OBJECTIVE

While naïve T cells circulate between peripheral blood and lymph nodes, memory effector T cells acquire certain surface molecules that enable them to travel to peripheral tissues and exert their effector function. We analyzed whether deficient numbers of effector-type T cells within the malignant effusion might contribute to tumor escape from immunosurveillance.

METHODS

We analyzed the expression of a broad range of adhesion molecules and chemokine receptors (CD62L, CD56, CCR4, CCR5, CCR7, CXCR3, CLA, and integrin alpha 4 beta 7) on tumor-associated lymphocytes in effusions and peripheral blood lymphocytes of patients with malignant ascites (n = 11) or malignant pleural effusion (n = 16). A tumor-associated lymphocyte:peripheral blood lymphocyte ratio was calculated as an indicator for homing of lymphocytes into the effusions and was compared with patients with nonmalignant ascites (n = 17).

RESULTS

Patients with malignancies show an increased enrichment of T cells expressing the phenotype of "naïve" (CD62L+ and CD45RA+CCR7+), "central memory" (CD45RA-CCR7+), and type 2-polarized (CCR4+) T cells within their effusions. In contrast, enrichment of "effector"-type (CD45RA-CCR7- or CD45RA+CCR7-) and presumably type 1-polarized T cells (CCR5+) at the tumor site is deficient. The same is true for natural killer cells and potentially cytotoxic CD56+ T cells.

CONCLUSIONS

Here we show for the first time that patients with malignant effusions show a deficient enrichment of T cells expressing the phenotype of type-1-polarized effector T cells at the tumor site. This mechanism is likely to contribute to the escape of tumor cells from immunosurveillance.

Bronchiolitis is characterized histologically by epithelial necrosis and peribronchial infiltration of leukocytes, with a high percentage of neutrophils in the airways. We investigated the expression of adhesion molecules (CD11a, CD11b, CD18, CD31, CD54, and CD62L) on neutrophils from nasopharyngeal aspirates (NPAs) and peripheral blood (PB) of infants with respiratory syncytial virus (RSV)-induced bronchiolitis. The expression of CD31 and CD62L on neutrophils from NPAs is decreased and the expression of CD11b, CD18, and CD54 on neutrophils from NPAs is increased compared with cells from PB of RSV-infected infants. The expression of CD18 and CD54 on neutrophils from PB of RSV-infected infants is also increased compared with cells from PB of control infants. Shedding of CD31 and CD62L on neutrophils in RSV infection may contribute to the neutrophil emigration from blood to airways; the upregulation of Mac-1 (CD11b/CD18) and CD54 on neutrophils may help explain the high percentage of neutrophils in the airways of RSV bronchiolitis; and the upregulation of Mac-1 may be involved in the increased neutrophil-airway epithelial adhesion in RSV infection.

We describe two modular protocols for immunostaining and multiparameter flow cytometric analysis of major human antigen-presenting cells (APCs; e.g., dendritic cells, monocytes and B lymphocytes) in minimally manipulated whole blood samples. Simultaneous detection of up to eight colors is enabled by careful selection and testing of cell-subset-defining monoclonal antibodies (anchor markers) in the appropriate fluorochrome combinations, in order to show the quantification of surface expression levels of molecules involved in chemotaxis (e.g., CX(3)CR1 and CCR2), adhesion (e.g., CD11b and CD62L), antigen presentation (e.g., CD83, CD86 and CD209) and immune regulation (e.g., CD101) on circulating APCs. Each immunostaining reaction requires as little as 50-100 microl of peripheral whole blood and no density-gradient separation, and the entire procedure from preparation of reagents to flow cytometry can be completed in <5 h.

