Blood T-lymphocyte Subpopulations Research Articles

Peripheral blood T-cell modulation by omalizumab in chronic urticaria patients.

Chronic spontaneous urticaria (CSU) is a highly prevalent and difficult to manage cutaneous disease characterized by the presence of recurrent urticaria, angioedema, or both, for a period of 6 weeks or longer. One of the biological treatments used for patients with CSU with an autoimmune background and bad control of the disease is omalizumab, an anti-IgE monoclonal antibody. The understanding of the mechanism of action of this biological drug in CSU along with the identification of potential biomarkers of clinical response can be helpful in the personalized management of the disease. The purpose of this study was to analyze the effect of omalizumab on peripheral blood lymphocyte subpopulations in patients with CSU in order to identify potential biomarkers of treatment response. We analyzed 71 patients with CSU [33 under omalizumab and 38 under non-immunomodulatory drugs (treated with antihistamines; NID)] and 50 healthy controls. An exhaustive immunophenotyping of whole blood T-cell subpopulations, including naïve, central memory, effector memory, effector cells, Th1, Th2, and Th17 was performed by multiparametric flow cytometry. Moreover, in CSU patients, we analyzed markers of inflammation (ESR, DD, CRP), atopy (prick test, IgE quantification), and autoimmunity (anti-thyroid antibodies and indirect basophil activation test).To evaluate the clinical activity, the Urticaria Activity Score 7 (UAS 7) test was used. In patients with CSU under treatment with omalizumab, there was a significant decrease in the percentage of naïve and an increase in the percentage of central memory CD4 T cells as well as a decrease in the percentage of naïve and increase in the percentage of effector CD8 T-cell subsets. Moreover, patients under treatment with omalizumab had higher percentages of Th1 and Th2 cells than patients under treatment with NID. The immune monitoring of T-cell subpopulations in patients with CSU starting omalizumab, may be a useful strategy to analyze treatment response in the clinical practice.

INCREASED CD4+ TO CD8+ T-CELL RATIO AS A DRIVER OF IMPAIRED ACHILLES TENDON HEALING IN HUMAN PATIENTS

Early identification of patients at risk for impaired tendon healing and corresponding novel therapeutic approaches are urgent medical needs. This study aimed to clarify the role of CD3+ T-cells during acute Achilles tendon (AT) healing. Blood and hematoma aspirate were taken from 26 patients during AT reconstruction, and additional blood samples were obtained during clinical follow-up at 6, 26 and 52 weeks after surgery. T-cell subsets were analyzed by flow cytometry using CD3, CD4, CD8, CD11a, CD57 and CD28 antibodies. Clinical follow-up included functional tests, MRI assessments, and subjective questionnaires. In vitro, the functional behavior of patient-derived tenocytes was investigated in co-cultures with autologous unpolarized CD4+ or CD8+ T-cells, or IFNy-polarized CD8+ or IL17-polarized CD4+ Tcells (n=5-6). This included alterations in gene expression (qPCR), MMP secretion (ELISA), migration rate (scratch wound healing assay) or contractility (collagen gels). Analysis revealed that elevated CD4+ T-cell levels and reduced CD8+ T-cell levels (increased CD4/CD8 ratio) in hematoma aspirate and pre-operative blood were associated with inferior clinical outcomes regarding pain and function at 26 and 52 weeks. Increased levels of CD8+ -memory T-cell subpopulations in blood 6 weeks after surgery were associated with less tendon elongation. In vitro, tenocytes showed increased MMP1/2/3 levels and collagen III/I ratio in co-culture with unpolarized and/or IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells. This coincided with increased IL17 receptor expression in tenocytes co-cultured with CD4+ T-cells. Exposure of tenocytes to IL17-polarized CD4+ T-cells decreased their migration rate and increased their matrix contractility, especially compared to IFNy-polarized CD8+ T-cells. The CD4+ /CD8+ T-cell ratio could serve as prognostic marker for early identification of patients with impaired AT healing potential. Local reduction of CD4+ T-cell levels or their IL17 secretion represent a potential therapeutic approach to improve AT healing and to prevent weakening of the tendon ECM.

Prebiotics Modulate Gut Microbiota-mediated T-cell Immunity to Enhance the Inhibitory Effect of Sintilimab in Lewis Lung Adenocarcinoma Model Mice.

