Cell Mitogenic Responses Research Articles

Responses to Mycobacterium leprae by lymphocytes from new and old leprosy patients: Role of exogenous lymphokines

Lepromatous leprosy patients generally have reduced response to Mycobacterium leprae antigens in an in vitro lymphocyte transformation test, which could be due to insufficient generation of reactions or to active suppression of any reaction generated. We could detect 3 types of lack of reactivity: one which could be restored by the addition of supernatants from healthy, PHA-stimulated lymphocyte cultures, one which could not thus be restored and one in which the culture supernatant contained factors able to suppress mitogen responses of healthy cells. We compared responses of cells rom untreated patients, patients treated for 12–20 months with multiple drug therapy and patients with up to 20 years of dapsone treatment; all types of the disease were represented. Untreated patients of all types had low responses which were not always reconstituted by lymphokine-rich supernatants, but they did not produce the non-specific soluble suppressive factors. In most cases, including BL/LL types, after the initial months of treatment, antigen response improved and was further increased by the addition of supernatants containing lymphokines. Most of the long-term-treated, stable patients had a lymphokine-reconstitutable antigen response, and in most cases also produced non-specific suppressive factor(s). The question as to why leprosy patients do not respond to M. leprae antigen is a complex one; our results suggest that it is related to the activity of the infection in each group of patients.

Synergistic Enhancement of Mitogen Responses of Human Lymphocytes by Inhibitors of Cyclo-Oxygenase and Lipoxygenase

Previously we reported that inhibitors of the enzyme cyclo-oxygenase, which reduce biosynthesis of prostaglandins, may enhance mitogenic responses of human blood lymphoid cells, whereas only marginal effects were observed with 5,8,11-eicosatriynoic acid (ETI), which inhibits 12-lipoxygenase and leukotriene biosynthesis. However, sharply enhanced mitogen responses were observed when lymphoid cells were cultured with both a cyclo-oxygenase inhibitor and ETI, suggesting a synergy between the two drugs. To test whether this is a more general phenomenon occurring between inhibitors of cyclo-oxygenase and lipoxygenase, we have now extended these studies to include the following lipoxygenase inhibitors: cirsiliol; esculetin; 5,8,11,14-eicosatetraynoic acid, and nordihydroguaiaretic acid. The results have shown that any of these drugs combined with a cyclo-oxygenase inhibitor may enhance mitogen responses more than one would expect by summing the effects of the inhibitors tested separately. We conclude that inhibitors of cyclo-oxygenase and lipoxygenase may synergistically enhance mitogen responses of lymphoid cells.

T-Cell Depletion by Monoclonal Antibodies Does Not Prevent Granuloma Formation in Mice

Effects of T-cell depletion on the formation of organized granulomas in mouse skin were investigated. Monoclonal antibodies (MoAbs) to L3T4 and Lyt-2.2 were injected into euthymic BALB/c mice in order to deplete both T-helper and T-suppressor cell populations. Four days after injection, hepatic schistosome egg granulomas were transplanted into the skin. Injection of MoAbs to L3T4 and Lyt-2.2 was repeated in the recipient mice 6 days later. At the time of transplantation, flow cytometric analysis showed that the number of T cells which were positively stained with surface markers decreased on average by 68% in both regional lymph nodes and spleen. The mitogen response of spleen cells was also markedly reduced. Despite their immunosuppressed condition, development of organized granulomas was observed in the skin of recipient mice by light and electron microscopy 3 weeks after transplantation. The findings indicate that T-cell function may not be essential for initiation of organized granuloma formation.

Early handling enhances mitogen responses of splenic cells in adult C3H mice

Experiments were done to investigate the effects of early postnatal handling in mice on humoral and cellular immune response at 21 and 60 days of age. In Experiment 1, C3H mice were either handled or left undisturbed from birth to weaning and their mitogen-induced, splenic B- and T-cell proliferative responses were measured at Day 21. No differences were found. In Experiment 2, mice were handled as in Experiment 1, except that at weaning the two groups were further subdivided into postweaning handled or nonhandled groups and their splenic B- and T-cell proliferative responses were measured at 60 days of age. These data showed significantly enhanced B- and T-cell proliferative responses due to preweaning handling and no effect of postweaning handling.

