Changes in blood lymphocyte numbers with age in vivo and their association with the levels of cytokines/cytokine receptors

Background

Alterations in the number and composition of lymphocytes and their subsets in blood are considered a hallmark of immune system aging. However, it is unknown whether the rates of change of lymphocytes are stable or change with age, or whether the inter-individual variations of lymphocyte composition are stable over time or undergo different rates of change at different ages. Here, we report a longitudinal analysis of T- and B-cells and their subsets, and NK cells in the blood of 165 subjects aged from 24 to 90 years, with each subject assessed at baseline and an average of 5.6 years follow-up.

Results

The rates of change of T-(CD4⁺ and CD8⁺) and B-cells, and NK cells were relative stable throughout the adult life. A great degree of individual variations in numbers of lymphocytes and their subsets and in the rates of their changes with age was observed. Among them, CD4⁺ T cells exhibited the highest degree of individual variation followed by NK cells, CD8⁺ T cells, and B cells. Different types of lymphocytes had distinct trends in their rates of change which did not appear to be influenced by CMV infection. Finally, the rates of CD4⁺, CD8⁺ T cells, naive CD4⁺ and naïve CD8⁺ T cells were closely positively correlated.

Conclusion

Our findings provide evidence that the age-associated changes in circulating lymphocytes were at relative stable rates in vivo in a highly individualized manner and the levels of selected cytokines/cytokine receptors in serum might influence these age-associated changes of lymphocytes in circulation.

     

Decrease of CD4+ T-lymphocytes in children exposed to environmental lead

The effects of environmental lead on the immune system of young children were assessed by determining the peripheral blood lymphocytes CD3⁺, CD4⁺, CD8⁺, B(CD19⁺) counts, and natural killer (CD16⁺ CD56⁺) cells in 35 preschool children whose mean blood lead level was 140.6 μg/L. The results were compared to an age- and sex-matched control group with a mean blood lead level of 64.3 μg/L. Compared to the controls, a significant reduction in the percentage of CD4⁺ cells and a significant increase of CD8⁺ cells were seen in the high-lead group. The negative correlation between the percentage of CD4⁺ cells and blood lead levels was found to be significant (p<0.01). These results suggest that exposure to environmental lead might result in alterations in the immune function of young children.     

The adaptive potential of human immunity during strength training

Quantitative and functional characteristics of human T-and B-cell-related immunity and natural cytotoxicity were studied during nine-week strength training. Long-term classic strength training and low-intensity strength training without relaxation did not change the peripheral blood contents of the main subpopulations of immunocompetent cells, the phytohemagglutinin-induced proliferative activity of T lymphocytes, serum levels of immunoglobulins A, M, and G (IgA, IgM, and IgG, respectively) and the cytotoxic activity of natural killer cells. At the same time, training was accompanied by activation of the immune system, which was evident from increased counts of CD25⁺ lymphocytes observed in the peripheral blood and in the mitogen-stimulated and nonstimulated cell cultures, as well as from the higher spontaneous and mitogen-induced productions of IgA, IgM, and IgG by B lymphocytes.     

Long-term implications of feed energy source in different genetic types of reproductive rabbit females. II. Immunologic status

