Robert F. Ashman

Use of intravenous γ-globulin in antibody immunodeficiency: Results of a multicenter controlled trial☆

Abstract A 2-year corssover study was performed in 34 patients with antibody-deficiency disorders characterized by normal T-cell function. Each patients was treated for 1 year with either ISG or MISG and then treated for an additional year with the other preparation. There were no significant differences in acute or chronic infections in the categories of otitis media, bronchitis, sinusitis, gastrointestinal tract, skin, or conjunctival infections. Although there were attendant increases in the number of mild side effects and the number of apparent acute upper respiratory tract illnesses during MISG treatment, the modified preparation was as effective as ISG in preventing life-threatening infections in these patients with antibody-deficiency syndromes. MISG appears to be suitable for the treatment of patients who require large amounts of intravenous γ-globulin, of individuals in whom a rapid increase in antibody levels is required, of patients with bleeding disorders such as the Wiskott-Aldrich syndrome, and of severely debilitated patients in whom intramuscular γ-globulin administration would be difficult.

Profile of multiple lymphocyte functional defects in acquired hypogammaglobulinemia, derived from in vitro cell recombination analysis

Immunoregulatory defects in patients can be assigned to B cells, T helper cells, or T suppressor cells by means of a modification of the in vitro PWM-stimulated Ig biosynthesis assay. When lymphocyte subpopulations from patient and normal are recombined, multiple internal comparisons within a single experiment reveal the functional activity of each subpopulation. By this technique we studied nine adult patients with acquired panhypogammaglobulinemia and one with isolated IgG deficiency. Low total Ig production by the B cell fraction was demonstrated in seven of the nine panhypogammaglobulinemic patients. In four of these seven, IgG and IgA secretion was markedly reduced compared with IgM. In the two patients with normal total Ig production, elevated IgM compensated for lower IgA and IgG. The one patient with no surface Ig+ cells had a B cell defect, but no T cell defect. Reduced T help and excessive T suppression characterized two of the four patients with the most severe B cell defects. Three patients were anergic by delayed hypersensitivity skin testing and failed to sensitize to DNCB, but the patient with the most severe T cell defects in vitro was not among them. One of these three patients showed a mild T cell help defect and suppressor excess and the other two had pure B cell defects. Thus, anergy and T regulator function were not correlated. In three instances where hypersuppression was more evident by adding patient unfractionated cells than by adding patient T cells, suppression by the T depleted fraction could be demonstrated. No cases of radiation-resistant T suppression were revealed among the seven patients tested. Subnormal total protein synthesis, noted in six of seven patients with low Ig production, was invariably less marked than the Ig defect and often affected both T and B cells. One additional patient with pure IgG deficiency of 2 yr duration was essentially normal in her in vitro lymphocyte function. The general applicability of this experimental design for analysis of positive or negative immunoregulatory abnormalities is emphasized.

Panhypogammaglobulinemia in systemic lupus erythematosus: in vitro demonstration of multiple cellular defects☆

Classically, systemic lupus erythematosus (SLE) is a disease of antibody overproduction, whereas the hallmark of acquired immune deficiency is antibody underproduction. Two patients are presented in whom panhypogammaglobulinemia developed during the course of SLE. In both patients, the levels of the major immunoglobulin (Ig) classes did not fall simultaneously. Anti-DNA antibodies were present, and exacerbations of SLE nephritis occurred in both cases 6 to 8 yr after Ig levels became subnormal. One patient still requires immunosuppressive therapy for renal disease; both patients are experiencing recurrent sinopulmonary bacterial infections. In the pokeweed mitogen--stimulated Ig biosynthesis assay, both patients showed abnormal Ig production due to defective function of three cell types: hyporesponsive B cells, excessive T suppression, and subnormal T help. The latter defect is rare in common variable hypogammaglobulinemia. One patient also showed extreme suppression of Ig production by phagocytic mononuclear cells. Thus, despite the similarity in the histories, the cellular function of these two patients was not identical in vitro.

