Normal CD5+ B lymphocytes are poor stimulators in the autologous mixed lymphocyte reaction (AMLR).

The AMLR is decreased in chronic lymphocytic leukaemia (CLL), which is characterized by a monoclonal expansion of CD5+ B lymphocytes. Since B cells are used to stimulate the AMLR, we investigated the capacity of normal CD5+ B cells to function as stimulators in the AMLR. We isolated B cells from the peripheral blood of normal individuals and fractionated them into subpopulations enriched for CD5+ and CD5- cells, which were used as stimulators in the AMLR. We found that the CD5- B cells were excellent stimulators, whereas stimulation of AMLR by B cells enriched for CD5+ cells resulted in significantly reduced proliferative responses (P < 0.005). This suggests that the reduced AMLR in CLL is due to the predominance of CD5+ B lymphocytes in the stimulating cell population.

Growth of B cell colonies independent of T cells: a new perspective.

Growth of human peripheral blood B cells in a B cell colony assay system is a useful technique to study the function of B cell biology. In the initial reports, T and B cells were admixed in the culture system, prior to which the T cells were treated with mitomycin or irradiation to prevent their proliferation. There were reports that optimal growth of B cell colonies required T cells to be in contact with the B cells. However, we were able to grow B cell colonies physically separated from T cells which were placed on a filter. We speculated then that T cells contacted B cells via pseudopods through the pores of the filter. We now report the growth of B cell colonies independent of T cells and conclude that B cell colony growth depends upon a critical number of B cells plated rather than on T cell help.

Feedback suppression of B cell colony formation by supernatants of B colony cells: role of immunoglobulin.

We have reported previously that CD5+ B cells from mature B cell colonies provide a negative feedback signal to the growth of autologous B cell colonies. Now we have observed that supernatants from mature B cell colonies also provide a negative feedback signal to the growth of autologous B cell colonies. We investigated the mechanism of this effect by growing B cell colonies physically separated by a 0.45 micron filter from T cells in millicell-CM chambers. Addition of colony supernatants to the T cell compartment reduced the number of B cell colonies by 28 +/- 6%. Colony numbers were reduced by 11 +/- 2 and 17 +/- 5% when the supernatants were added to the B cell or to both compartments, respectively. Pulsing T cells with the B cell colony supernatants before adding them to the colonies also decreased colony numbers by 33 +/- 13%. The addition of exogenous Ig classes and IgG subclasses to B cells decreased B cell colony numbers, although the effect was variable. In the presence of T cells, IgG had the greatest suppressive activity and the subclass IgG4 was most suppressive. In the absence of T cells, high concentrations of IgG almost abolished B cell colony formation. We conclude that these supernatants provide a negative feedback signal either directly to B cells, or via T cells which may be mediated at least in part by Ig.

Immunologic changes after blood transfusion in patients undergoing vascular surgery.

Immunologic changes after blood transfusions cannot be studied ethically in normal individuals. We therefore studied two comparable groups of patients with atherosclerotic cardiovascular disease who received similar drug treatment and experienced a similar degree of surgical trauma, except that one group received an average of 2.5 units of packed red cells at one time period during surgery. We conducted immunologic tests preoperatively and 5, 10, 45 to 60, 90, 180, and 360 days postoperatively. There was no significant difference in all indices tested preoperatively between the two groups. Five and 10 days postoperatively, the absolute numbers of CD3, CD4, CD8, and B cells decreased in both groups; however, the decrease was significantly greater in the transfused group than in the nontransfused group 5 days postoperatively. There was no significant difference in these parameters between the two groups at other time periods tested. At 5 and 10 days postoperatively, the lymphocyte responses to phytohemagglutinin, concanavalin A, and allogeneic lymphocytes in autologous serum were decreased in both groups. However, at 60 days postoperatively, the responses of the nontransfused group became significantly increased, whereas those of the transfused group remained relatively unchanged. By days 90, 180, and 360, the lymphocyte responses of the nontransfused group had dropped to levels seen at earlier time intervals and were comparable to responses in the transfused group. There were no significant differences between the groups in the number of T-cell colonies formed, the number of immunoglobulin-producing cells obtained, and the lymphokine responses (migration inhibitory factor/migration stimulation factor) at all times tested. The major immunologic perturbations attributed to blood transfusions were an exaggerated decrease in the numbers of circulating lymphocytes, particularly those with markers associated with T-helper cells, and failure to demonstrate a rebound increase in the proliferative response 45 to 90 days later.

