Lack of anti-tumour reactivity despite enhanced numbers of circulating natural killer T cells in two patients with metastatic renal cell carcinoma.

Natural killer T (NK T) cells play a central role as intermediates between innate and adaptive immune responses important to induce anti-tumour reactivity in cancer patients. In two of 14 renal cell carcinoma (RCC) patients, treated with interferon (IFN)-α, we detected significantly enhanced numbers of circulating NK T cells which were typed phenotypically and analysed for anti-tumour reactivity. These NK T cells were T cell receptor (TCR) Vα24/Vß11(+), 6B11(+) and bound CD1d tetramers. No correlation was observed between NK T frequencies and regulatory T cells (T(regs)), which were also enhanced. NK T cells expressed CD56, CD161, CD45RO and CD69 and were predominantly CD8(+), in contrast to the circulating T cell pool that contained both CD4(+) and CD8(+) T cells, as is found in healthy individuals. It is unlikely that IFN-α triggered the high NK T frequency, as all other patients expressed low to normal NK T numbers. A parallel was observed in IFN-α-related increase in activation of NK T cells with that in conventional T and non-T cells. Normal interleukin (IL)-7, IL-12 and IL-15 plasma levels were found. In one of the patients sporadic NK T cells were detected at the tumour site. α-Galactosylceramide (αGalCer) stimulation of peripheral blood mononuclear cells or isolated NK T cell lines from both patients induced IFN-γ, but no IL-4 and no response towards autologous tumour cells or lysates. The clinical course of disease in both patients was not exceptional with regard to histological subtype and extent of metastatic disease. Therefore, despite a constitutive high peripheral frequency and in vitroαGalCer responsiveness, the NK T cells in the two RCC patients did not show anti-tumour responsiveness.

Value of serum S-100B for prediction of distant relapse and survival in stage III B/C melanoma.

BACKGROUND: The biochemical marker serum S-100B has been proven to reflect the stage of melanoma and to be useful for disease monitoring and prediction of survival, mainly in stage IV disease. For stage III melanoma, limited data are available and its predictive value for relapse is unknown. Serum S-100B was evaluated prospectively for monitoring response and its predictive value for relapse and overall survival in stage IIIB/C melanoma patients. PATIENTS AND METHODS: Treatment consisted of one cycle of neoadjuvant and adjuvant chemo(immuno)therapy, around surgery. S-100B was measured at enrollment and prior to and following surgery. The levels of S-100B in serum were compared to the pattern and intensity of the expression of S-100B in the melanoma tissue. RESULTS: Some patients with normal initial S-100B values (n=18) showed responses (3 complete remission and 2 partial remission), in contrast to patients with elevated S-100B values. Distant relapse within one year was found in 11/23 (48%) patients with increased S-100B versus 2/18 (11%) patients with a normal value (p=0.01). Overall survival was decreased in patients with increased S-100B compared to those with normal S-100B (p=0.02). Correlations between the pattern and intensity of S-100B expression in the tumor specimen and the value of serum the S-100B did not reach statistical significance. CONCLUSION: Serum S-100B is a valuable biomarker for the evaluation of response to treatment and prediction of early distant relapse and survival in stage IIIB/C melanoma. The marginal correlation between serum S-100B values and expression of S-100B in the tumor specimens needs further study.

Autologous bone marrow transplantation for adult poor-risk lymphoblastic lymphoma in first remission.

PURPOSE: Adult patients with poor-risk lymphoblastic lymphoma (LBL) treated with intensive multiagent chemotherapy (acute lymphoblastic leukemia [ALL]-like regimens) have a poor prognosis, with a disease-free long-term survival rate of less than 20%, caused by a very high relapse rate. Thus, adult patients with poor-risk LBL are candidates for alternative intensive consolidation therapy. PATIENTS AND METHODS: Nine adult patients with poor-risk LBL in first remission after treatment with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP; six patients) or ALL-like regimens (three patients), were treated with high-dose cyclophosphamide and total body irradiation (TBI) followed by nonpurged autologous bone marrow transplantation (ABMT). RESULTS: Two of nine patients relapsed at 4 and 8 months, respectively, after BMT, and one patient died of acute myeloblastic leukemia (AML) 7 months after ABMT without recurrence of his lymphoma. Six patients are in unmaintained first remission with a follow-up of 12 to 113 months (median, 53 months) after transplantation. CONCLUSIONS: These results suggest that intensive consolidation therapy with high-dose cyclophosphamide and TBI followed by nonpurged ABMT may improve the long-term prognosis of this disease.

