Sheep Red Blood Cell Rosetting Research Articles

The Medawar Prize Acceptance Speech 2022.

The Medawar Prize Acceptance Speech 2022.

Haploidentical hematopoietic stem cell transplantation can lead to viral clearance in severe combined immunodeficiency

Severe combined immunodeficiencies (SCIDs) are inherited T lymphocyte differentiation defects,1 conventionally treated with hematopoietic stem cell transplantation (HSCT). For patients without an HLA-identical donor, T-lymphocyte–depleted (TCD) hematopoietic stem cell (HSC) transplantation from an HLA-mismatched donor is successful, with 75% to 87% survival.2,3 B-lymphocyte depletion reduces EBV-related lymphoproliferation. Various historical methods remove viable T lymphocytes, including in vitro anti-CD52 (CAMPATH-1M) antilymphocyte antibody, soy lectin, and sheep red blood cell rosetting.

Ex Vivo T Cell–Depleted versus Unmodified Allografts in Patients with Acute Myeloid Leukemia in First Complete Remission

This study was conducted to retrospectively compare the clinical outcomes after transplantation of T cell–depleted (TCD) and unmodified allografts in patients with acute myeloid leukemia (AML) in first complete remission (CR1). Patients received TCD grafts at Memorial Sloan-Kettering Cancer Center (MSKCC, N = 115) between 2001 and 2010 using the following preparative regimens: hyperfractionated total body irradiation (HFTBI)+thiotepa+fludarabine; HFTBI+thiotepa+cyclophosphamide; or i.v. busulfan+melphalan+fludarabine. TCD was performed by 1 of 2 immunomagnetic CD34+ cell selection methods for peripheral blood grafts or by soybean lectin agglutination followed by sheep red blood cell–rosette depletion for bone marrow grafts. No additional graft-versus-host disease (GVHD) prophylaxis was administered. Patients received unmodified grafts at M.D. Anderson Cancer Center (MDACC, N = 181) after conditioning with busulfan+fludarabine and GVHD prophylaxis with tacrolimus+mini-methotrexate. Patients with unrelated or human leukocyte antigen–mismatched donors received anti-thymocyte globulin (ATG) at both centers, with some recipients of matched related donor TCD transplants also receiving ATG, depending upon the preparative regimen. TCD graft recipients were more likely to be older, receive a mismatched transplant, and have peripheral blood used as the graft source. The incidences rates of grades 2 to 4 acute GVHD and chronic GVHD were significantly lower in the TCD graft group (5% versus 18%, and 13% versus 53%). Three-year relapse-free and overall survival rates were 58% and 57%, respectively, in recipients of TCD grafts, and 60% and 66% in recipients of unmodified grafts (P = not significant). Survival and relapse-free survival are similar after TCD and conventional transplants from related/unrelated donors in patients with AML in CR1, but TCD significantly reduces GVHD.

A novel method of CD34+ cell separation from umbilical cord blood

Umbilical cord blood (UCB) is rich in the heavily glycosylated CD34 antigen-bearing hematopoietic stem cells that are valuable for transplantation therapy of malignant and nonmalignant disease. CD34+ cell yields (0.13%-0.25%-0.3%) of mononuclear cells (UCMCs) isolated by anti-CD34 monoclonal antibody (MoAb) on immunomagnetic particles (e.g., Miltenyi particles) are insufficient to treat adults. We fractionated UCMCs by physicochemical charge-based methods. Avoiding Miltenyi particles and HESPAN yielded 30 x 10(6) to 54 x 10(6) UCMCs/20 mL UCB by Ficoll-Isopaque for serial depletion fractionation, using nylon wool column (NWC) or direct rosetting with sheep red blood cells (SRBCs) without incubation in the cold. CD34+ cell yields (approx. 5.12%) were 39 times greater than 0.13% (Korean study, 11,098 UCB units) and 10 to 20 times greater than 0.25% to 0.3% harvested by anti-CD34 Miltenyi particles. SRBC depletion of most high-specific-gravity T cells achieved considerable enrichment of CD34+ and BY55+ cells. Using NWC achieved 2.5-fold enrichment of CD34+ cells and twofold enrichment of BY55+ cells. Direct SRBC rosetting provided better or higher enrichment of CD34+ cells. Overall CD34+ cell yield in low-density fraction was more than twice after direct rosetting (38% vs. 16%) in contrast to separation by NWC followed by SRBC rosetting. CD3+ cell yields (by three CD markers) were approximately 8.83%, far below approximately 30 x 10(7) /kg considered acceptable to avoid graft-versus-host disease. Natural killer cell yields (CD16+/CD56+ and BY55+/ CD160+) are in perfect agreement. Achieving approximately 5% CD34+ cell yields from single UCB donations, a major advance, holds great promise for CD34+ cell therapy of adults and larger children, and cheaper cultured RBC manufacture.

