Estradiol differentially regulates calreticulin: a potential link with abnormal T cell function in systemic lupus erythematosus?

OBJECTIVE: Systemic lupus erythematosus (SLE) is an autoimmune disease that affects women nine times more often than men. The present study investigates estradiol-dependent control of the calcium-buffering protein, calreticulin, to gain further insight into the molecular basis of abnormal T cell signaling in SLE T cells. METHODS: T cells were purified from blood samples obtained from healthy females and SLE patients. Calreticulin expression was quantified by real-time polymerase chain amplification. Calreticulin and estrogen receptor-α were co-precipitated and analyzed by Western blotting to determine if the proteins associate in T cells. RESULTS: Calreticulin expression increased (p = 0.034) in activated control T cells, while estradiol decreased (p = 0.044) calreticulin in resting T cells. Calreticulin expression decreased in activated SLE T cell samples and increased in approximately 50% of resting T cell samples. Plasma estradiol was similar (p > 0.05) among SLE patients and control volunteers. Estrogen receptor-α and calreticulin co-precipitated from nuclear and cytoplasmic T cell compartments. CONCLUSIONS: The results indicate that estradiol tightly regulates calreticulin expression in normal human T cells, and the dynamics are different between activated and resting T cells. The absence of this tight regulation in SLE T cells could contribute to abnormal T cell function.

Cells involved in the immune response. XI. Identification of the antigen-reactive cell as the tolerant cell in the immunologically tolerant rabbit.

Rabbits were made immunologically tolerant to either human serum albumin or bovine gamma globulin by the neonatal administration of antigen. At 10 wk of age, they were challenged with the tolerogenic antigen and found to be non-responsive. However, these tolerant rabbits could respond with humoral antibody formation directed toward the tolerogenic antigen if they were treated with normal, allogeneic bone marrow or bone marrow obtained from a rabbit made tolerant toward a different antigen. They were incapable of responding if they were given bone marrow obtained from a rabbit previously made tolerant to the tolerogenic antigen. Irradiated rabbits were unable to respond if treated with tolerant bone marrow, but could respond well if given normal bone marrow. Since it has previously been demonstrated that the antibody-forming cell, in an irradiated recipient of allogeneic bone marrow, is of recipient and not donor origin, the data presented strongly indicate that the unresponsive cell in the immunologically tolerant rabbit is the antigen-reactive cell.

Cells involved in the immune response. X. The transfer of antibody-forming capacity to irradiated rabbits by antigen-reactive cells isolated from normal allogeneic rabbit bone marrow after passage through antigen-sensitized glass bead columns.

The antigen-reactive cells in normal rabbit bone marrow could be isolated from a suspension of marrow cells by passage of the cells through an antigen-sensitized glass bead column. The cells which passed through the column were deficient in antigen-reactive cells directed to the antigen used to sensitize the glass beads, whereas the cells eluted from the column could transfer antibody-forming capacity to irradiated recipients only with respect to the specific sensitizing antigen. Separation of the bone marrow antigen-reactive cells could not be achieved by passage of the cells through nonsensitized glass bead columns or in the presence of excess free antigen in the column. Cells which were retained by, and later eluted from, the antigen-sensitized glass bead columns were mostly small mononuclear cells, whereas cells which passed through the columns were morphologically similar to the original unfractionated bone marrow cell suspension. The data indicate the presence of an antibody or antibody-like structure, with defined immunological specificity, on the surface of the normal bone marrow antigen-reactive cell.

Cells involved in the immune response. VII. The demonstration, using allotypic markers, of antibody formation by irradiation-resistant cells of irradiated rabbits injected with normal allogeneic bone marrow cells and sheep erythrocytes.

Bone marrow cells obtained from rabbits of one allotype were injected into irradiated rabbits of a different allotype. The recipients were also injected with sheep red blood cells, and their spleen cells were tested for plaque-forming capacity 7 days later. Spleen cells of all recipients gave large numbers of plaques as did spleen cells incubated with antiserum, directed toward donor allotype. However, incubation of the recipient spleen cells with antiserum directed toward recipient allotype completely suppressed plaque formation. These results demonstrate that antibody-formation in irradiated recipients of transferred lymphoid cells is a property of the recipient animal and that the antibody-forming cell is relatively irradiation-resistant. It was also demonstrated that only viable normal bone marrow cells are capable of transferring antibody-forming capacity to irradiated recipient rabbits. Neither sonicates nor heat-killed preparations of normal rabbit bone marrow cells possessed this capacity.

