Cellular immunity to vaccinations and herpesvirus infections after bone marrow transplantation

The cellular immune response to herpesviruses was studied in 46 recipients of marrow grafts (23 autologous, 23 allogeneic). That study was performed in vitro by evaluating the degree of lymphocyte proliferative responses to herpes simplex virus (HSV), cytomegalovirus (CMV), and varicella zoster virus (VZV). No primary infections with any of those viruses were noted after bone marrow transplantation (BMT). The incidence of active infection in seropositive patients was significantly lower after autologous BMT than after allogeneic BMT (HSV, 2/22 vs. 11/22 patients, respectively, P = 0.007; CMV, 4/12 vs. 9/10 patients, respectively, P = 0.02; VZV, 3/23 vs. 11/23 patients, respectively, P = 0.02). After autologous BMT, the restoration of cellular immunity to the three viruses occurred at a clearly faster rate than after allogeneic BMT. That pattern may have contributed to the low incidence of active infections with those viruses after autologous BMT. Recipients of allogeneic marrow from donors with a positive lymphocyte proliferation test to HSV had a significantly increased incidence of active HSV infection post-BMT (8/9 patients) than those who received marrow from donors with a negative test (3/13 patients; P = 0.008). Acute or chronic graft-versus-host disease (GVHD) decreased the cellular immune response to the three herpes viruses, but not significantly. Our program of vaccinations with diphtheria and tetanus toxoids started in the fourth month post BMT. Chronic GVHD patients who were vaccinated had a clearly impaired cellular immune response to both toxoids as compared with those without chronic GVHD.     

Disordered salivary immunoglobulin secretion and sodium transport in human chronic graft-versus-host disease

Whole saliva samples and lip biopsies were collected from 12 allogeneic bone marrow transplant recipients who developed extensive chronic graft-versus-host disease (GVHD) and from 10 healthy allogeneic and syngeneic recipients without GVHD. Six of ten biopsies from patients with chronic GVHD had lichenoid stomatitis or sialadenitis, or both, with sialodochitis. Seven of nine biopsies from patients free of chronic GVHD were entirely normal, and two had either mild glandular or mucosal changes. Salivary gland involvement in chronic GVHD was associated with decreased or absent levels of salivary IgA and inorganic phosphate, decreased salivary flow rates, and increased concentrations of salivary sodium, albumin, and IgG. The most striking abnormalities were found in patients with histologic evidence of sialadenitis. In contrast, marrow transplant recipients without chronic GVHD had normal salivary immunoglobulin and electrolyte levels. Secretory IgA deficiency may contribute to the frequent sinobronchial infections observed in patients with chronic GVHD.     

Lymphocyte responses after cytomegalovirus infection in bone marrow transplant recipients--a one-year follow-up

A group of 46 bone marrow transplant (BMT) recipients were studied by lymphocyte stimulation with cytomegalovirus (CMV) antigen before, and repeatedly in the first year after, BMT. Of these, 25 patients developed CMV infection and 68% of these got a positive lymphocyte response to CMV. The recipients with an early response (up to 3 months) to CMV antigen after the CMV infection were less prone to develop chronic graft-versus-host disease (GVHD) than those who responded later or not at all (P = 0.002). After the CMV infection, the increased lymphocyte CMV reactivity remained in recipients without chronic GVHD, but recipients with chronic GVHD usually lost their reactivity. The results suggest that it may be possible to predict patients who are not going to develop chronic GVHD by studying lymphocyte responses to CMV infections.     

Faster immunological recovery after bone marrow transplantation in patients without cytomegalovirus infection

The following findings were noted among 45 bone marrow transplant recipients. The patients without cytomegalovirus (CMV) infection or chronic graft-versus-host disease (GVHD) showed normal lymphocyte stimulation in vitro by concanavalin A (Con A) more than 3 months after transplantation, and normal stimulation by phytohemagglutinin (PHA), anti-beta 2-microglobulin (A-beta 2m) and protein A (SpA) after 6 months. In contrast, the patients who had CMV infection without chronic GVHD had Con A and SpA responses within the normal range after 12 months and reduced lymphocyte responses to PHA and A-beta 2m more than 12 months after transplantation. The patients with chronic GVHD had reduced responses to all of these four mitogens after more than 12 months. In comparison with other patients those who later developed chronic GVHD showed an increased mixed lymphocyte culture stimulation during the first 3 months that decreased between 6-12 months. Patients with chronic GVHD still had reduced IgA levels at 12 months after transplantation. Patients with CMV infection, but without chronic GVHD, had higher percentages of lymphocytes with surface membrane Ig than healthy controls during the first 3 months after transplantation. The data suggest that CMV infection, regardless of chronic GVHD, delays immunologic recovery after marrow transplantation.     