Manipulation of macrophage migration inhibition/stimulation responses by adjuvants and interleukins.

C57 B1/6 mice were immunized with bovine serum albumin (BSA) and Freund's complete and incomplete adjuvants in various concentrations. Spleen cells from these animals were subsequently stimulated with concanavalin A (Con A), purified protein derivative or BSA, and lymphokine responses were measured in one-stage migration assays. Con A consistently produced macrophage migration inhibition factor (MIF) responses in nonimmunized animals and those immunized with complete adjuvant. This was switched to migration stimulation factor (MStF) responses by prior immunization with incomplete adjuvant. Immunization with complete adjuvant and with BSA alone was followed by MIF responses to antigenic stimulation whereas incomplete adjuvant promoted MStF responses. The MStF responses to both mitogenic and antigenic stimulation were inhibited by the addition of affinity purified interleukin-1 to the spleen cells. Interleukin-1 also inhibited MStF responses and potentiated MIF responses to Con A stimulation by human mononuclear cells whereas interleukin-2 did not influence these responses.

Feedback suppression of B cell colony formation in healthy individuals.

Proliferation and differentiation of B cells has been extensively studied and the study of feedback suppression of B cell proliferation has been limited to humoral factors. However, very little is known about feedback suppression of B cell proliferation by cellular influences. We have previously reported on the role of T cells and their subsets on B cell proliferation in that we did not observe suppression of B cell colony growth by T cells. We now report on the role of B cells in limiting B cell proliferation. B cell colonies were grown in methyl cellulose for either 3 days or 5-6 days utilizing 2 x 10(5) T cells irradiated with 9,000 rads, and 2 x 10(5) B cells. The B cells were then obtained from these colonies and increasing numbers of cells were added to fresh autologous B cells that were further cultured for 5 days to form new B cell colonies. At the end of this period, B cell colony numbers were determined. Our data show that addition of CD19- and CD20- positive B cells recovered from mature colonies after 5 days to fresh B cells suppressed further B cell colony growth in all cases tested, whereas addition of CD19-positive B cells recovered from immature colonies after 3 days of culture did not suppress further B cell colony growth. Elimination of CD 19- or CD20-positive cells with monoclonal antibody to CD19 and complement or by the technique of panning enhanced colony growth. Supernatants obtained from B cell colonies did not suppress B cell colony formation. Our data suggest that there is feedback suppression of normal progenitor B cell proliferation by constituent B cells and that this effect develops during maturation of colonies during the growth phase.

Growth of B cell colonies independent of T cell contact.

One of the methods of obtaining B cell colonies involves physically admixing B cells in growth medium containing mitogens with accessory T cells treated with mitomycin C or low dose irradiation to prevent T cell colony formation. However, under these conditions T cells still have the capacity to form colonies in methylcellulose, which is most frequently used for colony assays, and B cells recovered from the colonies are contaminated by the large number of added T cells. Therefore, we have developed a method of growing B cell colonies by physically separating the enriched B cells from the T cells by using millicell-HA inserts (Millipore). The requirement for mitogens, culture conditions, development, and number of B cell colonies formed were similar to those observed with admixture of T and B cells. The advantage of this system is that it permits growth of B cell colonies without the confounding influence of T cell proliferation or treatment of T cells with mitomycin or irradiation, and relatively pure B cell colonies can be recovered for further characterization or functional studies.

Familial systemic lupus erythematosus: evidence for separate loci controlling C4 deficiency and formation of antibodies to DNA, nRNP, Ro and La.

A family has been identified in the Canadian province of Prince Edward Island in which 2 sisters have systemic lupus erythematosus in a sibship of 14. Studies are reported on 11 of the siblings and 16 other family members. The affected siblings, and 4 other members of their sibship, are halfnull homozygotes for the C4A component of complement. We studied the distribution in family members of antibodies to ss and dsDNA, and to Ro(SSA), La(SSB), Sm and nRNP. Eight of 11 members of the affected sibship are antibody producers, compared to only 3 of 13 members of the parental generation. Our study provides further evidence for an association between null genes for C4A and familial lupus, and suggests, in an unusually large kindred, that several other genetic factors are involved in the production of antinuclear antibodies.

Presence of mitomycin resistant T cells in peripheral blood of normal individuals.

