Comparison of two immunomagnetic separation technologies to deplete T cells from human blood samples

The objective of this study was to compare the performance of two immunomagnetic separation technologies to deplete T cells from buffy coats of human blood. Specifically, two versions of the commercial MACS(R) Technology: MiniMACS and SuperMACS, and a prototype, flow-through system, the QMS, were evaluated. Peripheral blood mononuclear leukocytes (PBL) were isolated from buffy coats and an immunomagnetic separation of CD3(+) cells was conducted using company and optimized labeling protocols. To mimic peripheral blood containing bone marrow purged hematopoietic stem cells, HSC, CD34 expressing-cells (KG1a) were spiked into PBL prior to T-cell depletion once optimized depletion conditions were determined. Once the labeling protocol was optimized, the MiniMACS system performed well by producing a highly enriched CD3(+) fraction, and a respectable level of depletion of T cells and recovery of KG1a cells in the depleted fraction; an average log(10) depletion of T cells of 2.88 +/- 0.17 and an average recovery of the KG1a cells of 60.8 +/- 5.94% (n = 14). The performance of the SuperMACS system was very similar with an average log(10) depletion of T cells of 2.89 +/- 0.22 and an average recovery of KG1a of 63.1 +/- 8.55% (n = 10). In contrast, the QMS system produced an average log(10) depletion of T cells of 3.98 +/- 0.33 (n = 16) with a corresponding average recovery of 57.9 +/- 16.6% of the spiked CD34+ cells. The aforementioned QMS performance values were obtained using sorting speeds ranging from 2.5 x 10(4) to 1.7 x 10(5) cells per second. It is suggested that the lack of a 100% recovery of the unlabeled KG1a cells is the result of a previously reported "drafting" phenomena which pulls unlabeled cells in the direction of the magnetically labeled cells thereby resulting in loss of the unlabeled cells.     

Vaccine-induced tumor-specific immunity despite severe B-cell depletion in mantle cell lymphoma

The role of B cells in T-cell priming is unclear, and the effects of B-cell depletion on immune responses to cancer vaccines are unknown. Although results from some mouse models suggest that B cells may inhibit induction of T cell-dependent immunity by competing with antigen-presenting cells for antigens, skewing T helper response toward a T helper 2 profile and/or inducing T-cell tolerance, results from others suggest that B cells are necessary for priming as well as generation of T-cell memory. We assessed immune responses to a well-characterized idiotype vaccine in individuals with severe B-cell depletion but normal T cells after CD20-specific antibody-based chemotherapy of mantle cell lymphoma in first remission. Humoral antigen- and tumor-specific responses were detectable but delayed, and they correlated with peripheral blood B-cell recovery. In contrast, vigorous CD4(+) and CD8(+) antitumor type I T-cell cytokine responses were induced in most individuals in the absence of circulating B cells. Analysis of relapsing tumors showed no mutations or change in expression of target antigen to explain escape from therapy. These results show that severe B-cell depletion does not impair T-cell priming in humans. Based on these results, it is justifiable to administer vaccines in the setting of B-cell depletion; however, vaccine boosts after B-cell recovery may be necessary for optimal humoral responses.     

Development of a technology platform for large-scale clinical grade production of DC

BACKGROUND: Clinical studies require protocols where a sufficient number of well-characterized highly immunogenic DC are produced according to good manufacturing practice (GMP) guidelines. METHODS: In the present study, using leukapheresis products from 10 cancer patients, we validated an elutriation technology for large-scale clinical grade production of monocyte-derived DC. RESULTS: The elutriation method gave a very high purity (mean+/-SD) (86+/-5.3%) and recovery (66+/-10.4%) of monocytes. Specifically for the two monocyte-rich fractions (3 and 4,) the recovery was 42+/-13% of viable cells that could be further differentiated into immature DC in hydrophobic culture bags using GM-CSF and IL-4. The immature DC exhibited<1% CD83+ expression and >98% phagocytic activity. Maturation with TNF-alpha or poly I:C resulted in DC with expression of CD80+, CD86+ and HLA-DR+ (>99%) and CD83+ (80+/-11.9%), as well as producing IL-12p70 and lacking phagocytic activity (<5%). This cell product can be cryopreserved with cell viability >85% and cell recovery >80% after thawing. DISCUSSION: The elutriation procedure, when optimized and if the monocyte content of the starting material exceeds 5%, does not require further selection or depletion using affinity approaches.     