T-Cell Depleted (TCD) Hematopoietic Stem Cell (HSC) Boosts From the Original Donor without Conditioning for Poor Graft Function (PGF) Following Allogeneic Hematopoietic Stem Cell Transplants (HSCT)

Abstract 1942▪▪This icon denotes a clinically relevant abstract BACKGROUND:Poor graft function without immune rejection, defined as persistent cytopenias with a hypocellular marrow and full donor chimerism, is a life-threatening complication after allogeneic HSCT. Treatment options include supportive therapy with transfusions and growth factors, vs administration of additional HSCs from the same donor without any conditioning (stem cell boost). The use of unmanipulated boosts increases the risk of development or worsening of GvHD by virtue of infusing mature T cells. The aim of this retrospective study was to evaluate the efficacy and safety of TCD boosts in patients with PGF.PATIENT AND METHODS: Between January 1992 and December 2007, 35 patients from a single center with PGF following either an unmanipulated (10) or a TCD (25) allogeneic HCST received a TCD HSC boost collected from the original donor. T cells were removed ex-vivo from marrow grafts with soybean lectin agglutinin and sheep red blood cells (sRBC) rosetting and from peripheral blood stem cell grafts by positive selection with a CD34 antibody (Isolex) followed by sRBC rosetting. HSC donors were matched related (21), mismatched related (5) and unrelated (9). Indication for first transplant included: aplastic anemia (2), non-Hodgkin's lymphoma (5) and myeloid malignancies (28). The preparative regimen was myeloablative for all recipients of unmanipulated grafts and for 22 of 25 recipients of TCD grafts. With the exception of one patient, the cell dose as measured by total nucleated cell dose/kg or CD34 cells/kg was adequate. Following the initial transplant, all patients had partial or complete recovery of blood counts and full donor chimerism documented by karyotype, FISH analysis or DNA polymorphism.The median time from first transplant to the diagnosis of PGF was 4.5 months (0.72-90.7). Etiologies of PGF included: viral infection and anti-virals (10), bacterial sepsis (3), Mycobacterial infection (4), low cell dose (1), GvHD 12, and idiopathic (5). None of the patients had evidence of relapse or progression of their underlying disease. All patients received a TCD boost from the original donor; with 21 patients receiving bone marrow and 14 receiving PB HSC. RESULTS:Seven patients died before day 21 post boost and were not evaluable for blood count improvement. Improvement (PLT>50,000/μL, ANC>500/μL) occurred in 20 of the 28 evaluable patients (71.4 %) at a median time of 3.2 months; 17 of them (60.7%) had a more substantial improvement (PLT ≥100,000/μL and ANC31000/μL) at a median of 8.81 months. TCD boost infusions were well tolerated with no significant adverse events. Also, no new onset GvHD occurred after TCD boosts; although two patients with preexisting GvHD flared. The median survival for all patients following TCD boosts was 21.47 months (range: 1.84–208.45). The 2-year and 5-year survivals were 48.6% and 37.1% respectively. The 2-year survival for patients who had improvement of their counts was 90% and for those who remained pancytopenic despite the boost was 18%. The 2-year survival according to etiology was 52.9% for the infection group, 50% for the idiopathic and low cell dose, and 33.3% for the GvHD group. Patients were also analyzed according to their medical condition prior to receiving the boost and were separated into four groups based on organ function (serum creatinine ≥ 2; total bilirubin ≥ 2; need for mechanical ventilation) and infection status. Group A: outpatient with no infection and no organ dysfunction (9); group B: hospitalized but afebrile with no infection or organ dysfunction (7); group C: febrile or documented infection but preserved organ function (8); group D: organ dysfunction with or without a concurrent infection (11). The 2-year survival for each group was 55.6%, 85.7%, 50%, and 9%, respectively. Patients with organ dysfunction with or without concurrent infection had the lowest survival. Causes of death included: GvHD (10), infection (5), relapse (4), organ failure (3), and poor graft function (1). CONCLUSION:Treatment of PGF with a TCD HSC boost from the original donor is safe and effective with minimal risk of GvHD. Medical status at the time of the boost infusion had a significant impact on outcome. A TCD boost should be considered early in the course of PGF as once complications of persistently low blood counts occur the potential for benefit sharply declines. Disclosures:Small:Pfizer, Inc: Equity Ownership, family member employed by Pfizer, Inc. Perales:Pfizer, Inc: Equity Ownership.

