Anti-CD3-activated splenocytes enhance survival in lethally irradiated mice after transplant of syngeneic hematopoietic stem cells.

Although cytokines produced by activated T cells may accelerate immunohematopoietic reconstitution after autologous bone marrow transplantation (ABMT), there is no direct evidence that infusion of anti-CD3 mAb-activated T cells can accelerate engraftment by hematopoietic stem cells. This study tests the ability of anti-CD3-activated murine splenocytes (ASC) to enhance the rescue of lethally irradiated (9 Gy) BDF1 mice by transplant of a limiting dose of fresh unmanipulated syngeneic splenocytes (SC). A minority (14.8%, 10-25%) of mice could be rescued with 5 x 10(5) SC after 9 Gy total-body irradiation (TBI). When 10(6) or 10(7) ASC were added to 5 x 10(5) SC, survival increased to 50% in those that received 5 x 10(5) SC + 10(6) ASC (not significant [NS]) and to 81.4% (77.7-88.0%) in those that received 5 x 10(5) SC + 10(7) ASC (p < 0.001). Furthermore, adding a fixed dose of 10(7) ASC to increasing doses of SC (10(5), 5 x 10(5), and 10(6)) enhanced survival at the different doses of SC. ASC alone did not rescue mice. CD3+ cells were the predominant population (77.6 +/- 6.7%) in the ASC inoculum, while NK cells remained low (1.2 +/- 0.9%). Colony-forming unit-spleen (CFU-S) yield after injection of SC showed dose dependence, whereas injection of 10 x 10(6) ASC alone failed to show any CFU-S yield in 23 of 25 recipient spleens. These results show that ASC enhanced survival of mice rescued with limiting doses of SC and that this effect was ASC dose-dependent but not dependent on the addition of extra stem cells.

Signal transduction by B and T cells early after bone marrow transplantation: B cell calcium flux responses are intact whereas lack of CD4 cells accounts for impaired T cell responses.

We previously found that intracellular ionized calcium ([Ca2+]i) flux responses after anti-CD3 crosslinking of CD3/TCR on T cells from allogeneic and autologous bone marrow transplant (BMT) recipients were impaired, Yamagami et al. J Clin Invest 1990; 86: 1347-1351. In contrast to the earlier study, this study focuses on identifying the T cell subset(s) responsible for the defects and determining if B cell responses are defective in BMT recipients early after BMT. In 37 recipients after anti-CD3 stimulation of PBL, a mean of 25.9% responding T cells was observed. This was significantly lower than the mean of 43.6% responding T cells in PBL from 21 normals (P < 0.001). The proportion of responding T cells in PBL (T PBL) increased in the recipients with time after BMT. By 6 months after BMT, the mean percent of responding T PBL approached the normal range. On the other hand, a mean of 8.1% responding B cells in anti-IgM crosslinked PBL from 24 recipients was not different from the mean of 7.4% responding B cells in anti-IgM crosslinked PBL from 16 normals (P = 0.6). Four color flow cytometry was used to identify subpopulations of lymphocytes. Enriched B cells were tested by gating out CD3+ and CD56+ cells to confirm the results of unfractionated PBL. In 8 recipients, the mean percent responding B cells was 36.6% and was not different from 6 normals (mean = 41.0%).(ABSTRACT TRUNCATED AT 250 WORDS)

Allogeneic bone marrow transplantation for the treatment of leukemia.

Eighteen patients with leukemia have received HLA-identical allogeneic bone marrow transplantation (BMT) at our hospital since 1981. Fifteen of these patients have been living without relapse. for prophylaxis of GVHD, MTX was used in 8 patients, and cyclosporine (CSP) together with MTX in 6 patients, 3 received multiple agents at much smaller dosage, including monoclonal antibody. All patients received intravenous placental gamma-globulin, and 16 received garlic extract. Three patients died. One, who neither received MTX, nor CSP died of hyperacute GVHD, one who did not receive garlic extract died of GMV pneumonia, and the third one died of tuberculosis 18 months after BMT.

Preparation of red-blood-cell-depleted marrow for ABO-incompatible marrow transplantation by density-gradient separation using the IBM 2991 blood cell processor.