Depletion of T-cell receptor alpha/beta and CD19 positive cells from apheresis products with the CliniMACS device

Background aimsThe CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for depletion of T-cell receptor alpha/beta positive (TCRαβ⁺) and CD19 positive (CD19⁺) cells from apheresis products.MethodsInvestigators performed 102 separations. Apheresis products with a median 5.8 (minimum to maximum, 1.2–10.4) × 10¹⁰ mononuclear cells were used with a median 358 (92–1432) × 10⁶ CD34⁺ cells. There were 24.8% (6.1–45.7%) median TCRαβ⁺ cells and 4.4% (1.2–11.7%) median B cells in the apheresis product.ResultsAfter depletion, a median 0.00097% (0.00025–0.0048%) of TCRαβ⁺ cells could be detected, and B cells, as determined as CD20⁺ cells, were reduced to 0.0072% (0.0008–0.072%). TCRαβ⁺ cells were depleted by log 4.7 (3.8–5.5), and B cells were depleted by log 4.1 (3.0–4.7). Recovery of mononuclear cells was 55% (33–77%), and recovery of CD34⁺ cells was 73% (43–98%). Recovery of CD56⁺/3^? natural killer cells was 80% (35–142%), recovery of TCR gamma/delta positive (TCRγδ⁺) T cells was 83% (39–173%) and recovery of CD14⁺ cells was 79% (22–141%). Viability of cells was 98% (93–99%) after separation (all values median).ConclusionsProfound depletion of TCRαβ⁺ T cells can be achieved with the CliniMACS system. Recovery of CD34⁺ stem cells is in the same range than after CD34⁺ enrichment and CD3/CD19 depletion. Transplantation with >4 × 10⁶ CD34⁺ cells/kg can be performed for every patient with 1–5 × 10⁴ TCRαβ⁺ cells/kg and about 5–10 × 10⁶ TCRγδ⁺ cells/kg with two rounds of apheresis.     

Optimization of methodology for production of CD25/CD71 allodepleted donor T cells for clinical use

Background aimsImmunotherapy with allodepleted donor T cells improves immunity after T cell-depleted hematopoietic stem cell transplantation. We developed a methodology for selective depletion of alloreactive T cells after activation with host antigen-presenting cells by targeting T cells up-regulating CD25 and CD71. Combined depletion of these cells yields a pool of allodepleted donor T cells with antiviral properties with minimal capacity to cause graft-versus-host disease.MethodsMature dendritic cells were irradiated and used to stimulate donor peripheral blood mononuclear cells for 4 days. The co-culture was stained with anti-CD71-biotin followed by CliniMACS CD25 and Anti-Biotin Reagents (Miltenyi Biotec GmbH; Bergisch Gladbach, Germany) before depletion on the CliniMACS Plus (Miltenyi Biotec GmbH). Residual alloreactivity was tested by flow cytometry, a secondary mixed lymphocyte reaction and limiting dilution analysis, and specific anti-viral immunity with pentamer staining. The large-scale protocol was tested under current good manufacturing practice conditions in five donor-recipient pairs of human leukocyte antigen-matched volunteer donors.ResultsWe developed a closed-system methodology using cell differentiation bags for cell culture and the COBE2991 Cell Processor (CaridianBCT, Lakewood, CO, USA). We also validated an anti-CD71-biotin generated for ex vivo clinical use. In five large-scale runs, the depleted fraction demonstrated excellent viability (99.9%), minimal residual expression of CD3/CD25 and CD3/CD71 (<0.2%) and passed tests for Mycoplasma, endotoxin, bacterial and fungal sterility. In secondary mixed lymphocyte reaction assays, the median response to host after allodepletion was 0%, whereas responses to third-party peripheral blood mononuclear cells were preserved (median, 105%; range 37%–350%). Limiting dilution analysis assays also demonstrated a reduction in response to host (median, ?1.11 log) with preservation of third-party responses, and testing with human leukocyte antigen-restricted pentamers showed that populations of Epstein-Barr virus-specific and cytomegalovirus-specific CD8⁺ T cells were retained after depletion.ConclusionsWe optimized a protocol for the combined immunomagnetic depletion of alloreactive CD25/CD71 T cells under current good manufacturing practice conditions and tested the efficacy in five donor-recipient pairs.     

