CD25hi Cells Research Articles

High Proportions of CD4+CD25hi T Cells Pretransplant Correlate with Worse Overall Survival after Stem Cell Transplant.

Regulatory cells may have a modulatory effect on alloreactive phenomena following hematopoietic stem cell transplantation (HSCT), although their role in this setting in patients remains controversial. We have analysed the effect of pre-conditioning peripheral blood levels of T-regs on the outcome of 89 adult patients undergoing HLA-identical (10/10) unrelated donor (UD)-HSCT. Allografts were T-cell depleted with Alemtuzumab, which has been described to spare T-regs from, and we found that higher proportion of CD4+CD25hi T-cells correlated with worse survival (p=0.002). This higher proportion also correlated with higher incidence of relapse (p=0.0274) and with higher incidence of chronic (c) GvHD (p=0.033) but not with the incidence of aGvHD.Regulatory T cells (T-regs) are a naturally occurring regulatory population of CD4+CD25hi T-cells known to express the transcription repressor FoxP3 and to produce anti-inflammatory cytokines such as TGFβ and may therefore affect patient survival. We analyse the profile of the CD4+CD25hiT cells in our cohort. In our healthy donor control group (n=30), CD4+CD25hi-expression associated with both, FoxP3 mRNA-levels (r=0.649; p=0.001) and TGFβ production (r=0.912; p<0.001), as previously described for T-regs. In preconditioning patient samples however, CD4+CD25hi-expression had a strong correlation with TGFβ regulatory cytokine production (r=0.863; p<0.001) as expected. However, FoxP3 mRNA-expression correlated neither with the CD4+CD25hi T-regs phenotype (r=0.280; p=0.040), nor with the production of TGFβ (r=0.229; p=0.156), and appeared not be an accurate marker for regulatory T-cell function in this patient setting. In addition, an inverse correlation with TNFα expression (r=−0.458 p<0.001) was found that may argue that the CD4+CD25hi cells represent a true regulatory population and not activated cells. This suggests either that activated cells that are not expressing FOXP3 might be included in the CD4+CD25hi population or that CD4+CD25loFOXP3+ cells have not acquire the regulatory function (i.e. they are not expressing TGFβ).In summary, CD4+CD25hi Regulatory T cells may have an impact suppressing allo-responses against the tumour, decreasing the overall survival by increasing the rates of relapse.Although, in mice CD4+CD25hi population have been clearly identified by the expression of the FOXP3, which encodes a transcription repressor, in humans this remains controversial, where FOXP3 expression is not exclusive of the CD4+CD25hi population.Relying on the expression of CD4+CD25hi and FOXP3 in patients is insufficient to determine regulatory T cell numbers and suggests that other parameters of regulatory function should be taken in to account.

From Vanilla to 28 Flavors: Multiple Varieties of T Regulatory Cells

Numerous T cell subpopulations have now been claimed to exhibit regulatory activity. Shevach discusses the current understanding of the different subsets of T regulatory cells and provides a perspective on the current areas of uncertainly and controversy in the field.

Generation of CD4+ Regulatory T Cells by Retroviral Transduction of CD4+CD25− T Cells Either with the Full-Length or with a Common Exon-2 Negative Variant of Human Foxp3.