Ineffective cellular interaction and interleukin 2 deficiency causing T cell defects in human allogeneic marrow recipients early after grafting and in those with chronic graft-versus-host disease

Impairment in T cell proliferation in response to E. coli and in CML to unrelated alloantigens was usually observed in patients early after marrow grafting and persisted in long-term patients with chronic GVHD but not in those without chronic GVHD. We analyzed various cellular functions in the pathway of T cell activation and found that in patients with immunodeficiency, (1) their M phi usually could process and present antigens to normal T cells, (2) their T cells did not proliferate even in the presence of normal antigen-pulsed M phi, (3) IL-2 production by T cells was deficient, and (4) exogenous IL-2 restored CML activity in cells of most patients early after grafting but not in cells of most patients with chronic GVHD. Therefore, failure to induce proliferation and cytotoxicity in T cells of marrow recipients is not likely due to M phi defects but because of ineffective communication among T cell subsets, probably related to impaired IL-2 production and/or unresponsiveness to IL-2.     

Blood transfusions and changes in humoral and cellular immune reactivity in rhesus monkeys. Possible predictive value for kidney allograft prognosis

We have developed a murine model of acute and chronic forms of graft-versus-host disease (GVHD) as defined by clinical features that develop in response to minor histocompatibility antigen (minor HA) differences. C57BL/6J (B6) and LP/J mice were selected for serologic identity at H-2 and for mutual nonreactivity in mixed lymphocyte culture (MLC). Lethally irradiated B6 recipients were transplanted with anti-Thy-1.2-treated LP bone marrow cells plus various numbers of untreated LP spleen cells. The B6 recipient mice developed acute and chronic forms of GVHD that showed clinical and histological similarities to the acute and chronic forms of GVHD seen in human recipients of bone marrow transplants from HLA-identical and MLC-nonreactive donors. The definition of acute and chronic GVHD by clinical criteria appears to have biological significance in predicting subsequent survival patterns of recipient mice with GVHD. The incidence of acute and chronic GVHD in the mouse model was a function of the number of donor spleen cells transplanted. Using this model, we demonstrate that both acute and chronic forms of GVHD are initiated by donor lymphocytes. Based on these results, acute and chronic forms of GVHD induced to minor HA appear to be two manifestations of the same T-cell-mediated disease process rather than two different diseases.     

Cellular interactions in marrow-grafted patients. II. Normal monocyte antigen-presenting and defective T-cell-proliferative functions early after grafting and during chronic graft-versus-host disease

The proliferation of T cells of marrow donor origin in response to Escherichia coli, an ubiquitous antigen, presented by circulating monocytes of marrow donor origin was investigated in 30 human allogeneic marrow transplant recipients. Compared with cells from healthy marrow donors, T cell proliferation was found to be deficient in all recipients studied 36-71 days after grafting, regardless of the presence or absence of acute graft-versus-host disease and in most patients with chronic graft-versus-host disease studied 118-1804 days postgrafting . In contrast, lymphocytes from most long-term patients without chronic graft-versus-host disease studied 363-2673 days had immune reactivity comparable to that of lymphocytes from their marrow donors. Results of cell-mixing experiments showed that (1) monocytes from most marrow recipients were capable of presenting antigens to normal T cells of marrow donors, and (2) T cells from short-term patients and from long-term patients with active chronic graft-versus-host disease were not induced to proliferate by E-coli-pulsed monocytes from the marrow donors. This inability of T cells to proliferate was likely the result of ineffective interactions among T cell subsets.     