CD2 is expressed by a subpopulation of normal B cells and is frequently present in mature B-cell neoplasms

BACKGROUND: CD2 is expressed by T and natural killer (NK) cells and has been reported in T/NK cell lineage neoplasms as well as in immature B-lymphoblastic and myeloid leukemias. Although CD2+ B-cells have been identified in normal fetal and postnatal thymus, they have not been reported in adults. METHODS: We retrospectively reviewed flow cytometric immunophenotypic data on consecutive low-grade B-cell leukemias and lymphomas to investigate the frequency of CD2 expression. We also reviewed samples from normal healthy donors to determine whether there is a normal CD2+ B-cell population. RESULTS: CD2 expression (partial or complete) was observed in 13 of 83 (16%) chronic lymphocytic leukemias (CLL), 16 of 29 (55%) follicle center lymphomas (FCL), 3 of 12 (25%) hairy cell leukemias (HCL), 0 of 6 mantle cell lymphomas (MCL), 8 of 28 (29%) large cell lymphomas (LCL), and in 0 of 5 marginal zone/mucosa-associated lymphoid tissue lymphomas (MZL/MALT). We determined that 5.74 +/- 2.46% (mean +/- SD) of normal peripheral blood B cells and 6.48 +/- 1.62 % (mean +/- SD) of normal bone marrow B cells coexpress CD2. CONCLUSIONS: CD2 expression in B-cell neoplasia is a more prevalent phenomenon than previously appreciated. Normal CD2+ B-cell populations are observed in adults and may represent the nonmalignant counterpart of CD2+ B-cell neoplasms.     

Cutting edge: Rapid recovery of NKT cells upon institution of highly active antiretroviral therapy for HIV-1 infection

CD1d-restricted NKT cells play important regulatory roles in various immune responses and are rapidly and selectively depleted upon infection with HIV-1. The cause of this selective depletion is incompletely understood, although it is in part due to the high susceptibility of CD4+ NKT cells to direct infection and subsequent cell death by HIV-1. Here, we demonstrate that highly active antiretroviral therapy (HAART) results in the rapid recovery of predominantly CD4(-) NKT cells with kinetics that are strikingly similar to those of mainstream T cells. As it is well known that the early recovery of mainstream T cells in response to HAART is due to their redistribution from tissues to the circulation, our data suggest that the selective depletion of circulating NKT cells is likely due to a combination of cell death and tissue sequestration and indicates that HAART can improve immune functions by reconstituting both conventional T cells and immunoregulatory NKT cells.     

Depletion of CD4+ (L3T4+) lymphocytes in vivo impairs murine host defense to Cryptococcus neoformans

