CD57+ T lymphocytes are derived from CD57− precursors by differentiation occurring in late immune responses

CD3⁺ T cells expressing the 110-kDa CD57 antigen are found in survivors of renal, cardiac and bone marrow transplants, in patients with acquired immune deficiency syndrome and in patients with rheumatoid arthritis. They are also present in normal individuals and expand upon ageing. They do not grow in culture and their role in the immune response is poorly understood. The expression of the various isoforms of the leukocyte common antigen (CD45) identifies a spectrum of differentiation in CD4⁺ and CD8⁺ T cells ranging from naive (CD45RA⁺CD45RB^brightCD45RO^?) through early primed cells (CD45RA^?RB^brightRO^dull) to highly differentiated memory cells which are CD45RA^?RB^dullRO^bright. CD45 isoforms expressed by CD57⁺ T cells showed distinct differences between CD4⁺ and CD8⁺ populations, but in each case indicated an advanced state of differentiation. The expression of T cell receptor Vβ families was highly variable between individuals, but both CD57⁺ and CD57^? cells show a full range of the specificities tested. Vβ expression was more closely related within either the CD4⁺ or the CD8⁺ subsets, irrespective of CD57 expression, than between these subsets, suggesting a relationship between CD57⁺ and CD57^? cells within the same T cell pool. This possibility was supported by experiments showing that CD3⁺CD57⁺ lymphocytes were similar to CD3⁺CD57^? T cells in terms of the production of basic T cell cytokines [interleukin (IL)-2, IL-4, and interferon-γ]. Furthermore, in vitro stimulation of CD3⁺CD57^? T cells in secondary mixed leukocyte reaction or by co-culture with IL-2 and IL-4 induced the appearance of CD3⁺CD57⁺ cells with phenotypic and functional similarities to in vivo CD3⁺CD57⁺ cells. These data strongly suggest that the expression of CD57 is a differentiation event which occurs on CD57^? T cells late in the immune response.     

In vitro responses of human CD45R0brightRA− and CD45R0−RAbright T cell subsets and their relationship to memory and naive T cells

The cellular basis of immunological memory, particularly with respect to T cells is not understood. In humans, monoclonal antibodies to CD45 have been used to identify memory (CD45R0) and naive (CD45RA) T cells. However, this identification has been called into question by various studies which suggest that high molecular weight CD45 isoforms may be re-expressed by previously activated cells. In the present study, using cultures which supported responses of naive T cells, we examined the responses of purified CD45R0^brightRA^? or CD45R0^?-RA^bright T cell subsets. The former subset was found to respond preferentially to recall antigens with minimal responses apparent to neo-(or non-recall)-antigens. The inverse pattern was found for CD45R0^?RA^bright T cells, which converted to CD45R0^brightRA^? after stimulation with a neo-antigen. Moreover, the two populations of T cells exhibited distinct response kinetics with a faster response evident from the CD45R0^brightRA^?T cells compared to the CD45R0^?RA^bright subset. The poor responses of CD45R0^?RA^bright T cells to recall antigens compared to neo-antigens suggests that this putative naive population is specifically depleted of reactive T cells following an encounter with antigen. We propose that T cell priming results in the stimulation of many CD45R0^?RA^bright T cells with various T cell receptor specificities from which memory T cells are selected for survival. If re-expression of higher molecular weight isoforms does occur in humans in vivo, our results suggest that R0 expression would be retained (CD45R0⁺RA⁺). Alternatively, if primed CD45R0^?RA^bright T cells exist, they are not prevalent in peripheral blood and thus may be sequestered within lymphoid tissues. Our data support the view that in human peripheral blood, CD45R0^bright and CD45RA^bright expression identify memory and naive CD4⁺ T cells, respectively.     