Effect of auranofin treatment on aberrant splenic interleukin production in adjuvant arthritic rats.

Adjuvant-induced arthritis (AA) in rats is associated with a number of immunologic abnormalities which include a marked decrease in spleen cell mitogenic responses. In this study we investigated the altered production of interleukins in arthritic rats and evaluated the effects of auranofin treatment on disease progression and aberrant interleukin production. The capacity of the AA rat spleen cells to produce interleukin (IL) 2 and IL-3 was found to decrease during the development of the arthritic lesion, with maximum suppression occurring 16 to 17 days after adjuvant injection. In contrast, the production of IL-1 by splenic adherent cells from arthritic rats was markedly increased. Prophylactic treatment of AA rats with auranofin resulted in a slight reduction in paw edema, a complete normalization of the depressed IL-2 production, and a reduction of the elevated IL-1 production, but had no effect on the depressed IL-3 production. In contrast, auranofin administered to normal rats, in the same dosing regimen, did not affect interleukin production. Therapeutic administration of auranofin to AA rats with established disease resulted in normalization of IL-1 production without affecting the suppressed IL-2 and IL-3 levels. In contrast, while indomethacin treatment effectively decreased paw edema, it did not appreciably affect the systemic aberrant interleukin production. Taken together, these results suggest that disease-associated abnormalities in interleukin production may be mediated by different mechanisms with differential sensitivity to the effects of the disease-modifying drug auranofin. Furthermore, defining the relationship between drug-mediated normalization of aberrant immune parameters and clinical improvement will provide a basis for the elucidation of the mechanism of action of disease-modifying antiarthritic drugs as well as for assessment of clinical efficacy of drug treatment.

Effects of human growth hormone on immune functions: In vitro studies on cells of normal and growth hormone-deficient children

We have studied the in vitro effects of human growth hormone on cell surface markers and mitogenic responses of peripheral blood lymphocytes (PBL) of normal and growth hormone-deficient children before, during and after treatment with growth hormone. Growth hormone resulted in a decrease in B cell expression but it did not affect expression of T cell subsets. Growth hormone depressed the proliferation of PBL of normal and untreated growth hormone-deficient children. The proliferative responses to phytohemagglutinin (PHA) versus PHA with growth hormone were not statistically different, though the responses of most normal and on treatment children were diminished by the addition of growth hormone. PBL derived from growth hormone-deficient children during treatment with human growth hormone exhibited significantly greater spontaneous proliferation then the PBL of normal children. Growth hormone further significantly enhanced their proliferation. PHA and PHA with growth hormone resulted in significantly greater proliferation of these patients' PBL when compared to those of normal children. We demonstrated that human growth hormone had substantial in vitro effects on immune functions. These effects, some of which depend on the treatment status of the children, may need to be considered in the clinical use of human growth hormone.

Leucyl-leucine methyl ester treatment of donor cells permits establishment of immunocompetent parent----F1 chimeras that are selectively tolerant of host alloantigens.