Cells involved in the immune response. VI. The immune response to red blood cells in irradiated rabbits after administration of normal, primed, or immune allogeneic rabbit bone marrow cells.

Irradiated rabbits given allogeneic bone marrow cells from normal adult donors responded to an injection of sheep red blood cells by forming circulating antibodies. Their spleen cells were also capable of forming many plaques using the hemolysis in gel technique, and were also capable of undergoing blastogenesis and mitosis and of incorporating tritiated thymidine upon exposure to the specific antigen in vitro. However, irradiated rabbits injected with allogeneic bone marrow obtained from rabbits injected with sheep red blood cells 24 hr prior to sacrifice (primed donors) were incapable of mounting an immune response after stimulation with sheep red cells. This loss of reactivity by the bone marrow from primed donors is specific for the antigen injected, since the immune response of the irradiated recipients to a non-cross-reacting antigen, the horse red blood cell, is unimpaired. Treatment of the bone marrow donors with high-titered specific antiserum to sheep red cells for 24 hr prior to sacrifice did not result in any diminished ability of their bone marrow cells to transfer antibody-forming capacity to sheep red blood cells. The significance of these results, with respect to the origin of the antigen-reactive and antibody-forming cells in the rabbit, is discussed.

Cells involved in the immune response. 8. The relationship between the loss and reappearance of antigen-reactive cells and immune responsiveness after irradiation of normal adult rabbits.

By appropriate irradiation and cell transfer experiments, a direct correlation was observed between the presence of viable and immunologically active antigen-reactive cells and the capacity of the rabbits to respond following immunization. Rabbits given 800 R total body irradiation were unable to elicit a humoral immune response nor did they possess significant numbers of antigen-reactive cells. The ability to respond with humoral antibody formation did not reappear until antigen-reactive cells could be detected. These results strongly indicate that the presence of competent antigen-reactive cells are necessary for the successful induction of the humoral immune response in the rabbit.

Bone marrow: the bursa equivalent in man?

Human bone marrow lymphoid cells, particularly when enriched with plasma cells, as in multiple myeloma, respond to pokeweed mitogen and to antiserum to immunoglobulin but not to phytohemagglutinin. Cells of patients with X-linked agammaglobulinemia of the bursal deficient type failed to respond to either pokeweed or to the antiserum to immunoglobulin. Leukocytes of the agammaglobulinemia patients however responded in a normal fashion to phytohemagglutinin. Just as the in vitro response to phytohemagglutinin is taken as an index of the thymus-dependent system, the in vitro response to both antiserum to immunoglobulin and pokeweed may be considered an index for the bursaldependent system. Human bone marrow, therefore, contains bursal cells and probably very few or no thymus cells.

Comparative study of the in vitro proliferative responses of blood and synovial fluid leukocytes of rheumatoid arthritis patients.

Lymphocyte-rich suspensions from blood and synovial fluid (SF) of 20 patients with rheumatoid arthritis (RA) and from blood of 12 normal subjects, were cultured with heat-aggregated, aggregate-free, and native human gamma globulin (HGG), with autologous IgG separated from RA-SF by anion-exchange chromatography and with phytohemagglutinin (PHA). No significant differences were noted between the in vitro proliferative responses of blood lymphocytes of RA and normal controls to any of these preparations. Significant differences were noted between blood and SF lymphocytes of RA patients with respect to their responses to the aggregate-free HGG and to PHA. Incubation of RA-SF cells but not RA-blood cells with aggregate-free HGG before their culture with the aggregated HGG markedly suppressed the in vitro proliferative response to the latter. The observed differences between blood and SF lymphocytes and the suppression of blastogenic response of SF cells by exposure to the aggregate-free preparation raise the possibility of modulating the immune and/or the inflammatory responses in RA.

Suppressor-cell dysfunction in systemic lupus erythematosus. Cells involved and in vitro correction.