The forkhead/winged helix transcription factor, Foxp3 is a key element for the differentiation of CD4+CD25+ regulatory T (Treg) cells. While murine Foxp3 gene is expressed as a single full-length transcript, we observed that transcription of human Foxp3 gene usually reveals two different mRNA products with ~ 100 base pair difference. Sequencing of these products revealed that one transcript represents the full-length Foxp3, while the other transcript appears to be an alternative splicing product that lacks the exon-2. Using a specific primer set that amplifies the region between exon-1 and exon-3 we found that both exon-2pos and exon-2neg variants are preferentially expressed in CD4+CD25+ cells with high levels of expression in CD4+CD25hi cells. Analysis of individual Treg clones generated by limiting dilution of CD4+CD25hi cells revealed that both Foxp3 variants are simultaneously expressed in Treg clones. However, quantitative-real time PCR analyses, performed using a primer set that amplifies only the full-length Foxp3 and another set that amplifies both exon-2neg and exon-2pos variants, indicated that expression levels of the full-length Foxp3 gene do not correlate with expression levels of total Foxp3 in individual Treg clones, in PBMC of healthy donors and in leukemia patients after donor lymphocyte infusions. Remarkably, some donors expressed little or no full-length Foxp3 while expressing considerable amounts of total Foxp3, suggesting a predominant expression of the exon-2neg variant in CD4+CD25+ T cells of these donors. To determine the role of individual Foxp3 isoforms in Treg cell-differentiation, the full-length and the exon-2neg variants of the Foxp3 gene were cloned into retroviral vectors and transduced separately into highly purified CD4+CD25− cells. After a brief in vitro culture, T cells transduced with the Foxp3 isoforms were sorted to high purity by flow cytometry using the GFP marker gene and expanded to high quantities by nonspecific stimulation with allogeneic feeder cells, PHA and IL-2. Phenotypical and functional analyses of expanded cells revealed that T cells transduced with both Foxp3 variants expressed high levels of CD25, intacellular CTLA-4 and CD62L; were anergic to stimulation via CD3 and suppressed the CD3/CD28 triggered proliferation of autologous and allogeneic CD4+CD25− cells. These results reveal that both isoforms of human Foxp3 are functionally intact and can convert CD4+CD25− cells into Treg cells. Current efforts are focused on testing the in vivo capacity of in vitro expanded, Foxp3 transduced T cells to prevent Graft versus Host Disease (GvHD) in a xenogeneic model where GvHD is induced by administration of human T cells in Rag2, gamma chain double knockout mice.

Self-reactive memory-phenotype CD8 T cells exhibit both MHC-restricted and non-MHC-restricted cytotoxicity: a role for the T-cell receptor and natural killer cell receptors

AbstractWe have recently shown that interleukin-2 (IL-2)-activated CD8+CD44hi cells from normal mice express both adaptive and innate immune system receptors and specifically kill syngeneic tumor cells, particularly those that express NKG2D ligands. Here we show that CD8+ T cells from antigen-expressing H-Y T-cell receptor (TCR) transgenic mice also exhibit characteristics of both T cells and natural killer (NK) cells. Interaction with cognate self-antigen was required for the optimal expansion of these cells in peripheral lymphoid tissues. Although these cells possess a higher activation threshold relative to naive T cells, they can be activated by cytokine alone in vitro. They also undergo bystander proliferation in response to a bacterial infection in vivo. Interestingly, upon activation, the cells express the NKG2D receptor as well as the DNAX activation protein 12 (DAP12) adaptor protein. We provide evidence that NKG2D can act additively with the TCR in the killing of target cells, and it can also function as a directly activating receptor in non-major histocompatibility complex (MHC)-restricted killing of target cells. These properties of CD8+ T cells from H-Y TCR transgenic mice are remarkably similar to CD8+CD44hi cells that are found in normal mice. The H-Y TCR transgenic mice provide a well-defined system for characterizing the developmental biology and function of these cells. (Blood. 2004;104:2116-2123)

CD4+CD25+ T CELLS THAT SURVIVE AFTER T CELL ABROGATION BY POLYCLONAL ANTI-T CELL ANTIBODY (ALS) ARE POTENT INHIBITOR OF ALLORESPONSES

