Spontaneous immunoglobulin (Ig) secretion by cells from multiple sclerosis (MS) patients (in the progressive phase) treated with monthly pulse doses of cyclophosphamide (CY) (1000-1600 mg/M2) was measured using the protein A plaque assay, to evaluate the effect of CY treatment on B-cell function. Surprisingly, an increase, rather than a decrease, in Ig-secreting cells was seen following CY treatment. CY-treated MS patients averaged 1380 +/- 535 spontaneous total (IgM + G + A) Ig plaque-forming cells (PFC) per 1 X 10(6) peripheral blood mononuclear cells (MNC), measured at 15-22 days after monthly CY administration, while healthy adults had 280 +/- 47 Ig PFC/10(6) MNC, and MS patients not treated with CY had 300 +/- 43 Ig PFC/10(6) MNC. The observed increase was due to an increase in IgG and IgA PFC. PFC levels remained elevated for 4 weeks following CY treatment, decreasing to control levels by 7-8 weeks post-CY. A small increase in serum IgG level was noted after greater than 12 months of pulse CY therapy; no increase was seen in CSF IgG levels. A preferential decrease in the number of CD4+ T cells was also seen in the CY-treated MS patients. We propose that the observed increase in the number of spontaneous Ig PFC was due to the CY-induced disruption of the CD4+ T cell-mediated control of in vivo activated B cells.

The clonal selection theory, as formulated by Burnet, predicts that a lymphocyte bearing receptors for a given antigen will, upon activation, produce antibody to that same antigen. This prediction has for the first time been quantitatively confirmed, by precursor frequency analysis of highly purified sheep erythrocyte antigen-binding cells (SRBC-ABC), isolated from the spleens of non-immune mice, by means of an LPS-driven limiting dilution microculture system. These precursor frequencies indicated that virtually every non-immune SRBC-ABC that was a precursor for the secretion of IgM (or IgG) was also a precursor for the secretion of SRBC-specific IgM (or SRBC-specific JgG), after correction for non-SRBC-ABC B cells contaminating the SRBC-ABC preparations. These data help to clarify the relationship between antigen-binding cells and the antigen-reaetive cells of the clonal selection theory. In the course of making this observation, it was also shown that the precursor frequency of purified non-immune SRBC-ABC for JgG production was the same as that of unfractionated cells, whereas the precursor frequency for JgM production and LPS-induced thymidine incorporation were both lower in the ABC. The clone size estimates for IgM and IgG precursors in ABC and unfractionated cells all fell within a narrow range (t to 2 doublings).

Controversy concerning the immunologic role of antigen-binding cells (ABC) has prompted us to attempt to quantitate the proportion of stimulable ABC, in immunized animals, which are precursors for cells producing antibody specific for the antigen bound. Using a lipopolysaccharide (LPS)-driven limiting dilution analysis system, the precursor frequency (PF) of cells secreting IgM and IgG and sheep erythrocyte (SRBC)-specific IgM and IgG was established for highly purified SRBC antigen-binding cell (SRBC-ABC) and unfractionated populations taken from CBA/J mouse spleens on days 5, 12 and 180 of the in vivo primary immune response to SRBC. At all these times, almost all SRBC-ABC spontaneously secreting immunoglobulin (Ig) secreted SRBC-specific Ig, and almost all precursors of Ig-secreting cells in the ABC populations were precursors of cells secreting specific anti-SRBC antibody. In SRBC-ABC populations, the PF for total and SRBC-specific Ig secretion was seen to decrease on days 5 and 12 after immunization and to increase to 3.5 to 7 times nonimmune levels 180 days after immunization. The absolute number of precursors, within the SRBC-ABC population, for the secretion of SRBC-specific Ig decreased on day 12 after immunization. In the unfractionated population, the PF for SRBC-specific Ig secretion temporarily increased after immunization, reaching peak levels 5 days (IgM) and 12 days (IgG) after immunization. These two changes may be related, representing the progress of stimulated cells out of the ABC pool as they lose receptors en route to full maturation. The small clone sizes on days 5 and 12 indicate that ABC divide less in response to LPS when already engaged in a response to antigen. In contrast, the PF for total IgM and IgG secretion in the unfractionated population was not greatly affected by immunization.

Markedly reduced ecto-5'-nucleotidase activity was found in peripheral blood lymphocytes from 27 out of 30 homosexual men with the acquired immune deficiency syndrome (AIDS) in association with Kaposi's sarcoma (AIDS-KS; 2.67 +/- 1.70 U/10(6) cells; n = 13), opportunistic infections (AIDS-OI; 9.29 +/- 7.32; n = 7), or the AIDS-related complex (ARC; 9.82 +/- 6.12; n = 10). These values were significantly different from healthy controls (22.70 +/- 4.58; p less than 0.001). In AIDS-KS patients, both T cells and non-T cells exhibited significantly reduced ecto-5'-NT activity (p less than 0.001). AIDS-KS CD8 cells contained 20% of the mean ecto-5'-NT activity (7.04 +/- 3.53) displayed by control CD8 cells (34.07 +/- 4.86; p less than 0.001). No significant difference in enzyme level was observed between control and AIDS-KS CD4 cells (11.93 +/- 4.98 vs 7.98 +/- 3.28, respectively). In AIDS patients, lymphocyte ecto-5'-NT activity was inversely related (r = -0.518; p less than 0.01) to the absolute number of OKT10+ cells, but no correlation was found with the number of HLA-DR+ cells (r =-0.224). Two-color analysis of lymphocytes from AIDS-KS patients revealed that 75 +/- 12% of circulating CD8 cells expressed the OKT10 antigen, whereas only 10 +/- 6% of control CD8 cells did. HLA-DR antigens, which are not normally found on circulating resting T cells, were expressed in AIDS-KS CD8 cells, although to a lesser extent than OKT10. These data demonstrate that most AIDS CD8 cells differ from control CD8 cells. Although it has been suggested that these cells are activated cytotoxic or suppressor cells, the data presented here support the hypothesis they are immature. Reduced T cell ecto-5'-NT activity and enhanced expression of OKT10 and HLA-DR antigens on circulating CD8 cells, in conjunction with lack of transferrin receptor-(OKT9) and IL 2 receptor-(Tac) bearing lymphocytes, sustain this latter hypothesis. The correlation of the numerical reduction of CD4 cells with the reduced levels of ecto-5'-NT (r = 0.606; p less than 0.01) suggests that the abnormal maturation of CD8 cells seen in AIDS might be a consequence of the CD4 deficiency characteristic of this syndrome.

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