Membrane defects of the tolerant B cell: I. Failure of antigen-induced capping

Abstract In order to study the membrane function of tolerant B antigen-binding cells, tolerance to the trinitrophenyl (TNP) determinant was induced in mice by injecting the reactive form of the hapten, trinitrobenzene sulfonic acid (TNBS). By appropriate transfer experiments, Fidler and Golub ( J. Immunol. 112 , 1891, 1974) had previously shown that this form of tolerance is a B-cell property, induced and expressed in the absence of T cells. Hapten inhibition demonstrated the TNP-specificity of receptors on TNP-donkey erythrocyte(TNP-D)-binding cells in tolerant and nontolerant mice. About 88% of these cells were B cells by immunofluorescence, and the remainder were T cells. In the tolerant mice, challenge with TNP-sheep erythrocytes failed to expand the TNP-binding population, but sheep erythrocyte binders and anti-sheep plaque-forming cells expanded normally. Despite little or no change in TNP-binding cell numbers after tolerance induction, the TNP-binding cells of tolerant animals could not cap their receptors, in contrast to the sheep erythrocyte-binding cells from the same animals which capped normally. Although there is no anti-TNP plaque-forming cell response when tolerogen and immunogen are given simultaneously, capping failure is not evident until 2–4 days after tolerogen exposure. By Day 7, substantial recovery of immune responsiveness had occurred, yet even 12 months after a single dose of tolerogen there was no restoration of capping. Thus despite the association of both capping failure and unresponsiveness with tolerogen exposure, these lymphocyte functional defects appeared not to be causally related.

Membrane defects in the tolerant b cell. II. Mechanism of capping failure and reversal by antigen exposure.

Abstract The demonstration that TNP-binding B lymphocytes from animals whose B cells have been rendered tolerant to TNP by trinitrobenzene sulfonic acid cannot undergo antigen-induced capping of their TNP receptors for at least a year despite recovery of immune responsiveness has led to a search for the mechanism of the capping failure. Microtubule-dependent membrane “locking” analogous to that induced by concanavalin A appears to afflict the tolerant B cells, in that capping TNP receptors is restored after exposure to 10 −4 M colchicine or overnight incubation at 4 °C. Assignment of the defect to the cytoskeleton rather than the receptors themselves is also supported by the observations that enzymatic stripping and regrowth of receptors does not unlock the cell and that non-Ig membrane molecules recognized by antilymphocyte serum also cannot be capped on the tolerant cells. Cells which have remained locked for 4 days to 8 months after a single tolerogen exposure become unlocked 4 days after immunogen is given. Four days after immunogen, tolerogen fails to lock the membranes of TNP-binding cells. These results suggest that tolerogen contact interferes in a much broader range of functions in the TNP-binding cell than those which affect the immune response. Among these effects is a remarkably stable “locked” configuration of the cytoskeleton which is independent of immune responsiveness or receptor turnover, but which can be reversed by exposure to immunogen whether or not an immune response ensues.

The in vitro generation of antigen-binding cells

To analyze early events following the contact of antigen with specific receptor-bearing cells, antigen-binding cells (ABC) specific for sheep erythrocytes (SRC) were generated in a primary Mishell-Dutton culture system. The maximal frequency of ABC (a five-fold increase over Day 0 involving both T and B cells) occurred on Day 4 in culture conditions which yielded optimal numbers of plaque-forming cells (PFC) specific for SRC 1 day later. Assessment of the total Ig-secreting cells as well as ABC and PFC for ox and burro erythrocytes showed that the ABC as well as the PFC response to SRC was antigen specific. By autoradiography of [3H]thymidine-pulsed cultures and by inhibiting cell division with hydroxyurea, the continuous contribution of cell division to the generation of ABC was demonstrated. As with PFC, the decline in ABC numbers began at the point where their generation by cell division slowed. After removal of hydroxyurea, ABC frequency and thymidine incorporation recovered substantially, but PFC did not. However, division of ABC was not the only process involved in ABC generation. Indeed, 20% of the Day 4 ABC arose from cells dividing in the first 24 hr of culture which were not detectable as ABC at that time, i.e., some ABC were derived from dividing non-ABC. Thus both cell division and maturation contributed to the increase in specific ABC seen after primary in vitro immunization. Both processes can be added to the list of early antigen-stimulated events in specific ABC which can be analyzed in the primary culture system in future studies of cell interactions.

“Stop and go” kinetics of receptor movements induced by anti-Ig or antigen on individual lymphocytes

Abstract Serial observations were conducted on the time course of surface immunoglobulin (Ig) redistribution (capping) on individual mouse spleen lymphocytes. Capping of surface Ig by anti-Ig fluorescein and also antigen-induced capping of receptors on specific sheep erythrocyte antigen-binding cells were observed and the times required for individual cells to clear 90 and 180 ° of their circumference were recorded. There were striking differences between individual cells in both the onset and duration of receptor movements. Although the number of cells achieving complete clearing of first and second quadrants in successive time intervals declined, there was no correlation between the time required by a cell to clear the first quadrant and the time required by the same cell to clear the second quadrant. Thus, instead of observing gradual progressive migration of marker toward one pole of each individual cell at a rate resembling that of the whole population, we observed grossly discontinuous receptor movements, characterized by brief major shifts in receptors followed by a period of relative stability. Capping is thus viewed as a series of discrete contractile events related to the activity of membrane-associated cytoskeletal elements rather than a manifestation of “membrane flow”.