Genetic selection and nutrition management have played a central role in the development of commercial rabbitry industry over the last few decades, being able to affect productive and immunological traits of the animals. However, the implication of different energy sources in animals from diverse genetic lines achieving such evolutionary success remains still unknown. Therefore, in this work, 203 female rabbits housed and bred in the same conditions were used from their first artificial insemination until their fifth weaning. The animals belonged to three different genetic types diverging greatly on breeding goals (H line, hyper-prolific (n=66); LP line, robust (n=67) and R line, selected for growth rate (n=67), and were assigned to two experimental diets, promoting major differences in energy source (cereal starch or animal fat)). The aims of this work were to: (1) characterize and describe blood leucocyte populations of three lines of rabbit does in different physiological stages during their reproductive period: first artificial insemination, first weaning, second parturition and fifth weaning; and (2) study the possible influence of two different experimental diets on the leucocyte populations in peripheral blood. Flow cytometry analyses were performed on blood samples taken from females at each different sampling stade. Lymphocyte populations at both weanings were characterized by significantly lower counts of total, CD5⁺ and CD8⁺ lymphocytes (–19.8, –21.7 and –44.6%; P<0.05), and higher counts of monocytes and granulocytes (+49.2 and +26.2%; P<0.05) than in the other stages. Females had higher blood counts of lymphocytes B, CD8⁺ and CD25⁺ and lower counts of CD4⁺ at first than at fifth weaning (+55.6, +85.8, +57.5, –14.5%; P<0.05). G/L ratio was higher at both weanings (P<0.05), and CD4⁺/CD8⁺ ratio increased progressively from the 1AI to the 5 W (P<0.001). Regarding the effect of genetic type in blood leucocyte counts, LP animals presented the highest counts for total, B, CD5⁺ and CD8⁺ lymphocytes (+16.7, +31.8, +24.5 and +38.7; P<0.05), but R rabbits showed the highest counts for monocytes and granulocytes (+25.3 and +27.6; P<0.05). The type of diet given during the reproductive life did not affect the leucocyte population counts. These results indicate that there are detectable variations in the leucocyte profile depending on the reproductive stage of the animal (parturition, weaning or none of them). Moreover, foundation for reproductive longevity criteria allows animals to be more capable of adapting to the challenges of the reproductive cycle from an immunological viewpoint.     

Differential apoptotic response to ionizing radiation in subpopulations of human white blood cells

The purpose of this paper is to characterize the apoptotic response of various subpopulations of human white blood cells after in vitro exposure to ionizing radiation using the modified neutral comet assay (MNCA). White blood cells, isolated from human whole blood, were fractionated into granulocytes and mononuclear cells which were further separated into B-cells, natural killer (NK) cells, and CD4⁺ and CD8⁺ T-cells. The separated fractions were exposed to low doses of X-rays and then MNCA was used to measure the apoptotic fraction (AF) at different time points in irradiated and unirradiated aliquots of sorted cultures. The spontaneous AF in unirradiated control cells was the most critical determinant of whether an apoptotic response could be detected in irradiated cells. When cultured in isolation granulocytes and B-cells had the highest background AF, with NK cells having the next highest. CD4⁺ and CD8⁺ T-cells had a low, stable, spontaneous AF which gave them the highest signal-to-noise ratio. Although B-cells demonstrated the highest radiation-induced apoptotic response to 1 Gy of X-rays, CD8⁺ T-cells were the most radiation-responsive lymphocytes due to their low spontaneous AF. By generating dose response curves for CD4⁺ and CD8⁺ T-cells, the sensitivity of the MNCA for detecting apoptosis in these two cell types was also examined.     

Lymphocyte Gene Expression Signatures from Patients and Mouse Models of Hereditary Hemochromatosis Reveal a Function of HFE as a Negative Regulator of CD8+ T-Lymphocyte Activation and Differentiation In Vivo

Abnormally low CD8⁺ T-lymphocyte numbers is characteristic of some patients with hereditary hemochromatosis (HH), a MHC-linked disorder of iron overload. Both environmental and genetic components are known to influence CD8⁺ T-lymphocyte homeostasis but the role of the HH associated protein HFE is still insufficiently understood. Genome-wide expression profiling was performed in peripheral blood CD8⁺ T lymphocytes from HH patients selected according to CD8⁺ T-lymphocyte numbers and from Hfe ^-/- mice maintained either under normal or high iron diet conditions. In addition, T-lymphocyte apoptosis and cell cycle progression were analyzed by flow cytometry in HH patients. HH patients with low CD8⁺ T-lymphocyte numbers show a differential expression of genes related to lymphocyte differentiation and maturation namely CCR7, LEF1, ACTN1, NAA50, P2RY8 and FOSL2, whose expression correlates with the relative proportions of naïve, central and effector memory subsets. In addition, expression levels of LEF1 and P2RY8 in memory cells as well as the proportions of CD8⁺ T cells in G2/M cell cycle phase are significantly different in HH patients compared to controls. Hfe ^-/- mice do not show alterations in CD8⁺ T-lymphocyte numbers but differential gene response patterns. We found an increased expression of S100a8 and S100a9 that is most pronounced in high iron diet conditions. Similarly, CD8⁺ T lymphocytes from HH patients display higher S100a9 expression both at the mRNA and protein level. Altogether, our results support a role for HFE as a negative regulator of CD8⁺ T-lymphocyte activation. While the activation markers S100a8 and S100a9 are strongly increased in CD8⁺ T cells from both, Hfe ^-/- mice and HH patients, a differential profile of genes related to differentiation/maturation of CD8⁺ T memory cells is evident in HH patients only. This supports the notion that HFE contributes, at least in part, to the generation of low peripheral blood CD8⁺ T lymphocytes in HH.     

