potency assays.

Investigational cell-based therapeutics are rapidly heading towards pivotal clinical trials. The premise is that the scientific rationale is well defined, and that product quality reflects exactly this. In vitro potency assays are necessary tools for evaluating cell products, and with potency assays comes high demands for standardization and reproducibility of the methods involved. For demonstrating principles of cell therapeutics for allogeneic use or with claimed immunosuppressive efficacies, assays involving peripheral blood mononuclear cells (PBMC) are critical. Establishment of a cryopreserved bank of PBMC favors standardization, as it allows repeated use of a single donor and simultaneous testing of several donors. The first step to fulfil such potential is to ensure optimum conditions for preservation of PBMC function, and secondly to design assays which heightens the reproducibility. Emphasis should be put on application of the assay. The objective of the present study was to establish a methodological foundation for cell therapeutics to be tested, and several aspects were factored in, including cell concentrations and partial changes of medium. PBMC were isolated and cryopreserved in six formulations of cryoprotective medium consisting of fetal bovine serum (90%, 60%, and 30%) in combination with dimethyl sulfoxide (10% or 5%). The proliferative capacity of the cryopreserved cells was assayed by labeling with carboxyfluorescein succinimidyl ester and stimulation by phytohemagglutinin or in mixed lymphocyte reactions, analyzed by flow cytometry. To counter an eventual lag phase post thaw, the assays were designed to include two durations and to explore the possibility of reducing cell numbers, two cell concentrations. Qualitative and quantitative aspects of the staining were affected by formulation as well as design, stressing the importance of basic optimization for assay development. We conclude that the established methods allow for optimized preservation of function and will serve as a platform for further development of robust functional assays.

Activation-induced CD137 is a fast assay for identification and multi-parameter flow cytometric analysis of alloreactive T cells.

Detection and isolation of viable alloreactive T cells at the single-cell level requires a cell surface marker induced specifically upon T cell receptor activation. In this study, a member of the tumour necrosis factor receptor (TNFR)-family, CD137 (4-1BB) was investigated for its potential to identify the total pool of circulating alloreactive T cells. Optimal conditions for sensitive and specific detection of allogeneic-induced CD137 expression on circulating T cells were established. Thereafter, CD137(+) alloreactive T cells were phenotypically and functionally characterized by multi-parameter flow cytometry. Alloantigen-induced CD137 expression identified both alloreactive CD8(+) T cells (mean ± standard error of the mean: 0·21 ± 0·07%) and alloreactive CD4(+) T cells (0·21 ± 0·05%). CD137(+) alloreactive T cells were detected in different T cell subsets, including naive T cells, but were found preferentially in CD28(+) T cells and not in the terminally differentiated T cell subset. Upon allogeneic (re-)stimulation, the cytokine-producing as well as proliferative capacity of T cells resided mainly within the CD137-expressing fraction. About 10% of the CD137(+) alloreactive T cells produced any combination of interferon (IFN)-γ, interleukin (IL)-2 and TNF-α. Polyfunctional alloreactive T cells, defined by multiple cytokine expression, were observed infrequently. In conclusion, activation-induced CD137 expression is a fast assay allowing for detection and functional analysis of the total alloreactive T cell compartment at the single-cell level by multi-parameter flow cytometry.

Improved detection of clinically significant host-reactive antigens prior to HLA-identical sibling peripheral blood stem cell transplantation using a dendritic cell-based helper T-lymphocyte precursor assay.

SUMMARY: Graft-versus-host disease (GVHD) due to host-reactive antigenic differences between HLA-identical pairs remains an important cause of morbidity and mortality after allogeneic transplantation. The helper T-lymphocyte precursor (HTLp) assay, using peripheral blood mononuclear cells (PBMCs), has been variably shown to detect such host-reactive differences. We assessed whether using dendritic cells (DCs) as the stimulator cells would improve the ability of the HTLp assay to detect these differences. We used PBMCs (standard HTLp assay) or monocyte-derived DCs (DC-HTLp assay) as the stimulator cells for 12 HLA-identical sibling pairs undergoing allogeneic peripheral blood stem cell transplantation. HTLp frequencies were greater by the DC-HTLp assay (median 1:77 712 vs 1:727 514; P=0.008). The standard HTLp assay did not predict for acute GVHD (P=0.42), whereas a trend was noted for the DC-HTLp assay (P=0.095). Of note, of seven patients developing moderately severe to severe acute GVHD, four had a significantly greater DC-HTLp frequency compared to the standard HTLp frequency, whereas all four patients who developed no to moderate acute GVHD had similar HTLp frequencies whether PBMCs or DCs were used as the stimulator cells. Although the small number of donor/recipient pairs assessed limits the strength of any conclusions, our study suggests that the DC-HTLp assay is better able to detect clinically significant host-reactive antigenic differences between HLA-identical siblings.