O246* Aim: Antilymphocyte serum (ALS) is a complex mixture of antibodies with multiple specificities. In addition to the T cell depletion, ALS may also mediate immunosuppression by sparing or activating immunoregulatory T cells. Here, we investigated whether residual T cells after ALS treatment suppress alloresponses. Methods: C57BL/6 (B6) mice were given ALS twice three days apart. T cell phenotype changes were determined by flow cytometry. In vivo T cell proliferation was determined by flow cytometry following BrdU labeling for 6 days. CD4+CD25+ (CD25+) and CD4+CD25- (CD25-) T cells were separated from CD4+ cells by a Magnetic Cell Sorter. Immunoregulation by a post-ALS T cell subset was determined by addition of the cells to mixed lymphocyte culture (MLC) composed of CFSE-labeled B6 T cells (5x106) as responder cells and irradiated DBA/2 (D2) splenocytes (5x106) as stimulator cells in 24 well plates. After 2 – 5 day incubation at 37oC, proliferation was assessed by the CFSE profile and enumerated by mitotic events. Results: ALS administration caused ∼90% depletion of CD4+ and CD8+ T cells and, conversely, marked increase of CD44hi memory-type cells in the remaining CD4+ and CD8+ cell compartments. In contrast, the proportion of CD25+ cells remained minimally affected at 4 – 5%. ALS treatment also increased BrdU uptake in both CD4+ and CD8+ cells including CD25+ cells. When CFSE-labeled B6 cells were sensitized in vitro to fully allogeneic D2 cells, proliferation of CD4+ and CD8+ cells peaked on day 4 and 3, respectively. CD8+ cells proliferated much more vigorously than CD4+ cells, with majority of cells completing cell division by day 4, while CD4+ cells continued to proliferate. To test post-ALS immunoregulatory cell activity, splenic and lymph node cells were harvested from B6 mice 1 – 4 weeks after ALS treatment and separated into CD25+ and CD25- cells. Each subset (106) was added to B6 anti-D2 MLC. After 4 day incubation, surviving cells were pooled from 4 – 6 wells and assayed for CFSE profile of responder CD4+, CD8+ as well as memory-type CD4+CD44hi and CD8+CD44hi cells. At one week after ALS treatment, both CD25+ and CD25- cells suppressed proliferation of CD8+ and CD8+CD44hi cells while only CD25- cells exhibited suppression of CD4+OX40+ cells. CD25+ cells harvested 2 – 4 weeks after ALS treatment continued to suppress CD8+ and CD8+CD44hi cell response. Suppression was dose-dependent. In contrast, the same cells augmented the proliferative response of CD4+ and CD4+OX40+ cells. CD25- cells lost suppressive activity 3 weeks after ALS treatment. CD4+OX40+ and CD8+CD122+ cells, subsets of memory T cells, behaved virtually identical to CD4+CD44hi and CD8+CD44hi cells, respectively. CD25+ and CD25- cells (106) prepared from naïve B6 mice failed to suppress B6 anti-D2 alloresponse. Conclusion: T cell depletion by ALS not only results in homeostatic proliferation and emergence of memory-type cells in the residual T cells but also activates long-lasting, potent immunoregulatory CD4+CD25+ T cells capable of inhibiting alloresponses.

C-Jun NH(2)-terminal kinase (JNK)1 and JNK2 signaling pathways have divergent roles in CD8(+) T cell-mediated antiviral immunity.

c-Jun NH2-terminal kinases (JNK) play important roles in T helper cell (Th) proliferation, differentiation, and maintenance of Th1/Th2 polarization. To determine whether JNKs are involved in antiviral T cell immunity, and whether JNK1 and JNK2 bear biological differences, we investigated the immune responses of JNK1-deficient and JNK2-deficient mice to lymphocytic choriomeningitis virus (LCMV). After LCMV infection, wild-type (JNK+/+) mice had a 5- to 10-fold increase in splenic CD8+ T cells. In contrast, infected JNK1−/− mice showed a significantly lower virus-specific CD8+ T cell expansion. However, JNK1−/− mice cleared LCMV infection with similar kinetics as JNK+/+ mice. Splenic T cells from LCMV-infected JNK1−/− animals produced interferon γ after stimulation with viral peptides. However, fewer JNK1−/− T cells acquired an activated phenotype (CD44hi) and more JNK1−/−CD8+CD44hi cells underwent apoptosis than JNK+/+ cells at the peak of the primary response. In contrast, LCMV-infected JNK2−/− mice generated more virus-specific CD8+ T cells than JNK+/+ mice. These results indicate that JNK1 and JNK2 signal pathways have distinct roles in T cell responses during a viral infection. JNK1 is involved in survival of activated T cells during immune responses, and JNK2 plays a role in control of CD8+ T cell expansion in vivo.

Adhesion receptors on haematopoietic progenitor cells.

Adhesion receptors on haematopoietic progenitor cells.

Chemokine stromal cell-derived factor-1alpha modulates VLA-4 integrin-dependent adhesion to fibronectin and VCAM-1 on bone marrow hematopoietic progenitor cells.