Sintilimab (Sin) helps the body to restore the anti-tumor response of T lymphocytes. However, in clinical use, the treatment process is more complicated due to adverse effects and different dosing regimens. It is not clear whether prebiotics (PREB) have a potentiating effect on Sin for lung adenocarcinoma, and this study intends to investigate the inhibitory effect, safety and possible mechanism of Sin combined with PREB on lung adenocarcinoma from animal experiments. Lewis lung adenocarcinoma cells were inoculated into the right axilla of mice subcutaneously to prepare the Lewis lung cancer mouse model and treated in groups. The volume of transplanted tumors was measured, the histopathology of the liver and kidney of mice was observed by H&E staining, the levels of ALT, AST, UREA, CREA, WBC, RBC, and HGB in blood were analyzed biochemically; the ratio of T-cell subpopulations in blood, spleen, and bone marrow was detected by flow cytometry, the expression of PD-L1 in tumor tissue was detected by immunofluorescence staining, and 16S rRNA to analyze the diversity of fecal flora. Sin inhibited tumor growth and regulated immune cell homeostasis in lung adenocarcinoma mice, but liver and kidney histopathology showed different degrees of damage after Sin treatment, while the addition of PREB reduced liver and kidney damage in lung adenocarcinoma mice and promoted Sin's regulation of immune cells. In addition, the beneficial effects of Sin were associated with changes in intestinal flora diversity. The mechanism by which Sintilimab combined with prebiotics inhibits tumor volume and regulates immune cell subpopulation balance in lung adenocarcinoma mice may be related to gut microbes.

Evaluation of peripheral blood T-lymphocyte subpopulations features in patients with hepatitis B virus-related acute-on-chronic liver failure based on single-cell sequencing technology

Objective: T lymphocyte exhaustion is an important component of immune dysfunction. Therefore, exploring peripheral blood-exhausted T lymphocyte features in patients with hepatitis B virus-related acute-on-chronic liver failure may provide potential therapeutic target molecules for ACLF immune dysfunction. Methods: Six cases with HBV-ACLF and three healthy controls were selected for T-cell heterogeneity detection using the single-cell RNA sequencing method. In addition, exhausted T lymphocyte subpopulations were screened to analyze their gene expression features, and their developmental trajectories quasi-timing. An independent sample t-test was used to compare the samples between the two groups. Results: Peripheral blood T lymphocytes in HBV-ACLF patients had different differentiation trajectories with different features distinct into eight subpopulations. Among them, the CD4(+)TIGIT(+) subsets (P = 0.007) and CD8(+)LAG3(+) (P = 0.010) subsets with highly exhausted genes were significantly higher than those in healthy controls. Quasi-time analysis showed that CD4(+)TIGIT(+) and CD8(+)LAG3(+) subsets appeared in the late stage of T lymphocyte differentiation, suggesting the transition of T lymphocyte from naïve-effector-exhausted during ACLF pathogenesis. Conclusion: There is heterogeneity in peripheral blood T lymphocyte differentiation in patients with HBV-ACLF, and the number of exhausted T cells featured by CD4(+)TIGIT(+)T cell and CD8(+)LAG3(+) T cell subsets increases significantly, suggesting that T lymphocyte immune exhaustion is involved in the immune dysfunction of HBV-ACLF, thereby identifying potential effective target molecules for improving ACLF patients' immune function.

Immune profile of peripheral blood T-lymphocyte subpopulations in chronic hepatitis B

Background: The role of immune response to chronic hepatitis B virus (HBV) infection is complex; and the specific T-cell response to this infection can determine the duration and the extent of liver disease. Objective: This study aimed at assessing the profile of Tlymphocyte subpopulations in chronic hepatitis B (CHB) patients and its association with HBV replication. Methodology: The case group included 50 CHB patients with normal liver function tests (LFTs); and the control group included 50 age and sexmatched healthy individuals. The HBV markers, LFTs and serum viral load were measured in cases. Blood CD4 and CD8 T-lymphocyte subpopulations and the CD4/CD8 ratio were assessed in both groups by flow cytometry. Results: Our results showed significantly higher CD8 T-cells; significantly lower CD3 and CD4 T-cells; markedly reduced CD4/CD8 ratio in the cases as compared to the controls (P<0.001, for all). This T-cell impairment was also significantly linked to HBeAg positivity and elevated level of viraemia. The increased level of CD8 T-cells was significantly linked to both the HBeAg positivity (P<0.001) and the elevated level of viraemia (P=0.005), whereas the decreased levels of CD3, CD4 T-cells and CD4/CD8 ratio were significantly linked to both HBeAg positivity (P<0.001, in all) and the elevated level of viraemia (P<0.001, P=0.001& P=0.007, respectively). Conclusion: T-lymphocyte subpopulations imbalance could be expected by measuring the serum HBVeAg and the viraemia level in CHB patients exhibiting normal LFTs. These parameters are recommended to be measured regularly for the cellular immune function assessment.