The isotype cycle: Successive changes in surface immunoglobulin classes expressed by the antigen-binding B-cell population during the primary in vivo immune response

Abstract Using the antigen-binding inhibition method, capable of revealing any combination of three surface Ig (sIg) isotypes on a population of antigen-binding cells (ABC) (S. Kanowith-Klein, E. S. Vitetta E.L. Korn, and R.F. Ashman, J. Immunol. 122 , 2349, 1979) we have defined the sequence of antigen-induced changes in the expression of sIgM, sIgD, and sIgG on the sheep erythrocyte (SRC) antigen-binding B-cell population (SRC-ABC) throughout the in vivo primary immune response. The majority of nonimmune B-ABC simultaneously expressed M and D (M + D + G − ). By Day 3 sIgG had appeared, mainly on cells already bearing sIgM and sIgD. By Day 5, other G + populations appeared: M + D − G + and M − D − G + . By Day 12, M + D − G + ABC declined, while M − D − G + ABC remained predominant for another month. By 6 months, the sIg phenotypes on the ABC had returned to the original nonimmune pattern, mainly M + D + G − ; but the absolute number of 6-month immune ABC was four times greater than that of nonimmune ABC. This cyclical change in sIg expression was confined to the B-cell population expressing receptors specific for the immunizing antigen, and affected the large majority of such cells. Twelve days after immunization with SRC, ABC specific for a non-cross-reacting antigen still mainly expressed the nonimmune sIg phenotype, M + D + G − .

Physiology of receptor capping induced by erythrocyte surface antigens.

Abstract The capping of antigen-binding cell receptors by bound sheep erythrocytes (SRC) demonstrates that antigen mounted on a cell surface can generate a signal leading to the capping reaction. SRC-induced capping of ABC is: (a) highly dependent on both aerobic and anaerobic glycolysis, (b) unaffected by agents altering intracellular cyclic nucleotide concentrations, (c) slightly more vigorous in strain A than in CBA mice, (d) inhibited by calcium ionophore, (e) inhibited by the local anesthetic dibucaine and the tranquillizer chlorpromazine, (f) dependent on cytoskeletal activity (i.e., inhibited by the simultaneous presence of colchicine and cytochalasin B), (g) not dependent on the membrane ATPases inhibited by ouabain, (h) not dependent on motility, in that agents which inhibit motility (cytochalasin B alone) or stimulate motility (carbachol) do not alter capping behavior. These properties represent similarities between the capping of surface Ig by the cellular antigens on SRC and by proteins such as anti-Ig. SRC-induced capping is much slower than anti-Ig-induced capping, and only engages 30–40% of ABC, indicating that the nature of the crosslinking agent can influence the kinetics and extent of capping. But SRC cap with the rapid kinetics typical of anti-Ig-induced capping if the surface membrane of the ABC is first cleared of other glycoproteins with trypsin. The removal of negatively charged sialic acid residues by neuraminidase has no such effect. It is probable that the compression of bound SRC into a small area of the membrane requires more energy than does the capping of protein ligands, and that some cells cannot muster enough energy to achieve it.

Membrane defects of the tolerant B cell: III. Defective receptor replacement

Abstract When trinitrobenzenesulfonic acid (TNBS), the reactive form of trinitrophenyl (TNP) hapten, is injected into a mouse, a brief intrinsic B-cell tolerance to TNP has been shown to result. Yet antigen-binding cells (ABC) with receptors for TNP persist in the TNBS-treated animal. After treatment with Pronase under conditions preserving cell recovery and viability, 80–90% of TNP-ABC failed to bind antigen. After 2 hr in vitro , Pronase-treated 4-day immune TNP-ABC displayed significant recovery of antigen binding, whereas nonimmune TNP-ABC performed the same feat by 18 hr. However, TNP-ABC tested 2 to 11 days after TNBS failed to replace digested receptors by 18 hr in vitro . Thirty days after TNBS, they had recovered this ability. This defective receptor replacement by TNP-ABC was not reversed by colchicine, and was not shared by the sheep-erythrocyte ABC of the same animals, which replaced receptors normally. When challenged with antigen (TNP-sheep erythrocytes) simultaneously with TNBS, recovery by 2 hr was evident on Day 11. When challenged with antigen 4 days after TNBS, receptor regeneration had returned to normal by the next day, and partial recovery of the anti-TNP plaque-forming cell response was evident 4 days later. Thus, the inability to replace receptors and immune unresponsiveness coincides in time, so that a causal relationship between these two defects may be hypothesized. This result contrasts with the membrane locking defect, previously described in the TNP-ABC of TNBS-treated animals, which far outlasted the unresponsive state.