T cell colonies can be easily grown from peripheral blood, and are an index of cellular immunocompetence. Mitomycin-treated T cells are used as stimulator cells in mixed lymphocyte reactions and as feeder cells for growth of B cell colonies, the assumption being that mitomycin prevents proliferation of T cells. We tested this assumption by comparing the proliferation of mitomycin-treated T cells in response to stimulation with phytohemagglutinin (PHA) with that of untreated T cells in liquid cultures and in T cell colony assay. We found that incorporation of tritiated thymidine by cells from 11 healthy individuals pretreated with 25, 50 and 100 micrograms/ml mitomycin C was reduced to 13, 11 and 8%, respectively, of that of untreated cells when stimulated by an optimal concentration of PHA (10 micrograms/ml) in liquid cultures. However, parallel experiments with aliquots of the same cells showed that pretreatment with 25, 50 or 100 micrograms/ml mitomycin C merely reduced T cell colonies to 49, 45 and 45%, respectively, of untreated cells. In five additional experiments mitomycin 200 and 400 micrograms/ml reduced T cell colony numbers to 47 and 60%, respectively. Treatment of T cells with 9000 rad completely abolished T cell colony formation. Lower doses of radiation up to 6000 R did not abolish T cell colony formation, although it effectively blocked T cell proliferation to PHA in liquid cultures. 24 h preincubation of T cells with suboptimal doses of PHA and then treatment with mitomycin or radiation did not abolish T cell colony formation. T cells were recovered from the mitomycin-resistant T cell colonies and stimulated in liquid cultures with PHA, untreated or after exposure to 25 micrograms/ml mitomycin C. Incorporation of tritiated thymidine by the mitomycin-treated cells was reduced to 8% of the untreated controls. Our observations suggests: There may be inaccuracies in B cell colony assays using mitomycin-treated T cells because of significant T cell colony formation. There is a population of T cells in the peripheral blood of normal individuals which form colonies and are resistant to mitomycin.

The cellular basis for differential lymphokine responses to mitogen stimulation.

Human mononuclear cells from some individuals produce macrophage migration inhibition factor (MIF) when stimulated with Con A while those of others produce migration stimulation factor (MStF). T cells were responsible for these different responses but T4 cells produced MIF and T8 cells produced MStF regardless of the global response which was not explained by the individual T4:T8 ratios. Admixing the T-cell subpopulations in vitro revealed that MIF responses switched to MStF responses between T4:T8 ratios of 75:25 and 50:50 with MStF responders switching at higher ratios than MIF responders. Pulse exposure to supernatants from Con A-stimulated T4-enriched cells significantly reduced migration indices resulting from stimulation of fresh cells, promoting MIF responses regardless of the responder status of the supernatant donor. In contrast, supernatants from T8-enriched cells, when obtained from MStF responders, significantly increased migration indices while there was no effect when the supernatants were obtained from MIF responders. These results suggest that soluble factors from T8 cells are primarily responsible for determining whether an individual mounts a MIF or MStF response to Con A stimulation.

Immunoregulation of B lymphocyte colony formation by T cell subsets in patients with chronic lymphocytic leukemia.

Normal B lymphocytes are activated, proliferate, and then differentiate into plasma cells and secrete immunoglobulin (Ig). We have reported that chronic lymphocytic leukemia (CLL) T4 cells help and CLL T8 cells lack suppressor effects on Ig synthesis by normal B cells (Blood 62:767, 1983). We have now explored the earlier phase, proliferation, using B cell colony formation; in semisolid media. B lymphocyte colonies from normal individuals and from patients with CLL were grown in 0.3% agarose overlayed with T cells or T cell subsets and the B cell mitogen staphylococcal protein A. Enriched T cells, OKT4 or OKT8, were obtained either by sheep erythrocyte rosettes or depletion of OKT8 or OKT4 cells by monoclonal antibody or complement, respectively. Twenty thousand B cells from normal subjects yielded 65 +/- 9, 64 +/- 7, and 19 +/- 6 colonies with autologous unfractionated T-, OKT4-, or OKT8-positive cells, respectively. This compared to 29 +/- 11, 81 +/- 11, and 15 +/- 4 colonies from patients with CLL with added autologous unfractionated T-, OKT4-, or OKT8-positive cells. To determine whether the fewer number of colonies in both normal subjects and patients with CLL with OKT8-positive cells was due to suppression or lack of help, the number of OKT4-positive cells was held constant, and OKT8-positive cells were added in increasing numbers. No suppression of colony formation could be demonstrated. Furthermore, the addition of increasing numbers of concanavalin A (Con A)-activated OKT8-positive cells did not suppress colony formation. These results suggest that the CLL T cell subsets behave in a functionally similar manner to normal T cell subsets, namely, (1) that normal and CLL B cell colony growth is helped by OKT4 cells; and (2) in contrast to immunoglobulin secretion by B cells, neither normal nor CLL OKT8 cells, unstimulated or activated by Con A, suppress B cell colony growth.

