Experimental autoallergic sialadenitis in the LEW rat. III. Role of CD4+ T cells in EAS induction

Experimental autoallergic sialadenitis (EAS) in the LEW rat is an induced autoimmune disease of the salivary tissues. EAS is characterized by a lymphocytic infiltration that consists of both CD4+ (helper/inducer T-cell subset) and CD8+ (cytotoxic/suppressor T-cell subset) T cells and results in the immune-mediated destruction of the exocrine salivary glands. To investigate the role that each of the T-cell subsets may have in the pathogenesis of EAS, LEW rats sensitized with WF SMG homogenate were injected with monoclonal antibodies to deplete or inactivate, in vivo, the CD4, CD5 (OX19; pan T lymphocyte), CD8, or RT6 (70% of peripheral T cells) T-cell populations. Treatment with the OX8 (CD8), OX19 (CD5), or W3/25 (CD4) only partially reduced in vivo the respective splenic or lymph node T-cell subsets when analyzed on Day 14, while treatment with DS4.23 (anti-RT6) resulted in greater than 95% depletion of RT6+ spleen and lymph node T cells. EAS incidence and severity was significantly reduced in the W3/25 (CD4) treatment group (11% incidence rate; histologic score 1.0) as compared to medium-injected controls (88% incidence rate; histologic score 2.9). Although the incidence and severity of EAS in the OX19 (71%; histologic score 1.7), OX8 (55%; histologic score 1.7), and RT6 (67%; histologic score 1.6) treatment groups appeared decreased, the reduction was not statistically significant. These results provide evidence that CD4+ T cells have an important role in EAS induction and demonstrate that in vivo treatment with anti-CD4 can ameliorate and/or prevent EAS in the LEW rat.     

T cell subsets mediating lethal graft versus host disease: demonstration that synergy between CD4+ and CD8+ T cells is the predominant mechanism in low responder rat strains

T cell subsets from rat strains that have been characterized as high and low responders to alloantigen were examined for their capacity to mediate lethal graft versus host disease (GVHD) across strain combinations incompatible for class I, class II, and non-MHC antigens. Inocula of 5 X 10(7) lymph node and spleen cells (LC) from low responder DA (RT1a) and high responder W/F (RT1u) strains caused lethal GVHD in (W/F X DA)F1 hybrids given 6 Gy whole body irradiation. W/F CD4+ (W3/25+) cells (2 X 10(7], equal to the number in 5 X 10(7) LC mediated lethal GVHD but 10(8) DA CD4+ cells were required to cause lethal GVHD. CD8+ (MRC OX8+) cells (5 X 10(7] from W/F rats alone caused lethal GVHD but those from DA rats could not. Mixtures of CD4+ and CD8+ DA T cells, equivalent to the number in 5 X 10(7) LC, did mediate lethal GVHD, demonstrating that synergy between the subsets was the predominant mechanism with DA cells. These results suggest that differences in alloreactivity between the strains tested may be due to alternate requirements for the alloactivation of T cell subsets; the high responder subsets being self-sufficient and the low responder subsets being dependent upon each other.     

Cellular basis of allograft rejection in vivo. V. Examination of the mechanisms responsible for the differing efficacy of monoclonal antibody to CD4+ T cell subsets in low- and high-responder rat strains