Characterization of the protein tyrosine phosphatase CD45 on human epidermal Langerhans cells

Human Langerhans cells (LC) express CD45, but clear data about the isoform(s) and their function(s) are lacking. In the present study, double labeling experiments reveal that freshly isolated LC from normal skin are CD45RO⁺/RA^?/RB^?. However, after isolation and short-time culture where LC undergo an in vitro maturation resembling that to lymphoid dendritic cells, CD45RB emerges whereas CD45RO expression decreases. This evolution results from dynamic alternative RNA splicing. Addition of granulocyte-macrophage colony-stimulating factor or tumor necrosis factor-α to short-time cultures has no significant effect on CD45RB, but both cytokines accelerate the loss of CD45RO. LC isolated from lesional skin of atopic eczema highly express CD45RO and CD45RB. Cross-linking of CD45 on LC isolated from atopic individuals inhibits the calcium mobilization in response to activation via Fcε receptor type I(FcεRI). Hence, the protein tyrosine phosphatase CD45 from human LC is subjected to a splicing phenomenon related to the differentiation and activation stage of these cells and regulates their FcεRI-mediated activation.     

Peripheral blood T lymphocyte subsets in children with congenital asplenia

The aim of the current study was to examine whether a congenital lack of the spleen changes distribution, state of activation and function of peripheral lymphocyte T subsets. Seven children with congenital asplenia (CA) aged 1.5–17 years and seven age-matched controls were tested. By triple-color flow cytometry we examined: (1) the expression of CD3⁺, CD4⁺, CD8⁺, CD19⁺, and CD56⁺ on lymphocytes; (2) the distribution of CD45RA⁺ and CD45RO⁺ in CD4⁺ and CD8⁺; (3) the expression of CD27⁺ in the CD4⁺ and CD8⁺ T-cell-bearing CD45RA⁺, CD45RO⁺, or CD45RB⁺. Lymphocyte proliferative responses and cytokines production (IFN-gamma, IL-6, TNF-alfa, and IL-10) in anti-CD3-induced peripheral blood mononuclear cells were tested. The results indicate (1) a normal distribution of the basic lymphocyte subsets, (2) low CD3⁺/CD8⁺ percentage but expressing CD8^+high and non-significantly elevated CD4⁺/CD8⁺ ratio, (3) CD45RA^+high and CD27^+high in the CD4⁺ and CD8⁺ T cell, and (4) CD45RB^+high in the CD4⁺ and CD45RO^+high in the CD8⁺. The distribution of CD27⁺ in the CD45RA⁺ and CD45RO⁺ CD4⁺ T cells remained unchanged. However, the percentage of CD8⁺/CD45RO⁺/CD27⁺ T cells tended to be elevated. Altogether, these data indicate that CA is connected with (1) the presence CD4⁺ T cells expressing the “naive” phenotype (CD45RA^+high RB^+high and CD27^+high), (2) high numbers of activated CD8⁺ T cells shifted toward the memory phenotype (CD45RO^+high) but still showing high CD27⁺ expression, which may indicate failure in T CD8⁺ cytotoxic effectors differentiation, and (3) a tendency to the rather pro-inflammatory status of cells, low IL-10 expression, and suboptimal lymphocytes responses to mitogenic stimulation.     

Comparison of lymphokine secretion and mRNA expression in the CD45RA+ and CD45RO+ subsets of human peripheral blood CD4+ and CD8+ lymphocytes

Flow cytometric analysis of human peripheral blood T lymphocytes demonstrated that the majority of the CD4⁺ cells were CD29⁺ or CD45RO⁺ “mature” cells while the CD8⁺ cells were primarily CD45RA⁺ “naive” cells. After an initial separation into CD4⁺ and CD8⁺ cells and a secondary separation into CD45 subsets, lymphokine secretion was assessed after phorbol 12-myristate 13-acetate and ionomycin or fixed anti-CD3 stimulation. Within the respective CD45 subsets, CD4⁺ cells produced more interleukin (IL)-2, IL-4, and IL-6; but the CD8⁺ cells secreted more interferon-γ and granulocyte/macrophage-colony-stimulating factor. Tumor necrosis factor-α secretion was similar in the matched CD45 subsets. Northern analysis revealed a parallel pattern of lymphokine mRNA expression in the four lymphocyte subsets. These results suggest that human CD8⁺ peripheral blood lymphocytes have a significant capacity to secrete lymphokines, and that the low lymphokine production observed in unseparated CD8⁺ cells reflects the higher percentage of less functional CD45RA⁺ cells.     

CCR7+ Central and CCR7− Effector Memory CD4+ T Cells in Human Cutaneous Leishmaniasis

Purpose

The profile of central (=T_CM) and effector (=T_EM) memory CD4⁺ T cell subsets and the possible role as surrogate markers of protection is studied in the volunteers with history of cutaneous leishmaniasis (HCL).