Stromal cell-derived factor-1alpha (SDF-1alpha) is a potent chemoattractant for hematopoietic progenitor cells (HPC), suggesting that it could play an important role during their migration within or to the bone marrow (BM). The integrin VLA-4 mediates HPC adhesion to BM stroma by interacting with CS-1/fibronectin and VCAM-1. It is required during hematopoiesis and homing of HPC to the BM. As HPC migration in response to SDF-1alpha might require dynamic regulation of integrin function, we investigated if SDF-1alpha could modulate VLA-4 function on BM CD34(hi) cells.CD34(hi) BM cells and hematopoietic cell lines were tested for the effect of SDF-1alpha on VLA-4-dependent adhesion to CS-1/fibronectin and VCAM-1, as well as to BM stroma. CD34(hi) BM cells that adhered to VLA-4 ligands after SDF-1alpha treatment were characterized in colony-forming and long-term culture-initiating cell (LTC-IC) assays.SDF-1alpha rapidly (1 minute) and transiently upregulated the adhesion of CD34(hi) BM cells and hematopoietic cell lines to both CS-1/fibronectin and VCAM-1, and to BM stromal cells. The upregulation of VLA-4-dependent cell adhesion by SDF-1alpha targeted primitive LTC-IC as well as committed CD34(hi) cells. SDF-1alpha-triggered enhancement in VLA-4 function was inhibited by pertussis toxin (PTx) and cytochalasin D, indicating the involvement of G(i) protein downstream signaling and an intact cytoskeleton. Instead, activation of p44/42 MAP kinases by SDF-1alpha did not functionally correlate with enhancement of VLA-4-dependent cell adhesion. Modulation of VLA-4-mediated CD34(hi) BM cell adhesion by SDF-1alpha could play a key role in their migration within and to the BM and therefore influence their proliferation and differentiation.

Dysregulated expression of CD69 and IL-2 receptor alpha and beta chains on CD8+ T lymphocytes in flaky skin mice.

T-cell activation is considered to be an important element in the pathogenesis of psoriasis, a human skin disease characterized by keratinocyte hyperproliferation, altered keratinocyte differentiation and inflammation of the dermis and epidermis. Mice homozygous for the flaky skin (fsn) mutation develop a skin disorder that has histopathological and biochemical features resembling some forms of psoriasis. It has been reported recently that peripheral lymph nodes (PLN) in fsn/fsn mice exhibit various abnormalities in T-cell development suggestive of dysregulated T- and B-cell activation. In the present study, the expression of the inducible T-cell activation antigens CD69 and IL-2 receptor alpha chain (CD25) on PLN cells from fsn/fsn mice and their phenotypically normal littermates is examined. Expression of CD69 was significantly increased on PLN cells in fsn/fsn mice (mean +/- SD, 49.9 +/- 14.7% of cells) compared with control mice (14.6 +/- 4.2%). Analysis of CD4+ and CD8+ T cell subsets revealed that expression of CD69 in fsn/fsn PLN was significantly biased toward CD8+ cells. Although expression of CD25 was preferentially associated with CD4+ rather than CD8+ cells in both fsn/fsn and control PLN, with most CD4+ CD25+ cells being CD25hi, the proportion of CD4+ cells expressing CD25 was higher in fsn/fsn than control PLN. In contrast, CD25 was expressed by 2-3% of CD8+ PLN cells in both fsn/fsn and control mice and CD25hi cells accounted for < 1% of CD8+ cells in fsn/fsn PLN. The paucity of CD25 on CD8+ cells in fsn/fsn PLN did not appear to be due to a defect in the ability of these cells to upregulate CD25, because T cell receptor stimulation in vitro induced high expression of CD25 on both CD4+ and CD8+ cells. A striking and consistent finding was that most CD8+ cells in fsn/fsn PLN expressed high levels of IL-2R beta chain (CD122). In contrast, CD122 was expressed at low levels on CD8+ cells in control mice. Analysis of PLN cells from newborn fsn/fsn mice revealed that the high expression of CD122 on CD8+ cells was established by 2 weeks of age, prior to the appearance of clinical skin disease. These data indicate that large numbers of T cells in fsn/fsn mice are activated and reinforce the view that fsn is an important regulator of lymphocyte development and function. The relationship between T-cell activation and flaky skin disease in these mice remains to be established.

Characterization of the memory/activated T cells that mediate the long-lived host response against tuberculosis after bacillus Calmette-Guérin or DNA vaccination.

