Irradiated Peripheral Blood Lymphocytes Research Articles

Comparison of Isolated Lymphocyte and Whole Blood-Based CBMN Assays for Radiation Triage

Following a mass-casualty nuclear/radiological event, there will be an important need for rapid and accurate estimation of absorbed dose for biological triage. The cytokinesis-block micronucleus (CBMN) assay is an established and validated cytogenetic biomarker used to assess DNA damage in irradiated peripheral blood lymphocytes. Here, we describe an intercomparison experiment between two biodosimetry laboratories, located at Columbia University (CU) and Health Canada (HC) that performed different variants of the human blood CBMN assay to reconstruct dose in human blood, with CU performing the assay on isolated lymphocytes and using semi-automated scoring whereas HC used the more conventional whole blood assay. Although the micronucleus yields varied significantly between the two assays, the predicted doses closely matched up to 4 Gy – the range from which the HC calibration curve was previously established. These results highlight the importance of a robust calibration curve(s) across a wide age range of donors that match the exposure scenario as closely as possible and that will account for differences in methodology between laboratories. We have seen that at low doses, variability in the results may be attributed to variation in the processing while at higher doses the variation is dominated by inter-individual variation in cell proliferation. This interlaboratory collaboration further highlights the usefulness of the CBMN endpoint to accurately reconstruct absorbed dose in human blood after ionizing radiation exposure.

Establishment of in vitro Calibration Curve for 60Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays.

A rapid and reliable method for biodosimetry of populations exposed to ionizing radiation in the event of an incident or accident is crucial for initial triage and medical attention. DNA-double strand breaks (DSBs) are indicative of radiation exposure, and DSB-repair proteins (53BP1, γH2AX, ATM, etc.) are considered sensitive markers of DSB quantification. Phospho-53BP1 and γH2AX immunofluorescence technique serves as a sensitive, reliable, and reproducible tool for the detection and quantification of DSB-repair proteins, which can be used for biological dose estimations. In this study, dose-response curves were generated for 60Co-γ-rays induced phospho-53 Binding Protein 1 (phospho-53BP1) foci at 1, 2, 4, 8, 16, and 24 h, post-irradiation for a dose range of 0.05–4 Gy using fluorescence microscopy. Following ISO recommendations, minimum detection limits (MDLs) were estimated to be 16, 18, 25, 40, 50, and 75 mGy for dose-response curves generated at 1, 2, 4, 8, 16, and 24 h post-irradiation. Colocalization and correlation of phospho-53BP1 and γH2AX were also measured in irradiated peripheral blood lymphocytes (PBLs) to gain dual confirmation. Comparative evaluation of the established curve was made by γH2AX-immunofluorescence, dicentric chromosome assay (DCA), and reciprocal translocation (RT) assays by reconstructing the dose of 6 dose-blinded samples. Coefficients of respective in-house established dose-response curves were employed to reconstruct the blind doses. Estimated doses were within the variation of 4.124%. For lower doses (0.052 Gy), phospho-53BP1 and γH2AX assays gave closer estimates with the variation of −4.1 to + 9% in comparison to cytogenetic assays, where variations were −8.5 to 24%. For higher doses (3 and 4 Gy), both the cytogenetic and immunofluorescence (phospho-53BP1 and γH2AX), assays gave comparable close estimates, with −11.3 to + 14.3% and −10.3 to −13.7%, variations, respectively.

Therapeutic irradiation in the management of gynecological cancer and predictability of radiation-induced complications