Methods

Profile of T cell subsets based on CCR7/CD45RA expressions and phenotypic changes after soluble Leishmania antigen (SLA) stimulation were analyzed. Then, sorted CD4⁺CD45RO^?CD45RA⁺ naïve T, CD4⁺CD45RO⁺CD45RA^?CCR7^? T_EM, CD4⁺CD45RO⁺CD45RA^?CCR7⁺ T_CM subsets were cultured with SLA for proliferation, cytokine production and intracellular cytokine assays.

Results

In the HCL and control volunteers, the mean frequencies of CD4⁺CD45RA⁺CCR7⁺ naïve T cells and CD4⁺CD45RA^?CCR7^? T_EM cells were higher than the other subsets before culture. Frequency of naïve T cells and CD4⁺CD45RA^?CCR7⁺ T_CM cells was significantly decreased (P?=?0.01 for naïve T and P?<?0.05 for T_CM cells) and frequency of T_EM cells was significantly increased after SLA stimulation compared to before culture (P?<?0.001). By CFSE labeling, CD4⁺CD45RO⁺CD45RA^?CCR7⁺ T_CM cells showed more proliferation potential than CD4⁺CD45RO⁺CD45RA^?CCR7^? T_EM cells. Stimulation of the T_EM cells in HCL volunteers induced a significantly higher IFN-γ production (P?=?0.04) with higher number of intracellular IFN-γ positive cells (P?=?0.032) than the same cells from controls. A significantly higher number of T_CM cells produced IL-2 in HCL volunteers compared with controls (P?<?0.05). Most of the intracellular IFN-γ positive T_EM cells were proliferating CFSE-dim populations (P?<?0.05).

Conclusions

A combination of Leishmania-reactive IFN-γ producing CD4⁺CD45RO⁺CD45RA^?CCR7^? T_EM and Leishmania-reactive IL-2 producing CD4⁺CD45RO⁺CD45RA^?CCR7⁺ T_CM are identified in individuals with history of CL which might play a role in protective recall immune response against Leishmania infection.     

Specific patterns of CD4-associated immunosenescence in vertically HIV-infected subjects

Vertical transmission of human immunodeficiency virus (HIV) represents an important world-wide health problem although the incidence in developed countries has been drastically reduced by the extensive use of highly active antiretroviral therapy. Vertically HIV-infected subjects have been exposed to the virus during the maturation of their immune systems and have suffered a persistent chronic activation throughout their lifetime; the consequences of this situation for their immune system are not fully understood. The objective of this study was to analyse immunosenescence-related parameters in different CD4 T-cell subsets. Fifty-seven vertically HIV-infected subjects and 32 age-matched healthy subjects were studied. Activation (HLA^– DR⁺), senescence (CD28^– CD57⁺) and proliferation (Ki67⁺) were analysed on different CD4 T-cell subsets: naive (CD45RA⁺ CD27⁺), memory (CD45RO⁺ CD27⁺), effector memory (CD45RO⁺ CD27^–) and effector memory RA (CD45RA⁺ CD27^–). Compared with healthy subjects, vertically HIV-infected subjects showed increased naive and memory CD4 T-cell frequencies (p 0.035 and p 0.010, respectively) but similar frequencies of both effector subsets. Whereas naive CD4 T cells were not further altered, memory CD4 T cells presented increased levels of senescence and proliferation markers (p <0.001), effector memory CD4 T cells presented increased levels of activation, senescence and proliferation markers (p <0.001) and effector memory RA CD4 T cells presented increased levels of activation and senescence (p <0.001) compared with healthy subjects. Despite long periods of infection, vertically HIV-infected subjects show specific patterns of immunosenescence, revealing a preserved CD4 T-cell homeostasis for subset differentiation and distribution. Nevertheless, excepting the naive subpopulation, all subsets experienced some immunosenescence, pointing to uncertain consequences of the future aging process in these subjects.     