T cell-mediated immunity has been shown to play an important role in the host defense to Cryptococcus neoformans. Infections due to C. neoformans are increased in patients with AIDS who are deficient in the CD4+ subset of T lymphocytes. Thus, the effect of CD4+ (L3T4+) lymphocyte depletion on murine host defenses to C. neoformans was studied. The mAb GK 1.5 was administered to mice, and CD4+ T lymphocyte depletion was confirmed by the analysis of T cell subsets in blood, spleen, lymph node, and lung. Evidence of a functional defect was confirmed by demonstrating that the splenocytes of treated mice were unable to proliferate in response to class II incompatible spleen cells. Furthermore, delayed type hypersensitivity to C. neoformans was abrogated by CD4+ lymphocyte depletion. Mice depleted of CD4+ lymphocytes were inoculated with a virulent strain of C. neoformans by the i.v. or the intratracheal route. After i.v. inoculation of C. neoformans, the survival of mice depleted of CD4+ lymphocytes was reduced (27.8 +/- 1.8 vs 36.0 +/- 3.1 days, p less than 0.04). After intratracheal inoculation, C. neoformans disseminated from the lung to extrapulmonary organs. Dissemination occurred earlier in mice depleted of CD4+ lymphocytes compared to mice that received control antibody, and the burden of C. neoformans in extrapulmonary organs was greater in mice depleted of CD4+ lymphocytes than control mice. Surprisingly, there was no increase in the burden of C. neoformans in the lungs of CD4+ lymphocyte-depleted mice. Survival of mice inoculated with C. neoformans and depleted of CD4+ lymphocytes was reduced compared to control mice and was related to the increased rate of accumulation of organisms in the brains of treated mice. The mean survival of GK 1.5-treated mice was 34.1 +/- 0.9 days compared to control mice with a mean survival of 40.6 +/- 9 days (p less than 0.001). These data suggest that CD4+ lymphocytes play a prominent role in the host defense of infections due to C. neoformans, that CD4+ lymphocytes are required in extrapulmonary organs for optimal clearance of C. neoformans and that CD4+ lymphocytes are critical for survival of mice infected with C. neoformans.     

The dual effects of B cell depletion on antigen-specific T cells in BDC2.5NOD mice

B cells play a critical role in the pathogenesis of autoimmune diabetes. To investigate the mechanisms by which B cell depletion therapy attenuates islet β cell loss and particularly to examine the effect of B cells on both diabetogenic and regulatory Ag-specific T cells, we generated a transgenic BDC2.5NOD mouse expressing human CD20 on B cells. This allowed us to deplete B cells for defined time periods and investigate the effect of B cell depletion on Ag-specific BDC2.5 T cells. We depleted B cells with anti-human CD20 Ab using a multiple injection protocol. We studied two time points, before and after B cell regeneration, to examine the effect on BDC2.5 T cell phenotype and functions that included antigenic response, cytokine profile, diabetogenicity, and suppressive function of regulatory T (T(reg)) cells. We found unexpectedly that B cell depletion induced transient aggressive behavior in BDC2.5 diabetogenic T cells and reduction in T(reg) cell number and function during the depletion period. However, after B cell reconstitution, we found that more regenerated B cells, particularly in the CD1d(-) fraction, expressed immune regulatory function. Our results suggest that the regenerated B cells are likely to be responsible for the therapeutic effect after B cell depletion. Our preclinical study also provides direct evidence that B cells regulate both pathogenic and T(reg) cell function, and this knowledge could explain the increased T cell responses to islet Ag after rituximab therapy in diabetic patients in a recent report and will be useful in design of future clinical protocols.     

A novel high-yield volume-reduction method for the cryopreservation of UC blood units

BACKGROUND: For the application of umbilical cord blood (UCB) units as hematopoietic grafts, a dose of 3.7 x 10(7) nucleated cells (NC)/kg body weight is required. NC can be lost during volume-reduction processing and during thawing. A novel modification of the double-processing protocol with the aim of minimizing NC loss is described and evaluated. METHODS: One-hundred and fifty UCB were collected. The volume was reduced by a centrifugation step following double-processing in the presence of 2% HES 200/0.5. Pre- and post-processing cell counts and platelet parameters were measured with an automatic counter. The number of viable CD34+ hemopoietic stem cells was measured by flow cytometry. In 25 of the samples, colony-forming units (CFU) were also determined. The same samples were thawed 6 months after cryopreservation and re-evaluated. RESULTS: The volume was reduced to 6 +/- 1.5 mL. The recovery of NC, MNC, CD34+ hemopoietic stem cells, RBC depletion and CFU following double-processing was 93.6 +/- 3.2%, 95.8 +/- 2.2%, 98.4 +/- 1.5%, 96.8 +/- 1.1% and 107.1 +/- 6.1% (for 25 samples), respectively. The post-thaw recoveries of NC, MNC, CD34+ hemopoietic stem cells and CFU (for 25 samples) were 78.6 +/- 5.4%, 90.8 +/- 4.4%, 96.4 +/- 2.5%, 89.1 +/- 4.1%, respectively. No post-thaw cell aggregation was observed. A significant (P<0.05) post-thaw loss of platelets and signs of platelet activation was observed. DISCUSSION: The protocol uses non-expensive equipment and clinically approved materials and results in samples that can be used in patients with a mean weight of 32.7 kg.     