T-Cell Depletion Is An Alternative Approach for Patients >/= 55 Years Undergoing Allogeneic Stem Cell Transplantation as Curative Therapy for Hematologic Diseases

AbstractAbstract 1338Despite the risk of allogeneic hematopoietic stem cell (HSC) transplantation compared to chemotherapy, it is the only potentially curative therapy for many hematologic diseases. As a consequence, the medical community constantly strives to make this treatment modality available to an expanding array of patients (pts). Although the median age for development of many hematologic malignancies is 50–70 yrs, until the last 10 yrs publications on allogeneic transplantation in adult pts >50yrs and especially those > 65 were limited. The eligibility of such pts for transplant has been tempered by reports of increased toxicity to myeloablative conditioning regimens and higher rates of graft versus host disease (GvHD). The majority of these pts received grafts from matched related donors. The growing population of senior adults in the civilized world and the rising cost of healthcare have focused new attention on these groups. T cell depletion (TCD) of HSC grafts is a modality which has been shown to reduce the incidence and severity of GvHD. It has also made transplantation an option for older pts, who require an unrelated donor, for whom the risk of GvHD is even higher. We retrospectively reviewed a 15 year experience using TCD HSC transplants for pts > 55 years with hematologic malignancies at Memorial Sloan-Kettering Cancer Center (MSKCC). Data collection, which was by medical record and laboratory database review, and analysis were performed with the approval of the Institutional Review Board (IRB) of MSKCC. Protected health information was coded in accordance with requirements of Health Insurance Portability and Accountability Act. From February1995 thru February, 2010,151 pts were identified with only 25 pts transplanted prior to 2000. All patients had provided informed consent for treatment on an IRB approved protocol or a treatment plan. A diagnosis of acute leukemia (AML-29 primary, -48 secondary; ALL-7; ABL-3); CML/MPD (16); MDS (35) or NHL/MM/other (11) was pathologically confirmed at MSKCC prior to transplant. The majority of pts with AML and ALL were in a remission at the time of transplant. Median age was 60.5 yrs (range 55–72). Fifty-five percent were CMV+ pretransplant. Donors were matched (69) and mismatched (5) related, and matched (47) and mismatched (29) unrelated. Mismatched donors were > 8/10 HLA matched. The HCT comorbidity index scoring for the pts was 10% low, 35% intermediate and 55% high. The conditioning regimen used in 90/151 pts was busulfan (8-9.6 mg/kg), melphalan (140mg/m2) and fludarabine (reduced intensity), and 59/151 received a myeloablative TBI-containing regimen. Growth factor mobilization of peripheral blood stem cells (PBSC) provided 81% of the grafts, bone marrow (BM) was the source for 12% and 7% of pts received BM + PBSC. PBSC initially underwent CD34+ selection and BM was treated by soybean agglutination, and both were further depleted by sheep red blood cell rosetting. None of the pts received any posttransplant drug prophylaxis for GvHD. With a median follow up of 12.3 mos. (range 0.4–160.3), Kaplan Meier analysis for the OS and the DFS are 48.6% and 38.3% at two yrs, respectively. The 100d non relapse mortality was 11%. There were 35 relapses, 19 related and 16 unrelated. The incidence of acute (gr 2–4) and chronic GVHD was 15% and 10% (5% extensive), respectively. Deaths were almost equally divided between relapse, infection and GVHD. Notably, for the 27 pts > 65 yrs, with a median followup of 12.9 mos., all of the outcomes were similar to those of the entire group. Acknowledging the limitations of a retrospective review, this study represents one of the largest single center reports for pts > 55 yrs. It demonstrates that TCD HSC transplantation affords a curative therapy with minimal GVHD despite age and lack of a related donor. It provides support for continued investigation of TCD as an alternative approach to transplantation in this older population especially as the technology for TCD has been simplified and supportive care has improved. Disclosures:No relevant conflicts of interest to declare.