Coordinate expression of β1 and β2 integrin “activation” epitopes during T cell responses in secondary lymphoid tissue

The monoclonal antibodies (mAb) 15/7 and 24 recognize unique activation-dependent, conformational epitopes on β1 and β 2-integrins, respectively. The expression of both of these epitopes closely correlates with the ligand binding ability of their respective integrins, and thus serves as indicators of functional integrin “activation”. Here, we have used six-parameter flow cytometry to examine the expression of these epitopes and conventional β1- and β2-integrin epitopes during human T cell activation in secondary lymphoid tissues in vivo, focusing particularly on the virgin to memory/effector cell transition. Fresh tonsil lymphocytes were stained with mAb against conventional or activation-dependent integrin epitopes, followed by staining with mAb against CD3, CD45RA, and CD45RO, thus allowing the determination of integrin epitope expression on virgin (CD3⁺) T cells (CD45RA⁺/RO^?to±), memory/effector (CD45RA^?/RO⁺⁺) T cells, and T cells undergoing the virgin to memory/effector transition: transition region-1 (T1; CD45RA^+to++/RO⁺); -2 (T2; CD45RA⁺⁺/RO⁺⁺); and -3 (T3; CD45RA⁺/RO⁺⁺). Conventional β1- and β2-integrin epitopes progressively increase during the virgin to T3 stages of the transition in tonsil, in keeping with the generally higher levels of these adhesion molecules on memory/effector vs. virgin T cells. Expression of both the β1 (15/7)-and β2 (24)-integrin activation epitopes first appears on transitional T cells, and is maintained on a relatively constant number of cells (averaging 25-30%) throughout the T1-T3 stages. These epitopes are also noted on a subset of activated memory/effector T cells. Importantly, on both transitional and activated memory/effector T cell subsets, the expression patterns of the 15/7 and 24 epitopes vs. a variety of T cell activation antigens are identical, and the expression of these epitopes relative to each other is linearly correlated, findings strongly supporting the coordinate activation of β1 and β2 integrins duringT cell activation in vivo. These results provide the first evidence of integrin activation during an in vivo immunologic response, and demonstrate the usefulness of mAb recognizing conformational epitopes and multiparameter flow cytometry in delineating the dynamic interplay of adhesion molecules during complex physiologic processes.     

Expression in vivo of CD45RA,CD45RB and CD44 on T cell receptor-transgenic CD8+ T cells following immunization

We used mice transgenic for a major histocompatibility complex class I-restricted T cell receptor to study the changes of phenotype in vivo which follow priming by antigen of CD8 T cells. We show that following priming with peptide, CD44 on CD8 T cells is up-regulated. The change of phenotype was relatively stable, as primed CD8 cells isolated from thymectomized mice 6 weeks after priming still expressed increased levels of CD44. CD8 T cells in these mice are still responsive to peptide and could represent long-lived primed cells. No downregulation in vivo of the CD45RA or CD45RB isoforms was found, indicating that there is a differential regulation of the expression of CD44 and CD45RB by activated CD8 transgenic T cells. These results contradict earlier studies in vitro which showed that CD8 T cells which have been primed earlier belong to the CD45RA^? or CD45RB^? subset.     

CD8+ and CD45RA+ human peripheral blood lymphocytes are potent sources of macrophage inflammatory protein 1α, interleukin-8 and RANTES

The chemokines macrophage inflammatory protein 1α (MIP 1α), interleukin-8 (IL-8) and RANTES are potent regulators of leukocyte trafficking. Examination of chemokine secretion by human peripheral blood lymphocytes after stimulation with anti-CD3 or phorbol 12, 13 myristate acetate and ionomycin showed CD8⁺ cells were the dominant source of MIP 1α and RANTES. Although production of MIP 1α and IL-8 were similar in pharmacologically stimulated CD4⁺ CD45RA⁺, CD4⁺ CD45RO⁺, and CD8⁺ CD45RA⁺ cells, the largest amounts of MIP 1α and RANTES were secreted by CD8⁺ CD45RO⁺ lymphocytes. A parallel pattern of prolonged chemokine mRNA expression for at least 18 h after activation was observed in the T cell subsets. These results confirm that human T lymphocytes have a unique capacity for secretion of these three chemokines. In addition, CD8⁺ cells have an unrecognized role in recruiting cells to sites of inflammation, and adult human CD45RA⁺ cells have a physiologically significant secretory capacity.     

