Cytomegalovirus‐seropositivity has a profound influence on the magnitude of major lymphoid subsets within healthy individuals

Cytomegalovirus (CMV) infects most individuals and elicits a strong CMV‐specific immune response. We have studied the influence of CMV‐seropositivity on the size of lymphoid subsets in healthy donors and demonstrate that the virus substantially modulates the peripheral lymphoid pool. CD8⁺ T cell numbers are increased in all CMV‐seropositive individuals because of a striking 60% increment in the CD8⁺ T cell memory pool. The CD45RA⁺ resting memory pool is doubled after CMV infection and increases further with age. The magnitude of the naïve CD8⁺ T cell pool is dramatically reduced in CMV‐seropositive individuals at all ages, and this accelerates the physiological decline by approximately 40 years. The number of CD4⁺ effector memory T cells is increased in CMV‐seropositive individuals and is differentially accommodated by a reduction in the number of naïve and central memory CD4⁺ T cells in young and elderly donors respectively. CMV‐seropositivity also increases the total number of B cells in older donors and suppresses the number of CD5⁺ B cells. These data reveal that CMV has a profound influence on the immune system of all healthy individuals and add to growing concern regarding the clinical and immunomodulatory significance of CMV infection in healthy donors.     

High-frequency and adaptive-like dynamics of human CD1 self-reactive T cells

CD1 molecules present lipid antigens to T cells. An intriguing subset of human T cells recognize CD1‐expressing cells without deliberately added lipids. Frequency, subset distribution, clonal composition, naïve‐to‐memory dynamic transition of these CD1 self‐reactive T cells remain largely unknown. By screening libraries of T‐cell clones, generated from CD4⁺ or CD4^?CD8^? double negative (DN) T cells sorted from the same donors, and by limiting dilution analysis, we find that the frequency of CD1 self‐reactive T cells is unexpectedly high in both T‐cell subsets, in the range of 1/10–1/300 circulating T cells. These T cells predominantly recognize CD1a and CD1c and express diverse TCRs. Frequency comparisons of T‐cell clones from sorted naïve and memory compartments of umbilical cord and adult blood show that CD1 self‐reactive T cells are naïve at birth and undergo an age‐dependent increase in the memory compartment, suggesting a naïve/memory adaptive‐like population dynamics. CD1 self‐reactive clones exhibit mostly Th1 and Th0 functional activities, depending on the subset and on the CD1 isotype restriction. These findings unveil the unanticipated relevance of self‐lipid T‐cell response in humans and clarify the basic parameters of the lipid‐specific T‐cell physiology.     

Mineralocorticoid receptor signaling reduces numbers of circulating human naïve T cells and increases their CD62L,CCR7, and CXCR4 expression

The role of mineralocorticoid receptors (MRs) in human T‐cell migration is not yet understood. We have recently shown that the MR antagonist spironolactone selectively increases the numbers of circulating naïve and central memory T cells during early sleep, which is the time period in the 24 h cycle hallmarked by predominant MR activation. To investigate whether this effect is specific to spironolactone's blockade of MRs and to study the underlying molecular mechanisms, healthy humans were given the selective MR‐agonist fludrocortisone or placebo and numbers of eight T‐cell subsets and their CD62L and CXCR4 expression were analyzed. Fludrocortisone selectively reduced counts of naïve CD4⁺, central memory CD4⁺, and naïve CD8⁺ T cells and increased CXCR4 expression on the naïve subsets. In complementing in vitro studies, fludrocortisone enhanced CXCR4 and CD62L expression, which was counteracted by spironolactone. Incubation of naïve T cells with spironolactone alone reduced CD62L and CCR7 expression. Our results indicate a regulatory influence of MR signaling on human T‐cell migration and suggest a role for endogenous aldosterone in the redistribution of T‐cell subsets to lymph nodes, involving CD62L, CCR7, and CXCR4. Facilitation of T‐cell homing following sleep‐dependent aldosterone release might thus essentially contribute to sleep's well‐known role in supporting adaptive immunity.     

CD40 ligand‐expressing recombinant vaccinia virus promotes the generation of CD8+ central memory T cells

Central memory CD8⁺ T cells (T_CM) play key roles in the protective immunity against infectious agents, cancer immunotherapy, and adoptive treatments of malignant and viral diseases. CD8⁺ T_CM cells are characterized by specific phenotypes, homing, and proliferative capacities. However, CD8⁺ T_CM‐cell generation is challenging, and usually requires CD4⁺ CD40L⁺ T‐cell “help” during the priming of naïve CD8⁺ T cells. We have generated a replication incompetent CD40 ligand‐expressing recombinant vaccinia virus (rVV40L) to promote the differentiation of human naïve CD8⁺ T cells into T_CM specific for viral and tumor‐associated antigens. Soluble CD40 ligand recombinant protein (sCD40L), and vaccinia virus wild‐type (VV WT), alone or in combination, were used as controls. Here, we show that, in the absence of CD4⁺ T cells, a single “in vitro” stimulation of naïve CD8⁺ T cells by rVV40L‐infected nonprofessional CD14⁺ antigen presenting cells promotes the rapid generation of viral or tumor associated antigen‐specific CD8⁺ T cells displaying T_CM phenotypic and functional properties. These observations demonstrate the high ability of rVV40L to fine tune CD8⁺ mediated immune responses, and strongly support the use of similar reagents for clinical immunization and adoptive immunotherapy purposes.     