E rosette-forming lymphocytes in multiple sclerosis patients. Basic protein stimulation of rosette-forming cells

"Total" and "active" T cells were enumerated using sheep red blood cell (SRBC) rosetting technique. The percentages of "total" and "active" T lymphocytes were not significantly different in multiple sclerosis patients as compared to other neurological disorders and/or healthy controls. However, the number of "avid" T cells binding more than three SRBC was significantly increased in MS patients. Active E rosette test has shown hypersensitization to myelin basic protein in 50% MS patients and 31% OND patients. All healthy controls studied, except one had negative response.

Induction of Remission Prior to Transplantation Improves the Disease-Free Survival (DFS) of Patients with Advanced Myelodysplastic Syndromes (MDS) Treated with Myeloablative T - Cell Depleted (TCD) Stem Cell Transplants (SCT) from Histocompatible Unrelated Donors.

Disease relapse and transplant-related mortality (TRM) have been the two major problems limiting the success of unrelated donor SCT in patients with advanced MDS (RAEB 1 and 2 and AML evolved from MDS). Induction of remission is an approach that has been used to reduce the incidence of relapse, but its use remains controversial. T cell depletion of allografts has been used to reduce the incidence of graft-versus-host disease and TRM. From 1989 to 2004, 28 patients with advanced MDS underwent bone marrow or peripheral blood SCT from molecularly matched (HLA A, B, C, DRB1 and DQB1 - 18 patients) or partially mismatched (maximum 2 antigen mismatch allowed - 10 patients) volunteer donors following conditioning with myeloablative conditioning with total body irradiation (1375 cGy), thiotepa (10 mg/kg) and cyclophosphamide (120 mg/kg) or fludarabine 125 mg/kg) - 17 patients, or busulfan (8 mg/kg), melphalan (140 mg/kg) and fludarabine (125 mg/kg) - 11 patients. Twenty six patients received chemotherapy (2 low dose and 24 induction doses) prior to conditioning, and 2 patients did not receive any chemotherapy. Prior to transplant, 18 of the 28 treated patients were in hematologic remission (CR), 2 in a second refractory cytopenia phase (RCy2) (20 responders), and 6 had failed to achieve remission or their MDS had relapsed. The marrow grafts were depleted of T cells using the soybean agglutinin method and then sheep red blood cell rosetting (15 patients) and the G-CSF-mobilized peripheral blood stem cell (PBSC) grafts with CD34 selection and E-rosetting (13 patients). Rejection prophylaxis with anti-thymocyte globulin was used in all 28 patients. Posttransplant pharmacologic prophylaxis for GvHD was given only to 1 patient. The median age was 49.9 years (range 4–59.6), 14 patients were ≥ age 50. Two patients died before engraftment and 26 engrafted; 2 of them had late graft failure. Eight patients developed acute GvHD (grades 2 to 4) and 2 chronic GvHD (1 limited). The DFS in these three group of patients was significantly different (p= 0.0004). The DFS at 5 years was 55% for the patients in CR and RCy2, 33% for the failures, and no patients were alive in the untreated group. DFS was worse in patients with high risk IPSS. DFS was slightly better in patients cytoreduced with the busulfan-containing regimen, 72% versus 29% (p = 018). DFS was not statistically different in the recipients of matched (39%) or mismatched (60%) transplants. The cumulative incidence (CI) of relapse at 2-years posttransplant for the responders was 5.5%, for the failures 25%, and for the untreated group 50%. The CI of non-relapse mortality or TRM at 2-years posttransplant for the responders was 33.33%, for the failures was 62.5%, and for the untreated was 50%. All survivors have achieved ≥KPS 90%. These observations confirm the value of induction of remission prior to conditioning as a means to reduce the incidence of disease relapse in the posttransplant period and show that T cell depletion decreases the TRM in these patients receiving unrelated stem cell transplants.

Results of T Cell Depleted (TCD) Myeloablative Hematopoietic Stem Cell Transplants (HSCT) in Patients with Hematologic Malignancies ≥ 55 yrs of Age.

