Distinct patterns of transmembrane calcium flux and intracellular calcium mobilization after differentiation antigen cluster 2 (E rosette receptor) or 3 (T3) stimulation of human lymphocytes.

We evaluated CD2 (E rosette) and CD3 (T3)-triggered activation of resting lymphocytes by measuring the intracellular free calcium concentration ([Ca2+]i) of individual cells. The [Ca2+]i of indo-1-loaded cells was measured by flow cytometry and responses were correlated with cell surface phenotype. Stimulation with anti-CD3 antibody caused an increase in [Ca2+]i in greater than 90% of CD3+ cells within 1 min, and furthermore, the response was restricted to cells bearing the CD3 marker. In contrast, stimulation of cells with anti-CD2 antibodies produced a biphasic response pattern with an early component in CD3- cells and a late component in CD3+ cells. Thus, the CD2 response does not require cell surface expression of CD3. In addition, stimulation of a single CD2 epitope was sufficient for activation of CD3- cells, whereas stimulation of two CD2 epitopes was required for activation of CD3+ cells. Both the CD2 and CD3 responses were diminished in magnitude and duration by EGTA. However, approximately 50% of T cells still had a brief response in the presence of EGTA, indicating that the increased [Ca2+]i results in part from intracellular calcium mobilization, and furthermore demonstrates that extracellular calcium is required for a full and sustained response. Our results support the concept that CD2 represents the trigger for a distinct pathway of activation both for T cells that express the CD3 molecular complex and for large granular lymphocytes that do not.

Localization to chromosome 11 of a gene encoding a human minor histocompatibility antigen.

The graft-versus-host disease (GVHD) seen in human leukocyte antigen (HLA)-matched sibling bone marrow transplants is by definition due to the "minor" histocompatibility antigens (mHAs) encoded outside the HLA region of human chromosome 6. Few of these antigens have been characterized in humans, and in general the locations of the encoding loci are unknown. Genetic experiments performed in mice have identified many mHAs, but only a few genes have been identified. Using T lymphocyte clones reactive with specific mHAs, combined with genetic linkage analysis, we identified two distinct loci in a single patient, each locus encoding an antigen presented to a T cell clone by HLA-B7. The technique used in this study should allow a rough enumeration of the number of mHAs in humans that are capable of eliciting T cell responses in vivo. Whether these T cell responses correlate with clinical GVHD is not yet clear.

Results of allogeneic bone marrow transplantation with unrelated or mismatched donors.

As most patients are not fortunate enough to have an HLA-matched sibling to use as a bone marrow donor, attention has focused on the use of either HLA-matched but unrelated donors or HLA-mismatched family members. With the maturation of the field of histocompatibility testing, it is now possible to quantitate with relative precision the degree of disparity between patient and donor. In general, it appears that with respect to histocompatibility differences between donors other than HLA-matched siblings, there is an increased incidence of acute graft-versus-host disease, with the risk correlated with the degree of histoincompatibility. However, the overall disease-free survival is not always adversely affected, as a graft-versus-leukemia effect may counterbalance the increased death rate from graft-versus-host disease. To find donors for most patients, efforts are under way to recruit a large number of unrelated volunteers into the National Marrow Donor Program.

The prophylactic use of low-dose amphotericin B in bone marrow transplant patients.

PURPOSE: To evaluate the efficacy of prophylactic low-dose amphotericin B (0.1 mg/kg per day) (LDA) in preventing fungal infections in patients who have had a bone marrow transplant (BMT). MATERIALS AND METHODS: Double-blind, randomized, controlled trial in which patients undergoing bone marrow transplantation received intravenous LDA or similar-appearing placebo from the onset of neutropenia until the absolute neutrophil count remained > 0.5 x 10(9)/L, or until high-dose amphotericin B was initiated. Weekly surveillance cultures were obtained from all patients. RESULTS: Five of 18 patients (28%) randomized to placebo developed documented systemic fungal infections within the first 30 days after transplantation, compared to none of 17 patients who received LDA (P = 0.045). LDA recipients received fewer days of high-dose amphotericin B (P = 0.04) and fewer days of antibiotics (P = 0.008). There were trends towards fewer days of hospitalization (P = 0.14) and improved survival (P = 0.08); these differences were statistically significant among recipients of allogeneic BMT. No adverse effects occurred with LDA therapy. CONCLUSIONS: LDA appears to be safe and to reduce early systemic fungal infections in BMT recipients. Improved survival was observed among LDA recipients, but this was not directly attributable to the prevention of fungal infection.

Impact of racial genetic polymorphism on the probability of finding an HLA-matched donor.

