Analysis of T-lymphocyte subpopulations in inflammatory bowel diseases by three-color flow cytometry

In order to better define changes in the relative proportion of peripheral blood T-lymphocyte subpopulations in patients with inflammatory diseases of the bowel, we performed simultaneous three-color fluorescence-activated cytometric (FACS) analysis using fluorophore-conjugated monoclonal antibodies with specificity for CD4, CD8, Leu 8, and CD45RA on 22 normal control subjects, 28 patients with Crohn's disease (CD), 15 patients with ulcerative colitis (UC), and 11 patients with intestinal inflammation secondary to etiologies other than inflammatory bowel disease (NIBD). This staining combination allowed enumeration of distinct T-cell subpopulations as follows: virgin CD4⁺, recall antigen helper T cells, nonspecific B-cell helper T cell, virgin CD8⁺, cytotoxic effector and suppressor effector and recall antigen cytotoxic T cells based on a synthesis of published functional analyses. No differences in the proportion of CD4⁺ or CD8⁺ cells or in the CD4⁺/CD8⁺ ratios were evident when UC and NIBD patients were compared to normal subjects. A significant reduction in the proportion of CD4⁺ cells and an increase in CD8⁺ cells was observed, however, in the CD group. When two-color analysis was performed, several significant differences in the proportions of circulating lymphocytes were seen. Specifically, these included significant increases in the number of CD4⁺, Leu 8^– (P<0.01) cells in all disease groups and an increase in CD4⁺, CD45RA⁺ cells in the NIBD group. Conversely, significant decreases in the proportions of CD8⁺, Leu 8⁺ (P<0.01) cells were evident in the Crohn's disease group. Three-color FACS analysis revealed significant differences in the relative proportions of the defined T-cell subpopulations enumerated in the various groups as compared with the normal controls. These included a decrease in the proportions of Leu 8⁺, CD45RA⁺, (virgin) CD8⁺ T cells (P<0.05) and Leu 8^–, CD45RA⁺, (putative recall antigen helper) CD4⁺ T cells (P<0.01) in all patient groups as compared with normal controls. Conversely, an increase in the proportions of Leu 8^–, CD45RA⁺, (putative suppressor effector) CD8⁺ T cells (P<0.01), and Leu 8^–, CD45RA⁺, (function unknown) CD4⁺ T cells (P<0.05) was seen in all patient groups as compared with normal controls. CD patients but not UC or NIBD patients demonstrated a significant increase in the proportion of Leu 8^–, CD45RA⁺, (putative cytotoxic effector) CD8⁺ T cells (P<0.01). An increase in the ratio of Leu 8^–, CD45RA⁺, CD8⁺ (suppressor effector)/Leu 8⁺, CD45RA^–, CD8⁺ (putative cytotoxic effector) T cells was observed in all of the patient groups, but was most accentuated in those with Crohn's disease. Significant decreases in the ratios of Leu 8^–, CD45RA^–, CD4⁺ (putative nonspecific B cell helper)/Leu 8⁺, CD45RA^–, CD4⁺ (recall antigen helper) T cells and Leu 8⁺, CD45RA^–, CD4⁺ (recall antigen helper)/Leu 8^–, CD45RA⁺, CD4⁺ (function unknown) T cells were observed in all of the disease groups studied as compared with normal controls. These results suggest that the proportions of certain peripheral blood T-cell subpopulations are significantly altered in gastrointestinal inflammatory states. Further analysis of these T-lymphocyte subpopulations in the blood and tissues might provide valuable insights into immunological aberrations in inflammatory bowel diseases and might be of value in distinguishing among inflammatory diseases of the intestine.     

Selective loss of peripheral blood CD45RO+ T lymphocytes correlates with increased levels of serum cytokines and endothelial cell-derived soluble cell adhesion molecules in patients with chronic idiopathic neutropenia of adults

