Rhinovirus inhibits antigen-specific T cell proliferation through an intercellular adhesion molecule-1-dependent mechanism.

To determine whether binding of human rhinovirus (HRV) to intracellular adhesion molecule-1 might disrupt airway immune processes, effects of a major HRV group, HRV-16, on T cell proliferation and cytotoxicity were defined. HRV (1-10 TCID50/cell) significantly inhibited T cell proliferation induced by antigen but not proliferation secondary to mitogens, interleukin-2, or an irradiated allogeneic T cell line. Noninfectious (UV-irradiated) HRV had similar effects. Inhibition of T cell proliferation was dependent on HRV binding to intercellular adhesion molecule-1 on monocytes, indicating that the virus interferes with lymphocyte activation indirectly through effects on antigen-presenting cells. In addition, HRV inhibited T cell cytotoxic responses but not NK cell activity. If these effects also occur in vivo, the resulting disturbance in local airway immunity could increase the chances of successful viral replication, and might also be a factor in the pathogenesis of secondary viral or bacterial respiratory tract infections.

Factor V R506Q gene mutation analysis by PCR-RFLP: optimization, comparison with functional testing for resistance to activated protein C, and establishment of cell line controls.

Resistance to activated protein C (APC) has been recently identified as a highly prevalent risk factor for the development of venous thrombosis. In the majority of cases, APC resistance correlates with the presence of a single point mutation in the factor V gene (FV R506Q). The mutation is present in 3% to 5% of the general population and in up to 50% of patients with a personal and family history of venous thrombosis. In the current study, the authors have optimized and implemented for clinical diagnosis a method for detection of FV R506Q using the polymerase chain reaction coupled with restriction fragment length polymorphism analysis (PCR-RFLP). Forty-one healthy adults and 139 patients referred for hypercoagulability testing were genotyped and their APC resistance ratios determined using commercially available reagents (COATEST APC Resistance Kit). Comparative analysis indicated that if functional APC resistance was defined as per manufacturer's guidelines, a significant number of individuals with a normal factor V genotype were categorized as APC resistant and conversely, a significant number of individuals heterozygous for FV R506Q were categorized as non-APC resistant. These results indicate that comparative functional and genotypic analyses in the individual clinical laboratory setting are critical for establishing normal ranges and cut-off values for functional APC resistance due to FV R506Q. To facilitate molecular evaluation of APC resistance, Epstein-Barr virus (EBV) immortalized B-lymphocyte cell lines were established from individuals heterozygous and homozygous for FV R506Q.

Simultaneous demonstration of the Philadelphia chromosome in T, B, and myeloid cells.

A patient presented with lymphoblastic lymphoma in lymph-nodes and chronic myelogenous leukemia (CML) in narrow and peripheral blood. All marrow and unstimulated peripheral blood cells contained the Philadelphia chromosome[t(9:22)]. Lymphoma cells were analyzed by flow cytometry and were identified as T cells (CD2+CD5+CD7+CD34+). All fresh lymphoma cells contained the t(9:22) translocation. Cultures of purified peripheral blood T and B cells and specifically stimulated NK cells revealed that 59% of the B cells, 10% of the NK cells, and none of the normal T cells contained the translocation. The lack of translocation in normal peripheral T cells is attributed to their long lifespan. No rearrangement of immunoglobulin or T cell receptor beta or gamma genes was found in either the leukemia or lymphoma cells. Analysis of the DNA from cryopreserved lymphoma biopsy showed clonal rearrangement within the common breakpoint cluster region of the bcr gene identical to the bcr rearrangement in DNA from leukemia blood cells. The data support the concept that T and B cells originate in the patient's totipotent stem cell from which the CML is also derived.

In vitro preactivated human T cells engraft in SCID mice and migrate to murine lymphoid tissues.