As successful organ or marrow transplantation correlates with the degree of HLA-compatibility between patient and donor, registries have been developed to facilitate matching. However, racial minority groups have a lower chance of finding a match. We evaluate the impact of the biology of racial genetic polymorphism upon the probability of finding an HLA match for patients of different racial groups. The National Marrow Donor Program has compiled the HLA types of 20,449 patients and 1,625,159 potential volunteer donors. These HLA types were used to estimate the probability of finding an HLA-matched donor for patients of different racial groups. We estimated the HLA haplotype frequencies for different races, and then determined the probability of finding matched donors, given several hypothetical registry sizes. We confirmed that patients of minority races searching the current National Marrow Donor Program registry have low probabilities of finding matches. This was only partly due to the smaller number of donors from these racial minorities, as the observation persisted even when hypothetical donor registry sizes were the same for all racial groups. We demonstrate that African-Americans are more polymorphic with respect to HLA, and are hence less likely to find donors at any given registry size. An increase in the recruitment of minority racial groups for organ and marrow donors will only partially alleviate the problem of equal access to HLA matches for patients belonging to racial minority groups. It will therefore be important to attempt to improve methods for transplantation using HLA-mismatched donors.

Alloreactive T cell responses between HLA-identical siblings. Detection of anti-minor histocompatibility T cell clones induced in vivo.

Cytotoxic T cell (CTL) clones were isolated from the peripheral blood of a patient 13 days following marrow transplantation from her HLA-identical brother. Nineteen of the clones were specifically reactive with host--but not donor--cells, but one clone was reactive with donor but not host cells. Family studies using the 19 clones showed reactivity patterns consistent with three non-HLA, minor histocompatibility antigens (minor-HA). The frequency of reactivity on a large panel of unrelated cells indicated a relatively limited degree of polymorphism. Each of the clones was restricted in its activity by a class I epitope common to HLA-B7, B27, and B40. These data demonstrate that by clonal analysis it is possible to identify in vivo antidonor and antihost alloreactive CTL clones directed against HLA determinants following marrow transplantation.

HLA gene and haplotype frequencies in the North American population: the National Marrow Donor Program Donor Registry.

BACKGROUND: As of May 1, 1995, the National Marrow Donor Program had a donor registry consisting of over 1.35 million HLA-typed volunteers recruited from most major cities and states in the United States. This registry represents the largest single HLA-typed pool of normal individuals in the world. METHODS: We analyzed the HLA-A, -B, -DR locus phenotypes of the National Marrow Donor Program donors in order to estimate gene and haplotype frequencies for major racial groups of the United States: Caucasian American, Asian American, African American, Latin American, and Native American. The large size of the database allowed us to calculate the frequencies of relatively rare antigens and haplotypes with more accuracy than previous studies. RESULTS: We observed 89,522 distinguishable HLA-A, -B phenotypes in 1,351,260 HLA-A, -B-typed donors and 302,867 distinguishable HLA-A, -B, -DR phenotypes in 406,503 HLA-A, -B, -DR-typed donors. Gene and haplotype frequencies differed remarkably among the five racial groups, with African Americans and Asian Americans having a large number of haplotypes that were specific to their racial groups, whereas Caucasian Americans, Latin Americans, and Native Americans shared a number of common haplotypes. CONCLUSIONS: These data represent an important resource for investigators in the fields of transplantation and population genetics. The gene and haplotype frequencies can be used to aid clinicians in advising patients about the probability of finding a match within a specific ethnic group, or to determine donor recruitment goals and strategies. The information is also a valuable resource for individuals who are interested in population genetics, selection and evolution of polymorphic human genes, and HLA-disease association.

Treatment of acute graft-versus-host disease with a nonmitogenic anti-CD3 monoclonal antibody.