 The present study was designed to investigate the hypothesis that selective loss of peripheral blood CD45RO⁺ T lymphocytes in patients with chronic idiopathic neutropenia of adults (CINA), previously reported from our laboratory, may be due to enhanced extravasation into the tissues. Serum levels of endothelial cell-derived soluble cell adhesion molecules (sELAM, sICAM and sVCAM), usually used as indicators of endothelial cell activation, were measured in 73 CINA patients and 32 healthy volunteers using a micro-ELISA method. We found that patients had markedly elevated concentrations of all three soluble cell adhesion molecules studied compared to the controls, and serum levels of sELAM, sICAM and, more importantly, sVCAM correlated inversely with the numbers of both CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets. Using a micro-ELISA method, we also measured serum levels of two endothelial cell activators, interleukin (IL)-1β and TNF-α, and found that CINA patients had significantly higher cytokine concentrations than control subjects. Serum levels of IL-1β and TNF-α correlated positively with the values of all three soluble cell adhesion molecules and inversely with the numbers of CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets. Moreover, we measured serum levels of the chemokine RANTES by a micro-ELISA technique and found that CINA patients also had elevated concentrations of the molecule compared to controls. Serum RANTES correlated positively with IL-1β, TNF-α, sICAM, sVCAM and sELAM and inversely with the numbers of both CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets. These findings strongly suggest that CINA patients have an activated endothelium to which CD45RA⁺ and CD45RO⁺ T cells tether and roll, but firm adhesion and transendothelial migration are restricted to CD45RO⁺ T cell subsets, as endothelial VCAM-1 interacts with the vascular leukocyte adhesion molecule-4 (VLA-4) constitutively expressed on CD45RO⁺ but not on CD45RA⁺ T cells. Subsequent subendothelial and tissue migration of CD45RO⁺ T cells may be facilitated by the chemokine RANTES, which acts mainly on CD45RO⁺ T cells. We concluded that selective loss of peripheral blood CD45RO⁺ T lymphocytes in CINA patients is probably due, at least in part, to enhanced extravasation of both CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets into the tissues. Received: February 18, 1998 / Accepted: June 10, 1998     

Age-matched lymphocyte subpopulation reference values in childhood and adolescence: application of exponential regression analysis

Background: Normal values of lymphocyte subpopulations for healthy children and adults have been published in defined age groups exclusively, which results in difficult data interpretation for patients close to the limit of contiguous age group ranges. In addition, normal values for a number of lymphocyte subpopulations have not been established to date. Objective: The aim of this study was to develop a model which provides continuous age‐dependent reference values. This model was applied for lymphocyte subpopulations such as naïve and memory T cells as well as their activation profile with diagnostic relevance in children and adults. Study design: A total of 100 blood samples, obtained from 80 healthy children and 20 adults were analysed by means of four colour‐flow cytometry. Continuous age‐dependent reference values were computed based on the residual values in an exponential regression model. Results: We calculated a continuous age‐related regression model for both, absolute cell counts and percentages of CD3⁺CD4⁺ T helper (T_H) cells, CD3⁺CD8⁺ cytotoxic T cells, CD56⁺CD3^? natural killer (NK) cells, CD56⁺CD3⁺ T cells, CD3⁺CD4⁺CD45RA⁺ naïve T_H cells, CD3⁺CD4⁺CD45RO⁺ memory T_H cells, CD3⁺CD8⁺CD45RA⁺CD28⁺ naïve cytotoxic T cells, CD3⁺CD8⁺CD45RO⁺ memory cytotoxic T cells, CD3⁺CD8⁺CD69⁺ early activated cytotoxic T cells and CD3⁺CD8⁺HLA‐DR⁺ late activated cytotoxic T cells, respectively, to obtain reference values. Conclusion: Based on an exponential regression model, the obtained reference values reflect the continuous maturation of lymphocyte subsets during childhood.     

Age-related changes in the absolute number of CD95 positive cells in T cell subsets in the blood

Comparison of the absolute number of cells in distinct T cells subsets expressing CD95 (Fas) was carried out in two populations of healthy female volunteers. In one population, the average age was 30 ± 5 years, and in the second population the average age was 73 ± 13 years. No significant difference was noted in the total number of lymphocytes, CD3⁺, CD4⁺, or CD8⁺ cells per μL of blood between the two age groups, but major differences were noted in the number of cells expressing CD95. A significant reduction was seen in the number of cells per μL of blood in both the CD4⁺ CD45RA⁺ CD95⁺ and CD8⁺ CD45RA⁺ CD95⁺ populations in the older group compared with the younger group. Within the memory pool significantly fewer CD8⁺ CD45RO⁺ CD95⁺ cells were found in the older population compared with the younger group. No such difference were found in the number of CD4⁺ CD45RO⁺ CD95⁺ cells between groups. Such a significant decline in the number of CD95⁺ cells, whose expression is known to be linked with activation, may be implicated as a mechanism by which cells that have reached a stage of replicative senescence remain in the peripheral T cell pool. Anti-CD3–mediated activation of cells from both groups revealed much lower proliferative responses from the older group, supporting the idea that there is an age-associated increase in the number of cells that have reached their replicative limit. These cells may not be lost from the peripheral pool because they fail to express CD95.     