Background. Today, applying radiotherapy (RT) in management of both primary and secondary vaginal cancer (SVC) take pride of place in the spectrum of specialized treatments for cancer patients. Secondary vaginal tumors are more common (6% to 33%) in cervical cancer (CC) patients, while in uterine cancer (UC) occur in 8–10% of cases treated either surgically, or by means of radiotherapy, otherwise via a combination approach. Therefore, RT is administered in about 80% of primary vaginal cancer and particularly SVC patients. When using even the most advanced radiotherapy equipment implying the cutting-edge technologies, there is a risk of radiation-induced complications in healthy organs and tissues that fall under the irradiation area. Keeping in mind the key radiobiological paradigms makes it possible not only to predict the probability of tumor resorption upon radiation exposure, but also to assess the biological effectiveness of absorbed dose, as well as the risk of late radiation complications.
 Purpose – to enhance the effectiveness and assess the toxicity of SVC RT via ascertaining BT (brachytherapy) most suitable techniques depending on the type of ionizing radiation and exploring predictability of radiation-induced complications in terms of biomolecular cell properties.
 Materials and methods. Clinical study was performed at the National Cancer Institute Clinic (Radiation Oncology Department), using a high-energy BT unit with a HDR 192Ir source. The SVC patients (n = 106) were treated according to the developed BT methods. They had been pretreated for the CC (n = 65) with squamous cell carcinoma histologically diagnosed in the most cases or UC (n = 41) with histologically prevalent adenocarcinoma of a variable grade. Patients had a locally advanced pelvic tumor process with tumor staging II–III, T2-3N0-1M0. Along with clinical study the radiobiological research was conducted to count the apoptotic cells in both intact and irradiated peripheral blood lymphocytes (PBL), as well as the level of SH-groups of plasma proteins and peptides in gynecological cancer patients and healthy donors to predict the risk of radiation-induced complications.
 Results and discussion. Given the delayed effect of RT, the treatment effective- ness was analyzed immediately after RT session and also 3 months upon completion of the conservative therapy. Thus, positive tumor response upon three months of observation over time was registered in 67.9 ± 5.2% of patients in study group I, in 72.5 ± 6.9% in study group II, and in 51.3 ± 6.8% in comparison group. Hence the values in study groups were higher than in comparison group by 16.6% and 21.2% respectively. All patients tolerated BT satisfactorily. Neither general nor local rectum or bladder severe (above grade II) toxicities were noted both during treatment and over the next 3 months upon its completion in all the patients regardless of study group. Results obtained in radiobiological studies correlated with clinical and literature data.
 Conclusions. Decrease in manifestations of RT toxicity, namely of the ear- ly radiation reactions from interfacing critical organs was established in groups I and II vs comparison group I, especially in group I where a high- energy 192Ir source was used in the SBD irradiation mode of 3 Gy twice a week. The obtained results of the experimental study suggest that the content of SH-groups in blood plasma and the level of PBL apoptosis can be considered as additional predictive measures of radiosensitivity of non-malignant cells from the irradiated tumor environment.

ASTAXANTHIN MODIFIES CLASTOGENIC EFFECTS OF IONIZING RADIATION IN VITRO IN PERIPHERAL BLOOD LYMPHOCYTES OF THE PERSONS RECOVERED FROM ACUTE RADIATION SICKNESS

To assess radioprotective activity of astaxanthin toward radiation-induced in vitro cytogenetic effects in human peripheral blood lymphocytes (PBL). PBL from the cleanup workers exposed to ionizing radiation at high doses in 1986 during accident on Chornobyl nuclear power plant and who were diagnosed with acute radiation sickness of the first and second degrees, were cultured in vitro. Astaxanthin was added into the culture medium at a final concentration of 20.0µg/ml, prior to γ-irradiation of PBL in vitro at a dose of 1Gy. The slides of metaphase chromosomes were analyzed. Astaxanthin demonstrated considerable radioprotective effect in irradiated PBL manifested in significantly decreased levels of unstable cytogenetic markers of radiation exposure (dicentrics and centric rings). The data evidence on radioprotective capacity of astaxanthin toward radiation-induced cytogenetic effects in vitro in PBL of liquidators irradiated during Chornobyl nuclear power plant accident. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

The impact of astaxanthin on radiation-induced chromosome aberrations in human peripheral blood lymphocytes in vitro

Aim. Research objective is to establish the possibility of modifying the astaxanthin (a carotenoid from a xanthophyll group) radiation-induced cytogenetic effects in human peripheral blood lymphocytes (PBLs) in vitro. Methods. The cultivation of PBLs from four conventionally healthy volunteers, the preparation and analysis of uniformly stained slides of metaphase chromosomes. Astaxanthin in final concentrations of 2, 10 and 20 µg/ml was added into the culture of PBL prior to the incubation before irradiation with γ-quanta in a dose of 1 Gy. Results. Astaxanthin did not affect the level and spectrum of chromosome damage in non-irradiated PBLs both in individual persons, and along the group on average (P>0.05), indicating a lack of mutagenic activity. The effect of astaxanthin at a concentration of 20 μg/ml on irradiated PBLs resulted in a significant reduction of radioinduced cytogenetic effect in all donors. Medium-group level of chromosome aberrations decreased almost 3 times and was characterized by statistically significant (P<0.001) decrease in frequency of chromosomal type aberrations due to the classical unstable cytogenetic markers of radiation effect, dicentrics and ring chromosomes. Conclusions. Astaxanthin at a concentration of 20 µg/ml was found to reduce the mutagenic effect of ionizing radiation, thus suggesting its powerful radioprotective potential. Keywords: astaxanthin, culture of human peripheral blood lymphocytes, radiation mutagenesis, chromosome aberrations, radioprotective effect.