Allogeneic HSCT remains the only curative therapy for many patients(pts) with hematologic diseases. Studies have suggested that older pts experience greater toxicity from the intensive chemo-radiotherapy used in myeloablative conditioning regimens. As a consequence, many older pts are now offered non-myeloablative transplants (NMAT) for malignant conditions where a graft vs. tumor effect (GvT) is expected to provide the antitumor effect in place of the chemo-radiotherapy. Unfortunately, graft vs. host disease (GvHD) remains a common occurrence after conventional transplants, occurring more frequently in older pts and unrelated donor transplant recipients, resulting in significant morbidity and mortality. Furthermore, the efficacy of NMAT is limited by the disease status at time of transplant and by the susceptibility of the hematologic disease to a GvT effect. TCD of hematopoietic stem cell grafts offers an alternative to older pts, in particular those requiring unrelated donor transplants (URD), with the advantage of a reduced incidence of GvHD. From 1995–2005, 57 patients ≥55 yrs received myeloablative TCD transplants at our institution. The median age was 58.2 (range 55–69.2) yrs. Stem cell sources were TCD bone marrow, PBSC or both. Thirty-seven received transplants from related donors (RD), including two mismatched, and 20 received transplants from unrelated donors (URD), 9 of whom were mismatched. In addition to their advanced age, many of these pts were considered high risk based on the status of disease, HLA mismatch, and history of previous therapy. Twenty-three pts were considered “good risk” by disease status (CML-CP1, AML-CR1, CR2) and 34 pts were considered poor risk (>CML-CP1, >AML CR2, MDS, NHL, >ALL CR1, ABL.) BM was TCD by soybean agglutination followed by sheep red blood cell rosetting (E), and PBSCs by CD34+ selection and E-rosetting. Conditioning regimens included total body irradiation (TBI) in addition to thiotepa and cyclophosphamide, or thiotepa and fludarabine. The non-TBI preparative regimen consisted of busulphan, melphalan and fludarabine. Anti-thymocyte globulin was used as rejection prophylaxis for all TCD transplants until 2001 when it was eliminated from the TBI containing regimen for matched RD transplants. A total of 25 pts (15 matched RD and 10 URD, 6 of whom were mismatched) are alive following TCD transplants with a median follow up of 24 mos. for RDs and 12 for URDs. Of the survivors, 2/10 URD and 14/15 RD recipients received TBI containing regimens based on the triage system at our center. Three pts with CML-CP1, one with CML-acc and one with AML-CR1 showed evidence of minimal residual disease, received donor leukocyte infusions and subsequently achieved longterm continued CR. The incidence of post-transplant GvHD was low despite the high number of mismatched URD transplants - 1 Grade IV (RD), 2 Grade 3 and 1 Grade 1 (URD). The 100 day mortality was 15%. Overall and current disease free survival for ‘good risk' patients based on disease status is 58% for RD and 60% for URD. Although longer follow up is necessary to confirm these results, the promising DFS rates in association with a low incidence of GvHD in this older and relatively high risk patient population support further investigation of myeloablative TCD HSCT in these patients.

Ex vivo isolation protocols differentially affect the phenotype of human CD4+ T cells

Leukemic T cell lines have facilitated signal transduction studies but their physiological relevance is restricted. The use of primary T lymphocytes overcomes this limitation but it has long been speculated that methodological aspects of blood collection and the isolation procedure modify the phenotype of the cell. Here we demonstrate that several characteristics of human peripheral T cells are affected by the selection conditions. A significantly higher induction of the chemokine receptor CXCR4 was observed on CD4+ lymphocytes isolated by sheep red blood cell (SRBC) rosetting and CD4 MicroBeads as compared with positively selected CD4+ cells where the antibody/bead complex was immediately detached. These latter cells expressed CXCR4 at levels equivalent to that observed on CD4+ lymphocytes obtained by negative antibody-mediated selection. Furthermore, CD4+ cells isolated by SRBC rosetting and CD4 MicroBeads formed aggregates upon in vitro culture. CD4+ lymphocytes obtained by SRBC rosetting as well as those isolated following positive selection demonstrated basal phosphorylation of the extracellular signal-regulated kinase (ERK)-2. Altogether these data suggest that certain discrepancies concerning signal transduction in primary human T cells can be attributed to the selection conditions. Thus, it is essential to establish the parameters influenced by the isolation protocol in order to fully interpret T cell responses to antigens, chemokines, and cytokines.