Lymphopenia in patients with chronic idiopathic neutropenia is associated with decreased number of T-lymphocytes containing T-cell receptor excision circles

Objectives: Chronic idiopathic neutropenia (CIN) is a disorder of granulopoiesis characterized by the presence of activated T‐lymphocytes that induce/sustain apoptosis of bone marrow (BM) granulocytic progenitors. T‐cell lymphopenia is commonly found in CIN. The aim of the study is to probe the mechanisms underlying T‐cell lymphopenia in CIN. Methods: We investigated parameters of T‐cell homeostasis namely the proliferation/apoptotic rate of naïve and memory T cells, the T‐cell senescence by telomere measurement, the recent thymic T‐cell production through quantification of T‐cell receptor rearrangement excision circles (TRECs), and the production of interleukin (IL)‐7. Results: Patients with CIN (n = 44) displayed lower proportion of naïve CD45RA⁺ cells within the CD4⁺ and CD8⁺ cells compared with controls (n = 15). The proportion of apoptotic cells within the CD8⁺ fraction was higher in patients compared with controls and was correlated with the percentage of Ki‐67⁺ cells, indicating an activation‐induced accelerated CD8⁺ cell death. The TREC content of CD4⁺ and CD8⁺ cells was lower in patients compared with controls and was correlated with the proportion of CD45RA⁺ CD4⁺ and CD8⁺ cells and with the levels of serum and BM IL‐7, which were significantly decreased in the patients. The mean relative telomere length of CD4⁺ and CD8⁺ cells was significantly lower in patients with CIN compared with age‐matched controls. Conclusions: The aberrant T‐cell expansions associated with the pathogenesis of CIN result in increased proliferation/apoptosis and possibly exhaustion of peripheral blood T cells which, in association with the inadequate compensatory thymic export of new TREC expressing T cells partially because of IL‐7 deficiency, may contribute to lymphopenia in CIN.     

Pro- and anti-inflammatory cytokine production by autoimmune T cells against preproinsulin in HLA-DRB1*04, DQ8 Type 1 diabetes

Aims/hypothesis Preproinsulin is a target T cell autoantigen in human Type 1 diabetes. This study analyses the phenotype and epitope recognition of preproinsulin reactive T cells in subjects with a high genetic risk of diabetes [HLA-DRB1*04, DQ8 with Ab+ (autoantibody-positive) or without islet autoantibodies (control subjects)], and in HLA-matched diabetic patients.Methods A preproinsulin peptide library approach was used to screen for cytokine profiles and epitope specificities in human peripheral blood lymphocytes, and CD4⁺CD45RA^– and CD4⁺CD45RA⁺ T cell subfractions, representing memory and naive and recently primed T cells respectively.Results In CD4⁺ T cell subsets we identified immunodominant epitopes and cytokine production patterns that differed profoundly between patients, Ab+ subjects and non-diabetic HLA-matched control subjects. In Ab+ subjects, a C-peptide epitope C13–29 and insulin B-chain epitope B11–27 were preferentially recognised, whereas insulin-treated Type 1 diabetic patients reacted to native insulin and B-chain epitope B1–16. In peripheral blood lymphocytes of Ab+ subjects, an increase in T helper (Th) 1 (IFN, IL-2) and Th2 (IL-4) cytokines was detectable, wheras in CD45RA⁺ and CD45RA^– subsets, IL-4 and IL-10 phenotypes dominated, compatible with the contribution of non-CD4 cells to IFN content. In insulin-treated Type 1 diabetic patients, naive and recently primed CD4⁺ cells were characterised by increasd IFN, TNF, and IL-5.Conclusions/interpretation Our data show that T cell reactivity to preproinsulin in CD45RA subsets is Th2-dominant in Ab+ subjects, challenging the Th1 paradigm in Type 1 diabetes. Characteristic immunodominant epitopes and cytokine patterns distinguish diabetic patients and Ab+ subjects from HLA-matched healthy individuals. This could prove useful in monitoring of T-cell immunity in clinical diabetes intervention trials.Abbreviations PPI Preproinsulin - PBMC peripheral blood mononuclear cells - Th T helper cells - Ab+ autoantibody-positive - ICA islet cell antibodies - IA-2A islet thyrosine phosphatase - SI stimulation index - Tr T regulatory cells W. Karges and B.O. Boehm contributed equally to this article     

Autoreactive and immunoregulatory T-cell subsets in insulindependent diabetes mellitus