Intestinal lymphangiectasia,a disease characterized by selective loss of naive CD45RA+ lymphocytes into the gastrointestinal tract

Intestinal lymphangiectasia (InL) is a disease characterized by hypoproteinemia and lymphocytopenia resulting from blocked intestinal lymphatics and loss of lymph fluid into the gastrointestinal tract. This leads to immunologic abnormalities including hypogammaglobulinemia, skin anergy and impaired allograft rejection. In the present study, we evaluated whether the above immunologic abnormalities are secondary to a quantitative or qualitative disorder of T cells. In initial studies we demonstrated that adult InL patients' peripheral blood contain strikingly (and significantly) reduced numbers of CD4⁺/CD45RA⁺ T cells, whereas the numbers of CD4⁺ / CD45RO⁺ T cells were only moderately (and not significantly) reduced. In addition, the CD4⁺ / CD45RO⁺ T cell population contained an increased percentage of highly differentiated and previously sensitized cells, as demonstrated by decreased CD27 and CD31 expression and increased HLA-DR and CD69 expression. In subsequent functional studies, we showed that the InL CD4⁺ / CD45RO⁺ T cells, when stimulated in vitro, proliferate fivefold less than control CD4⁺ / CD45RO⁺ T cells and produce fourfold more IL-4 and threefold less IFN-γ and IL-2. Thus, this cytokine production profile also reflects the highly differentiated nature of the residual cell population. Overall, these studies provide new information on the trafficking of naive / mature and Th1 / Th2 T cell populations in this disease model.     

The progressive differentiation of primed T cells is associated with an increasing susceptibility to apoptosis

Recent studies have suggested that T cell memory for recall antigens resides in clones of primed T cells with a short inter-mitotic half-life. In humans such cells express an isoform of the leukocyte common antigen termed CD45RO. Nevertheless, little is known of the fate of these primed T cells after initial activation, since no markers are available to distinguish recently primed cells from long-established clones. This report is focused on a spectrum of primed CD4⁺ T cells characterized by an inverse relationship between the expression of two CD45 epitopes: CD45RB and CD45RO. We show that primed CD4⁺ T cells progress through many cycles of division from a CD45RB^brightO^dull to a CD45RB^dullO^bright state, resulting in a highly skewed distribution of the T cell receptor variable region usage within this particular population. The progressive differentiation defined by the shift from CD45RB^bright to CD45RB^dull is paralleled by the gradual loss of bcl-2 and gain of Fas expression, two features associated with an increased propensity for apoptosis. At the same time, the highly differentiated CD45RB^dull cells selectively lose the capacity to synthesize interleukin (IL)-2, a cytokine which is particularly effective in preventing T cell apoptosis, although they produce high levels of IL-4. The inability to produce adequate levels of IL-2 leads to the apoptosis of primed CD45RB^dull cells, when they are stimulated in the absence of exogenous IL-2. These observations show the crucial dependence of highly differentiated T cells on the availability of exogenous IL-2, and suggest both a major constraint for the persistence of T cell memory maintained by continually cycling primed cells, and an important mechanism contributing to the maintenance of T cell homeostasis in vivo.     

CD4+CD45RA+T cells from adults respond to recall antigens after CD28 ligation

The leukocyte common antigen isoforms CD45RA and CD45RO havelong been used to discriminate human naive and memory T cellsrespectively. This model was largely based on the observationthat CD45RO⁺ T cells respond preferentially to and show a higherfrequency of precursors specific for recall antigens. However,CD45RA⁺ T cells have more stringent requirements for stimulationand standard in vitro assays may favour CD45RO⁺ cells in thisrespect. We tested the hypothesis that CD45RAf T cells respondpoorly to in vitro stimulation with recall antigens becauseof inadequate stimulation rather than a lack of precursors.Limiting dilution analyses (LDA) for tetanus toxoid (lT)-specificT cells were performed in the presence or absence of exogenousantLCD28 antibody. Addition of antLCD28 yielded no proliferationin the absence of specific antigen. The precursor frequencyfor lT in the CD4⁺ CD45RO⁺ population was –1:4000, whilethe frequency of CD4⁺ CD45RA⁺ T cells specific for lT was 4-to >>20-fold lower. Addition of anti-CD28 antibody didnot significantly alter the apparent precursor frequency forCD45RA⁺ cells but yielded an enhancement of the value for CD45RA⁺cells by 3- to >>5-fold. No enhancement of antigen-specificproliferation by antLCD28 was observed with CD45RA⁺ T cellsderived from cord blood, although phytohemagglutinin responsesof these cells were amplified by CD28 antibody. These resultsindicate that conventional LDA underestimate the true precursorfrequency of antigen-specific cells within the adult CD45RA⁺population and support the possibility that a small number ofcells revert from a primed (CD45RO⁺) to an unprimed (CD45RA⁺)state. The majority of memory T cells, however, appear to residein the CD45RO⁺ population     