The complement receptor 2 (CR2, CD21) is part of a complex (CD21/CD19/CD81) acting as a co-receptor to the B cell receptor (BCR). Simultaneous triggering of the BCR and CD21 lowers the threshold for B cell activation. Although CD21 is important, B cells that express low amounts or lack surface CD21 (CD21(-/low) ) are increased in conditions with chronic inflammation, e.g. autoimmune diseases. However, little is known about the CD21(-/low) B cell subset in peripheral blood from healthy donors. Here, we show that CD21(-/low) cells represent approximately 5% of B cells in peripheral blood from adults but are barely detectable in cord blood, after excluding transitional B cells. The CD21(-/low) subset can be divided into CD38(-) 24(+) and CD38(-) 24(low) cells, where most of the CD38(-) 24(+) are CD27(+) immunoglobulin (Ig)M(+) IgD(+) and the CD38(-) 24(low) are switched CD27(-) . Expression levels of additional markers, e.g. CD95 and CD62L, are similar to those on classical memory B cells. In contrast to naive cells, the majority of CD21(-/low) cells lack expression of the ABCB1 transporter. Stimulation with a combination of BCR, Toll-like receptor (TLR)-7/8 and interleukin (IL)-2 induces proliferation and differentiation of the CD21(-/low) B cells comparable to CD21(+) CD27(+) memory B cells. The response excluding BCR agonist is not on par with that of classical memory B cells, although clearly above that of naive B cells. This is ascribed to a weaker response by the CD38(-) 24(low) subset, implying that some memory B cells require not only TLR but also BCR triggering. We conclude that the CD21(-/low) cells in healthy donors are memory B cells.

OBJECTIVE

Natural CD4(+)CD25(+) regulatory cells (nTregs) have been implicated in maintaining peripheral immune tolerance. This study aims to test whether immunotherapy using in vitro-expanded Treg (iTregs) could suppress allograft rejection in corneal transplantation model.

METHODS

Natural CD4(+)CD25(+) T cells were freshly purified from naïve mice and expanded in vitro by culturing with anti-CD3/CD28-coated Dynabeads, interleukin (IL)-2 and transforming growth factor (TGF-β1). Suppression ability of iTregs was assayed by co-culturing with CD4(+)CD25(-) T cells (Teff) in vitro and by targeting corneal allograft rejection in vivo. Tracking of iTreg after adoptive transfer in vivo were examined by CFSE labeling.

RESULTS

Natural Treg cells were expanded by culturing with anti-CD3/CD28-coated Dynabeads in the presence of IL-2 and TGF-β1. Compared with nTregs, iTregs had similar expression of CD62L, and PD- L1, lower expression of CD69, higher levels of PD-1, CD25, and Foxp3. iTreg cells exerted stronger suppression function than natural Treg cells when cocultured with CD4(+)CD25(-) T cells in vitro and prevented fully MHC-mismatched corneal allograft rejection. Survival of iTreg cells could suppress alloimmune reaction and most prone to migrate to graft draining LNs and spleens. Moreover, maintaining CD25 expression on iTregs was indicative for preservation of allosuppression.

CONCLUSIONS

Therapeutic use of in vitro-expanded CD4(+)CD25(+) T cells may be a effective and safe tool for controlling allograft rejection and may help induce allograft tolerance.

Although Leptospira can infect a wide range of mammalian species, most studies have been conducted in golden Syrian hamsters, a species particularly sensitive to acute disease. Chronic disease has been well characterized in the rat, one of the natural reservoir hosts. Studies in another asymptomatic reservoir host, the mouse, have occasionally been done and have limited infection to mice younger than 6 weeks of age. We analyzed the outcome of sublethal infection of C3H/HeJ mice older than age 10 weeks with Leptospira interrogans serovar Copenhageni. Infection led to bloodstream dissemination of Leptospira, which was followed by urinary shedding, body weight loss, hypothermia, and colonization of the kidney by live spirochetes 2 weeks after infection. In addition, Leptospira dissemination triggered inflammation in the kidney but not in the liver or lung, as determined by increased levels of mRNA transcripts for the keratinocyte-derived chemokine, RANTES, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-1β, inducible nitric oxide synthase, interleukin-6, and gamma interferon in kidney tissue. The acquired humoral response to Leptospira infection led to the production of IgG mainly of the IgG1 subtype. Flow cytometric analysis of splenocytes from infected mice revealed that cellular expansion was primarily due to an increase in the levels of CD4(+) and double-negative T cells (not CD8(+) cells) and that CD4(+) T cells acquired a CD44(high) CD62L(low) effector phenotype not accompanied by increases in memory T cells. A mouse model for sublethal Leptospira infection allows understanding of the bacterial and host factors that lead to immune evasion, which can result in acute or chronic disease or resistance to infection (protection).