Salvage therapy with ProMACE-MOPP followed by intensive chemoradiotherapy and autologous bone marrow transplantation for patients with non-Hodgkin's lymphoma who failed to respond to first-line CHOP.

PURPOSE: We used alternative chemotherapy immediately followed in early-response patients by high-dose chemoradiotherapy and autologous bone marrow transplantation (ABMT) to treat patients with non-Hodgkin's lymphoma (NHL) who had failed to respond to first-line chemotherapy. PATIENTS AND METHODS: Thirty-one patients with NHL of intermediate- or high-grade malignancy who had failed to respond to first-line cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy were treated. Seventeen patients had primary refractory disease and 14 had relapsed from first complete response (CR). The treatment consisted of prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, mechlorethamine, vincristine, and procarbazine (ProMACE-MOPP) salvage chemotherapy, followed, in case of responsive disease (at least partial response [PR]), by high-dose cyclophosphamide and total-body irradiation (TBI) with ABMT. RESULTS: Twenty-eight of 31 (90%) patients achieved PR (23 patients) or CR (five patients) with ProMACE-MOPP, and three failed to respond. Seventeen of 28 (61%) patients who responded underwent the ABMT procedure, which resulted in CR in 14 patients (82%); three failed to respond. Eleven responsive patients were not transplanted because of residual bone marrow infiltration (five patients), patient refusal (four patients), and ProMACE-MOPP-related mortality (two patients). To date, nine patients are alive and in CR, seven with a median follow-up of 41 months (range, 17 to 84 months). Referring to the original CHOP treatment, five of 17 (29%) patients with primary refractory disease remain free of disease at a median of 36 months after ABMT, and four of 14 (29%) patients in first relapse remain free of disease at a median of 33 months after ABMT. One patient died of AMBT-related toxicity. CONCLUSION: ProMACE-MOPP salvage chemotherapy produces a high early-response rate in patients who fail to respond to first-line CHOP, and more than half of the responding patients can be scheduled to receive ABMT, resulting in disease-free survival (DFS) at 3 years in 50% of the transplanted patients and in 25% of the original number of patients intended to receive this treatment.

Reinfusion of autologous lymphocytes with granulocyte-macrophage colony-stimulating factor induces rapid recovery of CD4+ and CD8+ T cells after high-dose chemotherapy for metastatic breast cancer.

PURPOSE: Repeated high-dose chemotherapy (HDCT) followed by peripheral-blood progenitor cell (PBPC) transplantation can induce a complete remission in patients with metastatic breast cancer sensitive to standard chemotherapy (CT), but the majority of patients relapse within 1 to 2 years. The immune system is seriously compromised after HDCT, which precludes the development of effective immunotherapy. We investigated whether autologous lymphocytes, reinfused after HDCT, could induce a rapid recovery of T cells. PATIENTS AND METHODS: Three patients were monitored for immune recovery without reinfusion of lymphocytes. In the next 11 patients, stem cells were harvested after CT + granulocyte colony-stimulating factor (G-CSF) and lymphocytes were harvested after CT + granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2. These patients received stem cells and G-CSF after the first HDCT; stem cells, G-CSF, and lymphocytes after the second; and stem cells, GM-CSF, and lymphocytes after the third HDCT. RESULTS: Patients not receiving lymphocyte reinfusion had a very slow recovery of lymphocytes. In particular, CD4 counts remained low (< 200/microL for 9 months). Lymphocyte reinfusion had a significant effect on the recovery of lymphocytes, T cells, and CD8+ T cells (normalized on day 25). Recovery of CD4+ T cells was significantly accelerated by lymphocyte reinfusion and GM-CSF, leading to counts of 500/microL at 25 days. CONCLUSION: Lymphocyte reinfusion with G-CSF had a significant effect on the recovery of CD8+ T cells, whereas rapid recovery of CD4+ T cells required lymphocyte reinfusion and GM-CSF, which possibly acts as a survival factor through activation of antigen presenting cells. Whether the rapid recovery of CD4+ and CD8+ T cells prevents or delays relapse of the disease should be further investigated.