慢性髓性白血病骨髓移植后的细胞遗传学改变及移植物抗白血病作用

对４４例由同胞供髓行骨髓移植的Ｐｈ染色体阳性慢性髓性白血病（ＣＭＬ）患者进行了系列的染色体研究。１２例移植后短期内，有２～９次检出３％～６８％的Ｐｈ阳性受者核型，但是并不伴有临床和血液学复发的迹象，患者在＋６月～＋１年后逐步由供者细胞完全代替造血，作者观察到其中７例的Ｐｈ阳性／Ｐｈ阴性的动态变化与临床发生的轻度移植物抗宿主病（ＧＶＨＤ）密切相关，患者的Ｐｈ染色体往往随着ＧＶＨＤ的发生而减少或消失，研究表明单纯放、化疗几乎不能完全清除白血病克隆，而且移植后短期内细胞遗传学的复发不一定说明血液学复发，持久植活病例的恶性细胞的消失过程提示，异基因骨髓移植物不仅具有代替受者造血的作用，而且其免疫活性细胞通过ＧＶＨＤ发挥着重要的抗白血病作用（ＧＶＬ）。     

Immunopathology of early graft-versus-host disease--a prospective study of skin, rectum, and peripheral blood in allogeneic and autologous bone marrow transplant recipients.

The immunopathological appearances of skin and rectum in 64 autologous and allogeneic recipients were determined before and after bone marrow transplantation. Patients who developed acute graft-versus-host disease were biopsied as soon as a clinical diagnosis was made. At the same time peripheral blood samples were collected for comparative analysis. Immunohistological and morphometric techniques were employed using a panel of monoclonal antibodies to T lymphocytes and subsets, B lymphocytes, natural killer cells, macrophages, and Langerhans cells. A reduction in the CD4/CD8 ratio after BMT was seen in skin and rectal biopsies from both autologous and allogeneic recipients with or without GVHD. The same pattern was observed in blood samples taken at the same time. Langerhans cells were reduced in the skin in all patients after BMT, probably by the conditioning regimen. Only a few cells expressing activation or natural killer cell markers were present and there were no changes observed in the macrophage population. This study has provided no evidence to implicate either CD4- or CD8-positive T lymphocytes as the initiators of the cellular damage in acute GVHD. The distribution of lymphocyte subsets in the blood was similar to that in the tissues, suggesting that the tissue changes reflect the pattern of lymphocyte repopulation after BMT and may have little bearing on the pathogenesis of GVHD.     

UVB pretreatment of rat bone marrow allografts. Prevention of GVHD and induction of allochimerism and donor-specific unresponsiveness

Ultraviolet B irradiation has been used to pretreat blood and islets to prevent subsequent graft rejection. In this study the optimal dose of UVB irradiation of bone marrow was determined in syngeneic recipients and was subsequently applied to in-vitro treatment of bone marrow allografts. UVB pretreatment of donor bone marrow inoculum led to complete prevention of GVHD in allogeneic rat recipients without major marrow or other toxicity. Long-standing recipients of allogeneic UVB-BM became stable adult chimeras. The recipients of allogeneic BM were populated by donor-type peripheral blood lymphocytes and did not reject host or donor-type heart grafts. The BM allograft recipients were immunocompetent as measured by their ability to normally reject third-party cardiac allografts. We suggest that the prevention of GVHD and induction of stable chimerism in adult recipients of allogeneic UVB-BM may be mediated by suppressor mechanisms.     

FLT3 ligand promotes engraftment of allogeneic hematopoietic stem cells without significant graft-versus-host disease

BACKGROUND: Graft-versus-host (GVH) reactions contribute to stable engraftment of allogeneic hematopoietic stem cell transplants. It was hypothesized that the in vivo expansion of recipient dendritic cells (DC) with the administration of ligand for Flt3 (FL) could promote allogeneic engraftment after reduced-intensity conditioning by enhancing the GVH effect. METHODS: FL was first administered to three nonirradiated healthy dogs for 13 days at a dosage of 100 microg/kg/day. Next, nine dogs received 4.5 Gy total-body irradiation (TBI) and unmodified marrow grafts from dog leukocyte antigen (DLA)-identical littermates without posttransplant immunosuppression. FL was administered to the recipients at a dosage of 100 microg/kg/day from day -7 until day +5. RESULTS: In normal dogs, FL produced significant increases in monocytes (CD14+) and neutrophils in the peripheral blood, a marked increase in CD1c+ cells with DC-type morphology in lymph nodes, and increased alloreactivity of third-party responders to peripheral blood mononuclear cells in mixed lymphocyte reactions (P<0.001). Sustained engraftment was observed in eight of nine (89%) FL-treated dogs compared with 14 of 37 (38%) controls (P=0.02, logistic regression). All engrafted FL-treated dogs became stable complete (n=2) or mixed (n=6) hematopoietic chimeras without significant graft-versus-host disease (GVHD). Recipient chimeric dogs (n=4) were tolerant to skin transplants from their marrow donors but rejected skin grafts from unrelated dogs within 7 to 9 days (median, 8 days). CONCLUSIONS: In this study, the authors showed that FL administered to recipients promotes stable engraftment of allogeneic marrow from DLA-identical littermates after 4.5 Gy TBI without significant GVHD.     