Malnutrition is associated with hyperinflammation and immunosuppression in COVID-19 patients: A prospective observational study.

BackgroundCoronavirus disease 2019 (COVID‐19) is spreading globally and has caused many deaths. This study investigated, for the first time, COVID‐19 patients’ nutrition status and its effects on their inflammatory and immune responses.MethodsForty‐seven COVID‐19 patients were recruited for this prospective study. According to the subjective global assessment at admission, patients were divided into the normal nutrition (NN), risk of malnutrition (RMN), or MN group. Serum cytokines and whole blood T‐cell subpopulations were measured to assess the inflammatory and immune responses in COVID‐19 patients. Analysis of covariance and χ2 tests were used.ResultsOn admission, the incidences of MN and the RMN in COVID‐19 patients were 17.0% and 38.3%, respectively. The MN group had a higher proportion with severe/critical COVID‐19 and a longer hospitalization duration than the NN group. Serum interleukin (IL) 6 concentrations were elevated in 97.9% of the patients and were the highest in malnourished patients. The IL‐4 and IL‐10 levels were elevated in 46.8% and 48.9% of the patients, respectively. The proportion of CD8+ T cells was significantly lower in the MN group than in the NN group.ConclusionA high proportion of COVID‐19 patients are malnourished or at risk of malnuourishment, especially those with severe disease. MN is associated with hyperinflammation and immunosuppression in COVID‐19 patients, and it may contribute to disease progression.

Sapropterin (BH4) Aggravates Autoimmune Encephalomyelitis in Mice

Depletion of the enzyme cofactor, tetrahydrobiopterin (BH4), in T-cells was shown to prevent their proliferation upon receptor stimulation in models of allergic inflammation in mice, suggesting that BH4 drives autoimmunity. Hence, the clinically available BH4 drug (sapropterin) might increase the risk of autoimmune diseases. The present study assessed the implications for multiple sclerosis (MS) as an exemplary CNS autoimmune disease. Plasma levels of biopterin were persistently low in MS patients and tended to be lower with high Expanded Disability Status Scale (EDSS). Instead, the bypass product, neopterin, was increased. The deregulation suggested that BH4 replenishment might further drive the immune response or beneficially restore the BH4 balances. To answer this question, mice were treated with sapropterin in immunization-evoked autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. Sapropterin-treated mice had higher EAE disease scores associated with higher numbers of T-cells infiltrating the spinal cord, but normal T-cell subpopulations in spleen and blood. Mechanistically, sapropterin treatment was associated with increased plasma levels of long-chain ceramides and low levels of the poly-unsaturated fatty acid, linolenic acid (FA18:3). These lipid changes are known to contribute to disruptions of the blood–brain barrier in EAE mice. Indeed, RNA data analyses revealed upregulations of genes involved in ceramide synthesis in brain endothelial cells of EAE mice (LASS6/CERS6, LASS3/CERS3, UGCG, ELOVL6, and ELOVL4). The results support the view that BH4 fortifies autoimmune CNS disease, mechanistically involving lipid deregulations that are known to contribute to the EAE pathology.