Functional Uncoupling of T-cell Receptor Engagement and Lck Activation in Anergic Human Thymic CD4+T Cells

Human thymic CD1a-CD4+ T cells in the final stage of thymic maturation are susceptible to anergy induced by a superantigen, toxic shock syndrome toxin-1 (TSST-1). Thymic CD4+ T-cell blasts, established by stimulating human thymic CD1a-CD4+ T cells with TSST-1 in vitro, produce a low level of interleukin-2 after restimulation with TSST-1, whereas TSST-1-induced adult peripheral blood (APB) CD4+ T-cell blasts produce high levels of interleukin-2. The extent of tyrosine phosphorylation of the T-cell receptor zeta chain induced after restimulation with TSST-1 was 2-4-fold higher in APB CD4+ T-cell blasts than in thymic CD4+ T-cell blasts. The tyrosine kinase activity of Lck was low in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, the Lck kinase activity increased in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Surprisingly, Lck was highly tyrosine-phosphorylated in both thymic and APB CD4+ T-cell blasts before restimulation with TSST-1. After restimulation, it was markedly dephosphorylated in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Lck from APB CD4+ T-cell blasts bound the peptide containing the phosphotyrosine at the negative regulatory site of Lck-505 indicating that the site of dephosphorylation in TSST-1-activated T-cell blasts is Tyr-505. Confocal microscopy demonstrated that colocalization of Lck and CD45 was induced after restimulation with TSST-1 in APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. Further, remarkable accumulation of Lck in the membrane raft was observed in restimulated APB CD4+ T-cell blasts but not in thymic CD4+ T-cell blasts. These data indicate that interaction between Lck and CD45 is suppressed physically in thymic CD4+ T-cell blasts and plays a critical role in sustaining an anergic state.

Mechanisms of cell-mediated myocytotoxicity in the peripheral blood of patients with inflammatory myopathies.

Cell-mediated cytotoxicity is thought to play an important role in the immunopathogenesis of inflammatory myopathies. We determined whether lymphocytes circulating in patient peripheral blood contain automyocytotoxic precursors. Peripheral blood mononuclear cells, sheep red blood cell rosetting (E+) and nonrosetting (E-) cells, were isolated from patients with polymyositis or dermatomyositis and tested for their ability to kill autologous-cultured myotubes derived from diseased muscle biopsies. Patient-derived as well as normal allogeneic mononuclear cells lysed both polymyositis-dermatomyositis and nonrelated myotubes. However, whereas patient E+ cells were preferentially cytotoxic to autologous myotubes, their E-cells did not discriminate autologous from allogeneic targets. Furthermore, cultures of patient E+ cells triggered by phytohemagglutinin and interleukin-2 maintained myocytotoxic potential. In these cultures, virtually all autologous but only 50% of allogeneic killing was mediated by CD3+ T cells. Moreover, autologous cell-mediated killing was abrogated by anti-CD3 monoclonal antibodies. In conclusion, both myocytotoxic CD3+ T-cell clones specific for autologous myotubes, as well as non-T cells, which are nonspecifically myocytotoxic, are present in the peripheral blood of patients with inflammatory myopathies.

Purification of functional T lymphocytes from splenocytes of the beluga whales ( Delphinapterus leucas)

In an effort to gain knowledge on immune functions in beluga whales, Delphinapterus leucas, we have used two physical methods for the purification of T lymphocytes of spleen cells. Isolation by sheep red blood cells (SRBC) rosetting and by adherence on nylon wool columns were tested. SRBC-rosetting gave unreliable results in obtaining purified T cells. Therefore, the purification of T cells was done using nylon wool columns. Less than 3% of the IgM + B cells remained in effluent populations. In the later population, 45% gave positive staining with mouse anti-human CD4 allowing us to verify functionality of the cells. The study of calcium mobilization and tyrosine kinase activation, mediated by CD4 cross-linking permitted verification of the functionality of cells. We also showed that upon activation with mitogens, beluga T cells upregulate the density of MHC class II molecules on their surfaces. CD4 cross-linking with a specific antibody inhibited the proliferation response. Overall, the activation of beluga whales lymphocytes did not differ markedly from what is known in other species. This study can help in the groundwork for functional investigation of the beluga whale’s immune system.