MRC OX35, an anti-CD4 mAb, was used to treat high responder Wistar Furth (W/F) (RT1u) and low responder DA (RT1a) rats which had been grafted with directly vascularized hearts from PVG (RT1c) rats across a full MHC plus non-MHC incompatibility. Four doses of mAb at 7 mg/kg given in the first 2 wk postgrafting induced indefinite graft survival (greater than 150 days) in DA hosts, but only delayed rejection to 18 to 42 days in W/F as compared to rejection times of 6 to 8 days in untreated rats. The extension of MRC OX35 treatment to 6 wk in W/F rats induced indefinite graft survival in three of six rats. During treatment MRC OX35 therapy only partially depleted CD4+ cells, and all circulating CD4+ cells were coated with MRC OX35. The capacity of naive CD4+ and CD8+ cells from W/F and DA to be activated to PVG alloantigen was compared both in vitro in an MLC assay and in vivo by an adoptive transfer assay of their capacity to restore rejection of PVG heart grafts in irradiated syngeneic hosts. CD4+ cells from both W/F and DA proliferated in MLC and restored graft rejection. W/F CD8+ cells both proliferated in MLC and restored rejection, but DA CD8+ cells neither proliferated nor reconstituted rejection. Examination of lymphocytes from MRC OX35 treated hosts with long-surviving grafts showed that they were neither depleted of CD4+ T cells nor did they lack the capacity to proliferate to PVG Ag in MLC, this response being similar to that to third-party Ag or by naive lymphocytes. Compared to first-set rejection, PVG skin graft rejection was delayed 2 to 3 days in W/F and 10 to 12 days in DA rats with long-surviving grafts after MRC OX35 therapy, whereas they rejected third-party skin grafts in first-set tempo. These studies show that differences in graft survival in anti-CD4 treated low and high responder strains may be due to the inherent capacity of CD8+ cells to be activated to effect rejection independent of CD4+ cells in W/F but not in DA. In those hosts that accept grafts, there is no evidence of clonal deletion, but there appears to be a form of unresponsiveness akin to that induced in adult rats by other immunosuppressive therapies that protects the graft from rejection.     

Phytohemagglutinin-lnduced acquisition of T-cell surface markers by rat bone marrow precursor cells in the absence of the thymic microenvironment

Two monoclonal antibodies specific for different rat T-cell subpopulations, the anti-helper-T-cell antibody, W3/25, and the OX8 suppressor cell antibody were used to investigate lectin-stimulated T-lymphocyte differentiation of F-344 rat bone marrow cells in culture. Cytofluorometric analysis of freshly isolated lymphocytes from thymus and spleen revealed that these tissues contained both W3/25? and OX8-positive populations but differed with respect to the number of cells and receptor density distribution. By contrast, bone marrow-derived lymphocytes exhibited negligible W3/25? or OX8-associated fluorescence. However, several days after stimulation of bone marrow lymphocytes with phytohemagglutinin (PHA), cells appeared bearing these markers. Two-parameter histogram analysis of light scatter measurements with cell surface immunoflu-orescence indicated that this phenomenon represented the appearance of a new population of cells, presumably mature T cells, bearing an increased density of marker. These findings suggest an induction of differentiation of bone marrow T precursor cells by nonthymic factors (PHA) since lymphocytes lacking mature T-cell marker expression developed this characteristic after several days in culture.     

Absence of RT6+ T cells in diabetes-prone biobreeding/Worcester rats is due to genetic and cell developmental defects

Diabetes-prone BB/Wor (DP) rats lack the RT6+ peripheral T cell subset whereas diabetes-resistant BB/Wor rats have normal numbers of RT6+ T cells. Lymphocyte transfusion experiments and in vivo depletion studies have demonstrated that RT6+ T cells have an important regulatory role in the pathogenesis of insulin-dependent diabetes mellitus in BB/Wor rats. In the present study, the results of genetic complementation studies indicate that the DP rat contains an intact RT6 gene, but fails to express the RT6.1 alloantigen in the functional absence of an accessory factor (provided by RT6+ cells). At the cellular level, irradiation chimeras demonstrate that the absence of RT6+ T cells in DP rats is due to an intrinsic defect that results in abnormal development and/or differentiation of prothymocytes into RT6+ T cells. The inability of DP prothymocytes to generate RT6+ T cells is not due to serum autoantibodies, lack of accessory cells, or to the presence of inhibitory cells. Inasmuch as DP bone marrow can transfer the susceptibility for diabetes to irradiated recipients, our present results suggest that an important predisposing factor for insulin-dependent diabetes mellitus in DP rats is the inability of DP prothymocytes to generate RT6+ T cells.     

Single expression of CD45RC and RT6 in correlation with T-helper 1 and T-helper 2 cytokine patterns in the rat

Previous studies involving the function and development of peripheral T cells have proposed that, in the rat, CD4(+)CD45RC(+)RT6(-) and CD4(+)CD45RC(-)RT6(+) T-cell subsets may represent Th1 and Th2 cells, respectively. Here we tested this hypothesis directly by analyzing frequencies of IFN-gamma- and IL-4-producing cells in these two subpopulations using ELISPOT assays. We found that the CD4(+)CD45RC(-)RT6(+) subset showed higher numbers of IL-4-producing cells than the CD4(+)CD45RC(+)RT6(-) subset and, though less pronounced, that the latter demonstrated higher numbers of IFN-gamma producers. Therefore, we conclude that our results provide evidence for the existence of phenotypically defined Th1 and Th2 cells in the rat. This is supported by the finding that the ratios of IFN-gamma/IL-4 and CD45RC/RT6 correlated positively among various rat strains. Finally, rat strains susceptible to induction of a Th1-mediated autoimmune disease showed the highest CD45RC/RT6 ratio, whereas the reverse was true for strains susceptible to a Th2-mediated autoimmune disease.     