Steroid-sensitive nephrotic syndrome (SSNS) is classically thought to be a T-cell disorder. The aim of this study was to examine whether or not thymus homeostasis was affected in SSNS. Mature and naive T cell recent thymic emigrants were quantified in the peripheral blood of nephrotic patients and controls. Because the generation of new T cells by the thymus ultimately depends on hematopoietic stem cells, CD34+ cells were also included in the study. Nineteen patients with SSNS during relapse, 13 with SSNS during proteinuria remission, and 18 controls were studied. Cell-surface markers (CD3, CD4, CD8, CD19, CD16, CD56, CD45RA, CD62L, CD34, and CD38) were analyzed by flow cytometric analysis. T-cell rearrangement excision circles (TRECs) were quantified in CD2+ cells by real-time polymerase chain reaction. Stroma cell-derived factor-1 (SDF-1) genotype and metalloproteinase-9 (MMP-9) plasma levels were also determined. Mature T cells (CD4+ and CD8+), circulating naive T cells (CD62L+ and CD3+ CD62L+), and recent thymic emigrants (CD45RA+) as well as TRECs, that measure thymus production, had a similar level in the three groups of patients. Conversely, CD34+ hematopoietic stem cells displayed a two-fold increase in SSNS patients during relapse either compared with controls or SSNS patients at remission. In addition, compared with controls, SSNS patients at remission displayed (1) a decrease in CD19+ cells (B cells) and (2) an increase in CD16CD56+ cells [natural killer (NK) cells]. In conclusion, thymus homeostasis is not significantly affected in nephrotic patients. Hematopoietic stem-cell mobilization at proteinuria relapse, as well as changes in B and NK cells during remission, suggest that SSNS might be due to a general disturbance of hematopoietic and immune cell trafficking.

BACKGROUND

After positive selection, the newly generated single positive (SP) thymocytes migrate to the thymic medulla, where they undergo negative selection to eliminate autoreactive T cells and functional maturation to acquire immune competence and egress capability.

RESULTS

To elucidate the genetic program underlying this process, we analyzed changes in gene expression in four subsets of mouse TCRαβ(+)CD4(+)CD8(-) thymocytes (SP1 to SP4) representative of sequential stages in a previously defined differentiation program. A genetic signature of the migration of thymocytes was thus revealed. CCR7 and PlexinD1 are believed to be important for the medullary positioning of SP thymocytes. Intriguingly, their expression remains at low levels in the newly generated thymocytes, suggesting that the cortex-medulla migration may not occur until the SP2 stage. SP2 and SP3 cells gradually up-regulate transcripts involved in T cell functions and the Foxo1-KLF2-S1P(1) axis, but a number of immune function-associated genes are not highly expressed until cells reach the SP4 stage. Consistent with their critical role in thymic emigration, the expression of S1P(1) and CD62L are much enhanced in SP4 cells.

CONCLUSIONS

These results support at the molecular level that single positive thymocytes undergo a differentiation program and further demonstrate that SP4 is the stage at which thymocytes acquire the immunocompetence and the capability of emigration from the thymus.

Exogenous dendritic cells display restricted trafficking when injected in vivo and stimulate CD8 T cell responses that are localized to a small number of lymphoid compartments. By examining these responses in the presence and absence of FTY720, a drug that causes sequestration of T cells in lymph nodes, we demonstrate that a significant fraction of divided CD8 T cells redistribute into Ag-free lymph nodes within 3 days of activation. Despite variation in the level of expression of CD62L, redistribution of these cells is CD62L-dependent. Redistributed CD8 T cells exhibit characteristics of differentiated effectors. However, when re-isolated from Ag-free lymph nodes 3 days after activation and transferred into naive mice, they persist for at least 3 wk and expand upon Ag challenge. Thus, CD8 T cells that redistribute to Ag-free lymph nodes 3 days after immunization contain memory precursors. We suggest that this redistribution process represents an important mechanism for establishment of lymph node resident central memory, and that redistribution to Ag-free nodes is an additional characteristic to be added to those that distinguish memory precursors from terminal effectors.