Ionizing Radiation–Induced γ-H2AX Activity in Whole Blood Culture and the Risk of Lung Cancer

Phenotypic biomarkers of DNA damage repair may enhance cancer risk prediction. The γ-H2AX formed at the sites of double-strand break (DSB) after ionizing radiation is a specific marker of DNA damage. In an ongoing case-control study, the baseline and ionizing radiation-induced γ-H2AX levels in peripheral blood lymphocytes (PBL) from frequency-matched 306 untreated patients with lung cancer and 306 controls were measured by a laser scanning cytometer-based immunocytochemical method. The ratio of ionizing radiation-induced γ-H2AX level to the baseline was used to evaluate interindividual variation of DSB damage response and to assess the risk of lung cancer by using unconditional multivariable logistic regression with adjustment of age, sex, ethnicity, smoking status, family history of lung cancer, dust exposure, and emphysema. The mean γ-H2AX ratio was significantly higher in cases than controls (1.46 ± 0.14 vs. 1.41 ± 0.12, P < 0.001). Dichotomized at the median in controls, high γ-H2AX ratio was significantly associated with increased risk of lung cancer [OR = 2.43; 95% confidence interval (CI): 1.66-3.56]. There was also a significant dose-response relationship between γ-H2AX ratio and lung cancer risk in quartile analysis. Analysis of joint effects with other epidemiologic risk factors revealed elevated risk with increasing number of risk factors. γ-H2AX activity as shown by measuring DSB damage in ionizing radiation-irradiated PBLs may be a novel phenotypic marker of lung cancer risk. γ-H2AX assay is a robust and quantifiable image-based cytometer method that measures mutagen-induced DSB response in PBLs as a potential biomarker in lung cancer risk assessment.

Analysis of miRNA and mRNA expression profiles highlights alterations in ionizing radiation response of human lymphocytes under modeled microgravity.

BackgroundIonizing radiation (IR) can be extremely harmful for human cells since an improper DNA-damage response (DDR) to IR can contribute to carcinogenesis initiation. Perturbations in DDR pathway can originate from alteration in the functionality of the microRNA-mediated gene regulation, being microRNAs (miRNAs) small noncoding RNA that act as post-transcriptional regulators of gene expression. In this study we gained insight into the role of miRNAs in the regulation of DDR to IR under microgravity, a condition of weightlessness experienced by astronauts during space missions, which could have a synergistic action on cells, increasing the risk of radiation exposure.Methodology/Principal FindingsWe analyzed miRNA expression profile of human peripheral blood lymphocytes (PBL) incubated for 4 and 24 h in normal gravity (1 g) and in modeled microgravity (MMG) during the repair time after irradiation with 0.2 and 2Gy of γ-rays. Our results show that MMG alters miRNA expression signature of irradiated PBL by decreasing the number of radio-responsive miRNAs. Moreover, let-7i*, miR-7, miR-7-1*, miR-27a, miR-144, miR-200a, miR-598, miR-650 are deregulated by the combined action of radiation and MMG. Integrated analyses of miRNA and mRNA expression profiles, carried out on PBL of the same donors, identified significant miRNA-mRNA anti-correlations of DDR pathway. Gene Ontology analysis reports that the biological category of “Response to DNA damage” is enriched when PBL are incubated in 1 g but not in MMG. Moreover, some anti-correlated genes of p53-pathway show a different expression level between 1 g and MMG. Functional validation assays using luciferase reporter constructs confirmed miRNA-mRNA interactions derived from target prediction analyses.Conclusions/SignificanceOn the whole, by integrating the transcriptome and microRNome, we provide evidence that modeled microgravity can affects the DNA-damage response to IR in human PBL.