Phase I clinical and pharmacologic study of chronic oral administration of the farnesyl protein transferase inhibitor R115777 in advanced cancer.

PURPOSE: To determine the maximum-tolerated dose, toxicities, and pharmacokinetics of R115777, a farnesyl transferase inhibitor, when administered continuously via the oral route. PATIENTS AND METHODS: Patients with advanced solid malignancies were treated with R115777 using an interpatient dose escalation scheme starting at 50 mg bid. Pharmacokinetics were assessed on days 1, 28, and 56. RESULTS: Twenty-eight patients were entered onto the study and the median duration of treatment was 55 days. The dose-limiting toxicities were myelosuppression and neurotoxicity. At a dose of 400 mg bid, grade 4 leukocytopenia and neutropenia were seen in two of four patients. Neurotoxicity grade 3 developed in one of five patients at 500 mg bid and in one of 13 at 300 mg bid after 8 weeks of treatment. Common nonhematologic toxicities were nausea, vomiting, and fatigue. The recommended dose for phase II/III testing in this scheme is 300 mg bid. The pharmacokinetic studies indicated dose proportionality. Little accumulation occurred and steady-state levels were reached within 2 to 3 days. Analyses of historic tumor material showed that five of 15 of patients had a K-ras mutation in codon 12. Three patients with pancreatic, colon, and cervix carcinomas had stable disease and one patient with a colon carcinoma had a minor response accompanied by a more than 50% decrease in carcinoembryonic antigen tumor marker. A fifth patient, with platinum-refractory non-small-cell lung cancer, showed a partial response that lasted for 5 months. CONCLUSION: Continuous dosing of R115777 is feasible with an acceptable toxicity profile at a dose of 300 mg bid.

Hepatocyte growth factor/scatter factor (HGF/SF) affects proliferation and migration of myeloid leukemic cells.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is produced by mesenchymal cells, including bone marrow (BM) stromal cells, and has mitogenic and motogenic effects on a variety of cell types. Recently, a role has been assigned to HGF/SF and its receptor, c-MET, in both normal and malignant hemopoiesis. We investigated the function of HGF/SF on hemopoietic mononuclear cells (MNC) from patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with circulating blasts. In contrast to results with normal MNC, HGF/SF alone stimulated the proliferation and colony formation of MNC from these patients. MNC from some (4/13) of the AML patients also produced HGF/SF (0.1-0.2 ng/ml/day), while we could not detect HGF/SF in cultures from normal MNC. Furthermore, it appeared that HGF/SF induced migration of leukemic cells in Boyden using KG1a cells as a model for leukemic blasts. The membranes dividing the two compartments of the Boyden chambers were coated with fibronectin. HGF/SF significantly promoted migration in 3/5 samples of MDS patients and in 5/7 samples of AML patients. Supernatant of human BM stromal cells, which is chemoattractive for normal human hemopoietic progenitor cells, also promoted migration of MNC from 4/5 MDS patients and 6/7 AML patients. Since HGF/SF is one of the growth factors produced by BM stromal cells, a neutralizing antibody directed against HGF/SF was added to the BM stroma supernatant, which reduced migration significantly in 2/3 MDS and in 3/6 AML responders to BM stroma supernatant. In conclusion, HGF/SF promotes proliferation and migration of hemopoietic cells from AML and MDS patients in vitro and may therefore contribute to the malignant potential of these cells.

Phase I trial of combined immunotherapy with subcutaneous granulocyte macrophage colony-stimulating factor, low-dose interleukin 2, and interferon alpha in progressive metastatic melanoma and renal cell carcinoma.