Heterogeneity of NK1.1+ T cells in the bone marrow: divergence from the thymus.

NK1.1+ T cells in the mouse thymus and bone marrow were compared because some marrow NK1.1+ T cells have been reported to be extrathymically derived. Almost all NK1.1+ T cells in the thymus were depleted in the CD1-/-, beta2m-/-, and Jalpha281-/- mice as compared with wild-type mice. CD8+NK1.1+ T cells were not clearly detected, even in the wild-type mice. In bone marrow from the wild-type mice, CD8+NK1.1+ T cells were easily detected, about twice as numerous as CD4+NK1.1+ T cells, and were similar in number to CD4-CD8-NK1.1+ T cells. All three marrow NK1.1+ T cell subsets were reduced about 4-fold in CD1-/- mice. No reduction was observed in CD8+NK1.1+ T cells in the bone marrow of Jalpha281-/- mice, but marrow CD8+NK1.1+ T cells were markedly depleted in beta2m-/- mice. All NK1.1+ T cell subsets in the marrow of wild-type mice produced high levels of IFN-gamma, IL-4, and IL-10. Although the numbers of marrow CD4-CD8-NK1.1+ T cells in beta2m-/- and Jalpha281-/- mice were similar to those in wild-type mice, these cells had a Th1-like pattern (high IFN-gamma, and low IL-4 and IL-10). In conclusion, the large majority of NK1.1+ T cells in the bone marrow are CD1 dependent. Marrow NK1.1+ T cells include CD8+, Valpha14-Jalpha281-, and beta2m-independent subsets that are not clearly detected in the thymus.     

Regulation of the immune response to alloantigens: suppressor and helper T cells generated in the primary MLR of the rat

The primary MLR of the rat was used to generate suppressor, cytotoxic, and helper T cells from lymph node cells of the WF (RT1 mu) inbred strain. They were assayed in 51Cr-release cytotoxic assays and by their effect on proliferation of fresh unprimed responder cells. Suppression by MLR cellular products was antigen-specific and generation and functional expression were directed to class II (RT1.B,D) antigens of stimulator cells in the strains tested. In contrast, help was not antigen-specific. The monoclonal antibodies OX8 and W3/25 were used to separate the primed products of the MLR into the constitutive subsets, suppressor/cytotoxic (OX8+) and helper/inducer (W3/25+). Gamma irradiation of OX8+ MLR-primed cells caused modest reductions in suppressive activity, but had no effect on the helper activity of W3/25+ cells. MLR-derived suppressor cells are effective only when added in the early stages of the test primary MLR, whereas helper cells can augment proliferation even when added late. Feedback suppression is not mediated by classical cytotoxic T cells, because of differences in kinetics of development, cell numbers required, susceptibility to freezing, and expression of the RT6 differentiation antigen.     

Host resistance to cyclosporine induced syngeneic graft-versus-host disease. Requirement for two distinct lymphocyte subsets

Cyclosporine is crucial for the prevention of organ allograft rejection and allogeneic graft-vs-host disease (GVHD). Despite its potent immunosuppressive activity, cyclosporine elicits a T cell-mediated autoimmune syndrome after autologous or syngeneic bone marrow transplantation, which has been termed syngeneic GVHD (SGVHD). Recent studies have shown that for disease manifestation, a cytoxan and radiation-sensitive T cell dependent host resistance mechanism must be eliminated, allowing the clonal expansion of autoreactive cells. This report characterizes the autoregulatory lymphocyte population, present in normal animals, capable of inhibiting the adoptive transfer of SGVHD. First, twice the number of unfractionated splenocytes from normal animals to those from autoimmune donors ensured complete inhibition of the adoptive transfer of immune reactivity. Second, the phenotype of this host resistance mechanism in normal splenocytes involves dual regulatory T cell subsets. A helper/inducer subset (W3/25+) must be cotransferred with a cytotoxic/suppressor subset (OX8+) in a ratio that approximates the normal ratio in normal unfractionated splenocytes in order to affect inhibition of the transfer of SGVHD. Moreover the specific inducer regulatory activity resides in the OX22-, W3/25+ subset of Th cells.     