Simian-Human Immunodeficiency Infection – Is the Course Set in the Acute Phase?

Identifying early predictors of infection outcome is important for the clinical management of HIV infection, and both viral load and CD4+ T cell level have been found to be useful predictors of subsequent disease progression. Very high viral load or extensively depleted CD4+ T cells in the acute phase often result in failure of immune control, and a fast progression to AIDS. It is usually assumed that extensive loss of CD4+ T cells in the acute phase of HIV infection prevents the establishment of robust T cell help required for virus control in the chronic phase. We tested this hypothesis using viral load and CD4+ T cell number of SHIV-infected rhesus macaques. In acute infection, the lowest level of CD4+ T cells was a good predictor of later survival; animals having less than 3.3% of baseline CD4+ T cells progressed to severe disease, while animals with more than 3.3% of baseline CD4+ T cells experienced CD4+ T cell recovery. However, it is unclear if the disease progression was caused by early depletion, or was simply a result of a higher susceptibility of an animal to infection. We derived a simple relationship between the expected number of CD4+ T cells in the acute and chronic phases for a constant level of host susceptibility or resistance. We found that in most cases, the depletion of CD4+ T cells in chronic infection was consistent with the prediction from the acute CD4+ T cell loss. However, the animals with less than 3.3% of baseline CD4 T cells in the acute phase were approximately 20% more depleted late in the infection than expected based on constant level of virus control. This suggests that severe acute CD4 depletion indeed impairs the immune response.     

A limited role for regulatory T cells in post-ischemic neovascularization

Recently, it was demonstrated that arteriogenesis is enhanced in mice deficient in regulatory T cells (CD4(+) CD25(+) FoxP3(+) T cell), which can suppress effector T cell responses. The present study investigates the effects of these regulatory T cells on arteriogenesis in more detail by either specific expanding or depleting regulatory T cells. Hind limb ischemia was induced by electro-coagulation of the femoral artery in mice. Regulatory T cells were either expanded by injecting mice with a complex of interleukin (IL)-2 with the IL-2 monoclonal antibody JES6-1, or depleted by anti-CD25 antibody or diphtheria toxin injections in DEREG mice (depletion of regulatory T cells). Blood flow restoration was monitored using laser Doppler perfusion imaging. Collateral arteries were visualized by immunohistochemistry. Regulatory T cell expansion led to a moderate though significant suppression of blood flow restoration after ischemia induction. Surprisingly, depletion of regulatory T cells resulted in minor increase on blood flow recovery. However, collateral and capillary densities in the post-ischemic skeletal muscle were significantly increased in DEREG mice depleted for regulatory T cells. The presence of regulatory T cells after ischemia induction when analysed in non-depleted DEREG mice could be demonstrated by green fluorescent protein staining only in lymph nodes in the ischemic area, and not in the ischemic muscle tissue. The current study demonstrates that, even under conditions of major changes in regulatory T cell content, the contribution of regulatory T cells to the regulation of the arteriogenic response is only moderate.     