Mitotic delay in lymphocytes from BRCA1 heterozygotes unable to reduce the radiation-induced chromosomal damage

Double strand breaks (DSB) are critical lesions involved in the formation of chromosomal aberrations. In response to DNA damage, the cell has mechanisms of repair and cell-cycle control to maintain the genome integrity in which BRCA1 gene is implicated. In the present study an evaluation of the radio-induced damage in G2 phase of the cell cycle in lymphocytes from BRCA1 heterozygotes is presented. For this purpose Calyculin-A-based premature chromosome condensation (PCC) combined with mitotic arrest has been applied to examine with conventional cytogenetics the damage in G2 and M phase cells, and to evaluate the G2-to-M phase transition. Irradiated peripheral blood lymphocytes from seven heterozygote females (BRCA1+/−) and seven control females (BRCA1+/+) have been analyzed. The mean proportion of G2 cells in BRCA1+/− was significantly higher than in BRCA1+/+, indicating a higher G2 delay after IR exposure in cells from BRCA1+/− females. On the other hand, whereas the mean frequency of chromatid breaks (chtb) in G2 cells was not statistically different between both groups, the mean frequency of chtb in M cells of the BRCA1+/− group was significantly higher than in the BRCA1+/+ one. Moreover, the mean proportion of M cells with aberrations was significantly higher in BRCA1+/− than in BRCA1+/+ suggesting that in spite of the higher G2 delay of BRCA1+/− more damaged cells are able to pass the G2-to-M transition.

Modeled microgravity affects cell survival and HPRT mutant frequency, but not the expression of DNA repair genes in human lymphocytes irradiated with ionising radiation

We analysed the possibility that a reduced gravitational force impairs the efficiency of DNA repair, increasing the risk of the exposure to conditions occurring during spaceflight: i.e., ionising radiation and microgravity. To obtain information on the effects of the reduced gravity in repairing DNA damage induced by radiation, we compared cell survival and mutant frequency at the hypoxanthine-guanine phosphoribosyl transferase ( HPRT) locus in human peripheral blood lymphocytes (PBL) irradiated and subsequently incubated for 24 h in 1 g or modeled microgravity. A weak decrease of the surviving fraction and a significant increase of the HPRT mutant frequency were observed in PBL incubated in modeled microgravity after irradiation compared with those maintained in 1 g. Given the increase of HPRT mutants observed in MMG, we investigated whether modeled microgravity can alter the transcription of 14 genes representative of the main DNA repair pathways: non-homologous end joining (NHEJ), homologous recombination (HR), base excision repair (BER) and nucleotide excision repair (NER). The transcriptional profiles of almost all BER and NER genes were up-regulated in irradiated PBL, whereas the expression of HR and NHEJ genes was only slightly or not affected by radiation. Incubation in modeled microgravity after irradiation did not significantly change the expression of genes involved in DNA repair, suggesting that transcriptional impairment was not responsible for the increase of mutant frequency observed in irradiated cells incubated in microgravity in comparison to the static 1 g condition.

Growth hormone protects human lymphocytes from irradiation-induced cell death.

1. Undesired effects of cancer radiotherapy mainly affect the hematopoietic system. Growth hormone (GH) participates in both hematopoiesis and modulation of the immune response. We report both r-hGH cell death prevention and restoration of secretory capacities of irradiated human peripheral blood lymphocytes (PBL) in vitro. 2. r-hGH induced cell survival and increased proliferation of irradiated cells. Western blot analysis indicated that these effects of GH were paralleled by increased expression of the antiapoptotic protein Bcl-2. 3. r-hGH restored mitogen-stimulated release of IL-2 by PBL. Preincubation of irradiated lymphocytes with the growth hormone receptor (GHR) antagonists B2036 and G120 K abrogated r-hGH-dependent IL-2 release. 4. These results demonstrate that r-hGH protects irradiated PBL from death in a specific, receptor-mediated manner. Such effect of r-hGH on PBL involves activation of the antiapoptotic gene bcl-2 and prevention of cell death, associated with preserved functional cell capacity. Finally, potential use of GH as an immunopotentiating agent could be envisioned during radiation therapy of cancer.