Treatment with the monoclonal antibody OKT3 specific for the CD3 complex associated with the T cell antigen receptor can reverse acute rejection of human renal allografts. However, efficacy of anti-CD3 antibodies for treatment of patients with acute graft-versus-host disease after marrow transplantation has not been established. The dose-limiting side effects resulting from T cell activation induced by some anti-CD3 antibodies in vivo have discouraged their use for this application. We now report a phase I-II study of GVHD treatment with the anti-CD3 antibody BC3, a monoclonal murine IgG2b that, unlike OKT3, does not activate T cells. Fourteen patients were treated with BC3 after progression of acute GVHD despite treatment with cyclosporine and corticosteroids, and three patients received BC3 as primary treatment for GVHD. BC3 was administered at a dose of 0.1 or 0.2 mg/kg/day for seven or eight days. Five patients achieved complete resolution of GVHD, eight patients had partial improvement, two patients had no change, and two patients had progression of GVHD on therapy. Responses were sustained in 8 of 13 patients. Mild chills, fever, hypertension, and chest discomfort occurred in various combinations following 6 of 17 (35%) initial infusions of BC3 and following 4 of 99 (4%) subsequent infusions. In each instance it was possible to continue BC3 therapy without adjusting the dose or treatment schedule. In each patient treated, the absolute count of peripheral blood lymphocytes decreased transiently but returned to baseline within 22 hr after the first infusion. Circulating T cells had surface CD3 molecules saturated by the infused antibody in all but one patient. Four patients survived longer than one year after treatment with antibody BC3, and 13 patients died of infection or organ failure. Administration of the nonmitogenic anti-CD3 antibody BC3 was associated with improvement in the clinical manifestations of GVHD with minimal acute toxicity. Efficacy of antibody treatment did not depend on depletion of circulating T cells. Therefore, antibody BC3 may be achieving therapeutic immunosuppression by modulating T cell function. Controlled studies in patients treated earlier in the course of GVHD should determine whether antibody BC3 can improve survival.

A phase I-II study evaluating the murine anti-IL-2 receptor antibody 2A3 for treatment of acute graft-versus-host disease.

A murine IgG1 antibody specific for the IL-2-binding site on the human lymphocyte IL-2 receptor beta chain (CD25) was evaluated in 11 patients who developed acute graft-versus-host disease following allogeneic marrow transplantation. All patients had received cyclosporine and methotrexate for prophylaxis of GVHD, either alone (4 cases), or in combination with antithymocyte globulin (4 cases) or with prednisone (3 cases). Patients had developed GVHD at 7-53 days (median 12) after transplantation and had failed treatment with corticosteroids for 3-44 days (median 19). Residual GVHD was of grade II severity in 4 patients, grade III in 5 patients, and grade IV in 2 patients. Sequential patients received monoclonal antibody in escalating doses from 0.1 mg/kg/day to 1.0 mg/kg/day for 7 days. Side effects were fever, respiratory distress, hypertension, hypotension, and chills occurring in 11 of 72 (14%) antibody infusions. Trough antibody levels greater than 6 micrograms/ml were achieved in patients treated with 0.5 or 1.0 mg/kg/day. Four of eight evaluable patients had an IgM antibody response, and one had an IgG response to the murine immunoglobulin. Clinical response of GVHD was evaluated in 10 patients who received the entire course of the antibody treatment. Among 7 patients treated within 40 days from transplantation, one patient had a complete response in the skin as the only involved organ, and 3 patients had a partial response, 2 in the skin and one in the gastrointestinal tract. No responses were achieved with liver disease at anytime or in any organ in patients treated beyond 40 days after transplantation. Since administration of this antibody was well tolerated and some efficacy was observed in patients with acute GVHD treated early after transplantation, there is a rationale for testing this antibody as an agent for prophylaxis of GVHD.

Probability of finding HLA-matched unrelated marrow donors.

Bone marrow transplantation has become the treatment of choice for certain hematologic diseases. However, only 30-40% of patients who might benefit from this procedure have a suitable family donor. Consequently, many centers have begun to explore the use of unrelated volunteer donors. Initial results have demonstrated the feasibility of this approach. As a result, a national effort has begun to recruit HLA-typed volunteers in order to establish a registry of individuals who would be willing to serve as bone marrow donors. This manuscript explores the potential impact of establishing such a registry. We find that a registry of attainable size could more than double the number of marrow transplants now being performed. However, even with a registry of enormous size, it will still not be possible to identify an HLA-matched donor for some patients.

Marrow transplantation from HLA-matched unrelated donors for treatment of hematologic malignancies.

Less than 40% of the patients who could benefit from marrow transplantation have an HLA-matched relative who can serve as a donor. For this reason, several centers have explored marrow transplantation from other categories of donors. This retrospective study analyzes the results of marrow transplantation for 52 patients receiving grafts from HLA-A,B,DR,Dw-phenotypically matched, MLC-compatible, unrelated volunteer donors compared to a disease, disease-stage, and age-matched cohort of 104 patients transplanted from HLA-genotypically identical sibling donors. The patients transplanted from unrelated donors had an increased incidence of grade II-IV acute graft-versus-host disease compared to patients transplanted from related donors (79% vs. 36%, P much less than 0.001). However, the probability of relapse-free survival appears similar in the two groups (P = 0.39 over all, with estimates of 41% vs. 46% at 1 year). We conclude from this preliminary data that marrow transplantation from HLA-matched unrelated donors should be considered in most, if not all, circumstances where transplantation from an HLA-matched sibling would be indicated if such a donor were available.