Evaluation of blood T-lymphocyte subpopulations involved in host cellular immunity in dogs with mammary cancer

Cancer-bearing patients are often immunosuppressed. In dogs with mammary or other cancers, various alterations in blood cell populations involved in host cellular immunity have been reported; among these cell populations some T-lymphocyte subsets play an important role against cancer. The purpose of the present study was to investigate any alterations in circulating T-lymphocyte subpopulations involved in cellular immunity in bitches with mammary cancer, in comparison to age-matched healthy intact bitches. Twenty eight dogs with mammary cancer and 14 control dogs were included in this study. Twelve out of the 28 bitches had mammary cancer of clinical stage II and 16/28 of stage III. Histological examination revealed that 23/28 animals had carcinomas, 3/28 sarcomas and 2/28 carcinosarcomas. White blood cell, neutrophil and lymphocyte absolute numbers were measured by complete blood count. Furthermore, blood T-lymphocyte population (CD3+) and the subpopulations CD4+, CD8+ and CD5low+ were assessed by flow cytometry. White blood cell and neutrophil but not lymphocyte absolute numbers were higher (P=0.003 and P=0.001, respectively) in cancer patients than controls. Flow cytometric analysis revealed that the relative percentage of T-lymphocytes (CD3+) and of CD4+, CD8+ subpopulations was lower (the CD4+/CD8+ ratio was higher), whereas the percentage of CD5low+ T-cells was higher, in dogs with cancer compared to controls; however, a statistically significant difference was found only in the case of CD8+ T-cells (P=0.014), whereas in the case of the CD4+/CD8+ ratio the difference almost reached statistical significance (P=0.059). Based on these findings, it can be suggested that, although the absolute number of blood lymphocytes is unchanged, the relative percentages of T-lymphocyte subpopulations involved in host cell-mediated immunity are altered, but only cytotoxic CD8+ T-cells are significantly suppressed, in dogs with mammary cancer of clinical stage II or III compared to age-matched healthy controls.

Allometric Dose Retranslation Unveiled Substantial Immunological Side Effects of Granulocyte Colony–Stimulating Factor After Stroke

Granulocyte colony-stimulating factor (GCSF) showed robust neuroprotective and neuroregenerative properties after stroke in rodents but failed to meet study end points in patients. Because immunologic side effects of GCSF may have escaped preclinical testing because of nonallometric dose translation, we hypothesized those as possible reasons. Stroke was induced in C57BL/6 mice by 45-minute filament middle cerebral artery occlusion. GCSF was administered at 50 and 832.5 μg/kg body weight. Treatment was controlled by vehicle injection, sham surgery, and naive animals. Immune cell counts were assessed in blood, spleen, and brain by multidimensional flow cytometry 1 day after stroke. High-dose GCSF significantly altered myeloid and T-cell subpopulations in blood and spleen and caused a tremendous increase of monocytes/macrophages infiltrating the ischemic brain. Dose-dependent immunomodulation superimposes central nervous system-specific effects of GCSF after stroke. Adaption of dose or treatment time may overcome this drawback.

Severe impairment of regulatory T-cells and Th1-lymphocyte polarization in patients with Gaucher disease.

We investigated peripheral blood T-lymphocyte subpopulations and intracellular expression of IFN-γ, IL-4, IL-10, and IL-13, by whole blood flow cytometry, in 22 type I Gaucher disease (GD) patients. Results were compared with those of 19 sex- and age-matched controls. Patients with GD exhibited decreased frequencies and absolute numbers of CD3+/CD4+ helper T lymphocytes (40.8 ± 9.8% vs. 49.4 ± 5.7%, p = 0.002, and 0.77 ± 0.33 vs. 1.04 ± 0.28 × 10(9)/μL, p = 0.011), as well as increased frequencies of CD3+CD8+ suppressor T lymphocytes (23.8 ± 8.0% vs. 18.4 ± 3.8%, p = 0.010), resulting in a significantly decreased CD4/CD8 cell ratio (p < 0.001). Moreover, they had significantly increased percentages of IFNγ-producing both CD4+ and CD8+ T cells (p = 0.0003 and p = 0.023, respectively), implying a TH-1 polarization pattern. Finally, patients with GD had decreased percentages and absolute numbers of CD4+CD25(dim) T lymphocytes (p = 0.033 and p = 0.007, respectively), of CD4+CD25(high) T lymphocytes (p = 0.039 and p = 0.016, respectively), and of CD4+CD25(high)FOXP3+ regulatory T cells (p = 0.036 and p = 0.019, respectively). Our results demonstrate that patients with GD have a significant numerical impairment of T-helper lymphocytes and a constitutive TH1 direction pattern of activation of both CD4+ and CD8+ cells, associated with a significant decrease of T-regs. Ineffective T-cell control may explain the chronic inflammatory reaction and the increased incidence of lymphoid malignancies, which have been repeatedly reported among patients with GD.