Mice with severe combined immunodeficiency (SCID) accept grafts of human T and B lymphocytes derived from resting peripheral blood mononuclear cells (PBMC). We wished to determine whether activated human T cells engraft and migrate into lymphoid tissues in SCID mice. PBMC (50 x 10(6)) activated in vitro in a 4-day mixed lymphocyte culture (MLC) were injected into the peritoneum of 12 SCID mice. In 11 of 12 animals killed at 3 or 4 weeks after injection, human cells were detected in cells pooled from lymphoid organs by flow cytometry and by immunohistochemical staining of frozen tissue sections. The percentage of CD45+ cells in the 11 mice ranged from 2% to 45% and the absolute numbers of CD45+ cells recovered from lymphoid organs ranged from 4 x 10(6) to 90 x 10(6). Up to 93% of the human cells expressed the CD3 antigen together with either CD4 or CD8. Human T cells were localized in periarteriolar areas in murine spleens, whereas in the lymph nodes and gut mucosa, the T cells did not show the pattern for T-dependent areas found in human lymphoid tissue. Numerous human plasma cells were detected in the spleen and gut mucosal crypts of engrafted SCID mice. Human IgG was detected in the serum of all 11 engrafted SCID mice. The functional activity of human T cells recovered from murine splenic tissue was very low 3-4 weeks after engraftment.

The effect of T-cell depletion on enhanced basophil histamine release after in vitro incubation with live influenza A virus.

A number of mechanisms participate in virus-induced asthma. Previously, we described enhanced basophil histamine release (HR) during an experimentally induced rhinovirus infection and after in vitro incubation of peripheral blood mononuclear cells (PBMC) with influenza virus. This study extends our previous observations and examines the effect of influenza A virus on basophil leukotriene C4 (LTC4) release as well as the effect of T-cell depletion on virus-enhanced basophil HR. PBMC were isolated from ragweed-allergic subjects and incubated with live influenza A virus or control medium (allantoic fluid). After incubation with influenza A, ragweed antigen (AgE) stimulated LTC4 and HR were enhanced (P less than 0.05). To further define the role of T cells in virus-enhanced basophil secretion, PBMC were isolated and divided into two aliquots. In one aliquot, T cells were removed by magnetic bead separation of mouse monoclonal anti-CD3-coated lymphocytes. T-cell-depleted and nontreated PBMC suspensions were incubated with influenza A or control medium, collected, and challenged with AgE to release histamine. Basophil HR was enhanced in the virus-treated group of PBMC that had not undergone T-cell depletion. In contrast, virus incubation did not enhance HR in the T-cell-depleted fraction. Finally, preliminary analysis of the supernate from virus-treated leukocytes indicates the presence of interferon-gamma. These findings suggest that T cells, and their cytokine products, play an integral role in the process by which viruses enhance basophil HR.

Acute B-lymphocytic leukemia with L1 morphology: a report of two pediatric cases.

Two pediatric cases of acute lymphoblastic leukemia with L1 morphology who also demonstrated immunoglobulin on the leukemic cell surface are presented. The first patient's disease progressed on standard induction therapy, despite the presence of good prognostic features at presentation. The second patient has achieved a sustained remission with an aggressive lymphoma regimen. These two cases represent the first report on the outcome of patients with this rare phenotype. They indicate the importance of assessing cell surface immunoglobulin in children with acute leukemia, even in the absence of the L3 phenotype, in addition to the currently utilized batteries of monoclonal antibodies. Failure to recognize this phenotype may have significant therapeutic implications.

Immunologic reconstitution after haploidentical bone marrow transplantation for immune deficiency disorders: treatment of bone marrow cells with monoclonal antibody CT-2 and complement.

Patients with congenital T lymphocyte deficiency disorders received transplants with parental bone marrow depleted of mature T cells by the use of an anti-T cell monoclonal antibody (CT-2) and complement. Our results with 16 consecutive patients (20 transplants) showed rapid engraftment of donor cells; cytoreduction (busulfan, cytosine arabinoside [ara-C], cyclophosphamide) was used in six transplants, and marrow ablation was used in six (ara-C, cyclophosphamide, 1,365-cGy total body irradiation). No patient received prophylactic anti-graft-v-host disease (GVHD) therapy posttransplant, and only one patient developed significant GVHD, which involved the skin, liver, and gastrointestinal tract. Seven others showed some manifestations of GVHD, but these were of minimal clinical significance and required only occasional steroid therapy. Overall, eight patients are alive and well; eight did not survive. Polyclonal immunoglobulin synthesis by donor memory B cells was seen shortly after transplantation, with peak donor-derived serum levels seen approximately 2 months after transplantation. After this initial immunoglobulin synthesis waned, another wave of B cell responses developed. This immunoglobulin response appears to be permanent. T cell functions appeared as soon as 3 weeks after transplantation. This experience in a variety of patients with combined immunodeficiency who received transplants with monoclonal antibody T cell-depleted marrow shows gratifying results with a consistent T and B cell benefit.