Human memory (CD45RO+) CD4+ T cells can be distinguished into two subpopulations on the basis of expression of the lymph node homing receptor, L-selectin (CD62L). In a prior study we showed that human L-selectin-positive memory T-helper (Th) cells promote the maturation of IgG- and IgA-producing cells by naive B cells. To further elucidate the contribution of memory CD4+ T cells to B-cell differentiation, human memory CD4+ T cells with or without L-selectin expression were evaluated for production of cytokines that participate in regulation of immunoglobulin production. It was found that L-selectin-positive human memory CD4+ T cells produce mainly interleukin (IL)-4 and IL-5, whereas L-selectin-negative CD4+ T cells produce mainly interferon-gamma (IFN-gamma). This profile of cytokine expression coincides with the profile that distinguishes Th1 and Th2 subsets. In contrast to the murine system, IL-10 production was similarly contributed by human L-selectin-positive and -negative memory CD4+ T-cell subpopulations. These results suggest that the human L-selectin-negative and -positive subpopulations of human memory CD4+ T cells contain Th1-like and Th2-like cytokine-producing cells, respectively.

Diversity in the peripheral T cell receptor repertoire of rhesus (Macaca mulatta) and pig-tailed macaques (Macaca nemestrina) has been studied by examining the profile of CDR3 lengths in TCR beta chains. Expressed CDR3 length distribution profiles for individual TCRBV families were obtained from total peripheral blood mononuclear cells (PBMC) and T cell subsets isolated from PBMC. These studies reveal that the T cell receptor repertoire of PBMC from healthy macaques often exhibits skewing in TCRBV family CDR3 profiles. The skewing of TCRBV family CDR3 profiles was evident as discrete expanded length(s) and was detected in up to 50% of the PBMC profiles. Analyses of separated T cell populations demonstrated that the CD8+ T cell subset was responsible for the majority of observed skewing in CDR3 length profiles. However, CD4+ T cells were also shown to contribute to the skewed peripheral PBMC repertoire in these animals. While certain TCRBV families frequently displayed skewed profiles, there was no concordance in the particular CDR3 lengths expanded among the different animals. Furthermore, an additional feature of the peripheral blood of the animals studied was the presence of an unusual population of extrathymic CD4 and CD8+ (double-positive) T cells (up to 9.6% in the PBMC of rhesus macaques). The double-positive T cells could be differentiated from CD4 single-positive and CD8 single-positive T cells by their increased surface expression of LFA-1 and decreased CD62L expression. The percentage of the double-positive T cells was higher in rhesus than pig-tailed macaques and contributed substantially to the peripheral T cell repertoire.

BACKGROUND

FTY720 (FTY), a novel immunomodulator with the potential to improve immunosuppressive therapy after organ transplantation, is currently under clinical investigation. Previous experimental animal studies have shown that FTY has a unique mechanism of action associated with altered lymphocyte recirculation.

METHODS

Participating in a phase I clinical trial, we studied the pharmacodynamic effects of FTY in stable renal allograft recipients. We analysed the effect of FTY on surface marker expression on T-cell subpopulations by flow cytometry.

RESULTS

A single oral dose of FTY (0.25-3.5 mg) significantly reduced peripheral lymphocyte counts by 30-70%. FTY reduced all T-lymphocyte subsets, CD4(+) cells more than CD8(+) cells. However, we observed that lower doses of FTY (0.25-2 mg, n = 11) did not affect peripheral CD4(+)CCR5(+) T-lymphocyte counts, while the highest FTY dose of 3.5 mg (n = 2) exerted a rapid reduction of CD4(+)CCR5(+) cells. Peripheral CD8(+)CCR5(+) T-lymphocyte counts were reduced by either low (0.25-2 mg) or high (3.5 mg) doses of FTY. In contrast to CCR5(+) cells, cells expressing CD62L were preferentially reduced after administration of FTY. In particular, CD4(+)CD62L(+) T cells declined after treatment. CD4(+) and CD8(+) T-lymphocyte subpopulations expressing the other chemokine and adhesion receptors (CXCR4, CD11a and CD49d) were reduced to a similar extent as compared with overall CD4(+) or CD8(+) T-lymphocyte counts.

CONCLUSIONS

Despite the limited number of patients, especially in the placebo (n = 3) and the high-dose groups (n = 2), our observations suggest that FTY exerts differential effects on T-cell subpopulations. FTY predominantly reduces CD4(+)CD62L(+) cells in the peripheral blood suggesting increased migration into lymph nodes. It seems that only FTY doses above 2 mg are able to reduce peripheral CD4(+)CCR5(+) T lymphocytes, which are potentially capable of infiltrating into the allograft during rejection.