Aims/hypothesis. Type I (insulin-dependent) diabetes mellitus is a T-cell mediated autoimmune disease. Several subsets of T-cells, in particular CD4⁺ and in vivo activate CD45RA⁺RO⁺ T-cells, have been shown to be increased at disease onset. The functional implications of these relative increases in CD4 T-cells were investigated. Methods. Subsets of T-cells were sorted on the basis of their activation status (CD45RA⁺ naïve cells, CD45RA⁺RO⁺ recently activated cells and CD45RO⁺ memory cells) and stimulated with autoantigens or recall antigen in vitro. Results. Proliferative responses to tetanus toxoid were primarily or exclusively observed in resting memory T-cells (CD45RO⁺). Autoimmune T-cell responses were, however, primarily measured in activated T-cells (CD45RA⁺RO⁺) in newly diagnosed Type I diabetic patients, whereas those with longer disease duration reacted to autoantigens with memory T-cells (CD45RO⁺) (p < 0.004). Interestingly, in non-diabetic control subjects not responding to autoantigens in the regular assay, considerable autoreactive T-cell responses were detectable after sorting in the CD45RO⁺ or CD45RA⁺RO⁺ lymphocyte subsets. Remixing these subsets showed that these autoimmune responses in activated cells could be down-modulated by CD45RA⁺ lymphocytes, whereas resting memory cells appeared unaffected by the suppressive CD45RA subset. Conclusion/interpretation. These results show that autoimmune T-cell responses can be linked to particular subsets which differ depending on clinical status. Furthermore, the CD45RA T-cell subset harbours lymphocytes potentially capable of suppressing autoimmune T-cell responses. The changes in responsiveness to exogenous insulin may help to unravel the mechanism by which isohormonal therapy could prevent the onset of Type I diabetes. [Diabetologia (1999) 42: 443–449]     

Differential activity of P-glycoprotein in normal blood lymphocyte subsets

To better understand the phenomenon of P-glycoprotein (P-170) expression we investigated lymphocyte subpopulations for P-170 function in healthy volunteers. Studies were based on three-colour flow cytometry including the fluorescent probe rhodamine 123 (Rh123), which is transported by P-170. Marked Rh123 efflux was detected in CD8⁺ T lymphocytes with CD8⁺/CD45RA⁺ T cells (naive cells) showing significantly higher P-170 activity as compared with CD8⁺/CD45RA^? cells (P < 0.04). Vice versa, CD8⁺/CD45RO⁺ T cells (memory cells) demonstrated less P-170 activity than CD8⁺/CD45RO^? cells (P < 0.04). P-170 function was less prominent in CD4⁺ T cells, however, Rh123 efflux was higher in the CD4⁺/CD45RA⁺ and CD4⁺/CD45RO^? subpopulations (P < 0.025) corresponding to the CD8⁺ results. Dye efflux differed significantly between activated and non-activated CD8⁺ and CD4⁺ as well as CD8⁺/CD11b⁺ and CD8⁺/CD11b^? T lymphocytes. Since CD16⁺ natural killer cells (NK) expressed the highest level of P-170, the NK cytotoxicity against ⁵¹Cr-labelled K562 target cells was assayed in the presence or absence of P-170 inhibitors. NK related cytotoxicity was significantly reduced in the presence of R-verapamil and dexnigaldipine-HCP in a dose-dependent manner. The differential expression of P-170 activity in naive and memory T cells together with the reduced NK related cytotoxicity in the presence of MDR-modulators suggest a physiological role of P-170 in immunological functions of these lymphocyte subsets. Consequently, the addition of MDR modulators to conventional chemotherapy as a strategy to overcome drug resistance should consider possible adverse immunosuppressive effects.     

Naïve subset develops the most important alloreactive response among human CD4+ T lymphocytes in Human Leukocyte Antigen‐identical related setting

In longitudinal clinical studies, receiving a high percentage of allogeneic donor‐derived CD4⁺CCR7⁺ T cells, which include naïve and central memory subsets have been correlated with increased incidence and severity of acute GVHD. Whether naïve and central memory CD4⁺ T‐cell subsets contribute more or equally to alloimmune responses are still unclear in human. The aim of this study was to investigate in vitro the alloreactive response of purified naïve, central memory, and effector memory CD4⁺ T‐cell subsets in HLA identical setting. By coculturing monocyte‐derived dendritic cells and purified CD4⁺ T‐cell subsets, from healthy HLA‐identical male and female sibling pairs, we found that naïve CD4⁺CCR7⁺CD45RA⁺ T cells developed the highest proliferative response upon stimulation by minor histocompatibility antigens and were progressively driven to produce high levels of interferon‐γ, tumor necrosis factor, and interleukin‐6. Comparatively, the central memory CD4⁺CCR7⁺CD45RA^neg subset proliferated to a lower extent and produced very low amounts of pro‐inflammatory cytokines while the CCR7^neg effector memory CD4⁺ subset was unresponsive. This study demonstrates the superior capacity of naïve CD4⁺ T cells to mount a primary alloreactive response as compared to central memory T cells. Their proliferative response associated with a pro‐inflammatory differentiation makes them potentially acute GVHD inducers. These in vitro results in line with what we have observed in clinical studies and may also lend support to approaches of partial selective T‐cell depletion for GVHD prevention.     