The memory/activated T cells, which mediate the long-lived host response against tuberculosis, in mice immunized with either bacillus Calmette-Guérin (BCG) or mycobacterium heat-shock protein 65 (hsp 65) antigen expressed from plasmid DNA (DNA-hsp 65), were characterized. Protection against Mycobacterium tuberculosis challenge by DNA-hsp 65 vaccination was associated with the presence of lymph node T-cell populations in which CD8+/CD44hi interferon-gamma (IFN-gamma)-producing/cytotoxic cells were prominent even after 8 or 15 months of plasmid DNA-mediated immunizations, whereas after BCG vaccination the majority were CD4+/CD44lo IFN-gamma-producing T cells. When the cells were separated into CD4+CD8- and CD8+CD4- and then into CD44hi and CD44lo types, CD44lo cells were essentially unable to transfer protection in adoptive transfer experiments, the most protective CD44hi cells were CD8+CD4- and those from DNA-vaccinated mice were much more protective than those from BCG-immunized mice. The frequency of protective T cells and the level of protection were increased up to 8 months and decreased after 15 months following DNA or BCG immunizations.

Regulatory role of CD95 ligation on human B cells induced in vivo capable of spontaneous and high-rate Ig secretion.

CD95 ligation elicits apoptotic signals in many cell systems. This study analyzes the effect of anti-CD95 mAb on human cells capable of spontaneous and high-rate Ig secretion. Such cells have been induced in vivo and represent a highly mature B cell stage. Addition of the anti-CD95 monoclonal antibody (mAb) CH11 to tonsil B cells inhibited 50-60% of their spontaneous Ig secretion. The effect was exerted early in the culture and could be reversed by a pre-treatment with a neutralizing mAb. N-acetyl-D-sphingosine (C2-ceramide), although not a close analog, also reduced Ig secretion to a similar extent. The inclusion of a tetrapeptide inhibitor for certain interleukin-1beta-converting enzyme proteases prevented the inhibitory effect of CH11 mAb on tonsil B cells. B cells capable of spontaneous Ab secretion obtained from blood of recently-immunized volunteers were also inhibited by CH11 mAb and C2-ceramide. In contrast, bone marrow (BM) B cells capable of spontaneous Ig secretion were unaffected by these agents. This CD95 ligation-mediated inhibition of tonsil and blood Ig-secreting B cells could not be reversed by cytokines with demonstrated activity on these B cells. Human mature B cells induced in vivo are identifiable as CD38hi cells. Flow cytometric analysis revealed that a fraction of tonsil CD38hi cells expressed low levels of CD95. Moreover, about 20% of these cells exhibited basal apoptosis, as defined by annexin V binding. This phenomenon was markedly increased by CD95 ligation. On the other hand, BM CD38hi cells showed neither CD95 expression nor CD95-induced annexin V binding. These data suggest that CD95 ligation might play a role in the control of human humoral responses by inducing apoptosis in susceptible mature B cells.

Granulocytic and Monocytic Differentiation of CD34hi Cells Is Associated With Distinct Changes in the Expression of the PU.1-Regulated Molecules, CD64 and Macrophage Colony-Stimulating Factor Receptor

The present study investigated the possibility that macrophage colony-stimulating factor (M-CSF) responsiveness of hematopoietic progenitor cells is regulated at the level of receptor expression and that M-CSF receptor (M-CSFR) may be used as an early marker of monocyte lineage commitment. Immunofluorescence measurements with an anti-M-CSFR antibody showed that 44% +/- 5% of CD34hi cells expressed the receptor. The M-CSFR was present on progenitor cells that were positive for the granulo-monocytic marker CD64, but not on primitive, erythroid, or lymphoid progenitors. The CD34hiCD64+ population could be divided into subsets of M-CSFRhi and M-CSFRlo cells. In addition, a subset of CD34hiCD64-M-CSFRhi cells was found. CD34+ cells that were positive for M-CSFR, CD64, or both gave rise exclusively to granulo-monocytic cells, and 65% of the granulomonocytic colony-forming cells in the CD34+ population were recovered from these cells. Approximately 70% of the colony-forming cells (CFCs) derived from CD34hiM-CSFRhi cells were macrophage colony-forming units (CFU-M), whereas 91% of the CFCs in the CD34hiCD64+M-CSFRlo population were granulocyte colony-forming units (CFU-G). The M-CSFRhi cells with the highest frequency of colony-forming and bipotent cells and largest average colony size were found in the CD64- subset, indicating that M-CSFR appears earlier than CD64 during monocyte development. After 60 hours in culture, a subset of the CD34hiM-CSFRhi cells had downmodulated M-CSFR (29% to 38%). This population gave rise almost exclusively to granulocytes, whereas the cells that remained M-CSFRhi gave rise exclusively to monocytes. In all experiments, the M-CSFRhi population responded to M-CSF, whereas minimal responses were observed among M-CSFRlo cells. These results suggest that M-CSF target specificity among human hematopoietic progenitor cells is determined by lineage-specific regulation of the M-CSFR and show that M-CSFR is a useful marker to discriminate between monocytic and granulocytic progenitor cells.