Change in functional phenotype of cloned human alloreactive cytolytic T cells.

Alloreactive T cell clones primed in vivo were tested for the expression of T cell differentiation antigens CD2, CD3, CD4, and CD8. Each of 29 different clones were found to express CD2 and CD3, but were variable in their expression of CD4 (7 positive clones) and CD8 (15 positive clones). Six clones were positive for both CD4 and CD8. One of the 29 clones expressed neither CD4 or CD8. Over a period of 12-18 weeks of culture, these clones began to lose their alloreactivity but acquired NK-like activity. By changing the concentration of TCGF, the "allo" and "NK-like" lytic activities could be modulated. After 18 weeks of culture, these clones lost their alloreactive specificity, but not their NK activity. The expression of surface markers was unchanged. CD2 and CD3 molecules were determined to play a role in both the alloreactive and NK activity of these clones.

Immune responses in an untransfused patient with aplastic anemia: analysis of cytolytic and proliferative T cell clones.

Unexpected unidirectional reactivity was noted in MLC by the cells of an untransfused patient with aplastic anemia against cells from her genotypically HLA identical brother. To analyze this reactivity, lymphocytes from the patient were primed in vitro for six days with irradiated lymphocytes from the HLA identical brother and then cloned by limiting dilution in the presence of interleukin-2. Following a period of clonal expansion, the patient's cells were tested for specific proliferative (PLT) and cytolytic (CTL) activity against cells of the brother. Thirty-six clones demonstrated proliferative activity, 30 clones demonstrated cytolytic activity, and 114 clones showed neither. No clone demonstrated both cytotoxic and proliferative activity. Several patterns of specificity were seen for the cytolytic T cell (CTL) clones, including both allo- and autoreactivity. Two distinct patterns of specificity were noted for the proliferative clones: one reactive to cells from DR3-positive males; the other reactive only to cells from certain DR2-positive males and females. The DR3-restricted clones are presumably directed towards the H-Y minor histocompatibility antigen while the DR2-restricted clones are directed toward an undefined minor histocompatibility antigen. It is thus possible to isolate both alloreactive and autoreactive T cells from the peripheral blood of some untransfused patients with aplastic anemia.

Probability of finding HLA-mismatched related or unrelated marrow or cord blood donors.

Given recent improvements in the technology of transplantation and histocompatibility testing, it is now possible to contemplate using related or unrelated allogeneic hematologic stem cell donors with high degrees of HLA disparity. This paper is a follow-up of an earlier publication on the probability of finding a matched donor (Transplantation 60:778-783, 1995) and addresses the probability of finding a partially mismatched donor. Assuming that a four of six antigen HLA-A, -B, -DR match is acceptable, it is possible to find unrelated donors for patients of any race from a putative registry with fewer than 10,000 potential donors. Further, storing cord blood from newborns in families with a known genetic disease would yield an acceptable future stem cell transplant product in nearly 40% of cases. These results show the potential impact of cord blood donors and emphasize the importance of improvements in transplantation using partially mismatched donors.

Effect of HLA incompatibility on graft-versus-host disease, relapse, and survival after marrow transplantation for patients with leukemia or lymphoma.

We analyzed the relevance of HLA incompatibility to acute graft-versus-host disease, relapse, and survival in 281 patients with hematologic neoplasms who underwent bone marrow transplantation. Each patient received marrow from a family member who shared one HLA haplotype with the patient but differed to a variable degree for the HLA-A, -B, and -D antigens of the haplotype not shared; 29 were phenotypically identical, 119 were incompatible for one locus, 104 for two loci, and 29 for three loci. These 281 patients were compared with 967 patients who received marrow from siblings with identical HLA genotypes. All patients were treated with cyclophosphamide and total-body irradiation followed by the infusion of unmodified donor marrow cells. Occurrence of severe acute graft-versus-host disease was evaluated in patients who achieved sustained engraftment. In recipients of haploidentical grafts occurrence of severe acute graft-versus-host disease was associated with (1) graft-versus-host disease prophylaxis containing the combination of methotrexate plus cyclosporine versus standard methotrexate, relative risk = 0.35; 95% confidence interval, 0.21-0.57, p less than 0.0001; and (2) the degree of recipient HLA incompatibility, relative risk = 1.95 for each locus incompatible; 95% confidence interval, 1.52-2.50, p less than 0.0001; (3) patient age, relative risk = 1.23 per decade; 95% confidence interval, 1.05-1.44, p = 0.0094. Acute graft-versus-host disease was associated with lower leukemic relapse after transplant in patients with acute lymphocytic leukemia, and chronic graft-versus-host disease was associated with lower relapse after transplant for acute nonlymphocytic leukemia in relapse or chronic myelogenous leukemia in blast crisis. After transplantation for acute nonlymphocytic leukemia in remission, the rate of leukemic relapse was 22% in 61 recipients of "one-locus" (A, B, or D)-incompatible grafts compared to 37% in 561 recipients of HLA-identical sibling grafts. Survival was decreased as the degree of HLA disparity increased. Survival of "one-locus"-incompatible transplant recipients, however, was equivalent to that of HLA-identical sibling transplant recipients.