Hydromorphone does not influence the immune response in patients affected by chronic pain.

Hydromorphone (HMP) is a semisynthetic opioid that has been widely used over many years in the treatment of chronic moderatesevere pain. A number of studies have demonstrated the use of HMP in patients with cancer pain as safe and affective, and more recently HMP has been also utilised in the treatment of chronic degenerative pain (CDP). Then, some opioids are able to influence several immune parameters. Aim of our study was to assess HMP in patients with CDP in terms of safety and efficacy, to evaluate effects of HMP, within 2 months of observation, on cutaneous cell-mediated immunity (CCMI) as well as other basic immune parameters, and the production of interleukin-2 (IL-2) and interleukin-6 (IL-6). Results have shown that HMP does not influence CCMI nor the sieric levels of immunoglobulins or other immune parameters. Furthermore, it also has no effects on the 3rd and 4th complement fractions, on white blood cells count and T-lymphocyte subpopulations, on presence of seric autoantibodies, nor on production of IL-2 and IL-6. Therapy with HMP has not effects on the studied parameters of the immune system and does not seem to have any influence on the immune competence and immune response in patients with chronic pain.

Antitumor and immunomodulating effects of polysaccharides isolated from Solanum nigrum Linne

We have examined the effects of the crude polysaccharides isolated from Solanum nigrum Linne (SNL-P) in vitro and in vivo against U14 cervical cancer. SNL-P showed no antiproliferative effects in vitro at a dose up to 1 mg/ml. In vivo administration with SNL-P (90, 180, 360 mg/kg b.w., p.o.) decreased the number of ascites tumor cells and prolonged the survival time of U14 cervical-cancer-bearing mice. FACScan flow cytometer analysis showed that most of the ascites tumor cells were arrested in G2/M phase of cell cycle and the ratio of CD4+/CD8+ peripheral blood T-lymphocyte subpopulations were restored following treatment of SNL-P. Furthermore, the treatment with SNL-P also caused a significant increment in IFN-gamma (p < 0.01, 90, 180 and 360 mg/kg b.w.) and a remarkable decrease in Il-4 (p < 0.01, 90, 180 mg/kg b.w.; p < 0.05, 360 mg/kg b.w.) by the method of ELISA. These data showed that SNL-P possess potent antitumor activity and SNL-P might exert antitumor activity via activation of different immune responses in the host rather than by directly attacking cancer cells on the U14 cervical cancer bearing mice. Thus, SNL-P could be used as an immunomodulator and an anticancer agent.

Impaired T-Lymphocyte Subpopulations and Deranged Profile of Immunologic Activation in Patients with Gaucher Disease Type I