Immunologic Control of Mus musculus Papillomavirus Type 1

Persistent papillomas developed in ~10% of out-bred immune-competent SKH-1 mice following MusPV1 challenge of their tail, and in a similar fraction the papillomas were transient, suggesting potential as a model. However, papillomas only occurred in BALB/c or C57BL/6 mice depleted of T cells with anti-CD3 antibody, and they completely regressed within 8 weeks after depletion was stopped. Neither CD4+ nor CD8+ T cell depletion alone in BALB/c or C57BL/6 mice was sufficient to permit visible papilloma formation. However, low levels of MusPV1 were sporadically detected by either genomic DNA-specific PCR analysis of local skin swabs or in situ hybridization of the challenge site with an E6/E7 probe. After switching to CD3+ T cell depletion, papillomas appeared upon 14/15 of mice that had been CD4+ T cell depleted throughout the challenge phase, 1/15 of CD8+ T cell depleted mice, and none in mice without any prior T cell depletion. Both control animals and those depleted with CD8-specific antibody generated MusPV1 L1 capsid-specific antibodies, but not those depleted with CD4-specific antibody prior to T cell depletion with CD3 antibody. Thus, normal BALB/c or C57BL/6 mice eliminate the challenge dose, whereas infection is suppressed but not completely cleared if their CD4 or CD8 T cells are depleted, and recrudescence of MusPV1 is much greater in the former following treatment with CD3 antibody, possibly reflecting their failure to generate capsid antibody. Systemic vaccination of C57BL/6 mice with DNA vectors expressing MusPV1 E6 or E7 fused to calreticulin elicits potent CD8 T cell responses and these immunodominant CD8 T cell epitopes were mapped. Adoptive transfer of a MusPV1 E6-specific CD8+ T cell line controlled established MusPV1 infection and papilloma in RAG1-knockout mice. These findings suggest the potential of immunotherapy for HPV-related disease and the importance of host immunogenetics in the outcome of infection.     

Accelerated memory cell homeostasis during T cell depletion and approaches to overcome it

Partial T cell depletion is used in solid organ transplantation as a valuable strategy of peritransplant induction immunosuppression. Using a murine cardiac allograft model, we recently demonstrated that this led to lymphopenia-induced (homeostatic) proliferation among the residual nondepleted lymphocytes. Rather than promoting tolerance, peritransplant T cell-depleting Abs actually resulted in resistance to tolerance induction by costimulatory blockade. In this study we show that memory T cells predominate shortly after subtotal lymphodepletion due to two distinct mechanisms: relative resistance to depletion and enhanced homeostatic proliferation. In contrast, regulatory cells (CD4+ CD25+ Foxp3+) are depleted as efficiently as nonregulatory cells and exhibit reduced homeostatic expansion compared with memory cells. The resistance to tolerance induction seen with subtotal T cell depletion can be overcome in two different ways: first, by the adoptive transfer of additional unprimed regulatory cells at the time of transplant, and second, by the adjunctive use of nondepleting anti-CD4 and anti-CD8 mAbs, which effectively block homeostatic expansion. We conclude that the resistance to tolerance induction seen after subtotal lymphocyte depletion can be attributed to alterations in the balance of naive, memory, and regulatory T cells. These data have clinically relevant implications related to the development of novel strategies to overcome resistance to tolerance.     

Normal development but differentially altered proliferative responses of lymphocytes in mice lacking CD81.

CD81 (TAPA-1) is a member of the transmembrane 4 superfamily (TM4SF) which is expressed on the cell surface of most cells of the body throughout their cellular differentiation. It has been recognized in several cell surface complexes of lymphocytes, suggesting that it may have diverse roles in lymphocyte development and activation regulation. Mice with a CD81 null mutation revealed normal T- and conventional B-cell development, although CD19 expression on B cells was dull and B-1 cells were reduced in number. However, both T and B cells of mutant mice exhibited strikingly enhanced proliferation in response to various types of stimuli. Interestingly, while proliferative responses of T cells following T-cell antigen receptor (TCR) engagement was enhanced in the absence of CD81, B-cell proliferation in response to B-cell antigen-receptor (BCR) cross-linking was severely impaired. Despite these altered proliferative responses, both tyrosine phosphorylation and intracellular calcium flux in response to cross-linking of cell surface antigen receptors were normal in mutant mice, reflecting apparently normal initial signaling of antigen receptors. In conclusion, though CD81 is not essential for normal T- and conventional B-cell development, it plays key roles in controlling lymphocyte homeostasis by regulating lymphocyte proliferation in distinct manners, dependent on the context of stimulation.     