Altered T-cell subtypes in spondyloarthritis, rheumatoid arthritis and polymyalgia rheumatica

The objective of the present study was to assess the prevalences of naive, memory, memory/effector, regulatory and activated T-cells in peripheral blood (PB) and synovial fluid (SF) of patients with spondyloarthritis (SpA), rheumatoid arthritis (RA), polymyalgia rheumatica/giant cell arteritis (PMR/GCA) and healthy controls (HC). Twenty-two patients with SpA, 15 patients with RA, 38 patients with PMR/GCA and 17 HC were prospectively enrolled. The expression of differentiation and activation markers (CD3, CD4, CD8, CD25, CD28, CD45RA, CD45RO) characterizing T-cell subsets were analyzed by flow cytometry. The frequency of CD3⁺CD4⁺CD28⁻ memory/effector T-cells was increased in PB of patients with SpA (median 1.1%, range 0.1–69.6), RA (2.5%, 0–42.9) and PMR/GCA (2.7%, 0–49.5) when compared with HC (0.7%, 0–38.0) and tended to be higher in SF of SpA patients (4.5%, 0.2–7.2, P = 0.084). CD28⁺CD45RA⁺CD4⁺ (9.6%, 4.1–10.3) and CD28⁺CD45RA⁺CD8⁺ naive T-cells (15.0%, 12.9–26.2) were reduced and CD28⁺CD45RO⁺CD4⁺ (93.5%, 51.0–99.0), CD28⁺CD45RO⁺CD8⁺ memory (81.2%, 38.9–83.5), CD8⁺CD25⁺ activated T-cells (10.9%, 2.7–13.8) and CD4⁺CD25^hi TREGs (10.2%, 7.0–13.3) were increased in SF compared to PB (P < 0.05 each). These findings demonstrate altered T-cell subsets in patients with immune-mediated disease, particularly at sites of inflammation.     

Disease trends over time and CD4+CCR5+ T-cells expansion predict carotid atherosclerosis development in patients with systemic lupus erythematosus

Background and Aim

Patients with Systemic Lupus Erythematosus (SLE) present increased cardiovascular mortality compared to the general population. Few studies have assessed the long-term development and progression of carotid atherosclerotic plaque in SLE patients. Our aim was to investigate the association of clinical and laboratory markers of disease activity and classical cardiovascular risk factors (CVRF) with carotid atherosclerosis development in SLE patients in a prospective 5-year study.

Distinctive CD8<Superscript>+</Superscript> T cell and MHC class I signatures in polycythemia vera patients

Polycythemia vera (PV) is a myeloproliferative neoplasm characterized by overproduction of red blood cells. We have performed a comprehensive characterization of blood immune cells for expression of naïve and memory receptors as well as β₂m-associated and β₂m-free MHC class I heavy chains, also known as closed and open conformers, respectively, in PV patients and age-matched controls (CTR). We show that the peripheral CD3⁺CD8⁺ T cell pool in PV patients is clearly divided into two discrete populations, a more granular CD3⁺CD8^high T cell population enriched in effector-memory CD45RA⁺ T cells (CD8⁺ TEMRA) when compared to CTR (P?<?0.001), and a less granular CD3⁺CD8^int T cell population that is completely absent in the CTR group (78 vs. 0%, P?<?0.001) and is a mixture of naïve (CD8⁺ T_N) and CD8⁺ TEMRA cells expressing intermediate levels of CD28, i.e., CD3⁺CD8^intCD28^int. While the percentage of CD3⁺CD8^int TN cells correlated positively with the number of erythrocytes, the percentage of CD3⁺CD8^int TEMRA correlated negatively with the number of platelets. Finally, we report that PV patients’ lymphocytes and monocytes display lower levels of closed (W6/32⁺) MHC-I conformers at the cell surface while exhibiting increased amounts of open (HC-10⁺) MHC-I conformers. The implications of this distinctive immune signature are discussed.     