Patients with Gaucher disease exhibit substantial evidence of impairment of their immune system, namely, increased serum levels of proinflammatory cytokines and immunoglobulins, and increased incidence of B-cell malignancies, such as non-Hodgkin's lymphoma, MGUS and multiple myeloma. We investigated peripheral blood T-lymphocyte subpopulations with dual color flow cytometry, as well as the direction of T-lymphocyte activation, by using intracytoplasmic immunostaining for IL-2, IL-4, IL-10 and IFN-gamma, on resting CD4+ and CD8+ T-lymphocytes and following activation with PMA- 1 with the presence of Brefeldin-A. Evaluations were performed on 16 patients with type I Gaucher disease and on 17 healthy controls. Patients had significantly decreased absolute lymphocyte count (1621±684 vs 2148±566/mm3, p=0.013), CD3+ (1197±478 vs 1508±431/mm3, p=0.045) and CD4+ T-lymphocytes (658±245 vs 945±253/mm3, p=0.021), but not CD8+ T-lymphocytes (491±331 vs 486±189/mm3, p: n.s.), resulting in a significant reduction of the CD4/CD8 ratio (1.59±0.68 vs 2.16±0.83, p=0.041). The populations of naive CD4+CD45RA+ and of memory CD4+CD45RO+ T-lymphocytes were also significantly decreased (218±128 vs 432±179/mm3, p=0.0005 and 484±185 vs 631±231/mm3, p=0.056 respectively), however, CD8+CD45RA+ and CD8+CD45RO+ subpopulations did nor differ significantly, when compared to controls. CD3−CD56+, but not CD3+CD56+ lymphocytes were also decreased (131±82 vs 199±97/mm3, p=0.037). Patients had higher percentages of CD8+ (29.2±9.7 vs 23.5±6.8%, p=0.042), CD8+CD45RA+ (22.1±6.2 vs 18.3±5.0%, p=0.046) and CD8+CD45RO+ T-lymphocytes (13.2±6.2 vs 9.6±3.7%, p=0.027), as well as of activated CD8+HLA-DR+ (0.93±0.68 vs 0.48±0.21%, p=0.008) and CD4+HLA-DR+ T-lymphocytes (1.77±0.93 vs 1.09±0.48%, p=0.008). Moreover, although both, the absolute number and the percentage of CD20+ B-lymphocytes were similar, patients exhibited significantly increased absolute number and percentage of CD5+CD20+ B-lymphocytes (1.63±0.55 vs 0.64±0.37% p=0.00002 and 29±20 vs 13±8/mm3, p=0.011, respectively). Finally, patients with Gaucher disease had significantly increased resting TH2-polarized CD4+T-lymphocytes (CD4+IL-10+: 0.41±0.29 vs 0.24±0.11%, p=0.045) and TH1-polarized CD8+ T-lymphocytes (CD8+IFNγ+: 0.15±0.07 vs 0.08±0.04%, p=0.005, CD8+IL10+: 0.22±0.08 vs 0.32±0.014, p=0.052, and IFNγ+/IL4+ ratio among the CD8+ population 2.54±2.1 vs 1.08±0.91, p=0.018). Following mitogenic activation a very significant impairment of obtaining the TH1 phenotype was observed (CD4+IL2+ lymphocytes 33.7±17.1 vs 65.4±6.1%, p<0.00001). The above findings suggest that in patients with Gaucher disease there is a significant numerical impairment of T-helper lymphocytes and a shift towards TH-2 direction of lymphocyte activation. These findings may explain the rarity of autoimmune manifestations despite the chronic inflammatory reaction, as well as the increased incidence of lymphoid malignancies, which has been reported among patients suffering from this disease.

Effects of ω-3 fatty acids on inflammatory mediators and splenocyte cytokine mRNA expressions in rats with polymicrobial sepsis

Objectives Many studies have shown that ω-3 fatty acids (FAs) have immunomodulatory effects. However, the influence of ω-3 FAs on septic conditions is not certain. This study examined the effect of fish oil (FO)–enriched diets before and/or ω-3 FA-containing total parenteral nutrition (TPN) after sepsis on the distribution of T-lymphocyte subpopulations and splenocyte cytokine mRNA expressions in rats with polymicrobial sepsis. Methods Rats were assigned to a control group and four experimental groups. The control group and groups 1 and 2 were fed a semipurified diet, and groups 3 and 4 had 20% soybean oil replaced by FO. After feeding the diets for 10 d, sepsis was induced by cecal ligation and puncture (CLP) in the experimental groups, whereas sham operation was performed on the control group. TPN was maintained for 3 d after CLP or sham operation. The control group and groups 1 and 3 were infused with conventional TPN, whereas the TPN solution of groups 2 and 4 was supplemented with FO. All rats were sacrificed 3 d after the operation to examine their immune responses. Results Messenger RNA expressions of interleukin-2 and tumor necrosis factor-α in splenocytes were higher in groups 3 and 4 than in the control group and group 1. Interleukin-10 mRNA expression in group 3 was higher than in the control group and group 2. Blood CD4 percentage and CD4/CD8 ratio in group 1 were significantly lower, whereas no differences were observed in FO-supplemented groups compared with the control group. Conclusion FO administration before and/or after CLP maintained blood T-lymphocyte subpopulations and modulated T-helper type 1 and 2 cytokine mRNA expressions in rats with polymicrobial sepsis.