Population dynamics of human activated natural killer cells in culture

The growth kinetics and population dynamics of recombinent interleukin-2 (rlL-2) stimulated human natural killer (NK) cell-enriched populations were studied in vitro. The NK-enriched populations was obtained from normal peripheral blood mononuclear cells (PBMNC) by immunomagnetic bead depletion of CD3(+) and CD5(+) T cells. The growth kinetics of NK cells, T cells, monocytes, and total cells are shown. In the absence of PBMNC accessory cells, the NK-enriched population showed limited expansion. In the presence of PBMNC accessory cells, the NK-enriched population expanded threefold more than in the absence of accessory cells due to increased NK cell growth rate and increased duration of exponential growth. Using a Transwell system, which separates two cell population by a polycarbonate membrane, the accessory cells were shown to act on the NK-enriched population via a diffusible factor. Accessory cell conditioned media was able to replace the accessory cell population to stimulate NK cell expansion. A monocyte-enriched population prepared by sheep red blood cell rosetting of T cells was extensively phenotyped and compared with the NK-enriched populations. Although the final cultured cells were phenotypically homogeneous for CD56(+)/CD3(-) NK cells, the initial NK precusor populations appear to be different. Namely, the NK cell precursors in the monocyte-enriched population were predominantly CD56(+)/CD2(-). Kinetic equations were formulated for this culture system and the effects of major culture variables are investigated.

Activation of HLA‐DR and Interleukin‐6 Gene Transcription in Resting T Cells via the CD2 Molecule: Relevance to Chronic Immune‐Mediated Inflammation

Only a minority of T cells at cell-mediated immune lesions are antigen specific. In the lesions of human autoimmune disease, such as the synovial membrane in rheumatoid arthritis, the T cells are activated as shown by a variety of phenotypic and functional changes including the expression of HLA-DR and the production of interleukin-6 (IL-6). The stimulatory pathway involved is unknown but does not seem to involve the T-cell receptor. Alternative pathways of activation which may be involved include the CD2 molecule. It is shown that the formation of sheep red blood cell (SRBC) rosettes with resting T cells from human peripheral blood, which is equivalent to CD2/LFA-3 binding, leads to the de novo transcription of the HLA-DR and IL-6 genes and the expression of HLA-DR on the surface of the T cells. There was no transcription of the interleukin-2 (IL-2) or the interleukin-2 receptor (IL-2R) genes and Tac expression was not seen. The rosetted T cells did not proliferate. These are all characteristics of T cells at chronic inflammatory sites. It is concluded that receptor-ligand interactions between CD2/LFA-3, which are expressed in increased amounts in the rheumatoid joint, may be one pathway by which antigen non-specific T cells are recruited as effector cells in lesions of human autoimmune disease.

Induction of HLA-DR Expression in Endometrial Epithelial Cells by Endometrial T-Cells: Potential Regulatory Role of Endometrial T-Cellsin Vivo

Endometrial epithelial cells express HLA-DR molecules of the major histocompatibility complex in vivo adjacent to aggregates of T-cells in the endometrial stroma. To test whether HLA-DR expression on endometrial epithelium is mediated by T-cells, endometrial T-cells were isolated from human endometrium by the sheep red blood cell rosetting technique. In contrast to the resting T-cells from peripheral blood and similar to the peripheral blood T-cells activated with Concanavalin-A, endometrial T-cells formed colonies in vitro. Direct addition of the endometrial T-cells to epithelial cell cultures derived from autologous glands induced both morphological changes as well as HLA-DR molecules in the epithelial cells. The intensity of the immunostained HLA-DR molecules in the epithelial cells as well as the percentages of the HLA-DR-positive epithelial cells correlated with the number of T-cells added to the epithelial cell cultures. T-Cells were bound to the HLA-DR-positive epithelial cells as single cells or aggregates, and they were not found bound to the HLA-DR-negative epithelial cells. The supernatant of the endometrial T-cells induced expression of HLA-DR molecules and morphological changes in cultures of HLA-DR-negative epithelial cells. This expression could be inhibited by a neutralizing antiserum to interferon-gamma. These findings are consistent with the hypothesis that endometrial T-cells are activated and suggest that the expression of HLA-DR molecules in glandular epithelium in vivo is mediated by the interferon-gamma secreted by the endometrial T-cells.