To characterize the cell(s) responsible for the suppressor-cell dysfunction in active systemic lupus erythematosus (SLE), we fractionated blood mononuclear cells into thymus-derived (T), bone marrow-derived (B), and monocyte-depleted populations. Various cell populations from active SLE, inactive SLE, or normals, were activated with Concanavalin A, washed, and then co-cultured with active SLE cells. Soluble immune response suppressor (SIRS) from culture supernates of the activated cells was also used for the possible correction of the suppressor-cell dysfunction. Suppression was tested by enumerating DNA-binding cells by radioautography and by quantitating anti-DNA antibody in culture supernates by radioimmunoassay; and immunoglobulin was tested in cells and supernates by the immunofluorescence and the immunofluor techniques, respectively. Except for the numbers of DNA-binding cells, which were not suppressed, all the other three parameters in co-cultures with cells from active SLE patients were suppressed by Concanavalin A-activated cells (P < 0.001), or by SIRS (P < 0.05) from normals or inactive SLE patients. Concanavalin A-activated autologous or allogeneic active SLE cells and nonactivated cells from active or inactive SLE failed to suppress the various B-cell functions. Nonactivated normal cells suppressed levels of anti-DNA and immunoglobulin in supernates (P < 0.05). In characterizing the cells responsible for the suppressor dysfunction, it was clear from the results that T cells responsive to Concanavalin A activation are deficient in active SLE and fail to generate SIRS. On the other hand, monocytes from active SLE patients are responsive to signals from the activated T cells of normals or inactive SLE donors. Because SIRS suppresses active SLE cells in vitro, it might be considered therapeutically for the in vivo modulation of SLE.

Suppressor-cell antibody in systemic lupus erythematosus. Possible mechanism for suppressor-cell dysfunction.

Circulating antibodies that could be responsible for the suppressor thymus-derived (T)-cell dysfunction in active systemic lupus erythematosus (SLE) were investigated. Sera from 14 active and inactive SLE patients were compared with a pool of 22 normal sera. All sera were adsorbed with a pool of normal platelets to exclude antihistocompatibility leukocyte antigen antibodies; with AB erythrocytes to exclude isohemagglutinins; and with a pool of normal bone marrow-derived (B) lymphocytes, monocytes, and neutrophils to deplete anti-B-cell antibodies, Fc-receptor antibodies, and antibodies directed against neutrophils or monocytes. Sera from active SLE patients were capable of inhibiting the activation of normal, blood lymphocytes by concanavalin A to become suppressor cells. The latter were assayed by coculturing the concanavalin A-activated cells with autologous lymphocytes, which were then activated with either phytohemagglutinin for proliferative response or with pokeweed mitogen for B-cell immunoglobulin (Ig) synthesis and secretion. Specific incorporation of cultures with phytohemagglutinin showed a value of 67+/-13 (mean+/-SD) for suppressor cells treated with adsorbed, active SLE sera. This value was significantly different (P < 0.001) from that of cells treated with the inactive SLE sera or with the pool of normal sera. Similar findings were seen with respect to the B-cell target parameters. Cytoplasmic Ig and IgG in supernates of cultures with pokeweed mitogen showed values of 17+/-5% and 717+/-134 ng/culture, respectively, for suppressor cells treated with the adsorbed, active SLE sera. This was significantly different from those treated with the inactive SLE sera or with the pool of normal sera. The antisuppressor-cell factor was shown to be IgG, complement independent, not cytotoxic, active at 37 degrees C and at room temperature, but not at 4 degrees C, and adsorbable with T cells. Suppressor T-cell antibody in sera of active SLE patients could be responsible for the observed suppressor T-cell dysfunction seen in active SLE. The mechanisms responsible for the induction of the antisuppressor-cell antibody are unknown.

Comparative study of bone marrow and blood B cells in infantile and acquired agammaglobulinemia. Possible role of circulating anti-IgM in pathogenesis.