Abstract Treatment of murine lymphocytes with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) selectively removes natural killer cells, cytotoxic T lymphocyte precursors, and the capacity to cause lethal graft-vs-host disease, whereas bone marrow stem cell function and alloantigen-induced L3T4+ T helper function remains intact. The present studies assess the immunocompetence of allogeneic bone marrow chimeras established by reconstituting irradiated (C57BL/6 X DBA/2)F1 (B6D2F1) mice with Leu-Leu-OMe-treated C57BL/6 (B6) bone marrow and spleen cells. Spleen cells from such chimeras were found to have normal B and T cell mitogenic responses. Furthermore, levels of natural-killer cell function were comparable to those observed in B6----B6 syngeneic radiation chimeras established without Leu-Leu-OMe treatment of donor cells. Spleen cells from B6----B6D2F1 mice were identical with B6----B6 or B6 mice in allostimulatory capacity and thus contained no discernible cells of non-H-2b phenotype. Whereas B6----B6D2F1 spleen cells demonstrated alloproliferative and allocytotoxic responses toward H-2k bearing spleen cells, no H-2d specific proliferative or cytotoxic responses could be elicited. B6----B6D2F1 spleen cells did not suppress the generation of anti-H-2d or anti-H-2k proliferative or cytotoxic responses from control B6 spleen cells. Furthermore, addition of rat concanavalin A supernatants did not reconstitute anti-H-2d responses of B6----B6D2F1 chimeric spleen cells. Thus, Leu-Leu-OMe treatment of B6 donor cells not only prevents lethal graft-vs-host disease, but also permits establishment of long-lived parent----F1 chimeras that are selectively tolerant of host H-2 disparate alloantigens, but fully immunocompetent with respect to natural killer cell function, B and T cell mitogenesis, and anti-third party alloresponsiveness.

Graft-versus-host reactions in the beige mouse. An investigation of the role of host and donor natural killer cells in the pathogenesis of graft-versus-host disease.

To investigate the role of natural killer (NK) cells in the induction and pathogenesis of graft-versus-host (GVH) disease, +/beige (+/bg; normal NK cell activity) and beige/beige (bg/bg; deficient NK cell activity) parental C57BL/6 (B6) lymphoid cells were used to induce GVH reactions in either B6 X C3H/Hej +/bg (+/bgF1) or B6 X C3H/HeJF1 bg/bg (bg/bg F1) hybrid mice. When B6 bg/bg parental lymphoid cells (PLC) were injected into bg/bg F1 mice, early splenomegaly, early severe suppression of the plaque-forming cell (PFC) response to sheep red blood cell (SRBC), and only partial suppression of T cell mitogen responses to concanavalin A (Con A) and phytohemagglutin (PHA) were observed on day 12 after GVH induction. In the same GVH combination, slightly augmented NK cytotoxic activity was induced and no GVH-induced moderate-to-severe pathological alterations in the liver and pancreas were observed. When bg/bg PLC were injected into +/bg F1 mice, early splenomegaly and pronounced immunosuppression of the PFC response to SRBC and partial suppression of Con A and PHA responses were observed on day 12 after GVH induction. In this combination (bg/bg----+/bg F1), significant NK cell activity was induced, but no moderate-to-severe histopathological alterations were observed. In contrast, when B6 +/bg PLC were injected into either +/bg F1 or bg/bg F1 hybrids, early splenomegaly, and severe immunosuppression of both the PFC response to SRBC and the T cell mitogen responses to Con A and PHA were observed by day 12--which persisted until day 30 after GVH induction. Furthermore, high NK cell activity was recorded and moderate-to-severe histopathological alterations appeared in both +/bg F1 and bg/bg F1 recipients. These results show that the bg/bg PLC can induce GVH-associated early splenomegaly and immunosuppression of the PFC response to SRBC in both the bg/bg F1 and +/bg F1 hybrids, but that it failed to induce moderate-to-severe histopathological alterations, even though NK cell activity of host origin was activated during GVH reactions. Conversely, when +/bg donor cells were used to induce the GVH reaction, splenomegaly and immunosuppression, as well as moderate-to-severe histopathological lesions were induced. These results suggest that donor NK cells rather than host NK cells play an active role in GVH-associated tissue damage, which in turn contributes to the long-term suppression.

Effect of immunodeficiency on diabetogenesis in genetically diabetic (db/db) mice.