The purpose of our study was to determine the maximally tolerated dose (MTD) and DLT of combined administration of granulocyte macrophage colony-stimulating factor (GM-CSF), low-dose interleukin 2 (IL-2) and IFN-alpha in patients with progressive metastatic melanoma or renal cell carcinoma (RCC). In addition, the activation and expansion of effector cells were measured. Cohorts of three patients were treated with increasing doses of IL-2 (1, 4, and 8 MIU/m2) and GM-CSF (2.5 and 5 microg/kg) with a constant dose of IFNalpha (5 million units) s.c. for 12 days every 3 weeks. An additional six patients were treated at the MTD. Immune activation was monitored during the first cycle. Response was evaluated after two cycles. The MTD was found to be 2.5 microg/kg GM-CSF, 4 MIU/m2 IL-2, and 5 mega units of IFNalpha. DLT was grade 4 fever, chills with hypotension, grade 3 fatigue/malaise, and fluid retention. Dose reduction of IL-2 to 2 MIU/m2 was necessary in three of nine patients who initially received the MTD. Treatment was initiated in the hospital but could be continued at home after 3-4 days. Significant increases in lymphocytes, (activated) T cells (CD4+ and CD8+), NK cells, monocyte DR expression, neutrophils, and eosinophils were found. CD8+ T-cell activation (sCD8) and NK cell expansion was mainly present in patients receiving 2 or 4 MIU/m2 IL-2. Of eight patients with progressive metastatic RCC after nephrectomy, three achieved a complete remission, and 1 of 7 patients with metastatic melanoma achieved a partial remission. In our study, the MTD of combined immunotherapy with GM-CSF, IL-2, and IFNalpha was established; DLT was: (a) grade 4 fever with hypotension needing i.v. fluid support; and (b) grade 3 fluid retention and/or fatigue/malaise. The scheme resulted in considerable expansion and/or activation of various effector cells. The complete responses in RCC patients are promising but need to be confirmed in Phase II studies.

Extended continuous oral temozolomide in patients with progressive metastatic renal cell carcinoma not responding to interferon alpha 2b.

The aim of the study was to evaluate the toxicity and efficacy of oral extended continuous temozolomide in patients with progressive metastatic renal cell carcinoma (RCC) not responding to immunotherapy after removal of the primary tumor. Patients with progressive metastatic RCC received protracted temozolomide 100 mg/m2 orally on days 1-21 every 28 days. Response was assessed after 2 cycles to be followed by another 2 cycles in the absence of progression. After 4 cycles only patients with further remission and acceptable toxicity were to continue. No objective responses were observed in 12 patients and the trial was stopped prematurely in stage 1. Six patients remained stable during 4 cycles of temozolomide (4 months), only one of these remained stable for another 2 months after having stopped treatment. Five patients progressed after the initial 2 cycles and one after the first cycle. Overall survival was 15.5 months (range 1-36 months). Repeated cycles of 3 weeks oral temozolomide 100 mg/m2 followed by one week rest proved tolerable though this regimen may only have limited activity against metastatic RCC.

Glycolytic enzymes of an erythroleukemic cell line, K562, before and after hemoglobin induction.

The enzyme activities and isozyme distribution of some glycolytic enzymes were studied in the K562 cell line before and after induction of hemoglobin formation. Special attention was paid to the three regulator enzymes of glycolysis, hexokinase, phosphofructokinase and pyruvate kinase. Results for the K562 cell line were compared with those for the mature red cell. K562 cells exhibit a relatively low phosphofructokinase and high pyruvate kinase activity. Electrophoresis of hexokinase shows the presence of two bands in the HK I region. HK II is also present, probably as a result of culture conditions. Only 15% of the total hexokinase activity is mitochondrial bound. Phosphofructokinase in K562 cells is mainly composed of the L- and F-types of which the F-type is characteristic for platelets and granulocytes and not for erythrocytes. In the electrophoretic pattern of pyruvate kinase a predominant K4 band besides three hybrids were found. The hybrids were demonstrated to contain L-type subunits of pyruvate kinase, which means a new erythroid marker of K562 cells, as the red cell is the only blood cell that contains L-type pyruvate kinase. Induction experiments with hemin, ARA-C and mitomycin-C gave rise to more than 85% benzidine positive cells after 11 days of culture. The isozyme composition of pyruvate kinase did not change after induction. HK II disappears after induction with ARA-C and mitomycin-C but not with hemin. The results support the idea of the multipotential features of the K562 cell line.