Th1 and Th17 cell subpopulations are enriched in the peripheral blood of patients with systemic juvenile idiopathic arthritis

Objective. The role of the adaptive immune system has not been explored in detail compared with the innate immune system in systemic JIA (sJIA) pathogenesis. The aim of this study was to examine the phenotype of circulating peripheral blood CD4(+) T-cell subpopulations in a cross-sectional study of sJIA patients during disease remission on medication and during acute flare of the disease. Methods. Flow cytometry was used to examine the phenotype and cytokine production of IFNγ-, IL-4- and IL-17-producing CD4(+) T cells in the peripheral blood of 10 sJIA patients with active disease, 9 sJIA with inactive disease, 14 JIA patients with oligoarticular onset, 10 adult control subjects and 10 age-matched control subjects. In parallel, we examined the proportion of FoxP3(+) Tregs. Results. IFNγ- and IL-17-producing CD4(+) T cells and IL-17-producing CD3(+)CD4(-) T cells were present at higher proportions in the peripheral blood of sJIA patients, irrespective of their disease status. Our data also confirm the known increase of the proportions of IFNγ-producing Th1 cells with increasing age and suggest an increase with age in the IL-17-producing CD4(+) T-cell population. Conclusion. This study is the first to describe significantly higher proportions of Th1 and Th17 T helper cell subsets in the peripheral blood of sJIA patients. These proinflammatory cells may play a pathogenic role in sJIA. Our data also emphasize the importance of using paediatric age-matched control subjects when evaluating the T-cell cytokine profile in JIA.     

TREC/KREC Levels and T and B Lymphocyte Subpopulations in COVID-19 Patients at Different Stages of the Disease

Background: T and B cell-mediated immunity can be assessed using T cell receptor excision circle (TREC) and Kappa-deleting recombination excision circle (KREC) analysis, respectively, and successful implementation of this method requires evaluation of the correlation between the TREC frequencies and T cell subsets as well as KREC levels and B lymphocyte subsets. The aim of the present study was to evaluate the correlation between the TREC/KREC concentrations and T/B lymphocyte subsets at different stages of COVID-19. Methods: We examined 33 patients in the acute stage of COVID-19 (including 8 patients with poor outcomes) and 33 COVID-19 survivors. TREC/KREC concentrations were measured using quantitative real-time PCR. T/B lymphocyte subsets were determined using flow cytometry. Results: Blood TREC and KREC levels were found to be significantly lower in the acute stage of COVID-19 compared to control values. Moreover, a zero blood TREC level was a predictor of a poor disease outcome. Reductions in CD3⁺CD4⁺CD45RO⁻CD62L⁻ and CD3⁺CD8⁺CD45RO⁻CD62L⁻ T cell counts (as well as in the main fractions of B1 and B2 B cells) indicated a favorable outcome in COVID-19 patients in the acute stage of the disease. Decreased CD3⁺CD4⁺CD45RO⁻CD62L⁺ and CD3⁺CD8⁺CD45RO⁻CD62L⁺ T cell frequencies and increased CD3⁺CD8⁺CD45RO⁻CD62L⁻ cell counts were found to indicate a poor outcome in patients with acute COVID-19. These patients were also found to have increased B1 cell counts while demonstrating no changes in B2 cell counts. The levels of effector T cell subsets an naïve B cells were normal in COVID-19 survivors. The most pronounced correlations between TREC/KREC levels and T/B cell subsets counts were observed in COVID-19 survivors: there were positive correlations with naïve T and B lymphocytes and negative correlations with central and effector memory T cell subsets. Conclusions: The assessment of correlations between TREC and T cell subsets as well as KREC levels and B cell subset counts in patients with acute COVID-19 and COVID-19 survivors has shown that blood concentrations of TREC and KREC are sensitive indicators of the stage of antigen-independent differentiation of adaptive immunity cells. The results of the TREC and KREC analysis correlated with the stages of COVID-19 and differed depending on the outcome of COVID-19.     