The pathogenesis of diabetes in C57BL/KsJ-db/db mice has been proposed to entail autoimmune mechanisms. We have combined immunodeficiency genes with the db mutation to determine whether beta cell necrosis and establishment of severe diabetes would occur in the absence of normal T and/or B lymphocyte functions. Inbred mice carrying the recessive mutations, severe combined immunodeficiency (scid), X-linked immunodeficiency (xid), nude (nu), and the Y-linked autoimmune accelerator (Yaa), were crossed with strains congenic for the db mutation. The diabetes syndrome was studied in double homozygotes produced in the F2 generation. In another experiment, C57BL/KsJ-db/db males were made T cell function deficient by adolescent thymectomy followed by lethal irradiation and bone marrow reconstitution. None of these manipulations served to prevent the induction of a severe diabetes syndrome in any of the model systems analyzed. Thus, diabetogenesis characterized by massive necrosis of the pancreatic beta cells and atrophy of the pancreatic islets was observed in both the absence of normal T cell function (as assessed by absence of T cell mitogen response) and humoral autoimmunity against beta cell antigens (insulin, retroviral p73). In conclusion, our data indicate that anti-beta cell autoimmunity is not a primary event in the etiopathogenesis of diabetes in the db/db mouse.

Thymic epithelium controls thymocyte expression of preleukemic phenotype and leukemogenic retroviruses.

Congenitally athymic AKR-streaker (nustr/nustr) mice were grafted separately with syngeneic or allogeneic, irradiated (1200 R) thymic reticuloepithelial (TRE) elements (stroma) or nonirradiated whole thymus grafts (control group) from N-tropic murine leukemia virus (MuLV) infection-susceptible (Fv-1n/n) or N-tropic-MuLV-infection-resistant (Fv-1b/n) murine strains. From 3 to 13 mo after grafting, the mononuclear cells repopulating the thymus grafts were stained with fluorescent monoclonal antibodies to thymocyte differentiation antigens, peanut agglutinin, and an antibody to MuLV antigens and were then analyzed by flow cytometry. Irradiated TRE of the Fv-1n/n genotype, whether from high or low leukemia-incidence strains, contained lymphoid cells of host (nustr/nustr) origin with alterations in thymocyte differentiation and MuLV antigen expression consistent with preleukemic changes. In contrast, transplanted TRE of the low leukemia-incidence Fv-1b/n genotype restricted preleukemic changes in thymocyte differentiation and MuLV antigen expression by lymphoid cells derived from the nustr/nustr host. Thus, nustr/nustr lymphocytes must infect susceptible TRE (Fv-1n/n) with N-tropic-MuLV before preleukemic changes occur in the mustr/nustr lymphocytes that later migrate to the thymus. Therefore, it was the radiation-resistant cells in the thymus that amplified or suppressed expression of AKR MCF retroviruses and the preleukemic phenotype, not the thymic lymphocytes. Thy-1.1+ splenocytes of ungrafted nustr/nustr mice were comparable in percentage to nustr/+ but were deficient in the Lyt-1+2- subpopulation and unresponsive to mitogens or alloantigens in vitro. Analysis of splenocyte cell surface markers, mitogen, MLC, and CML responses of Fv-1n/n-thymus-grafted nustr/nustr mice showed restoration of Lyt-1+2- cells to levels comparable to nustr/+ and reconstitution of in vitro proliferative and cytotoxic responses.

Alteration of immune function in mice following carcinogen exposure

Treatment of mice with the direct-acting alkylating agent methyl methanesulfonate produced marked suppression of the humoral response to sheep erythrocytes and suppression of T cell responses to foreign antigens. These effects occurred without loss of spleen, thymus or body weight and in the absence of peripheral blood, splenic or bone marrow cytotoxicity. In comparison, exposure to urethan decreased spleen weights, number and viability of spleen cells, and numbers of circulating lymphocytes. Significant suppression of T cell mitogen responsiveness was observed at all dose levels of urethan. Thymus weights, proliferative responses to the B cell mitogen lipopolysaccharide and delayed hypersensitivity responses were decreased at the highest urethan dose. Cyclophosphamide treatment significantly depressed thymic weight, lumphoproliferative responses of T and B cells, antibody production and delayed hypersensitivity responses. These results suggest differential sensitivity in components of the host defense system to weak carcinogens.

Natural suppressor activity in graft-vs-host spleen and normal bone marrow is augmented by IL 2 and interferon-gamma.