Although a role for CD8+ T cells in the pathogenesis of rheumatoid arthritis (RA) has been suggested, the precise nature of their involvement is not fully understood. In the present study we examined the central and effector memory phenotypes of CD4+ and CD8+ T cells in the peripheral blood of patients with RA and systemic lupus erythematosus. Terminally differentiated effector memory CD45RA+CD62L-CD8+ T cells were significantly decreased in RA patients, whereas the central memory CD45RA-CD62L+ CD8+ T-cell population was increased as compared with levels in healthy control individuals. Naïve and preterminally differentiated effector memory CD45RA-CD62L- CD8+ T cells did not differ between RA patients and control individuals. The CD45RA-CD62L+ central memory CD4+ T-cell subpopulation was increased in RA patients, whereas the naïve and effector memory phenotype of CD4+ T cells did not differ between RA patients and control individuals. In patients with systemic lupus erythematosus the distribution of naïve/memory CD4+ and CD8+ T cells did not differ from that in age- and sex-matched control individuals. These findings show that peripheral blood CD8+ T cells from RA patients exhibit a skewed maturation phenotype that suggests a perturbation in the homeostasis of these cells. The central memory CD45RA-CD62L+ CD4+ and CD8+ T-cell numbers were increased in RA, suggesting an accelerated maturation of naïve T cells. The decreased numbers of terminally differentiated CD45RA+CD62L- effector memory CD8+ T cells in peripheral blood of RA patients may reflect increased apoptosis of these cells or enhanced migration of these cells to sites of inflammation, which may play a role in the pathogenesis of RA.

The aging of the immune system, also named immunosenescence, affects vaccine responses. However, the onset of age-related immunosenescence has been uncertain, in particularly with regard to vaccine responses. Here, we show that the formation of antibodies in response to vaccination against hepatitis B virus infection was significantly reduced for donors with a mean age of 61 y compared with a group with a mean age of 33 y. Booster vaccination sero-converted the elderly donors, but only at a reduced level, while a stronger response was found for the group of young donors. Agreeing with these findings, the hepatitis B surface antigen-specific proliferative responses by donor-derived T cells were reduced for the elder donors. Interestingly, the association between expression of the adhesion molecule CD62L (L-selectin) on naïve and central memory T cells and the formation of antigen-specific antibodies differed significantly between younger and elder donors. This finding corresponds well with the observation made previously that CD62L gene ablation in animals alters the formation of antigen-specific antibodies. We suggest that a complex interplay between the expression of CD62L and its ligands is a determinant in early-age immunosenescence affecting the response to HBV vaccination.

We have previously identified 12 surface antigens whose differential expression represented the signature of B-cell chronic lymphocytic leukemia (B-CLL) subsets with different prognosis. In the present study, expression data for these antigens, as determined in 137 B-CLL cases, all with survivals, were utilized to devise a comprehensive immunophenotypic scoring system of prognostic relevance for B-CLL patients. In particular, univariate z score was employed to identify the markers with greater prognostic impact, while maximally selected log-rank statistics were chosen to define the optimal cut-off points capable to split patients into two groups with different survivals. A weighted immunophenotypic scoring system was developed by integrating results from these analyses. Six antigens were selected: three positive prognosticators (CD62L, CD54, CD49c) and three negative prognosticators (CD49d, CD38, CD79b), with cut-off values ranging from 30% to 50% of positive cells. By weighing the expression of each marker according to its statistical power, a complete scoring system, with point values comprised between 0 (complete absence of phenotypic conditions associated with good prognosis) and 9 (all the phenotypic conditions associated with good prognosis fulfilled), allowed to split the whole set of B-CLL patients, into three distinctive prognostic groups (P = 4.78 x 10(-11)) with high- (score 0-3), intermediate- (score 4-6), and low- (score 7-9) risk of death. The three risk groups showed different distribution of cases as for Rai's stages, IgVH mutations, and ZAP-70 expression. The proposed immunophenotypic scoring system may be an additional useful tool in routine diagnostic/prognostic procedures for B-CLL.

BACKGROUND

The objective of the current study was to illustrate the influence of neoadjuvant therapy on the local immune response in patients with cervical carcinoma.