Phenotypical and functional studies on a subtype of suppressor cells (CD8+/CD11+) in patients after bone marrow transplantation

Dual labeling with the monoclonal antibodies anti-Leu 2 (fluorescein-conjugated) and anti-Leu 15 (phycoerythrin-conjugated) was used to phenotype and sort the lymphocytes from 12 short-term (100 days postgrafting) recipients, 8 long-term (6 months postgrafting) stable, and 11 long-term patients with chronic graft-versus-host disease (GVHD). The proportion of Leu 2+ 15+ cells was increased in 11 of 12 short-term patients (mean + SEM: 31% +/- 3.6)-and in 6 of 11 patients with chronic GVHD (18% +/- 2.4) compared with normal controls (n = 8; 7.5% +/- 1.9). However, there was no correlation between proportions of Leu 2+ 15+ or Leu 2+ 15- cells and the presence of acute GVHD. Long-term patients with chronic GVHD tended to have a higher proportion of Leu 2+ 15- cells. To functionally characterize these two T cell subsets, Leu 2+ 15- and Leu 2+ 15+ subsets were sorted from purified T cells obtained from two recipients and one normal subject. Both Leu 2+ 15+ and Leu 2+ 15- cells from a short-term patient suppressed pokeweed mitogen--stimulated immunoglobulin production of normal B cells. Leu 2+ 15- cells from a long-term survivor with chronic GVHD and the normal control provided help, whereas the Leu 2+ 15+ cells also strongly suppressed immunoglobulin synthesis. The suppressor activity of the Leu 2+ 15+ subset in all three individuals was radiosensitive (12 Gy). These results illustrate the need for careful correlative phenotyping and functional studies. Furthermore, these studies clearly demonstrate that different functions may exist within a particular T cell phenotype after bone marrow transplantation.     

Evidence of donor-specific cellular suppressor activity in donor-specific cell-mediated lympholysis unresponsiveness in renal transplant patients

Twenty patients with well-functioning kidney grafts from one-haplotype-mismatched related donors, were studied 1-10 years after transplantation (A). Another group of six patients were studied at various times after transplantation (B). The presence of donor-specific transplantation tolerance, using mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) tests was investigated, as well as the possible existence of cells with suppressive activity. All recipients were transfused prior to transplantation and treated with conventional immunosuppression. The patients in group A showed MLC reactivity against donor and third-party cells, indicating a allogeneic response capacity. The CML activity against the donor was low, however, and remained low also following removal of adherent cells. The CML activity toward third-party cells was within the normal range of unmatched individuals. In group B, two of six recipients and high postoperative CML activity against the donor. Both recipients showed clinical signs of rejection. In the remaining four recipients, the antidonor CML reactivity one week after transplantation was lower than the preoperative level. The decrease was even more pronounced at 12 months, although the reactivity against third-party cells was unaltered. The CML reactivity from unrelated fourth-party individuals toward donors was suppressed when cells from recipients with long-term functioning kidneys were added to the cell cultures. The results suggest the presence of a donor-specific cellular suppressor mechanism underlying the donor-specific CML unresponsiveness in recipients with long-term-functioning kidney allografts.     

Bone marrow transplantation for severe aplastic anemia--a study of twenty-one Chinese patients in Taiwan.