Thymus and B cell reconstitution in severe combined immunodeficiency after transplantation of monoclonal antibody depleted parental mismatched bone marrow.

Two brothers were transplanted with bone marrow which was depleted of mature T cells with monoclonal antibody CT-2 plus complement. One child died of sepsis due to candida present prior to transplant. The other is alive and well with full T and B cell reconstitution over 36 months after transplant. Thymus biopsy taken after transplant demonstrated normal morphology and cellularity. Portions of the marrow that were radiolabeled permitted an assessment of traffic patterns of aliquots that were injected intravenously and directly into the marrow space. The studies reported here document that a one haplotype mismatch is not a sufficient disparity to preclude both B and T cell reconstitution, and that monoclonal antibody plus complement is an effective method for T cell depletion.

Immunologic studies of lymph node lymphocytes in the generalized lymphadenopathy syndrome.

A generalized lymphadenopathy syndrome (GLS) occurs in persons at high risk for development of acquired immunodeficiency syndrome (AIDS). The natural history and immunologic status of patients with GLS are not fully known, although in some persons GLS may progress to full AIDS. We present the clinical and immunologic findings in two children with severe hemophilia A with nonprogressive GLS for 18-24 months. The functional activity in vitro of lymphocytes from both peripheral blood and biopsied lymph nodes were compared. The peripheral blood lymphocytes responded normally to both mitogens and antigens; lymph node lymphocytes failed to respond to antigens, but did respond to mitogens. The implications of these abnormalities for understanding the pathogenesis of GLS are discussed.

Transplantation of HLA-haploidentical T-cell-depleted marrow for leukemia: autologous marrow recovery with specific immune sensitization to donor antigens.

Autologous marrow recovery without engraftment of donor marrow was observed after bone marrow transplantation (BMT) for two patients with acute lymphoblastic leukemia. Each had received marrow from a haploidentical mixed lymphocyte culture (MLC) reactive donor after pretransplant conditioning with total body irradiation and high-dose cyclophosphamide. To minimize graft-vs-host disease, the marrow was depleted of T cells in vitro by treatment with a monoclonal anti-T-cell antibody and complement. Two weeks after each transplant, reactive lymphocytes were noted transiently in the blood of each patient. Analysis of karyotype, HLA type, and in vitro MLC responsiveness proved the lymphocytes to be of host, not donor, origin. MLC studies showed rapid proliferative responses specifically to stimulating cells from the BMT donor, indicating in vivo sensitization to donor antigens. Return of hematopoietic function was markedly delayed, but it eventually normalized after several months, without evidence of chimerism. These studies confirm that some immune and hematopoietic stem cells of host origin survive the high-dose chemoradiotherapy used as transplant conditioning. Because these immune cells are specifically reactive to donor alloantigens, more potent suppression of host immunity may be needed to prevent nonengraftment of T-cell-depleted, HLA-mismatched bone marrow.

T-lymphoblasts with erythropoietic helper function in acute T-cell leukemia.

A patient with acute T-lymphoblastic leukemia was found to maintain a normal hemoglobin concentration both at presentation and preterminally several months later, despite a replaced bone marrow and over 80% circulating lymphoblasts on both occasions. Cell surface marker analysis demonstrated the T-lymphoblasts both at presentation and preterminally to belong to the T-helper subpopulation. In vitro culture studies demonstrated that the patient's T-lymphoblasts, as well as conditioned medium derived from these lymphoblasts, significantly stimulated normal bone marrow erythroid colony growth (CFU-E). These findings suggest that in this patient the preservation of erythropoiesis resulted from a helper effect exerted by his T-lymphoblasts.

Relapse of host leukemic lymphoblasts following engraftment by an HLA-mismatched marrow transplant: mechanisms of escape from the "graft versus leukemia" effect.

Leukemic relapses and graft versus host disease (GvHD) remain major complications following allogeneic bone marrow transplantation for leukemia. We present clinical and laboratory details for an eight-year-old boy who received a T-cell-depleted HLA-mismatched marrow transplant as therapy for acute lymphoblastic leukemia (ALL) in second remission. Engraftment with donor marrow was prompt and without any acute GvHD. Nevertheless, the patient's original ALL recurred and proved fatal. The patient remained a chimera with persistent donor lymphocytes present at the time of posttransplant relapse and subsequent to treatment with unsuccessful reinduction chemotherapy. In vitro immune studies showed that these leukemic cells could be recognized and destroyed by the donor's lymphocytes. The relapse itself suggests, however, that the donor's lymphocytes did not effectively destroy the patient's histoincompatible ALL cells in vivo following establishment of the chimeric state. Potential mechanisms are presented to account for this presumed "escape" from the postulated "graft versus leukemia" effect.