METHODS

Uninvolved tumor-draining lymph nodes (TDLN) (n=158 lymph nodes), including internal, external, and common iliac lymph nodes as well as obturator, presacral, and aortic lymph nodes from 15 nontreated (NT) patients, 4 chemotherapy (CT)-treated patients, and 19 chemoradiation (CR)-treated patients, were analyzed for lymphocyte subset distribution and for the proliferative response of T cells to polyclonal activation and interferon-gamma (IFN-gamma) production. Lymphocyte subsets in peripheral blood also were assessed.

RESULTS

TDLNs from CR-treated patients contained higher proportions of CD8+ cells and natural killer cells than NT and CT-treated patients (P values ranged from <0.05 to <0.01). TDLNs from CR-treated patients were enriched in activated-type CD4+ cells (HLA-DR+, CD134+, CD62L-, and CD25+ at an intermediate expression level; P values ranged from <0.05 to <0.01) and activated-type CD8+ cells (CD62L-, P<0.001) compared with NT patients. Concomitantly, there was a reduction in the proportion of naïve-type CD4+ and CD8+ cells (CD45RA+/CD62L+) (P<0.01 and <0.05, respectively). CR treatment increased the proportion of both CD4+ and CD8+ cells prone to produce IFN-gamma. All TDLNs contained suppressive CD4+ T regulatory (Treg) cells (CD25+ and CD152+ at a high expression level) whose frequency and suppressive activity was not influenced by the treatment. Therapy-induced changes in TDLN were mirrored only in part by respective alterations in peripheral blood.

CONCLUSIONS

To our knowledge, the current study is the first to show that neoadjuvant therapy produces an enhancing effect on the immune competency of TDLNs from patients with cervical carcinoma.

The quantitative and phenotypic changes of peripheral blood monocytes during the acute stage of simian immunodeficiency virus infection were investigated. We inoculated intravenously three cynomolgus monkeys (Macaca fascicularis) with 100 TCID50 of SIVmac239 and collected whole blood twice a week until 35 days postinoculation. We found that the relative number of monocytes in peripheral blood leukocytes significantly increased at 7-17 days postinoculation. This increase was concomitant with the peak of primary SIV antigenemia. To determine if the monocytes observed during the acute stage were phenotypically altered, they were periodically examined for the expression of surface markers (i.e., CD11b, CD14, CD16, CD29, D32, CD56, CD62L, CD64, CD80, and MHC-II-DR) by flow cytometry. The results showed that the expression levels of CD14 and CD56 on most of the monocytes were remarkably reduced at 7-17 days postinoculation, and a new subpopulation, CD14lowCD16+CD80+ monocytes, was clearly detected at 10 days postinoculation. These results indicate that the phenotypic alteration of peripheral blood monocytes occurs during the primary SIV infection.

Perturbations in naive T cell homeostasis and function may play a major role in the immunodeficiency that accompanies HIV infection. By examining naive CD4(+) T cell function on a single cell basis, we provide evidence that these cells have significant qualitative defects in HIV disease. Ki67, a molecule expressed during cell cycle progression, is induced less efficiently among naive CD4(+) T cells from HIV-infected individuals following activation with anti-TCR Ab. The impairment in Ki67 expression is evident even when a separate function, CD62L down-modulation, is within normal ranges. Moreover, the defects in Ki67 induction are only sometimes corrected by the addition of rIL-2 to cell cultures. An initial assessment of IL-2 unresponsiveness in cells from selected HIV-infected individuals suggests that the defect is not a consequence of impaired IL-2R expression or IL-2R signaling capability. Qualitative defects in naive T cells that cannot be routinely corrected by IL-2 have significant implications for disease pathogenesis and for strategies using IL-2 as a vaccine adjuvant in HIV disease.

BACKGROUND

Optimization of the culture environment for the ex vivo expansion of T cells is crucial for obtaining the large doses of cells needed for cellular immunotherapy. O2 tension is a key parameter that impacts the proliferation and quality of the expanded T cells.

METHODS

Peripheral blood mononuclear cells were stimulated with either PHA or an anti-CD3 monoclonal antibody under 5% (low) or 20% (high) O2 atmospheres. After stimulation, cells were cultured in the presence of IL-2 under either low or high O2 conditions.

