Analysis of the donor-specific cytotoxic T lymphocyte repertoire in a patient with a long term surviving allograft. Frequency, specificity, and phenotype of donor-reactive T cell receptor (TCR)-alpha beta+ and TCR-gamma delta+ clones

In the present study the transplant specific CTL repertoire of a patient (HLA:A1,3, B8,18, Cw5,7 DR3, DQw2, DPw3) with a long term surviving HLA mismatched kidney graft (HLA: A1,24 B8,27 Cw2,7, DR3, w13 DQw2,6 DPw1,3) has been investigated. This patient was unable to generate specific cytolytic activity against donor-derived PHA-blasts in the MLC in which donor spleen cells or B lymphoblastoid cell line were used as stimulator cells. In addition, the CTL precursor frequencies against donor alloantigens were very low (1/67,000). The patient had otherwise normal immune responses in vivo and in vitro and no signs of transplant rejection. Transplant specific CTL clones were generated in high frequencies (1/195) from T cell bulk cultures activated by PHA in the absence of any sensitization by donor Ag in vitro. The repertoire of 14 donor-reactive CTL clones (12 TCR-alpha beta+ and 2 TCR-gamma delta+) was analyzed. Two TCR-alpha beta+ CD8+ clones were specific for B27. Ten TCR-alpha beta+ CTL clones directed against class II HLA Ag were isolated. Seven of these were CD4+ and recognized DRw13 (3), DQw6 (3), and DPw1 (1), whereas three of these clones were CD4-CD8+ recognizing DRw13 (1) and DQw6 (2). In addition, two donor-specific TCR-gamma delta+ CTL clones were obtained recognizing HLA-A9(23,24) and DQw6. Our data indicate that the precursors of CTL clones specifically directed against donor class I or II HLA Ag are not deleted from the repertoire and that part of this reactivity resides in the TCR-gamma delta+ fraction.     

Interaction between des-Tyr1-γ-endorphin and HLA class I molecules: Serological detection of an HLA-A2 subtype

Preincubation of lymphocytes with des-Tyr¹--endorphin (DTE) inhibits the reaction between some HLA alloantisera and their corresponding antigens. One HLA-A2-specific antiserum was found which could detect a subtype of the HLA-A2 antigen on DTE-treated lymphocytes from some donors. Comparison with the HLA-A2 subtypes as defined by a combination of cytotoxic T lymphocyte typing and biochemistry showed a complete correlation with the previously described HLA-A2.3 subtype.     

A simple method to determine IgG light chain to heavy chain polypeptide ratios expressed by CHO cells

Objectives

To establish a high-throughput method for determination of antibodies intra- and extracellular light chain (LC) to heavy chain (HC) polypeptide ratio as screening parameter during cell line development.

Results

Chinese Hamster Ovary (CHO) TurboCell pools containing different designed vectors supposed to result in different LC:HC polypeptide ratios were generated by targeted integration. Cell culture supernatants and cell lysates of a fed batch experiment were purified by combined Protein A and anti-kappa affinity batch purification in 96-well format. Capture of all antibodies and their fragments allowed the determination of the intra- and extracellular LC:HC peptide ratios by reduced SDS capillary electrophoresis. Results demonstrate that the method is suitable to show the significant impact of the vector design on the intra- and extracellular LC:HC polypeptide ratios.

Conclusion

Determination of LC:HC polypeptide ratios can give important information in vector design optimization leading to CHO cell lines with optimized antibody assembly and preferred product quality.

     

Representation of lateral line and electrosensory systems in the midbrain of the axolotl,Ambystoma mexicanum

Summary The study focussed on the representation of the electrosensory and lateral line units in the midbrain of the axolotl Ambystoma mexicanum. In addition, the responses to photic and acoustic/vibrational stimuli were determined. Unit properties were characterized with respect to baseline activity, sensitivity, latency, directional specificity and number of input modalities. The anatomical arrangement of the units was determined using stereotactic and histological measurements of the electrode positions.Of 106 units recorded, 29 units were unimodal, 77 units responded to more than one modality. Most units discharged only in response to stimuli. Thresholds of electrosensory units were about 100 V/cm field strength; lateral line units had thresholds below 5 m pp amplitude. The shortest latencies (8–17 ms) were found for responses to visual stimuli. Lateral line and vestibular units responded after 35–58 ms, electroreceptive units after 79–150 ms. All electrosensory and about 50% of the lateral line units were sharply tuned to definite stimulus directions.Electrosensory and lateral line units formed topographical maps in the tectum. The map in each tectal hemisphere contained information about the contralateral surroundings. The electrosensory, lateral line and visual representations were only partly in register; especially in the caudal areas of the midbrain the alignment was poor.     