Non-suppressive regulatory T cell subset expansion in pulmonary arterial hypertension

Regulatory T cells (Tregs) have been reported to play a pivotal role in the vascular remodeling of pulmonary arterial hypertension (PAH). Recent studies have revealed that Tregs are heterogeneous and can be characterized by three phenotypically and functionally different subsets. In this study, we investigated the roles of Treg subsets in the pathogenesis of PAH in eight patients with PAH and 14 healthy controls. Tregs and their subsets in peripheral blood samples were analyzed by flow cytometry. Treg subsets were defined as CD4⁺CD45RA⁺FoxP3^low resting Tregs (rTregs), CD4⁺CD45RA^?FoxP3^high activated Tregs (aTregs), and CD4⁺CD45RA^?FoxP3^low non-suppressive Tregs (non-Tregs). The proportion of Tregs among CD4⁺ T cells was significantly higher in PAH patients than in controls (6.54 ± 1.10 vs. 3.81 ± 0.28 %, p < 0.05). Of the three subsets, the proportion of non-Tregs was significantly elevated in PAH patients compared with controls (4.06 ± 0.40 vs. 2.79 ± 0.14 %, p < 0.01), whereas those of rTregs and aTregs were not different between the two groups. Moreover, the expression levels of cytotoxic T lymphocyte antigen 4, a functional cell surface molecule, in aTregs (p < 0.05) and non-Tregs (p < 0.05) were significantly higher in PAH patients compared with controls. These results suggested the non-Treg subset was expanded and functionally activated in peripheral lymphocytes obtained from IPAH patients. We hypothesize that immunoreactions involving the specific activation of the non-Treg subset might play a role in the vascular remodeling of PAH.     

Microarray analysis reveals similarity between CD8+CD28- T cells from young and elderly persons, but not of CD8+CD28+ T cells

We isolated highly purified CD8⁺CD28⁺ and CD8⁺CD28⁻ T cell populations from healthy young and elderly persons for gene expression profiling using Affymetrix oligonucleotide microarrays. We demonstrate that the gene expression profile of CD8⁺CD28⁻ T cells is very similar in young and elderly persons. In contrast, CD8⁺CD28⁺ in elderly differ from CD8⁺CD28⁺ in young persons. Hierarchical clustering revealed that CD8⁺CD28⁺ in elderly are located between CD8⁺CD28⁺ in young and CD8⁺CD28⁻ (young and old) T cells regarding their differentiation state. Our study demonstrates a dichotomy of gene expression levels between CD8⁺CD28⁺ T cells in young and elderly persons but a similarity between CD8⁺CD28⁻ T cells in young and elderly persons. As CD8⁺CD28⁺ T cells from elderly and young persons are distinct due to a different composition of the population, these results suggest that the gene expression profile does not depend on chronological age but depends on the differentiation state of the individual cell types.     

Rudimentary signs of immunosenescence in Cytomegalovirus-seropositive healthy young adults

Ageing is associated with a decline in immune competence termed immunosenescence. In the elderly, this process results in an accumulation of differentiated ‘effector’ phenotype memory T cells, predominantly driven by Cytomegalovirus (CMV) infection. Here, we asked whether CMV also drives immunity towards a senescent profile in healthy young adults. One hundred and fifty-eight individuals (mean ± SD; age 21 ± 3 years, body mass index 22.7 ± 2.7 kg m²) were assessed for CMV serostatus, the numbers/proportions of CD4⁺ and CD8⁺ late differentiated/effector memory cells (i.e. CD27⁻CD28⁻/CD45RA⁺), plasma interleukin-6 (IL-6) and antibody responses to an in vivo antigen challenge (half-dose influenza vaccine). Thirty percent (48/158) of participants were CMV⁺. A higher lymphocyte and CD8⁺ count (both p < 0.01) and a lower CD4/CD8 ratio (p < 0.03) were observed in CMV⁺ people. Eight percent (4/58) of CMV⁺ individuals exhibited a CD4/CD8 ratio <1.0, whereas no CMV⁻ donor showed an inverted ratio (p < 0.001). The numbers of CD4⁺ and CD8⁺CD27⁻CD28⁻/CD45RA⁺ cells were ~ fourfold higher in CMV⁺ people (p < 0.001). Plasma IL-6 was higher in CMV⁺ donors (p < 0.05) and showed a positive association with the numbers of CD8⁺CD28⁻ cells (p < 0.03). Finally, there was a significant negative correlation between vaccine-induced antibody responses to the A/Brisbane influenza strain and CMV-specific immunoglobulin G titres (p < 0.05). This reduced vaccination response was associated with greater numbers of total CD8⁺ and CD4⁺ and CD8⁺CD27⁻CD28⁻/CD45RA⁺ cells (p < 0.05). This study observed marked changes in the immune profile of young adults infected with CMV, suggesting that this virus may underlie rudimentary aspects of immunosenescence even in a chronologically young population.     