Marrow transplantation using volunteer unrelated donors in a comparison of mismatched family donor transplants: a Seattle perspective.

Although allogeneic marrow transplantation has evolved into the treatment of choice for many otherwise fatal diseases, most patients are not candidates as they do not have an HLA-matched sibling donor. The Seattle Marrow Transplant Team, among others, has shown that patients who receive grafts from relatives who are partially matched for HLA (5 of 6 HLA-A,-B,-DR antigens) have an increased risk of graft-versus-host disease, but an overall survival that is comparable to that of similar patients receiving grafts from HLA-matched siblings. Similarly, patients receiving grafts from HLA-matched unrelated donors are at high risk for GVHD, but can look forward to favorable outcomes nearly as frequently as those who receive grafts from matched relatives. Grafts from more HLA-disparate donors, either related or unrelated, are at substantially increased risk of complications and death.

Representation of Hispanics in the National Marrow Donor Program.

Since less than one-third of patients in need of a BMT find related donors, most patients will rely on registries of volunteer donors. For patients from minority ethnic groups the chances of finding matched unrelated donors are lower, in part due to the smaller representation of minorities in the registries. Our purpose was to determine the representation of Hispanics in the National Marrow Donor Program (NMDP), the largest registry of volunteer marrow donors in the United States. We analyzed a database provided by the NMDP that contained information on minorities. The number of Hispanic volunteer donors has increased 110-fold in the last 6 years. The proportion of Hispanics in the registry has also increased from 1.1% to 6%. Nevertheless, the proportion of Hispanic patients that received unrelated marrow transplants facilitated by the NMDP has increased only from 2.8% to 3.9% since 1989. Only 19.7% of the formal searches initiated by Hispanic patients resulted in transplants compared to the 30.4% observed in the Caucasian population. Despite increments in the number and proportion of Hispanic volunteer donors, the proportion of Hispanics that receive BMT from unrelated donors remains low. We conclude that, in addition to increased recruitment efforts, other strategies will be necessary in order to find enough marrow donors to meet the needs of the Hispanic population.

Allogeneic bone marrow transplant for chronic myelogenous leukemia in a patient with multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system in which immune mechanisms appear to be an important component of the pathophysiology. Although the clinical manifestations are variable, a subset of patients develops a progressive clinical course associated with marked neurologic impairment and significant morbidity. BMT has been proposed as treatment for such patients based on preclinical data as well as clinical observations in other autoimmune diseases. We report clinical and MRI findings in an MS patient, later diagnosed with CML, and treated with an allogeneic BMT.

Treatment of severe Evans syndrome with an allogeneic cord blood transplant.

Immunosuppressive therapy is commonly used in the management of autoimmune disorders. As marrow-derived lymphocytes appear to play a key role in these diseases, lymphoid ablation followed by replacement with autologous or allogeneic stem cells may be a therapeutic option. We report a 5-year-old boy with severe Evans syndrome which consists of immune thrombocytopenia and Coombs-positive hemolytic anemia. He was rendered into complete remission with marrow ablation followed by rescue with an HLA-identical sibling cord blood transplant. He unexpectedly died 9 months following transplant from acute hepatic failure of unknown etiology.

The use of unrelated bone marrow donors in the treatment of patients with chronic myelogenous leukemia: experience of four marrow transplant centers.

Thirty-seven patients with chronic myelogenous leukemia who lacked an HLA-identical sibling were transplanted with bone marrow from an HLA-A,B,DR-matched, one locus-mismatched, or two locus-mismatched unrelated volunteer donor. Twenty-two were in chronic phase and 15 had advanced to either accelerated phase or blast crisis. The projected 1000-day survival is 55% for chronic phase patients and 22% for accelerated or blast phase patients. For patients transplanted during chronic phase, results appeared to be comparable whether the donor was fully HLA-matched or HLA one locus-mismatched. These results indicate that marrow grafting from either HLA-identical or HLA one locus-mismatched volunteer donors may be effective therapy for patients with chronic myelogenous leukemia who lack an acceptable related donor.