HLA-DRw6 and renal allograft rejection.

HLA-DRw6-positive patients are "high responders" to certain renal allograft antigens. A study was therefore conducted of the outcome of 247 first renal allografts in 74 DRw6-positive and 173 DRw6-negative recipients. The effectiveness of matching for HLA-DR determinants in both groups was also analysed. The one-year graft survival in DRw6-positive patients was 59% as compared with 75% in DRw6-negative recipients (p = 0.012). A striking difference between the two groups was that HLA-DR matching significantly improved renal allograft survival only in the DRw6-positive patients. In those patients the one-year survival of HLA-DR-identical grafts was 95% as compared with only 38% for 2-DR mismatched grafts (p = 0.009). In DRw6-negative patients only a slight beneficial effect of HLA-DR matching was observed (83% versus 72% at one year for the 0-DR and 2-DR mismatched grafts, respectively) (p greater than 0.05). These findings are clear evidence that DRw6-positive patients (about a quarter of the patients on the waiting list of Eurotransplant) should be given HLA-DR-identical kidney transplants only.     

Directional sensitivity of lateral line units in the clawed toadXenopus laevis Daudin

Summary Following lateral line stimulation with surface waves single unit activity was recorded from the periphery, torus semicircularis, and tecturn opticum ofXenopus laevis. The reaction of units to varying stimulus directions was examined.The directional specificity (DS) was calculated on the basis of spike counts per stimulus using circular statistics. It was expressed as the length of the mean vector.Discharges of primary afferents of the ramus supraorbitalis and ramus infraorbitalis were phase locked to the stimulus to a varying degree depending on the location of the corresponding groups of neuromasts. Their DS was not better than 0.26.Lemniscal fibers, representing the ascending output of the medulla and units of the torus semicircularis reached a DS of 0.10–0.24 and 0.11–0.36 respectively. Neurons in the tectum opticum were the most sharply tuned with DS ranging between 0.81 and 0.96.The surroundings were represented by the best directions of two arrays of tectal units forming a map which is in register with the representation of the corresponding visual field of the animal.Abbreviations DS directional specificity - r.i.o. ramus infraorbitalis - r.s.o. ramus supraorbitalis     

A proposed algorithm predictive for cytotoxic T cell alloreactivity

Previously, we showed that with an increasing number of amino acid differences in single HLA class I-mismatched molecules, the probability of T cell alloreactivity decreases. It is unlikely that every amino acid difference will affect T cell alloreactivity in a similar way; we hypothesized that the effect of an amino acid difference may be dependent on its position and/or physicochemical properties. We selected 131 patient/donor pairs with either a single HLA-A or -C mismatch in the graft-versus-host direction and that were compatible for HLA-B, -DRB1, and -DQB1. The alloreactive CTL precursor (CTLp) frequency was determined and associated with the amino acid differences between the single HLA class I mismatches. In the β sheet, only amino acids that are noncompatible in their physicochemical properties affect T cell alloreactivity, whereas in the α helices, both compatible and noncompatible amino acids affect CTLp outcome. Positions 62, 63, 73, 76, 77, 80, 99, 116, 138, 144, 147, and 163 were bivariately associated with CTLp outcome, irrespective of the total number of amino acid differences. In multivariate analysis, positions 62, 63, 73, 80, 116, 138, 144, and 163 were found to be most predictive for negative CTLp outcome. These results formed the basis for a weighted predictive mismatch score; pairs with the highest mismatch scores are estimated to be 13 times more likely to have a negative CTLp. This new algorithm may be a tool in donor selection for hematopoietic stem cell transplantation.