Thirty patients who had major ABO blood group incompatibility with their HLA-matched donors underwent allogeneic marrow transplantation after removal of red blood cells (RBC) from donor marrow by Ficoll-Diatrazoate (F-D) separation using the IBM 2991 blood cell processor. We selected the IBM 2991 because we were interested in obtaining information about RBC-depleted mononuclear cells for monoclonal antibody and complement incubation of marrow. The median residual marrow RBC volume was 2.6 ml (1.2% of the original volume) and marrow infusion was well tolerated in every instance. The median doses of nucleated and mononuclear cells were 8.7 X 10(7)/kg and 2.2 X 10(7)/kg recipient weight, respectively, representing median marrow total nucleated and mononuclear cell losses of 63.4% and 52.9%, respectively. The median CFU-GM loss was 52.4%. Four patients died 13-21 days after marrow infusion and were unevaluable for engraftment. One patient failed to achieve engraftment and received a second transplant on day 36 from a second donor. One patient with myelofibrosis had poor engraftment and died on day 177 with low peripheral blood counts. For evaluable patients, no significant differences were observed in the rate of recovery of peripheral blood granulocyte or platelet counts between those receiving RBC-depleted marrow or ABO-matched unprocessed marrow. However, posttransplant red cell transfusion requirements were increased and transfusion independence delayed in patients receiving RBC-depleted marrow as compared to patients receiving unprocessed marrow. We concluded that red cell depletion using the IBM 2991 was effective in removing red cell, but resulted in significant and variable hematopoietic cell losses which may have contributed to graft failure in at least one patient. Such cell losses appear to be inherent in both manual and semiautomated methods for F-D cell separation.

Adoptively transferred immunity persists in human marrow graft recipients.

This study was designed to determine if antibodies (Abs) to recall antigens (Ags) were produced after bone marrow transplantation (BMT). Sera from marrow recipients (recipients) were tested for Abs to recall Ags post-grafting, and T and B cells from recipients were tested for their ability to produce anti-tetanus toxoid antibody (anti-TT) using in vitro biosynthesis assays. Neither the donors nor recipients received booster immunizations with recall Ags before or after BMT. Serum Ab titers to tetanus toxoid (TT), diphtheria toxoid (DT), and measles virus (MV) were in the normal range for the majority of 235 short-term recipients (less than 100 days postgrafting) and for the majority of 149 long-term (greater than 6 months postgrafting) recipients. Anti-TT was produced in vitro by peripheral blood lymphocytes (PBL) from 6 of 14 long-term recipients after TT stimulation. In another system, purified B cells from 9 of 21 long-term recipients also produced anti-TT after Epstein-Barr virus (EBV) stimulation. The presence of Ab titers to TT, DT, and MV in the serum of recipients and the production of in vitro anti-TT by T and B cells from recipients show that Ag-specific memory was transferred via the marrow inoculum. These data show that adoptively transferred immunity persists in recipients for years postgrafting.

IgG anti-tetanus toxoid antibody production induced by Epstein-Barr virus from B cells of human marrow transplant recipients.

This investigation shows that Epstein-Barr virus (EBV)-activated human B cells from marrow transplant recipients can produce in vitro IgG anti-tetanus toxoid antibody (anti-TT) without booster immunizations with tetanus toxoid (TT). Purified B cells (E-rosette negative) from 8 normal subjects, 6 healthy long-term marrow graft recipients, and 15 long-term marrow graft recipients with chronic graft-vs-host disease (GVHD), were stimulated for 12 days with EBV to induce anti-TT production in culture supernatants. The amount of anti-TT in culture supernatants was quantitated using a enzyme-linked immunosorbent assay. B cells from all 8 normal controls produced in vitro IgG anti-TT after EBV stimulation. Five of 6 healthy recipients had B cells that produced anti-TT after EBV stimulation. Four of 15 recipients with chronic GVHD had B cells capable of producing anti-TT after EBV stimulation. The number of cultures making anti-TT responses was less in those with chronic GVHD than in those without chronic GVHD or normal individuals (P less than 0.001). B cells from patients with chronic GVHD had fewer responses exceeding the overall median of 0.7 ng/ml when compared with the other two groups (P less than 0.03). These data show that B cells of donor origin can produce in vitro IgG anti-TT antibody to tetanus toxoid antigen in a T-independent fashion.

Marrow harvesting for autologous marrow transplantation.

The results and complications of 224 marrow collections from 200 patients with malignant disease who underwent marrow aspiration and storage for subsequent autologous marrow transplantation (AMT) were analyzed. The median age of the patients was 35 years (range 1-68); 131 patients had hematologic malignancies and 69 had solid tumors. Thirty-one patients proceeded directly to AMT after marrow aspiration at a median of 4.5 days (range 0-10). A further 75 patients received AMT a median of 3.0 months (12 days-10 years) after marrow aspiration. The remaining 94 patients had marrow stored but not infused. When a second aspiration was performed from the same patient within seven weeks, the yield of marrow nucleated cells was significantly reduced (p less than 0.02). A negative linear correlation was observed between CFU-C/kg harvested and the day to achieve a posttransplant blood neutrophil count greater than 500/cmm (r = -0.3092, p less than 0.05). A total of 36 (17.4%) complications associated with marrow aspiration were observed including two (0.97%) life-threatening episodes. Postoperative fever accounted for 23 of 34 episodes of minor complications. There was no increased risk of serious complications with decreased time from aspiration to transplant. It was concluded that the morbidity and mortality from autologous marrow aspiration did not differ significantly from that observed in normal donors.