Cytotoxic T-cell capacity after autologous bone marrow transplantation.

A total of 19 patients, treated for aggressive tumors with high-dose chemo/radiotherapy and autologous bone marrow transplantation (BMT), were studied for concanavalin-A (Con A)-induced proliferation and Con-A-induced cytotoxicity. Ten patients with cytomegalovirus (CMV) antibodies before BMT showed increased Con-A-induced cytotoxicity before and from 100 days after BMT, while Con-A-induced proliferation decreased to less than 10% of control values after BMT and remained so. Nine CMV-negative patients showed normal cytotoxic capacity before and after BMT, while Con-A-induced proliferation recovered slowly from day +30 after BMT. Con-A-induced cytotoxicity was not significantly different between CMV-positive and CMV-negative patients, while Con-A-induced proliferation showed significant differences from day +100 onward.

Reduction and repopulation of T-lymphocytes after cytoreductive therapy with or without autologous bone marrow rescue.

The reconstitution of peripheral-blood T-lymphocytes following cytoreductive therapy in standard (11 patients) or in high dosages (ten patients) was compared with that after supralethal cytoreductive therapy followed by autologous bone marrow rescue (ABMR, 20 patients). Along with the increasing cytotoxic potential of the three therapy protocols, T-cell counts fell to lower levels. Following all three forms of cytoreductive therapy, T8+ T-cell counts decreased to lower levels than T4+ T-cell counts. The greater relative reduction of T8+ T cells may indicate that T8+ T cells are more sensitive to cytoreductive therapy than T4+ T cells, and/or that T8+ T cells have shorter survival times. The contribution of residual (mainly T4+) T cells to the T-cell repopulation was significant in the patients on standard-dosage chemotherapy, less important in those on high-dosage chemotherapy, and minor in those receiving supralethal cytoreductive therapy and ABMR. The repopulation rates of T8+ T cells following ABMR exceeded those observed after chemotherapy without ABMR. The T3- (T3 negative) T-cell subset, which comprises only 5%-10% of peripheral T cells in normal individuals, decreased rapidly to low levels and remained so for the entire six-week observation period in both chemotherapy groups. Following ABMR, however, those T3- T cells rapidly increased again to normal levels. Since the T cells in bone marrow biopsies have a large T3- fraction, that rapid recovery of T3- T cells may reflect the contribution of marrow precursors in the marrow grafts to the improved T-cell regeneration following ABMR.

Reduction and repopulation of recipient T4+ and T8+ T-lymphocytes in allogeneic bone marrow transplantation.

In eight recipients of allogeneic bone marrow grafts who had sex-mismatched donors, the reduction and subsequent repopulation of T4+ and T8+ T-lymphocytes of recipient origin were studied. The origin of the donor-recipient T4+ and T8+ T cells was studied using quinacrine staining of Y chromatin combined with T-cell typing for T4 and T8. Following chemoradiotherapy and bone marrow transplantation (BMT), T cells reached their nadir at a median of five (range 1-8) days after BMT. T8+ T cells decreased at a faster rate from the peripheral blood than T4+ T cells. The first T cells that appeared in the circulation at day 12 were predominantly T4+, and a large number of them were of recipient origin. Thereafter, they gradually decreased, and the numbers of T cells of donor origin increased. In the patients who had no or only minor complications, T4+ and T8+ T cells of donor origin repopulated the blood at similar rates. This pattern, however, was modified by severe graft-versus-host disease or by cytomegalovirus infection.

Influence of cytomegalovirus infection on the recovery of humoral immunity after autologous bone marrow transplantation.

Recovery of B-cell number and function was studied in 23 patients with hematological malignancies treated with high-dose chemoradiotherapy followed by autologous bone marrow transplantation (auto-BMT) in relation to the presence or absence of cytomegalovirus (CMV) infection. B cells recovered rapidly after auto-BMT and specific antibodies to herpes viruses remained nearly unchanged. Both were independent of the CMV status of the patients. However, the capacity of peripheral blood B cells to differentiate in vitro into cytoplasmic immunoglobulin (Ig)-positive cells (plasma cells) on pokeweed mitogen stimulation in the presence of normal T-cell help was significantly better in CMV-negative patients than in CMV-positive patients after auto-BMT, but was decreased in both groups. Serum Ig levels were, in contrast, higher in CMV-positive patients than in CMV-negative patients after auto-BMT.