The status of immunoglobulin (Ig) receptors of the bone marrow dependent (B) cells present in either the bone marrow (BM) or peripheral blood (PB) of three patients with infantile agammaglobulinemia (I-AGG), or seven patients with acquired agammaglobulinemia (A-AGG) is compared with those of 12 controls. Quantitative and qualitative changes of the different classes of Ig receptors on B cells were evaluated by their capacity to bind [(125)I]anti-Ig, to be stained with fluorescinated anti-Ig and their in vitro proliferative capacity upon incubation with the anti-Ig. Patients with I-AGG lacked B cells in both the BM and PB. Whereas BM cells of patients with A-AGG carried receptors similar to control cells, their blood B cells had fewer IgM, IgG, and IgA cells which failed to proliferate in vitro in the presence of the anti-Ig. An anti-IgM of the IgG class was detected in the sera of patients with A-AGG but not in sera of I-AGG. The isolated anti-IgM agglutinated human red cells coated with IgM. The anti-IgM partially blocked the binding of fluorescinated or radiolabeled anti-IgM to IgM peripheral blood lymphocytes of normal controls. The eluted anti-IgM in presence of complement was partially cytotoxic to normal cells. It is concluded that I-AGG-B cell defect is due to failure of B cell development in the bone marrow compartment whereas the peripheral exclusion of IgM cells by an anti-IgM with the subsequent failure of differentiation of both IgG and IgA cells could be an important mechanism in A-AGG-B cell defect.

Suppressor cell-mediated neutropenia in Felty's syndrome.

The mechanism of neutropenia in Felty's Syndrome (FS) was tested. The suppressor capacity of mononuclear cells from patients with FS on normal bone marrow granulopoiesis was tested by the in vitro colony forming unit in culture assay. Peripheral blood, bone marrow, and spleen cells from FS patients with marked neutropenia (less than 1,000 neutrophils/mm3) suppressed the colony forming unit in culture of normal bone marrow. Cells from rheumatoid arthritis patients without neutropenia, cells from patients with drug-induced neutropenia without rheumatoid arthritis, or plasma from FS patients failed to suppress the colony forming unit in culture. Though suppressor cells were predominantly thymus-derived (T) cells, monocytes were also effective in suppression. The suppressor efficiency of cells from the various compartments were spleen greater than bone marrow greater than peripheral blood. Splenectomy in FS transiently corrected the neutropenia and eliminated suppressor cell activity. Hyperactive suppressor cells may be responsible for the neutropenia in some patients with FS. Correction of neutropenia in these patients should be directed at modulating the suppressor cell subpopulation.

Network theory in autoimmunity. In vitro suppression of serum anti-DNA antibody binding to DNA by anti-idiotypic antibody in systemic lupus erythematosus.

Regulation of serum anti-DNA antibody in systemic lupus erythematosus (SLE) by an antiidiotypic antibody was evaluated. Various sera from SLE patients in active and inactive states of their disease, as well as sera from normal individuals, were first completely depleted of anti-DNA and of DNA by affinity chromatography. The suppressive capacity of equimolar concentrations of the various depleted sera (blocking sera) on target lupus sera were determined. The target sera were from lupus patients with known DNA-binding capacity. Blocking sera from inactive SLE suppressed the binding of autologous anti-DNA antibody to [(3)H]DNA (n = 19,P < 0.01). Blocking sera from active SLE (n = 19), as well as human serum albumin, did not suppress. Sera from normal donors who had no contact with lupus patients or with lupus sera did not suppress (n = 14, P > 0.5), whereas those from normal donors who had contact with lupus patients or sera did suppress the binding (n = 5,P < 0.02). The anti-anti-DNA antibody suppressive activity in the inactive lupus serum was shown to be localized within the F(ab')(2) portion of immunoglobulin (Ig)G and could not be removed upon adsorption by normal human gammaglobulin. Furthermore, immune complexes could be detected by a Clq binding assay when the inactive lupus blocking sera were incubated with the anti-DNA antibody containing target sera. The specificity of the suppressive serum factor was shown by its inability to block the binding of tetanus toxoid to antitetanus antibody and its ability to block the binding of DNA to F(ab')(2) fragments of active lupus IgG. Regulation of serum anti-DNA antibody levels by anti-antibodies could induce and maintain disease remission in lupus patients and prevent disease expression in normals.

Anti-tumor necrosis factor (TNF) therapy in rheumatoid arthritis: correlation of TNF-alpha serum level with clinical response and benefit from changing dose or frequency of infliximab infusions.