Immunomodulatory Effects of an Enzymatic Extract from Ecklonia cava on Murine Splenocytes

We investigated whether the brown seaweed Alariaceae Ecklonia cava (E. cava) has immunological effects on splenocytes in vitro. For that purpose, we prepared an enzymatic extract from E. cava (ECK) by using the protease, Kojizyme. Here, ECK administered to ICR mice dramatically enhanced the proliferation of their splenocytes and increased the number of their lymphocytes, monocytes and granulocytes. In flow cytometry assays performed to identify in detail the specific phenotypes of these proliferating cells after ECK treatment, the numbers of CD4⁺ T cells, CD8⁺ T cells and CD45R/B220⁺ B cells increased significantly compared to those in untreated controls. In addition, the mRNA expression and production level of Th1-type cytokines, i.e., TNF-α and IFN-γ, were down-regulated, whereas those of Th2-type cytokines, i.e., IL-4 and IL-10, were up-regulated by ECK. Overall, this dramatic increase in numbers of splenocytes indicated that ECK could induce these cells to proliferate and could regulate the production of Th1- as well as Th2-type cytokines in immune cells. These results suggest that ECK has the immunomodulatory ability to activate the anti-inflammatory response and/or suppress the proinflammatory response, thereby endorsing its usefulness as therapy for diseases of the immune system. Y-J. Jeon and Y. Jee contributed equally to this study.     

Effects of dietary conjugated linoleic acids on cellular immune response of piglets after cyclosporin A injection

The present study investigated the effects of dietary conjugated linoleic acid (CLA) on the cellular immune response of piglets after cyclosporin A (CsA) treatment. The experimental study had a 2×2 factorial design, and the main factors consisted of diets (0% or 2% CLA) and immunosuppression treatments (CsA or saline injection). CsA injection significantly increased feed : gain (F : G) of piglets (P<0.05); however, dietary CLA significantly decreased F : G of piglets (P<0.05). Dietary CLA partly ameliorated the deterioration of the feed conversion rate caused by CsA treatment (P<0.01). CsA treatment significantly decreased the percentages of CD4⁺ and CD8⁺ T lymphocytes in the thymus (P<0.01). Dietary CLA increased the percentages of CD4⁺ CD8⁺ double-positive and CD8⁺ single-positive T lymphocytes in the thymus (P<0.05), and had the trend to inhibit the decrease of CD4⁺ T lymphocytes in the thymus after CsA injection (P=0.07). CsA treatment significantly depleted the peripheral blood CD3⁺, CD4⁺ and CD8⁺ T lymphocytes (P<0.01). Dietary CLA significantly increased the number of peripheral blood CD8⁺ T lymphocytes and interleukin-2 (IL-2) production (P<0.05), and inhibited the decreases of peripheral blood CD3⁺, CD4⁺ and CD8⁺ T lymphocytes counts (P<0.01) as well as IL-2 production (P<0.05) after CsA treatment. Dietary CLA partly rescued the decrease of lymphocyte proliferation after CsA injection (P<0.05). In summary, dietary CLA effectively ameliorated CsA-induced cellular immunosuppression in piglets.     