Effect of blood storage on radiation-induced micronuclei in human lymphocytes

To evaluate the effect of blood storage on the yield of micronuclei (MN) in both irradiated (in vivo and ex vivo) and unirradiated peripheral blood lymphocytes (PBL), we applied the MN assay in cytokinesis-blocked (CB) PBL obtained from healthy subjects ( n=11), and from cancer patients ( n=10) who were undergoing fractionated partial-body radiotherapy (xRT). The heparinized blood samples were exposed to 137 Cs -irradiation (0 Gy or 2 Gy) immediately after blood collection and were stored upright in test tubes either at room temperature (22°C) or in the refrigerator (5°C). Duplicate whole blood cultures from each sample were set up at 0 h, 96 h, and 120 h after ex vivo irradiation. Giemsa (10%) stained slides were prepared from each culture. MN yield was determined per 1000 binucleated cells. As compared to that obtained from the corresponding fresh blood samples, we found that (1) the 22°C blood storage temperature did not affect MN yields in PBL of either healthy subjects or cancer patients up to 96 h, either with or without ex vivo irradiation; and (2) while blood samples were stored at 5°C, the MN yield increased significantly in PBL of healthy subjects (with or without ex vivo irradiation) at 120 h, and in cancer patients (with ex vivo irradiation) at 96 h and 120 h. Since handling of the blood sample is important for CBMN assay during shipment or in the laboratory, our findings showed that blood storage at 22°C or at 5°C up to 96 h appeared to provide insignificant variations of the MN results as compared to fresh blood samples. However, the 96 h of blood storage at 5°C elevated the MN frequency in ex vivo irradiated PBL of cancer patients who were undergoing xRT.

FLOW CYTOMETRIC SCORING OF APOPTOSIS COMPARED TO ELECTRON MICROSCOPY IN γ IRRADIATED LYMPHOCYTES

One of the early events occurring at the cell membrane during apoptosis is the translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer. These phosphatidylserine groups can be bound by fluorescein isothiocyanate (FITC)-labelled annexin V. The aim of this study was to evaluate the power of the annexin V flow cytometric assay in detecting apoptosis in γ irradiated peripheral blood lymphocytes and in differentiating between apoptosis and primary necrosis in these cells. Therefore, 5Gy and 20Gy γ irradiated peripheral blood mononuclear cells (PBMCs) were examined after a 24-h culture period. The terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) technique was performed as well. A comparison with an electron microscopic (EM) evaluation was made. EM is based on established morphological criteria allowing the classification of cells into four groups: viable, early apoptotic, secondary necrotic and primary necrotic cells. EM performed on annexin V positive sorted cells proved that a 5Gy γ irradiation of PBMCs mainly causes apoptosis, whereas a 20Gy γ irradiation mainly induces primary necrosis. Neither the annexin V flow cytometric assay nor the TUNEL assay were able to distinguish between primary and secondary necrotic cells. These results illustrate that if quantification of apoptosis is required, one should be careful in interpreting flow cytometric results obtained by annexin V or TUNEL staining in peripheral blood lymphocytes. Although in general primary necrotic cells show an increased forward scatter due to cellullar swelling, both early apoptotic and necrotic (primary or secondary) lymphocytes show a decreased forward scatter signal. Moreover, both primary and secondary necrotic lymphocytes are annexin V and propidium iodide (PI) positive and therefore indistinguishable. We conclude that if a new experiment focusing on apoptosis is set up, an initial EM evaluation is mandatory. If EM shows that the apoptosis inducing agent used in the design of the experiments is not causing primary necrosis, than the annexin V flow cytometric assay can provide rapid and quantitative information about apoptosis.

Sources of variability in the determination of micronuclei in irradiated peripheral blood lymphocytes

Following a dose of 4 Gy 250 kVp X-rays to the blood lymphocytes from three healthy donors the frequency of micronuclei was enumerated by three scorers. Eight slides were prepared from each blood sample with 1000 cytokinesis-blocked binucleate cells being scored for micronuclei and a recount undertaken 4 weeks later. Seven possible sources of variation were investigated: differences between donors, scorers, the four stages of sample preparation and time. A complete factorial analysis indicated that the major sources of variation were differences between donors and differences between scorers. One scorer identified fewer micronuclei than did the other two scorers in all slides of the irradiated blood samples and gave wide and inconsistent results for baseline counts. These results suggest that the performance of scorers should be screened before undertaking analysis of micronuclei. The most striking result, however, was the consistent performance of the scorers on the repeat counts compared with the very large differences between the scorers.