A total of 21 multiply transfused patients with severe aplastic anemia (SAA) were treated with bone marrow transplantation between March 1985 and September 1990: 20 allogeneic and one syngeneic transplants. A positive response in mixed lymphocyte culture (MLC) was also noted in 7 allogeneic recipients. Pregraft conditioning included high-dose cyclophosphamide (CY) 200 mg/kg over 4 consecutive days, followed by 300 cGy total-body irradiation the day before BMT. Seventeen patients older than 14 years received additional donor buffy-coat cells infusion for 5 days posttransplant. A combination of methotrexate and cyclosporine was used for prophylaxis of graft-versus-host disease. Seventeen patients were alive with a functional graft, and Kaplan-Meier product limit estimates showed a 80.95% probability of survival at 67.7 months. There were 4 deaths: two died of primary graft failure, one from secondary rejection, and the other from chronic GVHD-related complications. Acute GVHD, grade I was noted in only one patient (5.6%). In contrast, chronic GVHD was observed in 10 out of 18 (55.6%) evaluable patients. Venoocclusive liver disease and interstitial pneumonitis were not diagnosed. Our findings indicate that the combination of CY/TBI/BC is well tolerated and results in a low incidence of graft failure/rejection in multiply transfused Chinese patients who received transplants for SAA. The MTX/CsA combination was confirmed as being remarkable in reducing the incidence and severity of acute GVHD. For patients with SAA under the age of 40, with an HLA-identical sibling, we highly recommend BMT as the treatment of choice.     

Mechanisms of bone loss following allogeneic and autologous hemopoietic stem cell transplantation.

A significant proportion of patients will be long-term survivors of bone marrow transplantation (BMT) and little is known about their risk of late bony complications. We therefore evaluated bone mineral density (BMD) prior to BMT, post-transplantation changes in BMD, and mechanisms of bone loss in long-term survivors. We performed two analyses. The first was a cross-sectional study of 83 consecutive BMT patients (38 F, 45 M), examining the relationship between BMD and bone turnover, measured immediately prior to transplantation, and a number of disease and patient variables. The second was a prospective study of 39 patients (19F, 20 M) followed for a median of 30 months (range 5-64 months) following either allogeneic (allo, n = 29) or autologous (auto, n = 10) BMT to determine if bone loss was related to treatment of graft versus host disease (GVHD) with glucocorticoids and cyclosporine A, high bone turnover rates, or hypogonadism. Auto BMT recipients acted as a control group for effects of GVHD therapy on BMD. Prior to BMT, spinal and femoral neck (FN) BMDs were 8.6% and 14% lower in female auto BMT recipients than in female allo BMT recipients, respectively (p = 0.12 and p = 0. 003). Urinary bone resorption markers were higher than in normal gender- and age-matched control subjects. Patients treated previously with glucocorticoids also had 8% lower FN BMD. Glucocorticoid-pretreated women with amenorrhoea had lower lumbar spine (LS) and FN BMDs than eumenorrheic women and women receiving HRT. Post-allo BMT, patients lost 11.7% of FN BMD compared with a nonsignificant decrease of 1.1% post-auto BMT (p < 0.001). Spinal BMD and total body bone mineral content (TBBMC) decreased by 3.9% and 3.5%, respectively, post-allo, compared with an increase (1.5%, p = 0.03) or nonsignificant decrease (-3.7%, p = NS), respectively, post-auto BMT. Post-allo BMT bone loss correlated best with the cumulative prednisolone dose at the LS and FN, and with average daily prednisolone dose for TBBMC. At the spine, the rate of bone loss was 4%/10 g of prednisolone, while the rate of bone loss at the FN was greater (9%/10 g of prednisolone). Bone loss was also negatively related to the duration of cyclosporine therapy for GVHD and baseline deoxypyridinoline concentrations. Avascular necrosis of the femoral head occurred in four, and vertebral and rib fractures occurred in one of the allo BMT patients, but in no auto BMT patients. In conclusion, BMT recipients are at risk of osteoporosis secondary to bone loss associated with their underlying illness and/or chemotherapy, particularly in female autograft recipients, and in allograft recipients secondary to GVHD and its treatment.     