Ethyl-eicosapentaenoic acid ameliorates the clinical course of experimental allergic encephalomyelitis induced in dark agouti rats

Eicosapentaenoic acid (EPA), a fatty acid present in high amount in fish, modulates immune response and stimulates myelin gene expression. In the present paper, we investigated the effects of EPA in an established animal model for multiple sclerosis (MS): experimental autoimmune encephalomyelitis (EAE) induced in dark agouti rats. Diets supplemented either with 0.2% or 0.4% of EPA were administrated daily from the day of induction until the end of experiment. One group of rats received diet supplemented with 0.2% of EPA 10 days before induction. The control group (immunized rats) was fed with chow diet. The animals were analyzed at two different stages of the disease: during the acute phase (14 d.p.i.) and during the recovery phase (32 d.p.i.). We showed a delayed onset of clinical severity of disease in all groups of rats fed EPA-supplemented diets. This effect was associated to an increased expression of myelin proteins and an improved integrity of the myelin sheath as well as an up-regulation of FoxP3 expression in the central nervous system during the acute phase of EAE. No significant changes in T cell subsets were noted at the periphery. On the contrary, during the recovery phase of EAE, in animals assuming EPA-supplemented diet, an increase of CD4⁺CD25⁺ and CD4⁺CD25⁺FoxP3⁺ in peripheral lymphocytes was noted. Our results indicate that EPA-supplemented diets may provide benefits to MS patients.     

Contribution of CD8 T lymphocytes to the immuno-pathogenesis of multiple sclerosis and its animal models

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) characterized by multi-focal demyelination, axonal loss, and immune cell infiltration. Numerous immune mediators are detected within MS lesions, including CD4⁺ and CD8⁺ T lymphocytes suggesting that they participate in the related pathogenesis. Although CD4⁺ T lymphocytes are traditionally considered the main actors in MS immunopathology, multiple lines of evidence suggest that CD8⁺ T lymphocytes are also implicated in the pathogenesis. In this review, we outline the recent literature pertaining to the potential roles of CD8⁺ T lymphocytes both in MS and its animal models. The CD8⁺ T lymphocytes detected in MS lesions demonstrate characteristics of activated and clonally expanded cells supporting the notion that these cells actively contribute to the observed injury. Moreover, several experimental in vivo models mediated by CD8⁺ T lymphocytes recapitulate important features of the human disease. Whether the CD8⁺ T cells can induce or aggravate tissue destruction in the CNS needs to be fully explored. Strengthening our understanding of the pathogenic potential of CD8⁺ T cells in MS should provide promising new avenues for the treatment of this disabling inflammatory disease.     

Altered chemokine receptor profile on circulating leukocytes in human heart failure

Chemokines and their receptors have been implicated in the pathogenesis of different forms of heart failure (HF). We examined CC-and CXC-chemokine receptor expression in fresh peripheral blood leukocyte populations from 24 end-stage HF patients consisting of coronary artery disease (CAD; n=6) and hypertrophic cardiomyopathy (HCM; n=7) or idiopathic dilated cardiomyopathy (IDCM; n=8) or valvular disease (VD; n=3) and compared the data with 18 healthy controls. Levels of CCR1, 2, 3, 4, 5, and 7, and CXCR1, 2, 3, and 4 were measured by flow cytometry, and the expression profile was assessed as molecules of equivalent soluble fluorochrome units as well as frequency (percentage) of CD3⁺, CD4⁺, and CD8⁺ T cells and monocytes or granulocytes. Frequency of CD3⁺ CXCR4⁺, CD3⁺ CXCR1⁺, and CD3⁺ CXCR3⁺ cells was significantly increased in HF patients, whereas only CCR7 and CXCR4 expression levels were elevated on CD3⁺ cells. Both CD4⁺ CXCR4⁺ and CD8⁺ CXCR4⁺ cell frequencies were significantly increased irrespective of cardiac disease etiology. Elevated CCR7 expression was less pronounced on CD4⁺ than CD8⁺ cells in patients with CAD and IDCM. Expression of CXCR4 on CD8⁺ cells was upregulated substantially, regardless of the cause of disease. CD8⁺ CXCR1⁺ and CD8⁺ CXCR3⁺ but not CD4⁺ CXCR1⁺ or CD4⁺ CXCR3⁺ cells were increased in the HF patients with IDCM and CAD, respectively. Expression of CXCR1 or CXCR3 on both CD4⁺ and CD8⁺ cells did not differ in all the groups. For monocytes, frequency of CD14⁺ CCR1⁺ and CD14⁺ CCR2⁺ cells was significantly decreased in CAD patients, whereas, increase in CD14⁺ CXCR4⁺ cell frequency was accompanied with elevated CXCR4 expression. On granulocytes, CXCR1 and CXCR2 receptors were downregulated in all patients, compared with controls. Our results suggest that the altered expression profile of CC- and CXC-chemokine receptors on circulating leukocyte populations involves enhanced activation of the immune system, perhaps as part of the pathogenic mechanisms in HF. Modulation of the chemokine network could offer interesting novel therapeutic modalities for end-stage HF.     