Abrogation of Allospecific T Lymphocyte Responses in Swine by Ultraviolet Light-B Irradiation

Immunomodulatory effects of ultraviolet-light B (UV-B) irradiation were tested in pigs, using peripheral blood lymphocytes (PBL), with special emphasis on the generation of cytotoxic T lymphocytes (CTL). Stimulator PBL were irradiated with 300–1100 Joules/m 2 (J/m 2) of UV-B (experimental group) and cultured for 24 h prior to being placed in mixed lymphocyte culture (MLC) to determine proliferative responses against alloantigens, or placed in bulk culture with allogeneic PBL to determine the induction of CTL. PBL that received 3000 rads gamma irradiation were used as controls. Proliferative alloresponses against gamma irradiated PBL (control group) were high, with stimulation indices (S.I.) ranging from 13.46 to 81.93. The generation of allospecific CTL activity was also high, ranging from 50.78 % to 95.40 %. In contrast, proliferative responses and generation of allospecific CTL were markedly inhibited (> 49.77 %) against the same donor PBL irradiated with ≥ 500 J/m 2 UV-B. These results demonstrated that ultraviolet light treatment of pig PBL can reduce or prevent allospecific T cell proliferation as well as the generation of specific CTL, similar to results obtained with UV-B irradiation of lymphocytes in rodents.

Recognition of apoptotic cells by human macrophages: inhibition by a monocyte/macrophage-specific monoclonal antibody.

Cells undergoing death by apoptosis are rapidly engulfed by phagocytes in vivo, a highly efficient process which prevents leakage of potentially dangerous intracellular contents from dying cells to neighboring tissue. We have tested a panel of monoclonal antibodies (mAb) specifying a range of human monocyte/macrophage surface antigens for their capacity to inhibit the in vitro recognition of apoptotic cells by human peripheral blood monocyte-derived macrophages. The results identify the antigen defined by the 61D3 mAb, a widely-used marker of monocyte/macrophage lineage cells, as an important mediator of apoptotic cell recognition. In our system, apoptotic, but not viable, cells were recognized by the cultured macrophages and 61D3 was found to inhibit the recognition of all apoptotic cell types tested, including Ca2+ ionophore-treated or growth factor-depleted B and T lymphocyte lines, tonsillar germinal center B cells, irradiated peripheral blood lymphocytes and senescing neutrophils. Furthermore, the apoptotic cell recognition pathway specified by 61D3 could be distinguished from that involving the macrophage alpha v beta 3 vitronectin receptor which has been shown previously to play an important role in the recognition of apoptotic cells. These results provide further evidence that the mechanisms underlying rapid clearance of apoptotic cells involve multiple phagocyte receptors.

The proliferative immune response to autologous Epstein-Barr virus-transformed lymphoblastoid cells. I. Studies with HLA haplotype loss variants demonstrate a role for MHC-linked genes

The human Epstein-Barr virus transformed lymphoblastoid cell line (EBV-LCL) 721 and MHC haplotype loss variants derived from it were utilized to dissect the functional role of MHC genes in the proliferative response of autologous T lymphocytes to EBV-LCL. LCL-721 is heterozygous at all phenotypically defined MHC loci. One type of LCL-721 variant expresses only determinants encoded by the maternal (m) haplotype and the other expresses determinants encoded by the paternal (p) haplotype. Autologous (individual A) primary proliferative responses are strong to each type of haplotype deletant. The strong tertiary responses to the priming haplotype in comparison to the relatively weak responses to the reciprocal haplotype indicate that MHC linked genes encoded by each haplotype are important in the autologous response to EBV-LCL. Similar specific tertiary responses are observed when peripheral blood lymphocytes (PBLs) from the donor's mother are used as responding cells. Allogeneic responses were also studied by priming PBLs from unrelated donors with the haplotype deletants. Quantitative comparisons of the proliferation by primed allogeneic and autologous lymphocytes stimulated by irradiated PBLs from donor A and her mother, and by LCL-721 and its variants, show that some of the tertiary responses involve specific recognition of EBV-LCL while other detect recognition of alloantigens.