OBJECTIVE: To determine the relationship between serum TNF-alpha level and clinical response in rheumatoid arthritis patients treated by infliximab. This could be of value to predict clinical response to infliximab and to determine the optimal dose and interval between dosing of infliximab. RA patients who did not respond adequately to conventional doses (3 mg/kg) of infliximab were studied to see if increasing the dose or frequency of infliximab infusions would be more effective. METHODS: Fifty-five RA patients who fulfilled the American College of Rheumatology criteria and were receiving treatment by anti-TNF-alpha (infliximab 3 mg/kg body weight every 8 weeks) were evaluated by: clinical disease activity using the Richie score index before receiving their scheduled infliximab infusion. Serum level of TNF-alpha, as measured by competitive ELISA assay, was determined immediately before and 9-11 days after receiving infliximab. RA patients who did not respond adequately to treatment with infliximab were given either a larger dose of infliximab or given the infusion at six-week intervals. Their clinical response was then evaluated sixteen months later. RESULTS Patients were divided into 2 groups according to Richie score, active group with score > 10 (score 20.3 +/- 7.7 mean +/- standard deviation, n = 25) and inactive group with scores < or = 10 (score 4.1 +/- 3.2, n = 30). TNF-alpha serum levels pre-infliximab infusion were significantly higher in the active group 76.1 pg/ml than the inactive group 38.0 pg/ml (P < 0.02). Whereas TNF serum level significantly dropped post infliximab in the inactive group (P < 0.05), it did not drop in the active group. The mean level of the post-infusion TNF-alpha serum level was higher (76.6 +/- 93.4 ng/ml) in the-active than the mean level of the post-infusion serum TNF-alpha levels in the inactive group (26.4 ng/ml +/- 7.9) P < 0.01 using the t-test. Increasing the frequency was superior in RA patients' clinical outcome than increasing the dose of infliximab infusions. CONCLUSION: RA patients who responded well to infliximab and had inactive disease at the time of the study have lower levels of serum TNF-alpha which could be further suppressed by the recommended doses of infliximab. RA patients with active disease have higher serum levels of TNF-alpha which could not be suppressed after the recommended doses of infliximab infusion. Changing the frequency of infliximab infusions in the active group was more effective than increasing the dose of infliximab in inducing improved clinical outcome. We suggest that the lack of suppression of TNF-alpha in the active group could be due to inadequate dosing of infliximab or to the presence of a neutralizing antibody directed against infliximab. It remains to be seen if serum TNF-alpha levels could be used as a guide in determining the dose and intervals between dosing of anti-TNF therapy in RA in order to achieve the desired clinical response.

Effect of exchange transfusion with Fluosol DA 20% on splenic distribution and immunocompetence of rat lymphocytes.

T- and B-splenic lymphocyte frequency, immune response 4 days after immunization with sheep red blood cells (SRBC), and proliferative response to concanavalin A (Con A) were determined 1, 3, and 5 days after exchange transfusion with Fluosol DA 20% (FDA) in adult, male Sprague-Dawley rats vs sham transfused rats. T-cell, T-helper, T-suppressor, and B-lymphocyte were reduced 1 day after transfusion (P less than or equal to 0.001). T- and B-lymphocyte frequencies were still reduced at day 3 (P = 0.0372). By day 5, there were no significant reductions in T-cell, T-helper, T-suppressor, and B-cell frequencies in the FDA-transfused rats. The frequency of cells with cytoplasmic IgG was reduced (P less than or equal to 0.025) in cells harvested from spleens of FDA-transfused rats and tested fresh. Proliferative response of splenic lymphocytes to Con A was unaffected by transfusion with FDA (P greater than or equal to 0.078). Splenic hemolytic plaques in response to SRBC were unaffected if rats were transfused 3 days after immunization with SRBC and 1 day prior to study (P = 0.941), enhanced if rats were transfused 1 day after SRBC immunization and 3 days prior to study (P = 0.0015), and suppressed if rats were transfused 1 day before SRBC immunization and 5 days before study (P less than 0.0001). Transfusion with FDA causes transient decreases in identifiable T and B lymphocytes, depresses cytoplasmic IgG-positive B cells, does not affect proliferative response to Con A, does not affect an ongoing specific immune response, enhances an early specific immune response, and inhibits the induction of a specific immune response.

In vitro growth promotion of T-lymphocyte colonies from normal human bone marrow with 12-O-tetradecanoylphorbol-13-acetate (TPA).