小鼠乳腺表达抗PD-1抗体对其脾脏T细胞的影响

为从细胞水平研究小鼠乳腺表达抗PD-1抗体对转基因鼠脾脏T细胞表面抗原蛋白、细胞因子表达、脾脏CD4⁺T细胞增殖以及增殖相关通路的影响,将8周龄未经历过怀孕的和18周龄经历过哺乳的表达抗人PD-1抗体的转基因小鼠分成两组,每组以转基因阴性鼠为对照,提取脾脏淋巴细胞,检测脾脏淋巴细胞的变化。与转基因阴性小鼠相比,乳腺表达抗PD-1抗体的转基因小鼠的免疫系统中的脾脏T细胞的效应T细胞比例上升,Treg细胞比例下降,CD4⁺T细胞表达的IFN-γ、IL-17以及IL-2有不同程度的增加。IL-4、IL-10以及TGF-β都没有发生变化。与T细胞刺激相关的一些细胞表面的蛋白分子也没有引起变化。转基因阳性鼠和转基因阴性鼠中T细胞增殖没有显著性差异,转基因阳性鼠中PI3K/Akt/mTOR和Ras/MEK/ERK这两条通路上的磷酸化蛋白只有部分表达上调,整个通路没有完全上调。结果表明,转基因小鼠作为表达抗PD-1抗体这类免疫系统相关单克隆抗体的宿主是可行的。     

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+renal-cell-carcinoma-infiltrating lymphocytes

The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3⁺ tumor-infiltrating lymphocytes (CD3⁺ TIL) and in autologous CD3⁺ peripheral blood lymphocytes (CD3⁺ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4⁺ TIL and CD8⁺ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3⁺ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3⁺ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4⁺ TIL and simultaneously obtained CD8⁺ TIL revealed a significantly higher expression of IFN in the CD8⁺ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8⁺ TIL. Received: 14 January 1999 / Accepted: 30 April 1999     

基于免疫性PTR的血小板膜糖蛋白及T/B淋巴细胞表达水平研究

摘要 目的：探究血小板膜糖蛋白及T/B淋巴细胞免疫在免疫性血小板输注无效发生机制中的重要作用。方法：采用血小板特异性抗体检测试剂盒及流式细胞技术检测免疫性PTR患者血小板特异性抗体（抗GPIIb/IIIa、抗GPIa/IIa、抗GPIb/IX、抗GPIV）、血小板膜糖蛋白（CD36、CD61、CD41a）表达情况及外周血T/B淋巴细胞数量,运用SPSS19.0软件及R软件分析免疫性PTR与血小板膜糖蛋白、外周血T/B淋巴细胞的相关性。结果：免疫性PTR与血小板输注有效者比较,血小板特异性抗体的产生无显著差异,血小板膜糖蛋白CD36和CD61的表达具有显著差异,CD36对免疫性PTR发生风险具有极大的预测价值,外周血CD8⁺T细胞比例增高,而CD4⁺T细胞比例减低。结论：免疫性PTR患者产生血小板抗体具体机制不明确,了解患者细胞免疫状态有助于明确免疫性PTR的发生机制,为患者提供更优质的诊疗策略。     

Characterization of T-lymphocyte subpopulations infiltrating primary breast cancer