Persistent low thymic activity and non‐cardiac mortality in children with chromosome 22q11·2 microdeletion and partial DiGeorge syndrome

A subgroup of patients with 22q11·2 microdeletion and partial DiGeorge syndrome (pDGS) appears to be susceptible to non‐cardiac mortality (NCM) despite sufficient overall CD4⁺ T cells. To detect these patients, 20 newborns with 22q11·2 microdeletion and congenital heart disease were followed prospectively for 6 years. Besides detailed clinical assessment, longitudinal monitoring of naive CD4⁺ and cytotoxic CD3⁺CD8⁺ T cells (CTL) was performed. To monitor thymic activity, we analysed naive platelet endothelial cell adhesion molecule‐1 (CD31⁺) expressing CD45RA⁺RO^‐CD4⁺ cells containing high numbers of T cell receptor excision circle (T_REC)‐bearing lymphocytes and compared them with normal values of healthy children (n = 75). Comparing two age periods, low overall CD4⁺ and naive CD4⁺ T cell numbers were observed in 65%/75%, respectively, of patients in period A (< 1 year) declining to 22%/50%, respectively, of patients in period B (> 1/< 7 years). The percentage of patients with low CTLs (< P10) remained robust until school age (period A: 60%; period B: 50%). Low numbers of CTLs were associated with abnormally low naive CD45RA⁺RO^‐CD4⁺ T cells. A high‐risk (HR) group (n = 11) and a standard‐risk (SR) (n = 9) group were identified. HR patients were characterized by low numbers of both naive CD4⁺ and CTLs and were prone to lethal infectious and lymphoproliferative complications (NCM: four of 11; cardiac mortality: one of 11) while SR patients were not (NCM: none of nine; cardiac mortality: two of nine). Naive CD31⁺CD45RA⁺RO^‐CD4⁺, naive CD45RA⁺RO^‐CD4⁺ T cells as well as T_RECs/10⁶ mononuclear cells were abnormally low in HR and normal in SR patients. Longitudinal monitoring of naive CD4⁺ and cytotoxic T cells may help to discriminate pDGS patients at increased risk for NCM.     

The effect of allogeneic in vitro stimulation and in vivo immunization on memory CD4+ T‐cell APOBEC3G expression and HIV‐1 infectivity

Allogeneic immunity is one of the most potent natural immune responses. APOBEC3G (A3G) is an intracellular anti‐viral factor that deaminates cytidine to uridine. Allogeneic stimulation of human CD4⁺ T cells in vitro upregulated A3G mRNA and a significant correlation was found between the mixed leukocyte reaction and A3G mRNA. The mechanism of upregulation of A3G mRNA involves interaction between HLA on DC and TCR of CD4⁺ T cells, which is ZAP70 and downstream ERK phosphokinase signalling dependent and induces CD40L and A3G mRNA expression in CD4⁺ T cells. Alloimmune‐induced A3G was found to be significantly increased in CD45RA^?, CCR5⁺ and CD45RA^?CCR7^? subsets of effector memory T cells. In vivo studies of women alloimmunized with their partners' PBMC also showed a significant increase in A3G protein in CD4⁺ T cells, CD45RO⁺ memory and CCR7^? effector memory T cells. The functional effect of allostimulation upregulating A3G mRNA was demonstrated by a significant decrease in in vitro infectivity, using GFP‐labelled pseudovirus and confirmed by a decrease in HIV‐1 (BaL) infection of primary CD4⁺ T cells. The results suggest that alloimmunization offers an alternative or complementary strategy in inducing an innate anti‐viral factor that inhibits HIV‐1 infection.     