Differential incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and major histocompatibility complex class I and II molecules into human immunodeficiency virus type 1 virions and microvesicles: implications for viral pathogenesis and immune regulation

Human immunodeficiency virus (HIV) infection results in a functional impairment of CD4(+) T cells long before a quantitative decline in circulating CD4(+) T cells is evident. The mechanism(s) responsible for this functional unresponsiveness and eventual depletion of CD4(+) T cells remains unclear. Both direct effects of cytopathic infection of CD4(+) cells and indirect effects in which uninfected "bystander" cells are functionally compromised or killed have been implicated as contributing to the immunopathogenesis of HIV infection. Because T-cell receptor engagement of major histocompatibility complex (MHC) molecules in the absence of costimulation mediated via CD28 binding to CD80 (B7-1) or CD86 (B7-2) can lead to anergy or apoptosis, we determined whether HIV type 1 (HIV-1) virions incorporated MHC class I (MHC-I), MHC-II, CD80, or CD86. Microvesicles produced from matched uninfected cells were also evaluated. HIV infection increased MHC-II expression on T- and B-cell lines, macrophages, and peripheral blood mononclear cells (PBMC) but did not significantly alter the expression of CD80 or CD86. HIV virions derived from all MHC-II-positive cell types incorporated high levels of MHC-II, and both virions and microvesicles preferentially incorporated CD86 compared to CD80. CD45, expressed at high levels on cells, was identified as a protein present at high levels on microvesicles but was not detected on HIV-1 virions. Virion-associated, host cell-derived molecules impacted the ability of noninfectious HIV virions to trigger death in freshly isolated PBMC. These results demonstrate the preferential incorporation or exclusion of host cell proteins by budding HIV-1 virions and suggest that host cell proteins present on HIV-1 virions may contribute to the overall pathogenesis of HIV-1 infection.     

Generation of alloreactivity-reduced donor lymphocyte products retaining memory function by fully automatic depletion of CD45RA-positive cells

Background aims

For patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA⁺) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products.

Development of murine lupus in CD4-depleted NZB/NZW mice. Sustained inhibition of residual CD4+ T cells is required to suppress autoimmunity.

Chronic administration of anti-CD4 mAb prevents autoimmune disease in NZB/NZW F1 (B/W) mice. This may be due either to CD4 cell depletion or to inhibition of CD4 cell function. To evaluate the relative importance of these mechanisms, we devised a system in which the consequences of cell depletion could be analyzed independent of the inhibitory effects of chronic mAb therapy. This was accomplished by performing adult thymectomy before mAb administration. Specifically, female B/W mice underwent thymectomy or sham thymectomy at age 6 wk, followed at age 3 mo by a short course of either anti-CD4 (2 mg/wk for 3 wk) or saline. Treatment with anti-CD4 depleted 90% of circulating CD4 cells, but a small subpopulation (10%) of CD4 cells was refractory to depletion. In non-thymectomized mice, the CD4 population gradually reconstituted after cessation of therapy. In contrast, in thymectomized mice, recovery of CD4 cells was prevented by the absence of the thymus. Despite the striking reduction in CD4 cells in thymectomized mice, severe autoimmune disease developed, with autoantibody levels, proteinuria, and mortality comparable with non-thymectomized, nondepleted controls. The unexpected development of lupus nephritis in thymectomized, CD4-depleted B/W mice suggested that the thymus might be required to achieve the benefits of therapy with anti-CD4. To exclude this possibility, we demonstrated that chronic therapy with anti-CD4 prevents autoimmunity in thymectomized B/W mice. These findings imply that: 1) substantial depletion of CD4 T cells is not sufficient to suppress autoimmunity; 2) suppression of autoimmunity requires sustained functional inhibition of CD4 T cells; and 3) a small subpopulation of CD4 cells that is refractory to depletion by anti-CD4 is sufficient to promote the full expression of murine lupus in B/W mice.     