Natural suppressor (NS) cells isolated from the spleens of mice 34 or more days after induction of chronic graft-vs-host disease showed potent suppressor activity that was dependent on the presence of lymphokines such as those found in lectin-free Con A supernatant (CAS). Both IL 2 and IFN-gamma were found to enhance NS activity, with IFN-gamma being the most active molecule. To generalize these findings, normal adult bone marrow (BM), a second environment where NS activity has been reported, was examined. Normal adult BM cells nonspecifically suppressed both B and T cell mitogen responses. BM suppression could also be enhanced by the addition of CAS to the cultures. Removal of plastic-adherent cells, Thy-1.2-positive cells, or B cells did not significantly diminish suppression or remove the ability to enhance suppression with CAS. When isolated on discontinuous Percoll gradients, BM suppressor cells banded in the less dense fractions, and the suppression in these bands was also enhanced by lymphokines. The suppressive activity of these cells was greatly enhanced by the addition of CAS, IFN-gamma, or IL 2. However, IFN-gamma appears to be a more potent enhancing molecule, being active at levels between 1 and 10 U/ml.

Suppression of immune function in growth hormone-deficient children during treatment with human growth hormone

Inasmuch as growth hormone is known to interact with the immune system, we studied immune functions including immunoglobulins, cell surface markers, mitogen responses, and polymorphonuclear cell function in eight children with growth hormone deficiency, ages 1 to 17 years, before and during treatment with human growth hormone for 12 to 16 months. Before treatment immune functions were normal in all children. Treatment with human growth hormone did not significantly affect serum immunoglobulins, polymorphonuclear cell function, or percent T cells. However, percent B cells decreased to subnormal levels in seven of seven patients. T helper/suppressor ratios decreased in all patients, to subnormal values in seven of eight patients; and mitogen responses decreased to below normal in all. The decline of percent B cells was transient in all patients, of T helper/suppressor ratios in seven of eight, and mitogen responses in five of eight patients. In vitro incubation of lymphocytes with growth hormone resulted in no changes in cell surface markers or mitogen responses. Although the depression of immune functions resulted in no increased rate of infections during the observation period, we do not know the possible effects of prolonged treatment and therefore caution against the indiscriminate use of human growth hormone. The effects of biosynthetically obtained growth hormone on immune function remain to be determined.

The selective growth of murine newborn-derived suppressor cells and their probable mode of action.

Naturally occurring suppressor cells residing in the spleens of newborn mice of less than 5 days old are known to suppress various lymphocyte activities. A population of these suppressor cells can be maintained and expanded in the supernatants derived from Wehi-3 cells. These suppressor cells, designated as Wehi-3-expanded neonatal splenocytes (WENS), can suppress mixed lymphocyte reactions (MLR) and T and B cell mitogen responses without any genetic restrictions. The WENS bear the Ly-5, J11d, and class I molecules. WENS suppression is not mediated through an interleukin 1 or interleukin 2 absorptive mechanism. To achieve maximum suppression of MLR, WENS must be present for at least 24 hr. WENS inhibited the proliferation of Wehi-164 cells but not other tumor cells. The inhibition of Wehi-164 growth was due to the action of natural cytotoxic cells, because WENS lysed Wehi-164 cells but not the natural killer target cell YAC-1. Maximum lysis of Wehi-164 by WENS required 18 to 24 hr. Five WENS cell lines were cultured for more than 6 mo; three of the cell lines lost their capacity to lyse Wehi-164 targets (natural cytotoxicity) and simultaneously lost their natural suppressor activity. The two WENS lines that retained natural cytotoxicity also retained natural suppressor activity. Thus, natural suppressor cells may manifest their suppression through a natural cytotoxicity mechanism.