Age-related change in peripheral blood T-lymphocyte subpopulations and cytomegalovirus infection in the very old: the Swedish longitudinal OCTO immune study

Results from the previous times (Times 1–3) of the Swedish longitudinal OCTO immune study indicated that a combination of high CD8 and low CD4 percentages and poor T-cell proliferation in PBL was associated with a higher 2-year mortality in a sample of very old Swedish individuals. The combination of immune parameters was closely related to an inverted CD4/CD8 ratio. In the present study at Time 4 (T4) results are reported from the final follow-up of this longitudinal study, 8 years after it was initiated in 1989. An additional goal at this time point was to examine the immune system alterations in the very old in relation to evidence of lymphocyte activation and cytomegalovirus antibody status. In the present study immune system changes were identified that suggest a loss of T-cell homeostasis, as reflected by a decrease in the number of CD4 cells and a very significant increase in the number of CD8 cells in individuals with an inverted CD4/CD8 ratio. When considered over the duration of the OCTO study the inversion occurred in a high percentage (32%) of the individuals included in the original sample and was associated with non-survival. At T4 the changes were apparent in a number of the T-cell subsets, but particularly in the CD8 +CD28 − and CD57 + subsets. T-cell activation was significantly associated with the inversion of the CD4/CD8 ratio. In this very old sample the subset alterations were associated with evidence of cytomegalovirus (CMV) infection.

T-cell clonal expansion in patients with B-cell lymphoproliferative disorders.

We investigated whether T-cell clonal expansion could be found in the blood of 14 untreated patients with B-cell lymphoproliferative disorders [5 B-chronic lymphocytic leukemia (CLL), 4 myelomas, 5 non-Hodgkin lymphoma (NHL)]. The putative presence of T-cell clonotypes was analyzed with a polymerase chain reaction-based method determining V-D-J junction size patterns in 24 T-cell receptor (TCR) V beta subfamilies. This high-resolution method, analyzing CDR3 sizes of TCR transcripts, was used in conjunction with cytometric analysis of the corresponding T-cell subpopulations with 18 TCR V beta-specific monoclonal antibody. We found multiple dominant T-cell clonotypes in the blood of most patients with B-CLL or myeloma as well of a patient with stage IV NHL. In some cases, T-cell clonal expansion was so dominant that the percentage of these clonal T-cell subpopulations in blood represented more than the mean +2 SD value determined in a series of healthy controls. We conclude that a systemic antigen-specific (i.e., leading to clonotypic expansion) immune reaction involving few TCR clonotypes is a hallmark of disseminated B-cell malignancies. The nature of the putative antigens recognized is not known presently. Nonetheless, such insights into the T-cell repertoire of these patients may help to reassess the potential of immunotherapeutic strategies in B-cell malignancies.

Ovariectomy does not alter CD4+/CD8+ ratio in peripheral blood T-lymphocytes in the rat.

Alterations in peripheral blood T-lymphocyte subsets have been implicated to play a role in the pathophysiology of estrogen deficiency-induced bone loss in postmenopausal women. The aim of this study was to investigate this hypothesis further by flow cytometric analysis of peripheral blood lymphocyte subpopulations together with bone histomorphometry using ovariectomized (OVX) rats as an experimental estrogen deficiency model. 104 female 6-month-old Wistar rats were either OVX or sham-operated (SHAM). Eight rats served as baseline controls. Groups of 8 SHAM and 8 OVX rats were killed 1, 2, 4, 6, 9, and 12 weeks after surgery. Blood was collected prior to sacrifice. In whole blood samples, subpopulations of peripheral lymphocytes were analyzed by flow cytometry using FITC- or PE-labeled monoclonal antibodies against rat CD5, CD4, and CD8. CD4 and CD8 positive T-lymphocytes were determined by a double-labeling technique. Serum samples were analyzed for estradiol and progesterone. The cancellous bone of the distal femoral metaphysis was analyzed by quantitative bone histomorphometry. Ovariectomy caused a statistically significant fall in serum estradiol and progesterone levels from 2 weeks postovariectomy until the end of the trial. Deterioration of cancellous bone structure and osteopenia in the distal femur of OVX rats became evident at 2 and 4 weeks postovariectomy, respectively. Flow cytometric analysis of peripheral blood lymphocytes revealed that, except for a transient increase in CD4 positive T-lymphocytes in OVX rats relative to SHAM animals at 1 week post-surgery, the number of CD5, CD4, or CD8 positive lymphocytes or the mean fluorescence per cell for these antigens in OVX rats remained unchanged throughout the study. Also the CD4+/CD8+ ratio of peripheral blood T-lymphocytes was not influenced by ovariectomy, and none of these parameters were significantly correlated with serum estradiol or progesterone levels. These results suggest that consistent changes in peripheral blood T-lymphocyte subpopulations are not demonstrable in OVX rats by the techniques applied in this study, and do not seem to play a pathogenetic role in the development of estrogen deficiency-induced bone loss in the rat.