HLA typing and karyotyping of limited numbers of lymphocytes is facilitated by in vitro expansion with TCGF.

It is often difficult to obtain sufficient quantities of leukocytes from bone marrow (BM) transplant recipients for post-transplant immunological monitoring and evaluation of engraftment. We have developed a cell culture expansion technique that allows HLA-A,B, and DR typing as well as karyotyping on limited numbers (less than 10(7)) of lymphocytes. Lymphocytes from healthy control donors were allo-activated in vitro and further expanded by culturing in T-cell growth factor (TCGF). After 14 days of culture, these cells were sent for blind HLA-A,B and DR typing and for karyotyping. Surface marker studies showed that 90% of the T-cells expanded in growth factor are OK-la positive. HLA typing with these DR-bearing in vitro expanded T-cells showed excellent correlation with HLA typing on fresh unexpanded lymphocytes. Karyotyping of expanded T-cells showed normal male or female cells as expected. This technique may be especially valuable when testing pediatric or adult patients with very low lymphocyte counts.

Autoimmune hemolytic anemia as a manifestation of T-suppressor-cell deficiency.

This report describes two children in whom autoimmune hemolytic anemia was the initial clinical manifestation of an underlying T-cell deficiency. Further investigation revealed a profound deficiency of T suppressor cells in both children as detected by monoclonal T-cell antibody (OKT8) and/or functional assays. In vitro incubation of their lymphocytes with cultured thymus epithelium or thymic factors induced T suppressor cells. In vivo treatment with cultured thymus epithelium or calf thymosin fraction 5 resulted in increased T-suppressor-cell numbers and/or function in both and possibly decreased hemolytic activity in one. These results suggest that the autoimmune process in some patients with autoimmune hemolytic anemia is associated with T-suppressor-cell deficiency and that in vivo therapy with agents that modulate T-cell function may be of therapeutic value.

Leukemia presenting as central nervous disease without bone marrow involvement.

A case of a nine-year-old boy who presented with central nervous system leukemia is described. This case was unusual because he presented without initial bone marrow involvement. Bone marrow involvement was documented 15 months after central nervous disease was diagnosed. Immunologic marker studies revealed that the spinal fluid blasts lacked definable B-cell, pre-B cell, T-cell, and pre-T cell surface markers. The marker studies helped to define the nature of the disease process.

T-cell cloning to detect the mutant 6-thioguanine-resistant lymphocytes present in human peripheral blood.

Rare thioguanine-resistant T lymphocytes, present in vivo in human peripheral blood, were isolated and grown in vitro as thioguanine-resistant cultured T cells. The conditions for their selection in vitro were such that thioguanine resistance had to have arisen in vivo. The mutant cells bore T-cell surface markers, maintained their thioguanine resistance in vitro in the presence or absence of selection, and were deficient in hypoxanthine-guanine phosphoribosyltransferase activity.

Lymphocyte dysfunction in congenital hypoplastic anemia.

Congenital hypoplastic anemia (Diamond-Blackfan syndrome) is thought to involve the erythropoietic cell line alone. In this study, the evaluation of lymphocyte function in five patients with this syndrome revealed a number of abnormalities. Peripheral blood T lymphocyte percentages as assessed by monoclonal antibodies were decreased in three patients. T-helper/T-suppressor cell (OKT4:OKT8) ratios were almost unity in four of the five patients. We usually find a ratio of 2:1 in normal populations. Studies of lymphocyte-mediated suppression of lymphoproliferation demonstrated an inability to generate concanavalin A-induced suppressor cells in the same four patients and impaired prostaglandin-mediated suppression in two patients. Co-culture studies revealed a T lymphocyte-mediated suppression of erythropoiesis in a single patient, who also showed suppression of the mixed lymphocyte reaction. The four remaining patients showed no excessive suppressor effects either upon erythropoiesis or lymphoproliferation. These studies demonstrate that in congenital hypoplastic anemia, the cellular defect is not restricted to the erythroid progenitor cells, but extends to the lymphocytes.