Differential expression of type I insulin-like growth factor receptors in different stages of human T cells

Insulin-like growth factor I (IGF-I) has been implicated to play a regulatory role in T cell development and in T cell function. We investigated the expression of type I IGF receptors on human peripheral T cells related to the maturation and activation stage using the type I IGF receptor-specific monoclonal antibody αIR3. It appeared that 87% of the CD4⁺CD45RA⁺ cells and 66% of the CD8⁺CD45RA⁺ cells were αIR3⁺, whereas only 37% of the CD4⁺CD45RO⁺ cells and 38% of the CD8⁺CD45RO⁺ cells bound αIR3. We also found that the fraction of αIR3⁺ cells within in vivo or in vitro activated (HLA-DR⁺) T cells is markedly lower than in nonactivated (HLA-DR^?) cells. In vitro phytohemagglutinin-activated T cells and CD4⁺CD45RO⁺ cells activated with recall antigens also contained less αIR3⁺ cells (1–6%) than nonactivated cells (30–54%).     

Flow Cytometric Identification of CD93 Expression on Naive T Lymphocytes (CD4<Superscript>+</Superscript>CD45RA<Superscript>+</Superscript> Cells) in Human Neonatal Umbilical Cord Blood

Human CD93 has a molecular weight of about 100 kDa and is selectively expressed by myeloid cell lineages in peripheral blood (PB) mononuclear cells. Although CD93 was initially identified as a receptor for complement component 1, subcomponent q phagocytosis (C1qRp) involved in the C1q-mediated enhancement of the phagocytosis of various antigens, several recent studies have reported that CD93 is not a receptor for the C1q-mediated enhancement of phagocytosis. The expression patterns of CD93 have been previously investigated in PB mononuclear cells (lymphocytes, monocytes, and granulocytes) from adult PB and neonatal umbilical cord blood (UCB), and the expression of CD93 was not found on lymphocytes from either normal adult PB or neonatal UCB. However, the detection of CD93 expression in neonatal UCB using CD93 monoclonal antibodies (mAbs) that recognize different antigenic epitopes remains poorly understood. In this study, we examined the expression of CD93 on lymphocytes, monocytes, and granulocytes from neonatal UCB using four different types of CD93 mAb detection probes, mNI-11, R139, R3, and X-2, using flow cytometric and western blot analyses. We found that CD93, as defined using all four mAbs, was expressed on monocytes and granulocytes in PB mononuclear cells from adult PB and neonatal UCB. On the other hand, we observed for the first time that the expression of CD93 on lymphocytes in neonatal UCB can only be detected using the mNI-11 mAb, established in our laboratory, and not with commercially available CD93 mAbs (R139, R3, and X-2). However, CD93 expression on lymphocytes from normal adults was not detected using any of the four CD93 mAbs. Two-color flow cytometric analyses showed that the CD93 recognized by mNI-11 mAb was expressed on CD3⁺ T lymphocytes (mainly CD4⁺ helper T lymphocytes), but not on CD19⁺ B lymphocytes or on CD8⁺ suppressor/cytotoxic T lymphocytes from neonatal UCB. In addition, CD93 was expressed on CD45RA⁺ (naive antigen) lymphocytes from neonatal UCB, but not on CD45RO⁺ (memory antigen) lymphocytes from neonatal UCB or on CD45RA⁺ and CD45RO⁺ lymphocytes from normal adult PB. Three-color flow cytometric analysis showed that CD93 was co-expressed on naive T lymphocytes (CD4⁺CD45RA⁺) from neonatal UCB. In a western blot analysis, the CD93 mAb (mNI-11) immunoprecipitated at a molecular weight of 98 kDa, identified as a CD93 molecule, in the CD4⁺CD45RA⁺ cells from neonatal UCB but not from adult PB, similar to the results in the human monocyte-like cell line U937 (human CD93-positive cells). Taken together, these results provide the first direct evidence of a novel/naive cell population (CD4⁺CD45RA⁺CD93⁺) in neonatal UCB that may have an important role in cell biology, transplantation, and immature/mature immune responses.     