Efficacy of transient treatment with FK506 in the early phase on cyclophosphamide-induced bone marrow chimerism and transplant tolerance across MHC barriers

BACKGROUND: The use of mixed allogeneic bone marrow chimerism to induce donor-specific transplantation tolerance has been extensively demonstrated. In the present study, we assessed the effect of combined use of a short course of FK506 and a single-dose cyclophosphamide (CYP) on the induction of tolerance and development of GVHD after allogeneic BMT. MATERIALS AND METHODS: Lewis rat (RT1(l)) recipients received BMT from Brown Norway (RT1(n)) donors on the next day after injection of CYP at a dose of 200 mg/kg. The recipients were further treated with no FK506 (n = 8), 0.3 mg/kg/day FK506 on days 10-16 (n = 6), or the same dose of FK506 on days 0-6 (n = 6). In a subgroup of animals, heterotopic heart transplantation was performed to investigate transplantation tolerance. RESULTS: Six of eight recipient rats that did not receive FK506 died of severe GVHD, while high levels of chimerism were induced. Recipients of FK506 in the later phase developed mild transient GVHD around 2 to 3 weeks after BMT and recovered thereafter; however, the level of chimerism was significantly decreased (2.8 +/- 2.3% on day 100). Treatment with FK506 in the early phase completely prevented the development of GVHD and induced stable allogeneic chimerism in the long-term (13.8 +/- 8.3% on day 100). These recipients with stable chimerism accepted subsequent BN heart allografts indefinitely (>200 days x 5), while rejecting third-party (BUF) heart allografts by day 12. CONCLUSIONS: Early transient FK506 promotes the induction of stable bone marrow chimerism without GVHD after BMT with CYP pretreatment. The timing of treatment with FK506 is critical with a view to preventing GVHD and inducing stable long-lasting chimerism.     

Epithelial class II antigen expression in cutaneous graft-versus-host disease

Induced class II histocompatibility antigens have been observed in the target tissues of rodents and humans with acute graft-versus-host disease (GVHD). Possibly this expression triggers the target tissue phase, through antigen presentation, lymphocyte recruitment, or additional antigenic stimulus. We have tested whether the induced expression is required for cutaneous GVHD in human marrow recipients. Ninety-two skin biopsies from 37 allogeneic marrow recipients at the Johns Hopkins Bone Marrow Transplant Unit were stained for HLA-DR, OKT6 (Langerhans cells), and for surface markers of lymphocyte and monocytes. Of 22 biopsies taken at the onset of GVHD, 12 did not have detectable HLA-DR antigen, and 10 had patchy-to-diffuse expression. The biopsies with HLA-DR- GVHD consisted primarily of epithelial infiltrates of cytotoxic/suppressor cells (CD8+), while those with HLA-DR+ GVHD had a mixed infiltrate with more helper/inducer/class-II-reactive cells (CD4+) in the epidermis and dermis and more monocytes in the dermis. Eight of 9 patients with HLA-DR- GVHD had follow-up biopsies that later expressed epithelial DR antigen, but the epidermal and dermal infiltrates showed no significant changes. Most of those receiving cyclosporine (CsA) prophylaxis (7/9) developed HLA-DR- GVHD, while those receiving cyclophosphamide were split between the two groups (8 of 13 were HLA-DR+). HLA-DR antigen expression was evident in some biopsies with no GVHD or minimal GVHD but did not appear to predict the development of GVHD or the type of GVHD. HLA-DR antigen expression was not evident in 2 of 3 initial biopsies of lichenoid-type chronic GVHD. Class II antigen induction is clearly not necessary for the target phase in most patients of this study.     

A sensitive micromethod for generating and assaying allogeneically induced cytotoxic human lymphocytes.

A sensitive micromethod for generating and assaying allogeneically induced cytotoxic human lymphocytes in vitro is described. Responding lymphocytes are cultured with mitomycin-C treated allogeneic stimulating cells in wells of replicate microtrays for both one-way mixed leukocyte culture (MLC) and cell-mediated lympholysis (CML) assays. On day 5, MLC response is determined by measuring 3H-thymidine (3H-TdR) incorporation directly in wells of the MLC tray. On day 6 or 7 CML response is determined by measuring 51Cr released from labeled target cells added to replicate culture cells in the CML tray. It is thus possible to measure both MLC and CML responses of the 2.5 X 10(4) -U X 10(5) responding lymphocytes originally placed in replicate wells. 51Cr-labeled target cells can be added to wells containing dilutions of the stimulated cells and a log-linear relationship between the per cent specific 51Cr release and number of effector cells is observed. Significant levels of specific cytoxicity are detected at ratios as low as one effector cell per target cell; little cross-killing on third-party cells and no autokilling is observed. Lymphocytes purified from whole blood that is stored overnight at room temperature and purified lymphocytes stored overnight in the cold generate MLC and CML responses comparable to those of lymphocytes purified from fresh blood. Only 2 or 3 ml of whole blood are required to perform both MLC and CML assays, thus enabling the study of both proliferative and cytotoxic lymphocyte responses in young children and other individuals from whom only a few milliliters of blood can be obtained.     