Protection of hu-PBL-SCID/beige mice from HIV-1 infection by a modified oligonuleotide, RKS-1443

To study the canine immune system we generated a mouse model engrafted with canine lymphocytes using NOD SCID IL2R common gamma chain −/− (NSG) mice as recipients (Ca-PBL-SCID). Engraftment of canine peripheral blood lymphocytes (PBLs) was determined post-injection with 107 peripheral blood mononuclear cells (PBMCs) into irradiated NSG mice using flow cytometry and fluorescently labeled antibodies specific to canine helper T cells (CD45+ CD4+), cytotoxic lymphocytes (CD45+ CD8+), regulatory T cells (CD45+ CD4+ Foxp3+), and B cells (CD45+ Ig+ CD21lo). Canine CD45+ lymphocytes were detectable as early as day 1 in the peritoneal cavity, and beginning at 9 days in the blood, bone marrow, and spleen. CD4+ T cells, of which Foxp-3+ CD25hi cells constituted a minor percentage, were the predominant lymphocyte population at 9 days post engraftment contrasting with increasing proportions of CD8+ CTL's and Ig+ B cells beginning at 16 days. Canine immunoglobulin was initially detected in the serum of Ca-PBL-SCID mice at 9 days post-engraftment and peaked in concentration at day 36. From day 28 to 52 post-engraftment 30% of the Ca-PBL-SCID mice became markedly anemic and thrombocytopenic, yet gross and histopathologic examination of bone marrow, kidneys, spleen, liver, and intestine revealed no obvious lesions. Blood smear evaluation revealed agglutination of mature red blood cells, reticulocytes and a regenerative anemia. These findings demonstrate that NSG mice are capable of engraftment of canine PBLs yet develop graft versus host disease similar to Hu-PBL-SCID mice.

Identification of a Novel DNA Sequence Differentially Expressed Between Normal Human CD34+CD38hi and CD34+CD38lo Marrow Cells

We have applied the recently developed differential display method to extend the molecular characterization of the less mature CD34+CD38lo bone marrow progenitors in comparison with the CD34+CD38hi cells to better understand their functional differences. Immunomagnetic enrichment of CD34+ cells followed by flow cytometry was used to isolate CD34+CD38lo and CD34+CD38hi cells from human organ donor bone marrow. A limited set of the poly A+ RNA sequences present in these two cell populations was amplified by a combination of reverse transcription with an anchored oligo dT-based primer and polymerase chain reaction with the same oligo dT primer and arbitrary decamers. A radioactive tracer allowed these sequences to be displayed as a series of bands on a denaturing polyacrylamide gel. Eight bands were chosen that appeared in multiple displays to represent gene sequences differentially expressed between CD34+CD38hi and CD34+CD38lo cells. Comparison of the sequences with public DNA sequence databases available identified one sequence as myeloperoxidase. Two other clones matched sequence fragments of unknown function, whereas the remaining five are novel sequences not present in existing databases. The relative level of expression of all of the sequences was tested by an independent reverse transcriptase-polymerase chain reaction with sequence-specific oligonucleotide primers. The lower level of myeloperoxidase mRNA in CD34+CD38lo cells was confirmed, as was the higher expression of the novel sequence 345. Sequence 345 expression is highest in CD34+CD38- cells and decreases with increased CD38 expression. It is expressed in negligible amounts in hematopoietic cell lines and other sources of human tissue, suggesting it may have a functional role in normal hematopoiesis.

Benign Marrow Progenitors Are Enriched in the CD34+/HLA-DRlo Population But Not in the CD34+/CD38lo Population in Chronic Myeloid Leukemia: An Analysis Using Interphase Fluorescence In Situ Hybridization