HLA class II restriction repertoire of antigen-specific T cells. I. The main restriction determinants for antigen presentation are associated with HLA-D/DR and not with DP and DQ

In order to study the HLA class II restriction repertoire in antigen presentation to T cells, T lymphoblasts (T-LB) of ten different HLA class II donors were generated by a simple and rapid technique; peripheral blood lymphocytes (PBL) were restimulated in vitro with purified protein derivative (PPD) or tetanus toxoid (TET), and then propagated in interleukin-2 containing conditioned medium (IL2-CM). These T-LB appeared to be antigen specific and devoid of alloreactivity. Antigen was presented to these T-LB by allogeneic irradiated PBL as antigen-presenting cells (APC) in 179 combinations. T-LB proliferative responses were restricted mainly by determinants associated with HLA-DR and not with -DP or -DQ; in 102 fully DR mismatched T-LB/APC combinations matching for DP or DQ determinants had no significant influence on T-LB responses. For PPD, preferential DR1 restriction was observed, and the results suggest a preferential DRw11 vs. DRw12 restriction for TET. Moreover, DRw13 may be associated with low anti-PPD T-LB resposiveness.

Proliferative responses to a nonspecific factor produced by irradiated stimulating cells can simulate antigen-specific secondary responses in the primed lymphocyte test.

The primed lymphocyte test (PLT) was utilized to investigate the biologic relationship between alloantigens and soluble environmental antigens. Human lymphocytes primed in vitro to the soluble antigens Candida (CAN), Tetanus (TET), or Purified Protein Derivative of Tuberculin (PPD) gave a strong proliferative response when restimulated with the initial soluble antigen in the presence of irradiated peripheral blood lymphocytes (PBL) acting as antigen-presenting cells (APC). Surprisingly, the soluble antigen-primed cells as well as alloantigen-primed cells responded to other soluble antigens in the presence of APC. By testing primed cells with supernatants derived from irradiated lymphocytes plus soluble antigen, it was apparent that the responses observed were in part due to stimulation by a soluble factor produced by the irradiated "APC" in response to the soluble antigen rather than to recognition of widespread cross-reactivity by the primed cells. This "nonspecific" factor production could be diminished by increasing the dose of irradiation to the APC or by using a T lymphocyte depleted- (SRBC-E-) APC population. In addition, certain antigen-reactive T cell clones did not respond to the nonspecific factor to the same degree as the primed bulk cultures. Nevertheless, the recognition of nonspecific stimulation induced by factors produced by irradiated lymphocytes is critical in the interpretation of primed lymphocyte responses to alloantigens or soluble antigens.

Long-term maintenance of HLA-D restricted T cells specific for soluble antigens.

Lymphocytes from donors sensitized to soluble protein antigens tuberculin (PPD) and tetanus toxoid were stimulated in vitro with these antigens. The blasts were isolated on density gradients and maintained in long-term proliferating culture by the addition of supernatants from phytohaemagglutinin-stimulated (PHA) cultures. Blasts can be shown to retain specificity for the original stimulating antigen as measured by stimulation of DNA synthesis, but only when the antigen is presented in the company of a cooperating cell population. Autologous irradiated peripheral blood lymphocytes provide the best cooperation, but donors who share HLA-D antigens will also allow for continued proliferation in the presence of the appropriate soluble antigen. Donors sharing at HLA-A, -B, or -C show minimal ability to cooperate. The soluble antigen-specific blast cells do not manifest alloreactivity. The data are discussed with regard to possible application to clinical histocompatibility typing and to the implications of selfrecognition in the immune response.

Reduced in vitro response of CSF lymphocytes to mitogen stimulation in multiple sclerosis.

By means of a microculture technique and calculation of incorporation of 14C-thymidine, cerebrospinal fluid (CSF) lymphocytes from multiple sclerosis (MS) patients showed low or absent proliferation when stimulated with phytohaemagglutinin, concanavalin A, or pokeweed mitogen, in contrast to peripheral blood lymphocytes (PBL) obtained simultaneously and investigated in parallel. A lower proliferation of CSF lymphocytes compared with PBL was also found in acute aseptic meningitis, although it has been reported that CSF lymphocytes show greater proliferation than PBL when specifically stimulated. The low proliferation of MS CSF lymphocytes on mitogen stimulation may be a consequence of prolonged sensitization to an as yet unidentified antigen. The proliferation of MS CSF lymphocytes was not improved by adding irradiated PBL, making helper cell insufficiency less likely. MS CSF had no inhibitory effect on proliferation of PBL, arguing against an inhibitory effect of soluble factors in the CSF as an explanation for the depressed response of CSF lymphocytes.