An in vitro culture system for the growth of T-lymphocyte colonies from normal human bone marrow has been described. This culture system features the use of 12-O-tetradecanoylphorbol-13-acetate (TPA) at moderately high concentrations (10(-7)-10(-6) M) and daily feeding with growth medium containing leukocyte-conditioned medium primed with phytohemagglutinin. T-lymphocyte colonies in this system can reach a size of over 10,000 cells/colony, suggesting that cells assayed in this system are early progenitors of T-lymphocytes with extensive proliferative capacity. TPA, at a higher concentration (10(-5) M), is inhibitory to the growth of T-lymphocytes, but this level is 5 orders of magnitude higher than the minimum TPA concentration (10(-10) M) inhibitory to myeloid colony forming cells. This demonstrates marked difference in the sensitivity of T-lymphocyte colony forming cells and myeloid colony forming cells to TPA. It appears that this assay system should be useful for the study of the early progenitors of human bone marrow T-lymphocytes.

Lack of T-cell immune abnormalities in peripheral blood lymphocytes in patients with Graves' disease or hypothyroidism.

Lymphocytes capable of forming nonimmune (E) and immune (EAC) rosettes with sheep erythrocytes and lymphocyte reactivity to phytohemagglutinin were determined in normal subjects, in patients with hyperthyroidism due to Graves' disease and in patients with hypothyroidism with and without goiter. Neither patient group differed from the normal subjects either in regard to lymphocyte count, the proportion of thymus dependent (t) lymphocytes forming nonimmune or bone marrow-dependnet (B) lymphocytes forming immune rosettes or lymphocyte reactivity to phytohemagglutinin. These results fail to provide evidence that these disorders are associated with an immune abnormality with increased or abnormal T cells in peripheral blood.

Immunoregulation abnormalities in familial Addison's disease.

Three brothers, two with Addison's disease and a monozygous twin discordant for adrenal insufficiency, presented an unique opportunity to evaluate the endocrine and immunological abnormalities associated with this disorder. None of the brothers had clinical evidence of other autoimmune disease. However, each of the twins had elevated titers of antihydroglobulin and antiparietal cell antibodies, and both Addisonian siblings had cytoplasmic islet cell antibodies. We evaluated the ability of nonspecific Concanavalin A-activated suppressor cells from all three siblings to inhibit immunoglobulin biosynthesis by pokeweed mitogen-stimulated B cells, cell proliferation by phytohemagglutinin-stimulated T cells, and the proliferative response of an allogenic mixed lymphocyte culture. In comparison to normal controls, suppressor cells from the Addisonian siblings were less efficient in inhibiting both B and T cell activities. The nonAddisoninan twin had a lesser degree of impaired suppressor cell function to the B and T cell targets. Suppressor cell activity, as measured by the ability to inhibit proliferation within the mixed lymphocyte culture, was normal in all three siblings. The relationship of suppressor cell dysfunction, genetic predisposition, and the expression of the autoimmune state are discussed.

Histoplasma meningitis with hyperactive suppressor T cells in cerebrospinal fluid.

Histoplasma meningitis usually occurs in conjunction with disseminated histoplasmosis. We studied a patient with common variable hypogammaglobulinemia who manifested meningitis without disseminated histoplasmosis. No histoplasma antibody was detected in cerebrospinal fluid (CSF) or blood. Evaluation of lymphocyte function in the blood revealed normal numbers of T cells with increased numbers of B cells. Most blood lymphocytes were identifiable, but most lymphocytes in CSF were null cells. Lymphocyte proliferative response to phytohemagglutinin or pokeweed mitogen was poor. T cells in CSF suppressed proliferative responses to histoplasma antigen by cells from blood or CSF, whereas T cells from blood did not. This difference suggested compartmentalization of T-cell function. The lack of humoral and cellular response to histoplasma in CSF may have allowed meningitis to develop, while the cellular response to histoplasma elsewhere prevented development of disseminated histoplasmosis.

Lymphocyte studies in a patient with chronic polyradiculoneuropathy.

We studied immunologic abnormalities in one patient with chronic polyradiculoneuropathy and the effects of immune suppression and plasmapheresis on the clinical course and immune abnormalities. Increased helper T cells and B cells with deficiency of T suppressor cells to B-cell but not to T-cell targets were detected. The patient's blood lymphocytes, but not the controls' lymphocytes, proliferated in vitro on culturing them with P2 antigen in the presence of the patient's CSF. Plasmapheresis combined with corticosteroid and azathioprine reversed the majority of immune abnormalities to normal but did not decrease the patient's lymphocyte-P2 proliferative response; nor did it improve clinical status.