Memory and CD8 + are the predominant bovine bronchoalveolar lymphocyte phenotypes

Bovine lymphocytes obtained by bronchoalveolar lavage (BAL) of healthy calves were simultaneously analyzed and compared to peripheral blood lymphocytes using monoclonal antibodies specific for bovine leukocyte differentiation antigens. Phenotypic differences were observed between bronchoalveolar and peripheral blood T-lymphocyte subpopulations, demonstrating selective lymphocyte migration to the bovine lung. The bronchoalveolar and peripheral blood T-lymphocyte populations, defined by expression of CD2, were similar, but bronchoalveolar T lymphocytes were predominately CD8 + while peripheral blood T cells were predominately CD4 +. In addition, memory lymphocytes, characterized by low expression of CD45R and activated lymphocytes (CD25 +), were found in significantly higher proportions in the bronchoalveolar compartment. The proportion of γδ T lymphocytes was, however, significantly higher in peripheral blood. B cells were observed in similar proportions in the bronchoalveolar compartment and peripheral blood.

Changes in T-lymphocyte subpopulations and serum lymphokine concentrations after partial hepatectomy in rats

The relationship between liver regeneration and immunological response does exist. Extrathymic T cells and serum lymphokines are believed to be changed after partial hepatectomy, but there is still much controversy. This study evaluates the changes of peripheral blood T-lymphocyte subpopulations, natural killer (NK) cells, serum lymphokines, and NK cells in the remnant liver after partial hepatectomy. Male Wistar rats weighing around 200 g were used as subjects. Partial hepatectomies (67%) were done. Counting T-lymphocyte subpopulations (includes T3, T4, T8, T-IL2R, and NK cells) by using fluorescin isothiocyanate or phycoerythrin was performed preoperatively and on 1, 2, 3, 5, 7, and 10 d postoperatively. Serum gamma-interferon (γ-IFN), interleukin-2 and interleukin-2 receptor concentrations were measured. Absolute number of NK cells in the remnant liver was also counted. Rats with right-side nephrectomy, sham operation, and no operation were used as control groups. Comparing the difference between all groups, we found that all T-lymphocyte subpopulations in peripheral blood, serum γ-IFN, peripheral blood NK cells, and the absolute number of NK cells in the remnant liver of hepatectomized rats increased markedly, and were significantly higher than control groups rats, on the fifth and seventh post-hepatectomy day. It is suggested that γ-IFN and NK cells were activated markedly and might play important roles in the regulation of liver regeneration after partial hepatectomy.

Cellular and humoral immunity in rats after gestational zinc or magnesium deficiency

The effects of gestational Mg or Zn deficiency on the humoral or cellular immunity of newborn rats were investigated. Mg deficiency was induced by feeding a diet containing 180 ppm Mg from day 0 to day 21 of gestation and Zn deficiency was induced by feeding a diet containing 1.5 ppm Zn from day 0 to day 19. Controls were fed a diet with 1,000 ppm Mg and 100 ppm Zn from day 0 to day 21. Thereafter, all maternal rats and newborns were fed diets with normal amounts of Mg or Zn. Three and six weeks after birth, T-cell subpopulations in blood and thymus and B-cells in blood of the newborns were detected by flow cytometry. Plasma contents of IgG, IgM, and IgA were determined by radial immunodiffusion. Mg deficiency reduced litter size and pup weight. Three weeks after birth, the total number of leukocytes and lymphocytes in blood was significantly decreased, due to a reduction of T-helper and cytotoxic T-cells. Activated T-cells and B-cells were unchanged. Six weeks after birth, T-cell subpopulations approached controls values, whereas IgG content in plasma was slightly reduced. Gestational Zn deficiency reduced litter size and induced malformations. Three and six weeks after birth, body weight, number of leukocytes, lymphocyte, and T-cell subpopulations were not significantly changed. Plasma IgM was decreased 3 weeks after birth in correlation to the number of B-cells, which represented only 4% of total lymphocytes. These effects were repaired by the sixth week. Plasma IgG was reduced at 6 weeks. No effects on T-cell subpopulations in isolated thymocytes were detected after gestational Mg or Zn deficiency.