Imbalance between Subpopulations of Regulatory T Cells in Patients with Acute Exacerbation of COPD

Human regulatory T cells (Tregs) have been reported to be not significantly different in the peripheral blood of patients with chronic obstructive pulmonary disease (COPD) and healthy controls. Recent research has identified some new markers for Tregs and indicated that Tregs are composed of distinct subpopulations. The aim of the study was to describe the changing patterns of circulating Treg subpopulations in patients with acute exacerbation of COPD (AECOPD) and healthy controls, and to explore their potential roles in AECOPD pathogenesis. Blood samples were obtained from 30 never-smokers with normal lung function and 30 patients with COPD before and after they had an exacerbation. The proportions of Treg subpopulations were evaluated using flow cytometry. In the peripheral blood, decreased proportions of CD4⁺CD25⁺CD127^low Tregs, CD4⁺CD25⁺CD45RA⁺ Tregs, and CD4⁺CD25⁺CD62L⁺ Tregs and an increased proportion of CD4⁺CD25⁺CD45RO⁺ Tregs were found in patients with stable COPD compared with non-smokers with normal lung function. The patients showed further changes in Treg subpopulations when they had an AECOPD, with an overall decrease in a suppressive subset, indicating that the immune negative regulatory population of Tregs did not play an effective role. Immune homeostasis favored inflammation, and a negative correlation between the circulating tumor necrosis factor-alpha and the proportions of CD4⁺CD25⁺CD62L⁺ cells (r = ?0.698, p < 0.05) in patients with AECOPD was found. The imbalance between the suppressive subsets and the proinflammatory subset of Tregs and the decline of Treg subpopulations with immunosuppressive activity may play important roles in AECOPD progression.     

Strong increase in the percentage of the CD8bright+CD28- T-cells and delayed engraftment associated with cyclosporine-induced autologous GVHD

Abstract: Four children with acute lymphoblastic leukaemia had autologous bone marrow (BM) or peripheral stem cell (PSC) transplantation with low dose of cyclosporine (CsA, 1 mg/kg/d i.v. during the first 28 d) to induce an autologous GVHD (auto-GVHD). Two children did not have clinical auto-GVHD and they relapsed 3 and 4 months after treatment. The 2 other children had clinical signs of auto-GVHD (grade I and grade II): they both are in complete remission but after a first normal haematological recovery they had a prolonged period of aplasia until month 9 for 1 patient and still persistent at month 7 in the other case. We studied lymphocyte subsets reconstitution after transplantation in these patients. All patients had an important decrease in the CD4/CD8 ratio related both to a strong decrease in the CD4⁺ cells and a strong increase in the CD8⁺ cells. Most of the CD8⁺ cells were of the CD8^bright+CD28^- phenotype. These CD8^bright+CD28^- T-cells represented from 33% to 68% of the total lymphocytes. We discuss the role of these cells after autologous transplantation with CsA, and wonder if these cells could mediate cytotoxicity. In conclusion, among 4 children who received autologous BM or PBC transplantation with low dose of CsA, we observed a complete remission after an auto-GVHD and a prolonged period of aplasia in 2 patients and a relapse of leukaemia in 2 other patients. All these 4 patients had an increase in the CD8^bright+CD28^- T lymphocytes.     

Induction of CD4+CD8+ double positive T cells and increase in CD5+ B cells in efferent lymph in sheep infected with Trypanosoma evansi

The effects of Trypanosoma evansi on efferent lymphocyte phenotypes draining from a lymph node primed with Pasteurella haemolytica vaccine were studied in sheep. The prefemoral efferent lymphatic ducts of the infected sheep along with those of two uninfected sheep were surgically cannulated. Lymph was collected and lymphocytes recovered from it analysed by two-colour indirect immunofluorescence staining and cytofluoremetry in a fluorescence activated cell analyser (FACSCAN). The study showed the appearance and persistence of T. evansi in the efferent lymph for a long period of time and the appearance of CD4⁺CD8⁺ (double positive, DP) T lymphocytes in the efferent lymph of infected animals. The infection also resulted in increases in CD5⁺ B cells in the prefemoral efferent lymph. In addition, there were decreases in the output of conventional B cells, CD5⁺ and CD4⁺ T cell subsets but large increases in CD8⁺ cells followed by terminal depletion of all cell subsets. In contrast, inoculation of sheep with pasteurella vaccine antigen alone produced little alterations in the proportions, but large increases in the numbers of all T cell subsets except that of CD8⁺ cells which also showed little variation; and there was a concurrent increase in the numbers and proportions of efferent B cells. In addition, the abnormal expression of DP and CD5⁺ B cells did not occur in the uninfected vaccinated sheep. It is concluded that these abnormal changes in the kinetics of efferent lymphocyte phenotypes are likely to play a role in the genesis of the generalized immunosuppression seen in trypanosome-infected hosts.