Hepatocyte growth factor/scatter factor (HGF/SF) is produced by human bone marrow stromal cells and promotes proliferation, adhesion and survival of human hematopoietic progenitor cells (CD34+).

The fate of hematopoietic progenitor cells (HPCs) in the bone marrow (BM) microenvironment is determined by two different interactions: 1) they adhere (via integrins) to both extracellular matrix molecules and BM stromal cells; and 2) stromal cells produce cytokines that influence their survival, proliferation, differentiation, and mobilization. The ligands for the protein tyrosine kinase receptors c-KIT and FLT3/FLK2, stem cell factor (SCF), and FL are produced by BM stromal cells and are known to affect several facets of hematopoiesis. We studied another protein tyrosine kinase receptor, c-MET, and its ligand hepatocyte growth factor (HGF), also known as scatter factor (SF), which play a similar role in hematopoiesis. c-MET mRNA is expressed in immature human BM HPCs (CD34+CD33- or CD34+CD38-), but not in more mature HPCs (CD34+CD33+ or CD34+CD38+). The ligand HGF/SF is predominantly produced by BM stromal cells at both the mRNA and protein levels. We confirmed functionally that HGF/SF alone has no effect on proliferation of HPCs, but that when combined with granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin-3 it acts as a synergistic proliferative factor, although not as potently as kit-ligand or FLT-3/FLK-2 ligand. Furthermore, HGF/SF promotes adhesion of HPCs to immobilized fibronectin. HGF/SF-induced adhesion to fibronectin is probably caused by activation of the integrins alpha4beta1 and alpha5beta1, insofar as we were able to block this interaction by using monoclonal blocking antibodies directed against these integrin subunits. Addition of the tyrosine-phosphorylation inhibitor genistein inhibited HGF/SF-induced adhesion, supporting the idea that HGF/SF-induced effects are the result of signaling via the receptor c-MET after ligand binding. The enhanced adhesion of HGF/SF to fibronectin proved to be beneficial for the maintenance of the colony-forming potential of HPCs. HGF/SF alone and especially in combination with fibronectin prolongs survival of GM colony-forming cells in liquid culture. Our data indicate that HGF/SF is a polyfunctional cytokine in the BM microenvironment. It is produced by human BM stromal cells and directly or indirectly promotes proliferation, adhesion, and survival of human HPCs.

Marrow donor immunity to herpes simplex virus: association with acute graft-versus-host disease.

The interactions between humoral and cellular immunity to herpes simplex virus (HSV) in bone marrow donors, the occurrence of active HSV infections, and the development of grades II-IV acute graft-versus-host disease (GVHD) in their HLA-A,B,C,DR-identical sibling recipients were studied. The absence of IgG-class HSV antibodies in the marrow donors was associated with a low incidence of GVHD: 38 of 53 recipients (72%) of marrow from HSV-seropositive donors developed GVHD versus only two of 15 recipients (13%) with HSV-seronegative donors (p = 0.0004). The cellular immunity to HSV was studied in vitro by evaluating the degree of lymphocyte proliferative responses to that virus and was also significantly associated with GVHD: 30 of 43 recipients (70%) of marrow from donors with a positive test developed GVHD versus 10 of 25 recipients (40%) of marrow from donors with a negative test (p = 0.03). The previously reported risk for GVHD attributed to donor CMV antibodies increased the risk of GVHD due to donor HSV antibodies. Of 31 recipients of marrow from donors who were both HSV- and CMV-seropositive, 27 (85%) developed GVHD versus 11 of 22 recipients (50%) of marrow from HSV-seropositive but CMV-seronegative donors (p = 0.008).

Response of leptomeningeal metastases from breast cancer to hormonal therapy.