Multilaboratory evaluation of serum analysis for HLA antibody and crossmatch reactivity by lymphocytotoxicity methods.

CONTEXT: This report presents results of the serum antibody analysis and crossmatch challenges in the proficiency testing program for histocompatibility testing jointly sponsored by the American Society for Histocompatibility and Immunogenetics and the College of American Pathologists. OBJECTIVE: To obtain information about consensus rates among participating laboratories that reported antibody screening and crossmatch results by direct complement-dependent lymphocytotoxicity (CDC) and/or anti-human globulin (AHG)-augmentation methods. DESIGN: We analyzed responses from approximately 165 laboratories participating in 32 surveys during 1993-2000. Most of the testing was done by CDC methods, but increasing proportions of laboratories are using AHG augmentation of these techniques; almost one half of the serum screenings and crossmatches were done by AHG. RESULTS: A total of 40 serum specimens were screened to determine the percent panel-reactive antibody (PRA) and identify HLA-specific antibodies. Participants often reported very wide ranges of PRA values. Panel-reactive antibody ranges exceeded 60 percentage points for 16 (40%) of the serum screening results by CDC and for 31 (77%) of the results by AHG. The interlaboratory variability of PRA values suggests that in many laboratories, the CDC or AHG procedures were often too insensitive or overly sensitive. The antibody identification results revealed inconsistent patterns among the participants performing CDC or AHG screening. Most participants reported the same primary antibody specificities by both methods. The consensus levels were generally high for the monospecific sera. On the other hand, there was much less agreement among the participants if the sera reacted with 2 or more HLA antigens. Participants using the more sensitive AHG method reported additional antibody specificities in many specimens, but invariably the consensus levels were rather low. A total of 192 serum-cell combinations were used for the crossmatch challenges. There was considerable interlaboratory variability; 21% of the CDC crossmatches and 36% of AHG crossmatches failed to reach the 90% consensus threshold. CONCLUSIONS: This experience demonstrates considerable inconsistencies in serum screening and crossmatching among laboratories participating in the American Society for Histocompatibility and Immunogenetics/College of American Pathologists surveys. A lack of uniformity in test results may limit the efficient application of these methods in a clinical setting. Standardization of crossmatch and antibody screening techniques is highly desirable.

Whole blood assay for assessment of the mixed lymphocyte reaction.

When lymphocytes from genetically different individuals are mixed together in tissue culture blast transformation occurs, a reaction known as the mixed lymphocyte reaction (MLR). The MLR is a clinically relevant in vitro assay where lymphocytes from one individual (effector, E) are incubated with the lymphocytes of another individual (stimulator, S) which have been previously rendered incapable of blast transformation by gamma-irradiation. We have standardised a whole blood (WB) MLR assay where the E lymphocytes were provided by 20 microl of WB and the S lymphocytes were provided by irradiated peripheral blood mononuclear cells (PBMCs) either as a mixed pool of 20 donor PBMCs or as single donor PBMC. The optimum number of S lymphocytes needed was comparatively higher than in the standard PBMC MLR: the optimum calculated E:S ratio was 1:20 compared to a E:S ratio of 1:1 or 3:2 in the standard PBMC MLR. In ten normal individuals the WB/PBMC MLR was similar to the standard PBMC/PBMC MLR. As a clinical example, the WB/PBMC MLR proliferative capacity of 13 patients with malignant mesothelioma was no different from the proliferative capacity of their age-sex matched controls. This standardised WB/PBMC MLR assay is a simple and more practical assay than the standard MLR assay and can be incorporated easily in clinical studies with biological end-points.