THE GRAFT-VERSUS-HOST REACTION AND IMMUNE FUNCTION

Studies were conducted to determine whether a functional B cell defect occurred in the bone marrow of mice experiencing a GVH reaction (GVHBM). GVH reactions were induced in AxCBA F1 adult mice by an injection of A strain lymphoid cells. The GVH reaction was confirmed by immunosuppression and thymus histology. At various intervals after GVH induction, GVHBM was tested for its ability to restore B cell function in adult thymectomized irradiated mice reconstituted with normal thymocytes. GVHBM cells obtained seven days after GVH induction restored but slightly the plaque forming cell (PFC) response to sheep erythrocytes and the mitogen response to lipopolysaccharide (LPS). GVHBM cells obtained 10 days or later failed to reconstitute the PFC or LPS responses. GVHBM cells suppressed neither the T or B cell function of normal spleen cells nor the LPS mitogen response of normal bone marrow cells. In addition, the splenic colony-forming units (CFU-s) in GVHBM were slightly decreased by day 10 after GVH induction and markedly depressed by day 22 after GVH induction. These results suggest that the GVH reaction may affect two different events in B cell differentiation. The early decrease in functional B cells that occurs before there is any change in the CFU-s population suggests a direct effect on B cell production, whereas the later absence of functional B cells could be due to the marked decline in stem cell production (CFU-s).

Suppression of antibody response by excess dietary zinc exposure during certain stages of ontogeny.

A study was made of the effects of excess dietary zinc on the antibody response to sheep red blood cells (SRBC) in mice. C57BL/6J mice were divided into 10 different dietary groups and exposed to diets containing zinc in normal (50 ppm) or excess (2000 ppm) concentrations during gestation/lactation/postweaning development in the sequences (1) 50/50/50; (2) 50/50/2000; (3) 2000/50/50; (4) 2000/2000/50; (5) 2000/50/2000; (6) 50/2000/50; (7) 50/2000/2000; (8) 2000/2000/2000; (9) 50/50/50 (pair-fed); and (10) chow/chow/chow. Mice in group 8 had severe signs of copper deficiency at 8 weeks of age, such as reduced plasma copper, lowered plasma hematocrit, and achromotrichia. Mice receiving 2000 ppm zinc during gestation had fewer offspring per litter (measured at 2 weeks of age) and more nonviable births than mice given 50 ppm zinc during gestation. The growth curve of mice exposed to excess zinc in the 50/50/2000 group was identical to that of the control (50/50/50) group. Growth curves for all other groups were reduced by varying amounts. The plaque-forming cell response to SRBC was reduced only in the groups receiving 50/2000/2000 and 2000/2000/2000 ppm zinc (P less than 0.05); this reduced response was not associated with atrophy of the lymphoid organs. Splenic cell surface markers and mitogenic responsiveness were similar in the 50/50/50 and 2000/2000/2000 groups. These results suggest that the immune response is more susceptible to dietary manipulation during development than after the immune response has been developed.

Differential Effect of Dietary Protein Type on the B-Cell and T-Cell Immune Responses in Mice

The effect of 20 g/100 g diet of lactalbumin (L), casein (C), soy (S) and wheat (W) protein on the immune responsiveness of C3H/HeN mice has been investigated by measuring the humoral immune response to the T cell-independent antigen, TNP-Ficoll. The humoral immune response of mice fed the L diet was found to be higher than that of mice fed the C, S and W diets. On the other hand, delayedtype hypersensitivity, and splenic cell mitogen responses to phytohemagglutinin and concanavalin A did not differ among mice fed the various diets. Similarly, the type of diet did not appear to influence host resistance to Salmonella typhimurium. It is postulated that the type of protein in the diet influences directly the intrinsic capacity of the B lymphocytes to respond to an immunogenic stimulus.

Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. II. Development of natural suppressor cell activity.