Age-related accumulation of LFA-1high cells in a CD8+CD45RAhigh T cell population

The differential expression of CD45 isoforms has been suggested as a marker for stages of post-thymic T cell development, that is, CD45RA+CD45R0^? T cells and CD45RA^?CD45R0⁺ T cells are supposed to be virgin and memory cells respectively. Recently, several adhesion molecules have been shown to be up-regulated on the cell surface of memory T cells, and have been suggested to serve as a memory marker. In this study, we investigated the levels of LFA-1 expression on T cells in various subpopulations defined by CD45 isoform expression in donors of various ages. In CD4⁺ T cells, the proportion of LFA-1^high cells among CD45RA^highCD45R0-T cells remained low in all age groups and did not show significant accumulation with age. CD4⁺CD45RA^?CD45R0^highTcells expressed LFA-1 at a higher level than CD4⁺CD45RA^highCD45R0^? T cells. Thus, the currently prevailing view that CD45RA and CD45R0 can be markers for virgin and primed cells was consistent with LFA-1 expression in CD4⁺ T cell population. In CD8⁺ T cells, however, CD45RA^highCD45R0^? T cells consisted of two distinct subpopulations, LFA-1^low and LFA-1^high cells, whereas CD45RA^?CD45R0^high T cells were almost exclusively LFA-1^high When CD29 expression was examined in place of LFA-1 expression, similar results were obtained; CD45RA^high CD45R0^? T cells consisted of two distinct subpopulations, CD29-^{to low} and CD^29high cells, while CD45RA-CD45R0^high T cells were mostly CD29^high. The proportion of LFA-1^high cells in the CD8+CD45RA^high T cell subpopulation increased significantly as a function of age (r = 0.9, p < 0.001). It ranged from 8% in a 14-year-old donor to 94% in a 79-year-old donor. Furthermore, the proportion of CD8⁺CD45RA^highLFA-1^high cells in the CD8⁺ T cell population increased significantly as a function of age (r = 0.85, p < 0.001). On the other hand, the proportion of LEA-1^high cells in CD8⁺CD45RA^? T cell subpopulation exceeded 90% in most donors irrespective of age. These results indicate that the CD8⁺CD45RA^highCD45R0^? T cell subpopulation contains a considerable number of LFA-1^high cells and CD29^high cells, phenotypically similar to previously activated cells. Thus, in terms of LFA-1 and CD29 expressions, the simple scheme that CD45RA is a marker of virgin cells is not applicable to the CD8⁺ T cell population.     

Cytomegalovirus infection induces the accumulation of short-lived, multifunctional CD4+CD45RA+CD27+ T cells: the potential involvement of interleukin-7 in this process

The relative roles that ageing and lifelong cytomegalovirus (CMV) infection have in shaping naive and memory CD4⁺ T-cell repertoires in healthy older people is unclear. Using multiple linear regression analysis we found that age itself is a stronger predictor than CMV seropositivity for the decrease in CD45RA⁺ CD27⁺ CD4⁺ T cells over time. In contrast, the increase in CD45RA⁻ CD27⁻ and CD45RA⁺ CD27⁻ CD4⁺ T cells is almost exclusively the result of CMV seropositivity, with age alone having no significant effect. Furthermore, the majority of the CD45RA⁻ CD27⁻ and CD45RA⁺ CD27⁻ CD4⁺ T cells in CMV-seropositive donors are specific for this virus. CD45RA⁺ CD27⁻ CD4⁺ T cells have significantly reduced CD28, interleukin-7 receptor α (IL-7Rα) and Bcl-2 expression, Akt (ser473) phosphorylation and reduced ability to survive after T-cell receptor activation compared with the other T-cell subsets in the same donors. Despite this, the CD45RA⁺ CD27⁻ subset is as multifunctional as the CD45RA⁻ CD27⁺ and CD45RA⁻ CD27⁻ CD4⁺ T-cell subsets, indicating that they are not an exhausted population. In addition, CD45RA⁺ CD27⁻ CD4⁺ T cells have cytotoxic potential as they express high levels of granzyme B and perforin. CD4⁺ memory T cells re-expressing CD45RA can be generated from the CD45RA⁻ CD27⁺ population by the addition of IL-7 and during this process these cells down-regulated expression of IL-7R and Bcl-2 and so resemble their counterparts in vivo. Finally we showed that the proportion of CD45RA⁺ CD27⁻ CD4⁺ T cells of multiple specificities was significantly higher in the bone marrow than the blood of the same individuals, suggesting that this may be a site where these cells are generated.