Membrane antigen phenotype of sensitized T lymphocytes mediating tuberculin-delayed hypersensitivity in rats

The membrane antigen phenotype of immune lymph node cells (LNC) which mediate tuberculin-delayed hypersensitivity (DH) in Lewis rats was examined. The results show that the T-cell population which expresses the RT7.1 alloantigen defined by the BC 84A monoclonal antibody contained cells capable of transferring DH. Separation of the RT7.1-positive T-cell population with the monoclonal antibodies W3/25, MRC OX-8, or DS 4.23 (which defines the RT6.1 alloantigen) revealed that either the W3/25-positive or the RT6.1-negative T-cell subpopulations contained DH effector cells, whereas the corresponding MRC OX-8-positive or RT6.1-positive T-cell subpopulations did not. Moreover, when the W3/25-positive T-cell subpopulation was divided into either RT6.1-positive or RT6.1-negative T-cell subsets, only the W3/25-positive, RT6.1-negative subset transferred DH. These results indicate that the effector cells that mediate tuberculin DH are contained within the immune T-cell subset which bears both the RT7.1 and the W3/25 markers, but lacks both the MRC OX-8 and the RT6.1 markers.     

Developmental potential of CD4-8- thymocytes. Peripheral progeny include mature CD4-8- T cells bearing alpha beta T cell receptor

We have used the intra-thymic transfer system to investigate the population dynamics of thymocyte and mature T cell subsets in the absence of continuing precursor input from the bone marrow. We have followed the development and life span of CD4+ and CD8+ thymocyte subsets and mature peripheral T cells from intra-thymically injected adult or fetal CD4-8- thymic precursors. Both precursor types proliferated, differentiated, and exported to peripheral lymphoid tissues alpha beta-TCR+CD4+8- and CD4-8+ progeny which formed a stable, long-lived component of the peripheral T cell pool. The production of phenotypically mature thymocytes and peripheral T cells occurred more rapidly from fetal CD4-8- precursors. CD4+8-:CD4-8+ ratios among peripheral progeny of intra-thymically-injected CD4-8- precursors were initially normal, but they steadily declined among progeny of the fetal precursors. Thus, there appear to be differences in the life span and/or proliferative capacity of mature T cells derived from embryonic vs adult progenitors. In addition to the predominant CD4+8- and CD4-8+ subsets of peripheral T cells, a minor (1 to 20%) population of Thy-1+CD3+4-8- T cells was identified among peripheral progeny of intra-thymically-injected CD4-8- thymocytes, as well as in lymph nodes of unmanipulated animals. A total of 20 to 34% of this subset expressed V beta 8+ TCR and the majority were CD5hi, Pgp-1+, and J11d-. The function and specificity of this newly identified population of thymically derived peripheral T cells remains to be investigated.     

The mediators of acquired resistance to Listeria monocytogenes are contained within a population of cytotoxic T cells

T cells from peritoneal exudates induced in rats convalescing from a recent infection of Listeria monocytogenes were fractionated into two subsets based on their ability to bind monoclonal antibodies to cell-surface determinants that are expressed on some but not all peripheral T cells. Two phenotypically distinct subsets, one recognized by the antibody MRC OX8 and the other by W3/25, were assayed for their protective capacity in Listeria-challenged recipients, and for their ability to kill unmodified syngeneic fibroblasts in vitro. The two activities were mediated by the OX8+ subset which comprised approximately half the T cells in the exudates.     