Identification of a Secretory IgA Receptor on Breast-Milk Macrophages: Evidence for Specific Activation Via These Receptors

Binding of secretory IgA (sIgA) to human milk macrophages was identified by rosette formation with sIgA-sensitized indicator cells and competitive inhibition binding studies with [125I]-sIgA. Macrophages formed rosettes with sIgA-sensitized sheep red blood cells that were inhibited by sIgA (87%) but not IgG. Both IgA1 and IgA2 inhibited sIgA-sensitized sheep red blood cell rosette formation. Rosette formation was completely inhibited by galactose, partially inhibited by asialofetuin, and unaffected by mannose. [125I]-labeled sIgA binding to macrophages reached a plateau after 60 min, was dependent on the number of macrophages in culture, and was specifically inhibited by unlabeled sIgA. Incubation of macrophage monolayers with increasing concentrations of sIgA-anti-IgA immune complexes resulted in a progressive increase in the oxidative burst and increased secretion of prostaglandins. These studies indicate that human milk macrophages have a receptor for sIgA and that activation of these macrophages may occur via these receptors.

Immunohistochemical Studies on Ontogeny of Bovine Lymphoid Tissues.

Developing lymphoid tissues of bovine fetuses ranging from 70 to 270 days of fetal age were examined by histological and immunohistochemical procedures. In the peripheral blood, surface membrane immunoglobulin bearing cells (B-lymphocytes) and sheep red blood cell rosette forming cells (T-lymphocytes) had already appeared by 70 days of fetal age. In the lymph nodes intracytoplasmic IgM positive cells appeared at 90 days of fetal age. The cells positive for IgG appeared at 150 days of fetal age and IgA positive cells appeared at 180 days of fetal age. The spleen contained intracytoplasmic immunoglobulin positive cells at almost the same time as those in the lymph nodes. In the ileocecal region, IgM positive cells and IgG positive cells were present at 180 days of fetal age and IgA positive cells were present at 210 days of fetal age. The tonsils contained IgM positive cells and IgG positive cells at 240 days of fetal age. In the thymus, terminal deoxynucleotidyl transferase positive cells appeared at 90 days of fetal age.

Mapping the dextran sulfate binding site on CD2

This study has analysed the binding of a series of anti-CD2 monoclonal antibodies (MoAbs) to T cells in the presence of the sulfated polysaccharide dextran sulfate (2.3 sulfates/monosaccharide, 500 kDa) (DXS) to define the DXS binding site on CD2. The results show that DXS interacts primarily at the T11(2) epitope. Thus five anti-CD2 MoAbs which bound to the T11(2) epitope were inhibited in their binding by DXS. In contrast, seven anti-CD2 MoAbs that totally inhibited sheep red blood cells (SRBC) rosetting (identifying the T11(1) epitope) were unaffected in their binding to T cells in the presence of DXS. Three MoAbs which partially inhibited SRBC rosetting and thereby defining only part of the T11(1) epitope, were also inhibited in their binding by DXS. Consistent with the conclusion that the DXS binding site on CD2 is associated with the T11(2) epitope was the observation that interaction of DXS with CD2 resulted in augmented binding of the four MoAbs defining the T11(3) epitope, possibly reflecting an increased expression of the T11(3) (activation, CD2R) epitope of CD2. Collectively, the data presented support the notion that a natural ligand for the T11(2) epitope of CD2 will be identified as a sulphated carbohydrate structure.

Sheep erythrocyte rosetting induces multiple alterations in T lymphocyte function: Inhibition of T cell receptor activity and stimulation of T11/CD2

When T lymphocytes from human blood or lymphoid organs are prepared by the sheep red blood cell (SRBC) rosetting procedure, glycoproteins of the SRBC membrane interact intimately with the CD2 (T11) molecule on the T cell surface. We now show that rosette formation has measurable short- and long-term effects upon the T cells. First, for a period of 24–48 hr after rosetting, the signal transducing and activation functions of the T3/Ti T cell antigen receptor complex is paralyzed for anti-T3-induced calcium mobilization, with a concomitant decrease in proliferative response to mitogens or stimulatory anti-T3 antibodies. Calcium mobilization through the alternate pathway of T cell activation, the T11/CD2 SRBC receptor, was also inhibited by rosetting. Second, rosetting appears to confer a partial stimulatory signal through the T11/CD2 pathway. Thus, 72 hr or more after rosetting, there was increased expression of the T11 3 activation epitope, and rosetted T cells were stimulated to proliferate in the presence of anti-T11 3 antibodies alone. These results provide further details on the effects of SRBC-T cell interactions, with important methodological implications. Moreover, they suggest a hitherto unrecognized down-regulatory effect of engaging the CD2 molecule, and provide further evidence that the T cell receptor is functionally interconnected to the CD2 activation pathway.