Lymphokine responses to concanavalin A stimulation: association with HLA DR antigens.

Stimulation of human lymphocytes with concanavalin A (Con A) resulted in variable lymphokine responses as indicated by factors inhibiting macrophage migration (MIF) or stimulating macrophage migration (MStF), or resulted in negligible responses. These responses were consistent for a given individual when repeated after several months. MIF responses were observed more frequently than MStF responses in patients with renal failure who had demonstrable alloantibodies. MStF responses were statistically associated with the presence of HLA DR1 antigens in patients with renal failure and two separate groups of healthy individuals, while MIF responses were associated with DR7 in the three groups studied. There was no correlation between immunoglobulin allotypes and lymphokine responses. These results suggest that lymphokine responses to Con A are indicators of nonspecific immunological responsiveness and are influenced by genes associated with the major histocompatibility complex.

Normal T cell colony numbers in untreated patients with chronic lymphocytic leukaemia (CLL).

T cell colony formation in normal individuals and untreated patients with chronic lymphocytic leukaemia (CLL) was studied to determine if there is a deficiency in the number of T cells capable of forming colonies in this disease. In normal individuals, assays using enriched T cells did not reveal the total number of potential colony forming cells, preventing accurate assessment in patients with B cell CLL where enriched T cells are mandatory. Conditions were therefore optimized to obtain the maximal number of colonies, so that accurate comparisons could be made between the potential of normal individuals and patients with CLL. Addition of normal autologous non-T cells to enriched T cells enhanced colony numbers in normal individuals to those seen in the whole mononuclear population. However, addition of normal non-T cells to CLL T cells would have caused a mixed lymphocyte reaction (MLR), which influences T cell colony numbers. Therefore the MLR was bypassed by adding to the enriched T cells, supernatants having characteristics of Interleukin-1 or commercially obtained Interleukin-2. The supernatants with Interleukin-1 activity enhanced T cell colony numbers comparably in both normal individuals and patients with CLL. Interleukin-2 did not increase T cell colony numbers in normal individuals and the increase seen in patients with CLL were not significant. Thus the effect of the lymphokines on T cell colony numbers was comparable in both normal individuals and untreated patients with CLL. We therefore concluded that there are normal numbers of cells which have the potential to form T cell colonies in untreated patients with CLL.

Immunoglobulin secretory function of B cells from untreated patients with chronic lymphocytic leukemia and hypogammaglobulinemia: role of T cells.

The mechanism of the hypogammaglobulinemia in patients with chronic lymphocytic leukemia (CLL) was studied by determining the generation of specific immunoglobulin-secreting cells in response to mitogen and antigen stimulation in culture. Normal peripheral blood B lymphocytes from 18 normal subjects cocultured with equal numbers of autologous T cells generated cells secreting 2,542 +/- 695 IgG, 2,153 +/- 615 IgA, and 2,918 +/- 945 IgM. Normal B lymphocytes cocultured with normal allogeneic T cells generated similar numbers. However, B lymphocytes from patients with chronic lymphocytic leukemia cocultured with T cells from the same patient generated only 0.5% as many cells secreting IgG and 11% and 23% as many secreting IgA and IgM, respectively. The reason for this markedly defective generation of immunoglobulin-secreting cells was investigated by evaluating T-helper, T-suppressor, and B-cell function using B cells from tonsil and T and B cells from peripheral blood of normal and leukemic individuals. T cells from patients with chronic lymphocytic leukemia provided somewhat greater help than did normal T cells to normal peripheral blood B cells and normal help to tonsil B cells, whether stimulated with mitogen or antigen. T cells from patients with chronic lymphocytic leukemia did not demonstrate increased suppressor function compared to normals with B cells from normal peripheral blood. The hypogammaglobulinemia in these patients therefore was associated with a markedly defective generation of immunoglobulin secreting cells, and as there was normal or increased T-cell helper activity without excessive suppressor activity, it seems likely that this was due to an intrinsic B-cell defect.