RESULTS

T cells stimulated and grown under 5% O2 exhibited higher proliferation rates and a mean (n = 11) of 5.8-fold greater total expansion over T cells grown under 20% O2. Stimulation under 5% O2 produced a lasting proliferative effect even after a switch to 20% O2. Examination of apoptosis by the flow cytometry-based TUNEL assay showed a mean (n = 9) of 2.9-fold greater percentage of apoptotic cells under 20% O(2). Flow-cytometric analysis of the IL-2 receptor (CD25) showed that the normal downregulation kinetics - following stimulation-induced CD25 upregulation - were slowed under 5% O(2), such that the 5% O2 cultures had a greater number of CD25+ cells, and those CD25+ cells expressed an average (n = 6) of 41% higher levels of CD25 receptor per cell. No significant O2 tension effects were observed on other surface antigens (CD3, CD28, and CD62L) examined. The key metabolic parameters, specific glucose uptake rate, q(glu), and specific lactate production rate, q(lac), were both increased by a mean (n = 5) of 47% under 5% O2.

CONCLUSIONS

Beyond the physiological significance, improved T-cell proliferation under 5% O2 would allow for decreased culture times in expanding T cells for cellular immunotherapies. Evidence of increased IL-2R expression and reduced apoptosis levels under 5% O2 may help explain this phenomenon.

Peptide immunotherapy (PIT) offers realistic prospects for the treatment of allergic diseases, including allergic asthma. Much is understood of the behavior of naive T cells in response to PIT. However, treatment of patients with ongoing allergic disease requires detailed understanding of the responses of allergen-experienced T cells. CD62L expression by allergen-experienced T cells corresponds to effector/effector memory (CD62L(lo)) and central memory (CD62L(hi)) subsets, which vary with allergen exposure (e.g., during, or out with, pollen season). The efficacy of PIT on different T helper 2 (Th2) cell memory populations is unknown. We developed a murine model of PIT in allergic airway inflammation (AAI) driven by adoptively transferred, traceable ovalbumin-experienced Th2 cells. PIT effectively suppressed AAI driven by unfractionated Th2 cells. Selective transfer of CD62L(hi) and CD62L(lo) Th2 cells revealed that these two populations behaved differently from one another and from previously characterized (early deletional) responses of naive CD4(+) T cells to PIT. Most notably, allergen-reactive CD62L(lo) Th2 cells were long-lived within the lung after PIT, before allergen challenge, in contrast to CD62L(hi) Th2 cells. Despite this, PIT was most potent against CD62L(lo) Th2 cells in protecting from AAI, impairing their ability to produce Th2 cytokines, whereas this capacity was heightened in PIT-treated CD62L(hi) Th2 cells. We conclude that Th2 cells do not undergo an early deletional form of tolerance after PIT. Moreover, memory Th2 subsets respond differently to PIT. These findings have implications for the clinical translation of PIT in different allergic scenarios.

BACKGROUND

In animal and human autopsy studies of sepsis, CD4+ splenocytes either undergo apoptosis or are polarized to the Th2 effector subtype. In mice, these changes occur within 24 hours of the onset of sepsis. Preventing the loss of CD4+ T cells and the Th2-polarization of CD4+ T cells provides a significant survival advantage in mouse models of sepsis. The molecular mechanism(s) for the phenotypic changes of splenic CD4+ T cells in sepsis are not well understood.

METHODS

CD4+ splenocytes were enriched by negative selection from disaggregated spleens of septic and sham-operated mice at 6 and 24 hours after surgery. Phenotypic analysis using cell surface markers (CD25, CD44, CD62L, CD69), cytokine secretion in response to CD3/CD28 coligation, and whole genome microarray gene expression profiles were obtained for these cells.

RESULTS

Consistent with previous reports, sepsis induced a progressive decrease in the number of CD4+ splenocytes and a time-dependent alteration in CD4+ T-cell phenotype. At 6 hours, when no differences in cell number or surface marker expression were observed, significant alterations in RNA abundance were measured for 498 probe sets. Ontologic classification of these genes indicated changes in cellular physiology. Pathway analysis indicated that T-cell receptor signaling and mitogen-activated protein kinase signaling were significantly altered by sepsis.

CONCLUSIONS

These data demonstrated a sepsis-specific transcriptional program that precedes sepsis-induced phenotypic changes in CD4+ splenocytes.