Histocompatibilidad / Histocompatibility

El Complejo Mayor de Histocompatibilidad humano consiste en una serie de genes fuertemente enlazados presentes en el brazo corto del cromosoma 6, normalmente heredados en bloque y constituyen el haplotipo HLA. La mayoría presenta alto polimorfismo, lo que permite la presencia de numerosas variantes alélicas. La herencia de este segmento puede seguirse dentro de una familia por tipificación de ADN celular o de los antígenos HLA expresados en las membranas celulares, permitiendo estudios de filiación o poblacionales. La función más importante es el reconocimiento de la identidad: las células se reconocen entre sí como propias de un individuo. Juegan un papel esencial en la selección clonal de linfocitos, en la presentación antigénica y en la regulación del sistema inmune. El estudio de su asociación con enfermedades representó un importante avance en la Inmunología clínica. En la actualidad numerosos trabajos reafirman su papel en los procesos autoinmunes, en la respuesta inmune según la interacción del sitio de unión del HLA y el epitope antigénico presentado y probablemente en la susceptibilidad a determinados tumores. Objetivos: Introducir al conocimiento del Sistema Mayor de Histocompatibilidad y su aplicación clínica, comprender los fundamentos de los estudios más frecuentes fomentando la autoevaluación y educación continua

Histocompatibilidad / Histocompatibility

El Complejo Mayor de Histocompatibilidad humano consiste en una serie de genes fuertemente enlazados presentes en el brazo corto del cromosoma 6, normalmente heredados en bloque y constituyen el haplotipo HLA. La mayoría presenta alto polimorfismo, lo que permite la presencia de numerosas variantes alélicas. La herencia de este segmento puede seguirse dentro de una familia por tipificación de ADN celular o de los antígenos HLA expresados en las membranas celulares, permitiendo estudios de filiación o poblacionales. La función más importante es el reconocimiento de la identidad: las células se reconocen entre sí como propias de un individuo. Juegan un papel esencial en la selección clonal de linfocitos, en la presentación antigénica y en la regulación del sistema inmune. El estudio de su asociación con enfermedades representó un importante avance en la Inmunología clínica. En la actualidad numerosos trabajos reafirman su papel en los procesos autoinmunes, en la respuesta inmune según la interacción del sitio de unión del HLA y el epitope antigénico presentado y probablemente en la susceptibilidad a determinados tumores. Objetivos: Introducir al conocimiento del Sistema Mayor de Histocompatibilidad y su aplicación clínica, comprender los fundamentos de los estudios más frecuentes fomentando la autoevaluación y educación continua (AU)

In vitro effects of ultraviolet B radiation on human Langerhans cell antigen-presenting function.

The effects of ultraviolet B radiation (UBV) on the immune function of human epidermal Langerhans cells (LC) were studied by using the mixed epidermal cell-lymphocyte reaction (MELR). Exposure of both enriched LC suspensions (eLC, 8-20% LC) and purified LC suspensions (pLC, 70-90% LC) to increasing doses of UVB radiation (25 to 200 J/m2) decreased the proliferative T cell response in a very similar dose-dependent way, suggesting that keratinocytes did not play a major role in the UVB-induced inhibition of MELR. Supernatants from irradiated cultured eLC or pLC failed to inhibit T cell proliferation induced by untreated pLC. Furthermore, addition of irradiated eLC to untreated pLC did not affec the allogeneic T cell response. Taken together, these results provide evidence that in vitro UVB-induced immunosuppression was not mediated by inhibitory soluble factors that could affect either LC allostimulatory property or T cell proliferative response. UVB irradiation of human LC inhibited the capacity of these cells to induce CD4+ as well as CD8+ T cell proliferation. UVB-irradiated LC also induced a decreased T cell response to recall antigen or mitogen. Moreover, addition of exogeneous cytokines such as IL-1 beta, IL-1 alpha, or IL-2 did not reverse the defective function of UVB-irradiated LC in MELR. The inhibitory effect of UVB radiation on human LC was not related to a decreased HLA-DR expression. Because cultured LC appeared to be less sensitive than freshly isolated LC to UVB-induced suppressive effects, the deleterious effects of UVB radiation on human LC allostimulatory properties may be associated with an impaired development of LC accessory function.