Fluorescence in situ hybridization (FISH) was used to discriminate between benign and malignant cells in sorted populations of chronic myelogenous leukemia (CML) marrow. FISH has the advantage of allowing for a cell by cell analysis of the breakpoint cluster region (BCR) gene rearrangement immediately after flow sorting in nondividing G0/G1 cells that are potentially transcriptionally inactive. We initially selected CD34+ cells with very low expression of the activation antigen CD38 as a candidate phenotype for an immature and hypothetically more benign cell population, but found no enrichment for Ph negativity in that subtype. In five CML samples, 55% +/- 3.3% (mean +/- SE) of CD34+/CD38hi cells had the BCR gene rearrangement, similar to 57% +/- 3.7% seen in the CD34+/CD38lo population. In contrast, subsequent experiments (n = 4) determined that the CD34+/HLA-DRlo population in CML marrow does contain an increased proportion of benign cells: 15% +/- 1% of the CD34+/DRlo cells were BCR rearranged, compared with 52% +/- 5.8% of the CD34+/DRhi cells (P = .001). Our results indicate that benign progenitors in CML are enriched within the CD34+ cells with low DR antigen expression, but not low CD38 expression. One possible interpretation of these observations is that low CD38 antigen expression is not as useful as low HLA-DR expression for isolating immature cells.

Cell Cycle and Functional Differences Between CD34+/CD38hi and CD34+/38lo Human Marrow Cells After In Vitro Cytokine Exposure

The proliferation kinetics and clonogenic activity of CD34+/38hi (CD38hi) and CD34+/38lo (CD38lo) human marrow cells were measured before and after culturing the cells in vitro over a 6-day period in serum-deprived medium containing recombinant growth factors (interleukin-1 [IL-1], IL-3, IL-6, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage [GM]-CSF, kit ligand, and erythropoietin). Before in vitro culture, 3% +/- 3% of the CD38lo and 13% +/- 2% of the CD38hi cells were in the S-phase of the cell cycle. The clonogenic activity of CD38hi cells was twofold greater than that of the CD38lo cells, as measured by colony-forming units (CFU) in short-term assays. However, CD38hi cells contained fewer pre-CFU than did the CD38lo cells, generating only 3 +/- 2 colonies per 1,000 cells after 4 weeks of culture on competent stromal layers, compared with 107 +/- 46 colonies per 1,000 cells from the CD38lo population. CD38hi and CD38lo cells exhibited distinctly different responses when cultured in serum-deprived medium supplemented with recombinant growth factors. After culturing cells for 24 hours, CD38lo cells essentially remained a noncycling population with only 5.1% +/- 3.0% of the cells cycling, whereas 44.2% +/- 6.9% of the CD38hi cells were in DNA synthesis. Gradually CD38lo cells were recruited into cycle, such that by 72 hours, approximately 28% of the CD38lo cells were in S-phase. However, during 6 days of culture, the percentage of cycling CD38lo cells never exceeded the proliferative response observed for CD38hi cells. Phenotype analysis conducted at day 6 indicated that 86% of the CD38hi population were no longer phenotypically CD34+/38hi, while 60% of CD38lo cells maintained a CD34+/38lo phenotype. Long-term cultures initiated with 6-day in vitro-expanded CD38lo cells showed approximately a twofold decrease in clonogenic activity attributable to a loss of erythroid precursors and a decrease in GM colonies. Thus, a proportion of CD38lo cells capable of generating CFU was maintained even after exposure to growth factors.

Human fetal bone marrow early progenitors for T, B, and myeloid cells are found exclusively in the population expressing high levels of CD34

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

Human Fetal Bone Marrow Early Progenitors for T, B, and Myeloid Cells Are Found Exclusively in the Population Expressing High Levels of CD34