OBJECTIVE

Numerous studies have examined arterial occlusion followed by reperfusion but few studies have reported about venular occlusion which, in contrast to arterial occlusion, is associated with elevation of the capillary blood pressure. Here we examine leukocytes infiltration and tissue injury in rat mesentery during local venular occlusion and venous hypertension followed by reperfusion, and determine the level of protection offered by pretreatment with micronized, purified flavonoid fraction (MPFF).

METHODS

Leukocyte rolling, adhesion, migration, and parenchymal cell death as detected by propidium iodide labeling were determined during venular occlusion using a micropipette followed by reperfusion in the rat mesenteric microcirculation pretreated with 0, 50, or 100 mg/kg MPFF for 7 days. Spontaneous leukocyte activation by nitroblue tetrazolium reduction and expression of CD18 and CD62L on naive donor neutrophils incubated with plasma from each treatment group were determined.

RESULTS

Venous occlusion led to elevated levels of leukocyte rolling, adhesion, and migration as well as parenchymal cell death. These injurious processes were significantly inhibited by MPFF in a those-dependent fashion. MPFF reduced spontaneous leukocyte NBT reduction and the neutropil expression of CD62L, even though CD18 was not affected.

CONCLUSIONS

These results suggested that microvascular occlusion in venules with elevation of the micropressure followed by reperfusion is a highly cytotoxic process in the rat mesentery which can be attenuated by MPFF pretreatment.

CD4 and CD8 lymphocyte numbers in the gut lamina propria are grossly altered in HIV-1 infection, out of proportion to alterations in the circulation. Such alterations in lymphocyte counts in the tissues may be due to altered leucocyte migration from the blood. One factor affecting leucocyte migration is adhesion molecule expression. Levels of adhesion molecule expression on peripheral CD4 and CD8 lymphocytes, monocytes and neutrophils from HIV-1-infected (AIDS and non-AIDS) and low-risk control individuals were compared. CD11a, CD62L, CD44, CD49d and beta7 integrin expression were examined by FACS analysis of fresh whole blood. Significant alterations in adhesion molecule expression were detected in HIV infection. The most striking alterations were observed in the CD8 lymphocyte population. CD11a expression was increased and CD62L and CD44 decreased. The CD4 lymphocyte population followed a similar, though less striking, pattern of alteration in adhesion molecule expression. Neutrophils displayed significantly reduced expression of both CD11a and CD62L, but only after onset of AIDS. Monocytes from infected individuals without AIDS displayed a different pattern of altered adhesion molecule expression compared with individuals with AIDS. These findings suggest that in HIV infection, leucocyte functions, such as migration, which require adhesion molecules are abnormal.

Th2-biased immune responses characterizing neonates may influence the later onset of allergic disease. The contribution of regulatory T cell populations in the prevention of Th2-driven pathologies in early life is poorly documented. We investigated the potential of CD8(+) T cells stimulated at birth with alloantigens to modulate the development of allergic airway inflammation. Newborn mice were immunized with semiallogeneic splenocytes or dendritic cells (DCs) and exposed at the adult stage to OVA aeroallergens. DC-immunized animals displayed a strong Th1 and Tc1/Tc2 alloantigen-specific response and were protected against the development of the allergic reaction with reduced airway hyperresponsiveness, mucus production, eosinophilia, allergen-specific IgE and IgG(1), and reduction of lung IL-4, IL-5, IL-10, and IL-13 mRNA levels. By contrast, splenocyte-immunized mice displayed a Th2 and a weak Tc2 alloantigen-specific response and were more sensitive to the development of the allergen-specific inflammation compared with mice unexposed at birth to alloantigens. DC-immunized animals displayed an important increase in the percentage of IFN-gamma-producing CD8(+)CD44(high), CD8(+)CD62L(high), and CD8(+)CD25(+) subsets. Adoptive transfers of CD8(+) T cells from semiallogeneic DC-immunized animals to adult beta(2)m-deficient animals prevented the development of allergic response, in particular IgE, IL-4, and IL-13 mRNA production in an IFN-gamma-dependent manner, whereas transfers of CD8(+) T cells from semiallogeneic splenocyte-immunized mice intensified the lung IL-4 and IL-10 mRNA level and the allergen-specific IgE. These findings demonstrated that neonatal induction of regulatory CD8(+) T cells was able to modulate key parameters of later allergic sensitization in a bystander manner, without recognition of MHC class I molecules.