Bone marrow transplantation depleted of T cells followed by repletion with incremental doses of donor lymphocytes for relapsing patients with chronic myeloid leukemia: a therapeutic strategy

BACKGROUND: For patients with chronic myeloid leukemia (CML), long-term survival after stem cell transplantation requires adequate control of graft-versus-host disease (GVHD) and disease recurrence. Relapsing patients respond to donor lymphocyte infusion (DLI) but develop life-threatening complications. METHODS: Patients with CML in first chronic phase received bone marrow (n = 14) or peripheral blood progenitor cell transplants (n = 4) from HLA-identical siblings. GVHD prophylaxis was by ex vivo T-cell depletion with CAMPATH 1G. If disease recurred, donors' mononuclear cells were collected by apheresis, the CD3 samples commencing at 10(6)/kg were aliquoted at half-log increment intervals, cryopreserved, and infused until disease clearance. RESULTS: Eighteen patients (median age: 32.5 years) received transplants. All engrafted without procedure-related mortality. Fourteen patients relapsed, and 13 entered the DLI program. Two developed extensive GVHD after single schedule infusions ranging from 89x10(6) to 670x10(6) mononuclear cells/kg, and one survives in complete remission (CR). The rest, treated with incremental dose DLI, experienced no acute toxicities. One, who had developed grade III steroid-responsive GVHD, died in CR2 from opportunistic infections. Steroids reversed limited cutaneous GVHD and elevated liver enzymes in five patients. Three others developed pancytopenia, and two restored blood counts only after donor peripheral blood progenitor cell infusions. Molecular CR2 was established in 12/13 patients, occurring in 10/11 (91%) on the incremental program at a median accumulation of 67 (range: 5-166) x10(6) CD3 cells/kg. Sixteen of 18 (89%) survive at median of 854.5 days from bone marrow transplantation, 4 in CR1 and 10 in CR2 at a median disease-free survival (for remission 2) duration of 341 days. The median combined disease-free survival of the 14 patients in CR 1+2 is 660 days, with 99% average performance status. CONCLUSIONS: Escalating DLI leads to safe new molecular CR in most CML relapse patients. These results raise the possibility of using "safe" transplantation programs of T-cell depletion, that include graded DLI as prevention against disease recurrence.     

The Impact of Bone Marrow Fibrosis on the Outcome of Hematopoietic Stem Cell Transplantation

We retrospectively analyzed the data of 175 patients who underwent autologous (n = 69) or allogeneic hematopoietic stem cell transplantation (HCT) (n = 106) including 19 (27.5%) and 38 (35.8%) recipients who had bone marrow fibrosis (BMF) prior to transplantation, respectively. We investigated the effects of BMF on engraftment, graft-versus-host disease (GVHD), early posttransplant complications, and survival. Pretransplantation BMF did not delay engraftment and showed no impact either on early posttransplant complications or on the development of acute and/or chronic GVHD. Probability of 1-year overall survival (OS) and progression-free survival (PFS) of autologous HCT recipients were similar, namely 76.7% versus 88.6% (P > .005) and 26.33% versus 16.5% (P > .05) among patients with versus without fibrosis, respectively. In allogeneic HCT recipients, the probability of 1-year OS was 35.2% among patients with versus 48.9% among those without fibrosis (P = .004) PFS at 1 year was inferior among allogeneic HCT recipients with BMF: 27.8% versus 51.2% (P = .0008). Cox regression analysis revealed BMF to be independently associated with age, Sorror comorbidity index, primary disease, and disease status during HCT (P = .045).