Human lymphokine-activated killer (LAK) cells: identification of two types of effector cells

We analyzed the antigenic phenotype of lymphokine-activated killer (LAK) effector cells. Human blood lymphocytes were cultured for 3 days with 100 U/ml recombinant interleukin 2 (rIL 2), subpopulations isolated with monoclonal antibodies and a fluorescence-activated cell sorter (FACS) and assayed for cytotoxic activity against 51chromium labeled noncultured melanoma tumor cells. Initial experiments compared the LAK effector function of CD5+ T lymphocytes vs CD5- cells (predominantly CD16+ NK cells). The mean percent specific release at a 10:1 effector:target (E:T) ratio was 25% +/- 16 for CD5- cells, 10% +/- 6 for CD5+ cells, and 22% +/- 9 for unsorted cells. In contrast, when lymphocyte subpopulations were isolated before rIL 2 culture (LAK precursors), CD5- cells but not CD5+ cells developed LAK activity (28% +/- 12 vs 1% +/- 1, mean percent specific release, 10:1 E:T ratio), confirming our previous results showing that only CD16+ cells were LAK precursors. The discrepancy between LAK effector and precursor phenotypes suggested that LAK precursors acquired CD5 determinants during rIL 2 culture; however, double label immunofluorescence of rIL 2 cultured CD16+ cells showed that this was not the case. The data suggested that in the presence of other cell types, some T lymphocytes may develop LAK activity, but purified blood T lymphocytes do not develop LAK function when cultured with rIL 2 alone. We also analyzed LAK effector function in lymphocyte subpopulations defined by CD4 and CD8 antigens. The data showed that lymphocytes with a low density expression of CD8 and no expression of CD4 were enriched for LAK effector cells, whereas CD4+ and CD8- had less activity than unsorted cells. Lymphocytes with a high density expression of CD8 had activity similar to unsorted cells. We also assessed the contribution of Leu-7 (HNK-1) granular lymphocytes to LAK effector function. After culture with IL 2, lymphocytes were depleted of Leu-7+ cells by antibody and complement treatment and then were sorted into CD5+ and CD5- fractions. The cytotoxic activity of Leu-7-CD5+ cells was a mean 5% +/- 5 vs a mean 14% +/- 8 for the total CD5+ population (20:1 E:T ratio). The activity of Leu-7- CD5- was slightly less than the total CD5- fraction (21% +/- 9 vs 28% +/- 14, 10:1 E:T ratio). In conclusion, LAK effector function was highest in non-T cell (CD5- CD16+) populations and some activity was also present in T cell populations (CD5+ and predominantly Leu-7+).     

Longitudinal Analysis of T and B Cell Phenotype and Function in Renal Transplant Recipients with or without Rituximab Induction Therapy

Background

Prevention of rejection after renal transplantation requires treatment with immunosuppressive drugs. Data on their in vivo effects on T- and B-cell phenotype and function are limited.

Methods

In a randomized double-blind placebo-controlled study to prevent renal allograft rejection, patients were treated with tacrolimus, mycophenolate mofetil (MMF), steroids, and a single dose of rituximab or placebo during transplant surgery. In a subset of patients, we analyzed the number and phenotype of peripheral T and B cells by multiparameter flow cytometry before transplantation, and at 3, 6, 12, and 24 months after transplantation.

Results

In patients treated with tacrolimus/MMF/steroids the proportion of central memory CD4⁺ and CD8⁺ T cells was higher at 3 months post-transplant compared to pre-transplant levels. In addition, the ratio between the percentage of central memory CD4⁺ and CD4⁺ regulatory T cells was significantly higher up to 24 months post-transplant compared to pre-transplant levels. Interestingly, treatment with tacrolimus/MMF/steroids resulted in a shift toward a more memory-like B-cell phenotype post-transplant. Addition of a single dose of rituximab resulted in a long-lasting B-cell depletion. At 12 months post-transplant, the small fraction of repopulated B cells consisted of a high percentage of transitional B cells. Rituximab treatment had no effect on the T-cell phenotype and function post-transplant.

Conclusions

Renal transplant recipients treated with tacrolimus/MMF/steroids show an altered memory T and B-cell compartment post-transplant. Additional B-cell depletion by rituximab leads to a relative increase of transitional and memory-like B cells, without affecting T-cell phenotype and function.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00565331","term_id":"NCT00565331"}}NCT00565331