Summary Characterization of T-lymphocyte subpopulations adjacent to and infiltrating the primary tumor of breast cancer was carried out using a direct immunofluorescence procedure with the antibodies anti-(Leu-2a) for suppressor/cytotoxic (CD8⁺) and anti-(Leu-3a) for helper/inducer (CD4⁺) T-lymphocytes. Fifty-six primary malignant tumors with lymphoid infiltration were studied. The majority (58.9%) were infiltrating duct carcinoma. There were metastases to axillary lymph nodes in 6.67% of the patients. Massive lymphoid infiltration (>40 lymphocytes per ×400 microscopic field) was found in 19.6% of the tumors and moderate infiltration (20–40 lymphocytes per field) in 51.8%. In all the tumors studied there was a reversed CD4⁺/CD8⁺ ratio as compared to that found in normal peripheral blood. In 66.1% the CD4⁺/CD8⁺ ratio (helper/suppressor) was less than 1.0. The reversed ratio was due to a significant decrease in the number of helper cells (P<0.0005). The most significant drop was in the stroma area (P<0.0001) as well as in the tumor tissue (P=0.001). Of particular interest was the significant positive correlation between the age of the patients and an increased number of CD4⁺lymphocytes in the stroma (P=0.02). Significant negative correlations were found between a reduced number of CD4⁺ lymphocytes or CD4⁺/CD8⁺ ratio and several histological parameters: tumor diameter, pleomorphism, nucleus/cytoplasm ratio. There was also a significant positive correlation between the total number of CD8⁺ lymphocytes infiltrating the tumor tissue and the number of axillary lymph nodes with metastatic disease (P=0.03). It is suggested that the reversed ratio of CD4⁺/CD8⁺ lymphocytes may significantly affect the host/tumor immune surveillance.     

Mesenteric adipose tissue B lymphocytes promote local and hepatic inflammation in non‐alcoholic fatty liver disease mice

Mesenteric adipose tissue (MAT) inflammation is associated with non‐alcoholic fatty liver disease (NAFLD), and immune cells play pivotal roles in the inflammation of adipose tissue. Here, we investigated the roles of MAT B lymphocytes in NAFLD. Mice fed with high‐fat diet (HFD) and normal diet (ND) were killed in time gradients (4, 8 and 12 weeks). Compared with ND‐fed mice, intra‐hepatic CD45⁺CD19⁺ B lymphocytes increased after 4 weeks (P < 0.01) of HFD feeding, and lasted until the 12th week, infiltrated earlier than CD45⁺CD3⁺ T lymphocytes and CD45⁺F4/80⁺ macrophages. The mRNA expression of tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and monocyte chemotactic protein (MCP)‐1 decreased in MAT of B^null HFD‐fed mice compared to that in wild‐type HFD‐fed mice, along with lesser macrophages. Mesenteric adipose tissue B cells from HFD‐fed mice promoted macrophage differentiation to type‐Ι macrophages and expression of pro‐inflammatory cytokines in vitro. Macrophages pre‐treated with MAT B cells from HFD‐fed mice showed elevated mRNA expression of IL‐6 and TNF‐α and declined IL‐10 levels in adipocytes compared to ND MAT B cell pre‐treated macrophages. Besides, internal near‐infrared scanning and external transwell assay showed that HFD MAT B cells migrated to the liver more than ND MAT B cells. High‐fat diet MAT B cells induced higher MCP‐1 and lower IL‐10 expression in primary hepatocytes compared to ND MAT B cells in co‐culture experiment. These data indicate that B lymphocytes infiltrate early in MAT during the development of NAFLD, which may not only promote MAT inflammation by regulating macrophages but also migrate to the liver and induce hepatocytes inflammation.     

Early postnatal development of the immune system in piglets: the redistribution of T lymphocyte subsets

In this study, we have characterised postnatal changes in T lymphocyte subsets, especially γδ T lymphocytes, in blood, spleen and lymph nodes. Detection was carried out using two-colour flow cytometry and three-colour immunohistochemistry. During ontogeny, there was a significant increase in the total percentage of γδ T cells in the spleen and blood. In the lymph nodes, there were no age-dependent changes in the total percentage of γδ T cells, but the percentage of the γδTCR⁺CD8⁺ subpopulation significantly increased. The tissue distribution of γδTCR⁺CD8⁺ and γδTCR⁺CD8⁻ cells in the lymph nodes is random and not collocated with a particular area of the organ. Furthermore, postnatal development was characterised by an increasing frequency of CD8⁺CD3⁺CD4⁻γδTCR⁻, which was compensated by a decreasing proportion of CD4⁺ lymphocytes. Double positive CD4⁺CD8⁺ lymphocytes were rare during the first month of life and a significant age-dependent increase of these cells was found in all the compartments monitored.