Clonogenic potential of murine CD4+8+ thymocytes. Direct demonstration using a V beta 6-specific proliferative stimulus in Mlsa mice

Although considerable indirect evidence supports the hypothesis that CD4+8+ thymocytes are developmental intermediates in the generation of mature (CD4+8- or CD4-8+) T cells, the ability of these cells to proliferate in vitro has been highly controversial. We demonstrate here that a fraction of purified murine CD4+8+ thymocytes can be induced to proliferate in response to immobilized anti-TCR mAb. To exclude possible proliferation by trace mature T cell contaminants, we have exploited our recent finding that in Mlsa mice mature V beta 6-bearing thymic T cells are virtually absent (less than or equal to 0.5%) due to clonal deletion, whereas V beta 6 +CD4+8+ thymocytes are present in much higher numbers (approximately 3%). Proliferation of sorted CD4+8+ thymocytes from Mlsa mice was therefore induced at limiting dilution with immobilized anti-V beta 6 mAb to select against any contaminating mature T cells. Under optimal culture conditions, the frequency of CD4+8+ thymocytes proliferating specifically to anti-V beta 6 mAb (1/1000) was higher than those obtained for purified CD4-8+ (1/2000) or CD4+8- (1/5000) subsets, thus demonstrating directly that a proportion (in this case 3%) of CD4+8+ thymocytes are potentially clonable. During culture, V beta 6 +CD4+8+ thymocytes gave rise to a mixture of phenotypically "immature" (CD4-8-) and "mature" (CD4-8+) T cells. This system should be valuable for further analysis of the elusive CD4+8+ thymocyte subset.     

Analyses of acute graft-versus-host-like reaction in [MRL/lpr----MRL/+] chimeric mice using MRL/lpr-Thy-1. 1 congenic mice.

When MRL/Mp(-)+/+(MRL/+) mice are lethally irradiated and then reconstituted with MRL/Mp-lpr/lpr (MRL/lpr) bone marrow and/or spleen cells, these MRL/+ mice develop "lpr-GVHD" which is similar to acute graft-versus-host disease (GVHD). Using a Thy-1 congenic strain of MRL/lpr mice (MRL/lpr-Thy-1.1), we analyzed T cell subpopulations in the thymus and spleen of MRL/+ mice suffering from lpr-GVHD. lpr-GVHD was induced in MRL/+ mice by transplantation of bone marrow cells (BMC) from MRL/lpr-Thy-1.1 mice; severe lymphocyte depletion associated with fibrosis was observed in the spleens after 7 weeks of bone marrow transplantation (BMT). Thymocytes of the host MRL/+ thymus were replaced with donor-derived cells from the early stage of lpr-GVHD, whereas in the spleen, a small number of host T cells (Thy-1.2+) (4-5%) were retained until the late stage of lpr-GVHD. Donor-type (Thy-1.1+) T cell subsets were not different from those of nontreated MRL/+ mice in the thymus, whereas in the spleen. CD8+ T cells (Thy-1.1+) reached a peak at 5 weeks after BMT, and CD4+ T cells (Thy-1.1+), a peak at 6 weeks. The elimination of T cells from MRL/lpr BMC had no evident effect on the prevention of lpr-GVHD. T cell subpopulations showed a similar pattern to GVHD elicited by MHC differences. Analyses of autoreactive T cells expressing V beta 5 or V beta 11 revealed that autoreactive T cells were deleted from the peripheral lymph nodes. Interestingly, the levels of IgG anti-ssDNA antibodies markedly increased, and both IgM and IgG rheumatoid factors slightly increased 5 to 7 weeks after BMT. These findings are discussed in relation to not only GVHD elicited by MHC differences but also autoimmune diseases.     

Selective induction of OX19+ (CD5+) or OX19- (CD5-) alloreactive cytolytic lymphocytes in the rat

The phenotypes of alloselective cytolytic lymphocytes of the rat are defined by staining of peritoneal cells of alloimmunized donors with monoclonal antibodies, sorting in a cytofluorometer and evaluating cytolytic capacity in a 51Cr-release assay. We demonstrate that alloimmunization of BN rats can result in either OX19+ (CD5+) or OX19- (CD5-) cytolytic alloselective lymphocytes and show that the OX19- (CD5-) cytolytic cells are OX34+ W3/25- (CD4-) OX8+ (CD8+) lymphocytes not exposing surface Ig. It is further demonstrated that the appearance of CD5+ and CD5- cytolytic alloselective lymphocytes are mutually exclusive; immunization with (WF X BN) F1 cells leading exclusively to appearance of OX19+ effector cells while immunization with WF cells leads to OX19- effector cells. Alloimmunization of WF rats only results in appearance of OX19+ cytolytic lymphocytes.     