We explored the immunoincompetence of mice undergoing a chronic graft-vs-host reaction (GVHR) across minor histocompatibility barriers. BALB/c and B10.D2 mice are H-2d and mls b, and differ only with regard to minor histocompatibility antigens (MiHA). A large number of BALB/c mice were unirradiated or were irradiated with 300, 600, or 900 R. They then were injected with 5 X 10(7) spleen cells from either allogeneic B10.D2 or syngeneic BALB/c mice. The spleen cells from these recipient mice were assayed at various times post-irradiation/injection for their proliferative response to Con A and LPS, their ability to suppress the mitogen responses of normal spleen cells, and for the genetic specificity of this suppression. Spleen cells from BALB/c mice that had received 600 or 900 R (but not 0 or 300 R), and allogeneic B10.D2 lymphocytes, became very hyporesponsive to mitogens and became suppressive in vitro by days 7 to 10 post-irradiation/injection. These phenomena persisted for the entire 49 days of the experiment. After an initial period of splenomegaly, the spleens of these mice gradually became depleted of viable lymphocytes. Initial characterization of suppressor cells found in the spleens of GVH mice showed that they were not removed by treatment with anti-Thy-1.2 plus complement. GVH suppressors also were not adherent to plates coated with antiserum directed towards murine Ig. In addition, these cells did not adhere to plastic plates. Thus, we believe that the suppressor cells found in mice undergoing GVHD across MiHA are not mature T cells, B cells, or macrophages, but belong to a class of suppressor cells termed natural suppressor (NS). Genetic analysis of NS cell activity showed that as early as 10 days post-irradiation/injection, NS cells inhibited mitogen responses of all mouse strains tested, the exception being the relative difficulty in suppressing the LPS response of B10.D2 (syngeneic with donor cells). By day 42, this had developed into an almost complete inability to suppress a B10.D2 LPS response, although at this time NS cells were still capable of inhibiting all the other mitogen responses of all strains tested, including the Con A response of B10.D2 spleen cells. Moderate amounts of mitogen unresponsiveness and suppressor activity were seen in the syngeneic groups (BALB/c----BALB/c) but only if recipients received 600 or 900 R. This was a transient phenomenon that was maximal at day 14, and which we believe to be a similar but less severe degree of immunoincompetence when compared with that seen with allogeneic stimulation in the B10.D2----BALB/c GVH model.(ABSTRACT TRUNCATED AT 400 WORDS)

A procedure for the isolation of highly purified populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood

A sequential separation protocol is described which reproducibly yields biologically active populations of B cells, T cells and monocytes from human peripheral and umbilical cord blood. The purification protocol employed T lymphocyte rosetting with unmodified sheep red blood cells, monocyte adherence to microexudate-coated flasks and anti-immunoglobulin panning of B cells. Population purity was determined by cytofluorographic light scatter analysis, expression of cell surface markers, esterase activity and mitogen responsiveness. The sequential protocol reproducibly yielded viable and functionally active cell populations of greater than 95% purity from a single starting population of mononuclear cells.

939 PHENOTYPIC AND FUNCTIONAL CHANGES OF LYMPHOCYTES IN HEMOPHILIACS

Changes in cellular immunity have been reported in hemophiliacs receiving lyophilized factor concentrate, T cell subsets, mitogenic responsiveness, and degree of hypergammaglobulinemia in healthy hemophiliacs were examined in order to determine the effect of product therapy on immune responsiveness of children with hemophilia (age range, 3-21 years). The study population was comprised of 21 hemophilia A patients receiving factor VIII concentrate, 10 hemophilia A patients receiving cryoprecipitate, 8 hemophilia B patients receiving factor IX concentrate and 20 healthy children receiving no blood products. The percentage of T4+ lymphocytes was decreased in all hemophiliacs regardless of therapy (p<0.05). Only patients with hemophilia A receiving factor VIII concentrate exhibited a concomitant increase in the percentage of T8+ lymphocytes and showed a significantly decreased T4/T8 ratio (p<0.05). Depressed mitogenic responsiveness to phytohemagglutinin and concanavalin-A was observed in both hemophilia A and B patients receiving factor concentrate (p<0.05). No correlation was observed between changes in the T4/T8 ratio or mitogen responsiveness relative to the amount of product received. Hypergammaglobulinemia of the IgG class was demonstrable in all hemophilia groups and correlated with age (p<0.05). Phenotypic and functional alternations of lymphocytes demonstrated in hemophiliacs are probably the result of chronic factor exposure, which becomes more pronounced with increasing age.