Erythrocyte-rosetting properties of feline blood lymphocytes and their relationship to monoclonal antibodies to T lymphocytes

Rosette formation of feline peripheral blood leukocytes with guinea pig (GP) and gerbil (G) erythrocytes (E) has been shown in an earlier study to identify T lymphocytes expressing helper and suppressor cell activity, respectively. This T lymphocyte distinction was based on the removal of the E-rosetting populations from peripheral blood leukocytes (PBL) and the subsequent functional evaluation of the remaining cells in a pokeweed mitogen (PWM)-induced synthesis of immunoglobulin (Ig). In the present study, we demonstrate a direct helper and suppressor function of GPE- and GE-rosetted cells, respectively, wherein the induction of Ig synthesis is altered in a positive or negative way by the addition of the cells to a control target population. A pan-T monoclonal antibody (mAb), CT843, and mAbs to the CD4 (CT248) and CD8 (CT87) subsets are also described; their specificities are established in functional assays, the PWM-induced Ig synthesis and the production of interleukin-2 following Concanavalin A stimulation of PBL, and a biochemical analysis of the surface membrane antigens detected by the mAbs. Immunoprecipitation and SDS-PAGE analyses showed CT248 to react with a approximately 60-kDa protein under both reducing and nonreducing conditions. Under reducing conditions, CT87 reacted with one subunit at approximately 35 kDa; a second faint band at approximately 39 kDa was poorly resolved. mAb CT843 detected a heterodimer of approximately 70 and approximately 60 kDa under both reducing and nonreducing conditions. The relationship of the mAbs to E-rosetting was examined in FACScan analyses and rosette inhibition studies. The percentage of GE-rosetting cells agreed with the percentage of cells stained with the CD8 mAb, whereas a comparison of GPE-rosetting and staining with the CD4 mAb showed variability. The binding of GE to PBL was blocked by pretreatment of PBL with the CD8 mAb, whereas no inhibition of GPE rosettes was observed with any of the mAbs. In a previous study, we had shown that an overnight culture of feline PBL at 37 degrees C leads to the development of a second population of GPE-rosetting cells, also having a helper function. The relationship of the two GPE-rosetting populations to the CD4 mAb, CT248, was examined in rosette depletion studies and FACScan analyses. It was found that depletion of the GPE-rosetting cells from fresh, i.e., Day 0 cells, removed only a small percentage of cells reactive with the CD4 mAb, whereas GPE-rosette depletions performed on Day 1 PBL, which contained both populations of GPE-rosetting cells, removed almost all cells reactive with this antibody. The latter study suggests that the GPE-rosetting phenomenon is detecting two subsets of CD4 cells with T helper function, those present in fresh blood and those acquiring the GPE receptor after an overnight culture.     

Epitope mapping of conformational monoclonal antibodies specific to NhaA Na+/H+ antiporter: structural and functional implications

The recently determined crystal structure of NhaA, the Na ⁺/H ⁺ antiporter of Escherichia coli, showed that the previously constructed series of NhaA-alkaline phosphatase (PhoA) fusions correctly predicted the topology of NhaA's 12 transmembrane segments (TMS), with the C- and N-termini pointing to the cytoplasm. Here, we show that these NhaA-PhoA fusions provide an excellent tool for mapping the epitopes of three NhaA-specific conformational monoclonal antibodies (mAbs), of which two drastically inhibit the antiporter. By identifying which of the NhaA fusions is bound by the respective mAb, the epitopes were localized to small stretches of NhaA. Then precise mapping was conducted by targeted Cys scanning mutagenesis combined with chemical modifications. Most interestingly, the epitopes of the inhibitory mAbs, 5H4 and 2C5, were identified in loop X-XI (cytoplasmic) and loop XI-XII (periplasmic), which are connected by TMS XI on the cytoplasmic and periplasmic sides of the membrane, respectively. The revealed location of the mAbs suggests that mAb binding distorts the unique NhaA TMS IV/XI assembly and thus inhibits the activity of NhaA. The noninhibitory mAb 6F9 binds to the functionally dispensable C-terminus of NhaA.     

Two criteria for the comparison of multiple arrangements of experiments and their use to answer the question: Is it better to compare p treatment groups with p controls or with a single control

In experimental situations examining the effectiveness of p(p>1) treatments by comparing treated groups and untreated controls the following question arose: Which experimental arrangement is more appropriate, an arrangement A₁ using for the p comparisons only one single control, or an arrangement A₂ using for every comparison another control. To answer this question two criteria are formulated. The first criterion takes into consideration the expected numbers of type I and type II errors, and the second criterion the probabilities of type I and of type II errors. According to the first criterion A₁ and A₂ do not differ, generally. According to the second criterion A₁ with the comparison of means of normal distributed variables is better than A₂. Pooling of all p controls is better than A₂ according to both criteria. A₁ is better also according to the often used experimentwise type I error rate. The formulated criteria have some analogy to those of MILLER (1966). It may be possible to use them not only for comparing experimental arrangements but also tests.