Mechanisms maintaining antibody-induced enhancement of allografts. II. Mediation of specific suppression by short lived CD4+ T cells

In DA rats grafted with PVG hearts, the injection of 1 ml of Wistar-Furth x DA)F1 anti-PVG serum on the day of grafting prevents rejection and induces a state of specific unresponsiveness. An adoptive transfer assay was used to test the capacity of T cell subsets, taken from rats given enhancing serum, to either restore rejection or to transfer unresponsiveness to syngeneic hosts irradiated with 9 Gy and grafted with donor (PVG) or third party (Wistar-Furth) hearts. W3/25+ (CD4+) cells from these animals retained some capacity to restore rejection until 50 days posttransplant, after which they invariably failed to restore PVG graft rejection but retained the capacity to effect Wistar-Furth rejection. At this time CD4+ cells were also capable of inhibiting naive but not specifically sensitized CD4+ cells capacity to restore PVG graft rejection in irradiated hosts. The development of CD4+ suppressor cells was concurrent with the appearance of clinically evident unresponsiveness in the host. MRC Ox8+ (CD8+) cells from enhanced rats when mixed with naive CD4+ cells delayed rejection in adoptive recipients but did not reestablish unresponsiveness. Paradoxically, the CD4+ cells that transfer unresponsiveness to the adoptive host proliferate such as normal cells in MLC to both donor and third party alloantigen. Unfractionated cells, CD4+ or CD8+ cells did not proliferate to relevant idiotype in vitro. The CD4+ cells after 3 days in culture, with either alloantigen or idiotype-bearing stimulator cells, lost their capacity to suppress in the adoptive transfer assay. The maintenance of specific unresponsiveness was thus shown to be due to a CD4+ suppressor T cell whose function was lost in culture, and therefore could not be detected in MLC or idiotype assays.     

Lymphoid precursors in intestinal cryptopatches express CCR6 and undergo dysregulated development in the absence of CCR6

Small intestinal cryptopatches (CP) are the major anatomic site for extrathymic differentiation by precursors destined to become intestinal intraepithelial T lymphocytes (IEL). We found that mice deficient in CCR6 exhibited a 2.7-fold increase in the number of alphabeta TCR IEL, but little or no expansion of gammadelta TCR IEL. Among the alphabeta TCR IEL subsets, the CD4- CD8alphaalpha+ and CD4+ CD8alphaalpha+ subsets were preferentially expanded in CCR6 null mice. Because some CD8alphaalpha+ IEL can arise through extrathymic differentiation in CP, we investigated CCR6 expression by T lymphocyte precursors undergoing extrathymic differentiation in intestinal CP. In sections of CP, 50-60% of c-kit+ precursors were CCR6+. CD11c(+) cells concentrated at the periphery of CP did not express CCR6. A subset of c-kit+, Lin- cells in lamina propria suspensions was CCR6+, but CCR6 was absent from c-kit+ precursors in bone marrow. CCR6 was absent from the vast majority of mature IEL. CCR6 is present on lymphocyte precursors in cryptopatches, expressed transiently during extrathymic IEL development, and is required for homeostatic regulation of intestinal IEL.     

Human herpesvirus 6 infection of CD4+ T-cell subsets

The immune system includes CD4+ regulatory T (T reg) cells that play a role in self-tolerance and demonstrate functional variations that govern immune responses. HHV-6 is an important immunosuppressive virus that completely replicates in vivo and in vitro in only CD4+ T cells. However, there have been no reports of the specific T-cell subpopulation that permits the replication of this virus. Here, we evaluated the infectivity of HHV-6 to specific T-cell populations such as CD4+CD25 high, which includes the majority of T reg cells, and CD4+CD25(-). These cells were isolated from peripheral blood and then expanded. The expanded cell fractions were then infected with the HHV-6 variant B strain, and the spreads of infected cells were evaluated by immunofluorescence. Viral growth was also quantified by real-time PCR. The effects of virus infection on cytokine production from these T-cell subsets were examined using ELISA. Our results revealed that both these fractions permitted complete HHV-6 replication. Virus infection enhanced the production of both Th1- and Th2-type cytokines from CD4+CD25(-) T cells; however, only Th2-type cytokine release was augmented from viral-infected CD4+CD25 high T cells. Further, while virusinfected CD4+CD25 high T cells shift their antiviral immunity toward Th2 dominance by producing IL-10, the role of virus-infected CD4+CD25(-) T cells remains obscure.     