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

THE EFFECT OF AGING ON CYTOKINE RELEASE AND ASSOCIATED IMMUNOLOGIC FUNCTIONS

An in vivo switch in T-cell subpopulations from those of naive to those of activated / memory subsets may offer some benefit to the aged by counteracting the overall deleterious effect of the aging process on the immune system. Although there is no question that immune responses are dampened by the aging process, the enhanced production of lymphokines by aged T cells in the presence of a decrease in proliferative responses to both mitogens and antigens (of the T-helper variety) is likely to be advantageous to the aged. The shift during the aging process from the naive subset to the activated/memory subset and the associated loss in the proliferative response of CD4+ T cells are accompanied by a decrease in intracellular calcium mobilization.51 It has been suggested that the primary cause of the dampening of immune function during the aging process is the inability of individual cells to undergo significant [Ca2+]i mobilization. Because the aging process also is associated with a marked increase in secretion of lymphokines involved in T helper cell function, however, it is questionable whether the loss of [Ca2+]i mobilization in either CD4+ or CD8+ T cells is as detrimental to immune function as originally hypothesized. Differences in the function of CD44hi cells isolated from young adult and aged mice, although perhaps only quantitative, suggest that there must be some underlying difference in the events involved in either the activation or regulation of lymphokine production by these two groups. The enhanced production of lymphokines from CD4+ T-cell subsets from aged mice cannot be explained by their enhanced production of the regulatory lymphokine IL-10; if IL-10 down regulation was playing a major role, the production of lymphokines such as IFN$gM would be even more pronounced in the aged. A more effective use of a particular population of APCs by the aged T cell could result in enhanced lymphokine signals in the absence of additional T-cell expansion. The loss of MHC restriction58 during the aging process or the more effective use of activated B cells such as APC in aged mice also may account for increased lymphokine production. Additional expansion may not be required in these activated/memory cells, which already may have undergone maximum expansion. There are marked differences in the pathways of both T-cell activation and T-cell tolerance in Th1 (IL-2-producing) and Th2 (IL-4-producing) T-cell clone studies in vitro.22 It has been clearly established in mice, for example, that cloned CD4+ helper cells of the Th2 type are characterized by a T helper pattern of lymphokine secretion and do not mobilize intracellular calcium.22 It is likely that different types of T cells exist in vivo as well as in vitro, and that one of these subpopulations is of the activated/memory type (CD44hi, IL-4-producing). If that interpretation of the available data is correct, then the aged animal is adequately equipped with memory CD4+ and CD8+ T cells that can be activated to release lymphokines required to drive T helper cell activity, but is likely to be deficient in precursor T cells directed against new antigens that were not encountered during early life. Such new antigens could be associated with new strains of influenza virus or a malignant neoplasm of recent onset. It appears likely that, as a result of repeated expansion of T cells to environmental antigens throughout life, there is an accumulation of memory cells to these antigens with adequate lymphokine potential and a concomitant loss of naive cells. This scenario, applicable in the normal aging process, may be influenced directly by other events during the aging process. Exposure to infections, tumors, or various drugs may cause further down-regulation of the immune system in the aged patient. Future studies of cytokine synthesis and release should provide deeper insights into these, as well as other, immunologic changes noted during aging. Those insights may well lead to new therapeutic approaches, including cytokine therapy or administration of cytokine inhibitors or antagonists for treatment of immunologic problems in the elderly.

Patterns of cytokine gene expression by CD4+ T cells from young and old mice.

We have analyzed the patterns of induced cytokine gene expression and cell cycle activity by CD4+ cells from mice, and have examined how these response patterns change during the aging process. CD4+ cells were isolated from spleens of young adult and old C57BL/6NNia mice and were stimulated in vitro with plate-bound anti-CD3 epsilon mAb. The cells were then assessed over time for the capacity to accumulate transcripts for IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IFN-gamma, TNF-alpha, and TNF-beta; to secrete IL-2, IL-3, IL-4, IL-5, IL-6, and IFN-gamma; and to progress through S phase. Before the first major cell division in culture (< 32 h), stimulated CD4+ cells of the old group contained similar peak levels of IL-2, TNF-alpha, and TNF-beta transcripts relative to young adult controls, whereas IL-3, IL-4, IL-5, and IFN-gamma transcripts accumulated to significantly higher peak levels in the old group. These findings were consistent with the patterns of cytokine secretion later in culture (24 to 72 h): the peak IL-2 levels were similar between age groups, but the old group exhibited an enhanced capacity to release IL-3, IL-4, IL-5, and IFN-gamma. In contrast, CD4+ cells of the young group were superior in the hyper-expression of the housekeeping gene, rpL32, before cell division and in the levels of S phase activity throughout 3-day cultures. Similar analyses of CD4+ cells from mice of intermediate ages showed that the alterations in cytokine profiles occurred gradually from young adulthood to old age, whereas the reductions in proliferative capacity were late life changes. Consistent with previous reports, we found that the splenic CD4+ cell group also underwent a progressive, age-dependent increase in the proportions of cells expressing high levels of membrane CD44 (a phenotype associated with memory or effector cells). Moreover, the analysis of IL-3, IL-5, and IFN-gamma production by isolated CD4+CD44lo and CD4+CD44hi cells revealed that the capacity to produce these cytokines segregated predominantly with the CD44hi subset, regardless of donor age. Taken together, our data suggest that gradual age-associated shifts in the subset composition of the splenic CD4+ cell pool underlie progressive changes in the patterns of cytokine gene expression by this cell group.