Intraventricular chemotherapy with radiotherapy is the standard treatment of leptomeningeal metastasis (LM) from breast cancer; this treatment increases median survival only to about 3 months and is frequently complicated by serious side effects. The authors describe two patients with LM from breast cancer who were treated with hormonal therapy, which provided a neurologic response of at least 12 months and a survival of 14+ and 19 months. Hormonal therapy can be effective and nontoxic for patients with LM from breast cancer.

PHA-induced cytotoxicity of human lymphocytes against adherent hela cells.

The conditions for a phytohaemagglutinin(PHA)-induced cytotoxicity test of human peripheral blood lymphocytes were investigated. [3H]thymidine prelabelled HeLa cells were used as target cells. Stimulation with 10 microliter PHA/ml during 24 h gave the best measure of lymphocyte cytotoxic capacity. Supernatants of PHA-activated lymphocytes showed no cytotoxicity against adherent HeLa cells. Mitomycin treatment did not influence cytotoxic capacity. Removal of phagocytizing mononuclear cells reduced spontaneous cytotoxicity, but increased PHA-induced cytotoxicity. Adherent cells showed high spontaneous cytotoxicity, with little increase on addition of PHA. The method was evaluated for clinical applicability by testing mononuclear cells from 19 normal subjects and purified lymphocytes from 15 normal subjects. Purified lymphocytes showed a higher PHA-induced cytotoxicity with a smaller variation and greater ratio dependent increase in cytotoxicity than unseparated mononuclear cells. Results with fresh purified lymphocytes were reproducible.

Differentiation between irreversible and reversible rejection in renal transplant patients by monitoring of phytohemagglutinin-induced cytotoxicity.

Phytohemagglutinin (PHA)-induced cytotoxicity against adherent HeLa cells, a reproducible test for primary T cell cytotoxicity, was used in the followup of 34 renal transplant recipients and related to the outcome. During the 1st week, two uncomplicated cases showed a decrease in cytotoxicity of more than 20% and it remained low. One patient with a cytomegalovirus (CMV) infection showed a marked increase in cytotoxicity (37%) and it remained high. In 31 patients with a rejection episode, a change of -20 to +20% was observed. During the 2nd week, all 10 patients who developed an irreversible rejection showed an increase of more than 20% (mean, 29 +/- 8%), in contrast to only 2 of 21 patients with a reversible rejection (mean, -1 +/- 15%, P less than 0.001). There were no differences between these two groups in lymphocyte and T lymphocyte counts, or in prednisone or azathioprine dose or blood urea nitrogen levels. These results indicate that regular assessment of PHA-induced cytotoxicity may be useful in the followup of renal transplant patients.

The influence of T cell depletion on recovery of T cell proliferation to herpesviruses and Candida after allogeneic bone marrow transplantation.

To test the influence of T cell depletion of the marrow in allogeneic bone marrow transplantation on functional T cell recovery, in vitro lymphocyte proliferation tests (LPTs) to microbial antigens were regularly performed in 23 recipients of normal BM and in 25 patients receiving BM with a fixed low number of T cells (1 x 10(5) T cells/kg body weight; recipients of T-depleted BM). The long-term recovery of positive LPT to at least 1 of the 4 tested microbial antigens--Candida, herpes simplex virus (HSV), varicella-zoster virus (VZV), and cytomegalovirus--was nearly similar in both groups: 16/23 versus 18/25. Recovery of LPT to Candida and HSV in the first 3 months appeared to be greatly influenced by prophylactic measures; only 2/23 recipients of normal BM, receiving amphotericin B, showed a positive LPT to Candida versus 13/25 recipients of T-depleted BM (P less than 0.01). In contrast, only 1/23 seropositive recipients of T-depleted BM, receiving acyclovir, showed a positive LPT to HSV versus 9/22 recipients of normal BM (P less than 0.05). A positive LPT to CMV in the first 3 months was found in 9/9 seropositive recipients of normal BM, versus in 5/11 seropositive recipients of T-depleted BM (P less than 0.05). Five of the 6 patients with a negative LPT died of CMV-interstitial pneumonia versus 1/14 with positive LPT (P less than 0.01). We conclude that in CMV-seropositive recipients of allogeneic BM, T cell depletion of the graft affects the early recovery of T cell proliferation to CMV, which is associated with a higher risk of fatal CMV-interstitial pneumonia.