Phenotypic analysis of a large number of normal human bone marrow sample by flow cytometry

Bone marrow aspirates from 48 healthy donors (34 adults, 14 children) were analyzed by flow cytometry (FACS Analyzer) after purification of low-density bone marrow cells (Ld BMC) on a density gradient (d = 1,077) and labelling with 23 anti-hematopoietic cell monoclonal antibodies. Based on physical properties, these Ld BMC could be divided into four different populations called E, My, Mo and L, which comprised 14% +/- 9%, 31% +/- 16%, 10% +/- 5% and 45% +/- 14% of these cells, respectively. The phenotypic analysis of these different populations enabled the identification in E, of erythrocytes (Glycophorin A+, Rhesus D+, but negative for early erythroid differentiation markers such as the transferrin receptor (Tf. R) and the FA6-152 antigen); in My of cells of the myeloid lineage (VIM2+, HLA DR-); in Mo of cells of the monocytic lineage (VIM2+, CD14+) plus some myeloblasts (VIM2+, CD14-, HLADR+) and finally in L of a heterogeneous population including: 1. T lymphocytes labelled to the same extent by CD2, CD3, CD5 and CD6 (28% +/- 10%), B lymphocytes assessed by CD19 and CD20 (12% +/- 8%), Pre-B cells (CD10+ = 8% +/- 7%), less than 5% of "natural killer" cells (CD16+ or Leu7+) and finally, less than 6% of myelomonocytes (CD14+ and/or VIM2+). 2. The erythroid lineage (rhesus D+ = 42% +/- 20%, Tf.R+ and FA6-152+ = 32% +/- 12%). 3. Undifferentiated cells or progenitor cells (CD34+ = 7% +/- 5%). 4. Cells unlabelled by any antibodies (approximately 6%). We observed no difference between bone marrow samples from adults or children, with respect to physical properties, and with all but four immunological markers. A significantly higher proportion of B cells (CD19+ and CD10+) (P less than 0.001) and undifferentiated cells (CD34+ and HLADR+) (P less than 0.02) was observed in children. These data, obtained from a large number of bone marrow samples, could be used to quantify the imbalance of some bone marrow disorders.     

Differential recovery of polymorphonuclear neutrophils,B and T cell subpopulations in the thymus,bone marrow,spleen and blood of mice following split-dose polychemotherapy

In these studies, we examined the effect of a maximum-tolerated, split-dose chemotherapy protocol of cyclophosphamide, cisplatin, and 1,3-bis(2-chloroethyl)-1-nitrosourea carmustine on neutrophil and lymphocyte subpopulations in the peripheral blood (PBL), thymus, bone marrow and spleen. It was found that this protocol of polychemotherapy, modeled after the induction protocol used with autologous bone marrow transplantation for breast cancer, suppressed both B and T cell populations and T cell function at times when the absolute neutrophil count had returned to normal or supernormal numbers. In the peripheral blood, 7 days following initiation of chemotherapy, there was a twofold increase in the percentage of granulocytes as compared to the level in control animals on the basis of a differential count. The polymorphonuclear neutrophil (PMN) frequency in the bone marrow was increased on day 14 and statistically identical to that in control mice on all other days analyzed. In contrast to the bone marrow cells and PBL on day 7, the frequency of PMN in the spleen and thymus was depressed. B cells (B220+) were depressed in the PBL, spleen and bone marrow and took 18–32 days to return to their normal frequency, while the frequency of B cells in the thymus was increased owing to a loss of immature T cells. The percentage of CD3+ cells in the thymus, spleen and bone marrow was significantly increased and required 10–18 days to return to normal levels, while the absolute number of CD3+ cells in the blood varied around the normal value. The ratio of CD4+ to CD8+ cells in all the organs studied varied only slightly owing to a similar reconstitution of CD4+ and CD8+ cells. In contrast to the phenotypic recovery of the CD3+, CD4+ and CD8+ cells, the ability of the splenic lymphocytes to respond to concanavalin-A was depressed and remained depressed, despite the phenotypic reconstitution of the T cell subsets, on the basis of both percentage and absolute cell number. These results show a selective T and B cell depression following multi-drug, split-dose chemotherapy in tissue and blood leukocyte populations and a chronic depression in T cell function.