Strong and sustained effector function of memory‐ versus naïve‐derived T cells upon T‐cell receptor RNA transfer: Implications for cellular therapy

Current protocols used to select CMV‐specific T cells for adoptive immunotherapy focus on virus‐specific memory T cells from seropositive donors. However, this strategy is not feasible in patients undergoing allogeneic haematopoietic stem‐cell transplantation (HSCT) from CMV‐seronegative donors. Here, we redirected T cells of CMV‐seronegative donors with a human genetically engineered TCR recognizing an HLA‐A*0201‐binding peptide epitope of CMVpp65. To facilitate clinical translation of this approach, we used a non‐viral expression system based on in vitro transcribed RNA and electroporation. Although memory and naïve‐derived T‐cell subsets were both efficiently transfected by TCR‐RNA, memory‐derived T cells showed much stronger levels of HLA‐A*0201‐restricted cytolytic activity to CMV‐infected fibroblasts and maintained acquired function for 5–10 days. In addition to redirection of CD8⁺ cytotoxic T cells, TCR‐RNA transfection was capable of redirecting CD4⁺ T cells into potent Ag‐specific Th cells that efficiently triggered maturation of DCs. Our data suggest that memory rather than naïve‐derived T cells are the preferred subset for transient TCR expression by RNA electroporation, providing more efficient and sustained virus‐specific CD4⁺ and CD8⁺ T‐cell function. CMV TCR‐RNA may represent a suitable therapeutic ‘off‐the‐shelf’ reagent to be used in severe CMV infections of HSCT patients when endogenous CMV‐specific T‐cell immunity is insufficient.     

IL‐15 is critical for the maintenance and innate functions of self‐specific CD8+ T cells

IL‐15 is a pleiotropic cytokine involved in host defense as well as autoimmunity. IL‐15‐deficient mice show a decrease of memory phenotype (MP) CD8⁺ T cells, which develop naturally in naïve mice and whose origin is unclear. It has been shown that self‐specific CD8⁺ T cells developed in male H‐Y antigen‐specific TCR transgenic mice share many similarities with naturally occurring MP CD8⁺ T cells in normal mice. In this study, we found that H‐Y antigen‐specific CD8⁺ T cells in male but not female mice decreased when they were crossed with IL‐15‐deficient mice, mainly due to impaired peripheral maintenance. The self‐specific TCR transgenic CD8⁺ T cells developed in IL‐15‐deficient mice showed altered surface phenotypes and reduced effector functions ex vivo. Bystander activation of the self‐specific CD8⁺ T cells was induced in vivo during infection with Listeria monocytogenes, in which proliferation but not IFN‐γ production was IL‐15‐dependent. These results indicated important roles for IL‐15 in the maintenance and functions of self‐specific CD8⁺ T cells, which may be included in the naturally occurring MP CD8⁺ T‐cell population in naïve normal mice and participate in innate host defense responses.     

Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor

Mesenchymal stem cells (MSCs) inhibit T‐cell activation and proliferation but their effects on individual T‐cell‐effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4⁺ T cells toward the Th17 phenotype was examined. CD4⁺ T cells exposed to Th17‐skewing conditions exhibited reduced CD25 and IL‐17A expression following MSC co‐culture. Inhibition of IL‐17A production persisted upon re‐stimulation in the absence of MSCs. These effects were attenuated when cell–cell contact was prevented. Th17 cultures from highly purified naïve‐ and memory‐phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX‐2 inhibitor. Media from MSC/Th17 co‐cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC‐mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation‐induced IL‐17A secretion by naturally occurring, effector‐memory Th17 cells from a urinary obstruction model was also inhibited by MSC co‐culture in a COX‐dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T‐cell precursors and inhibit naturally‐occurring Th17 cells derived from a site of inflammation. Suppression entails cell‐contact‐dependent COX‐2 induction resulting in direct Th17 inhibition by PGE2 via EP4.     

HIV‐1 impairs in vitro priming of naïve T cells and gives rise to contact‐dependent suppressor T cells

Priming of T cells in lymphoid tissues of HIV‐infected individuals occurs in the presence of HIV‐1. DC in this milieu activate T cells and disseminate HIV‐1 to newly activated T cells, the outcome of which may have serious implications in the development of optimal antiviral responses. We investigated the effects of HIV‐1 on DC–naïve T‐cell interactions using an allogeneic in vitro system. Our data demonstrate a dramatic decrease in the primary expansion of naïve T cells when cultured with HIV‐1‐exposed DC. CD4⁺ and CD8⁺ T cells showed enhanced expression of PD‐1 and TRAIL, whereas CTLA‐4 expression was observed on CD4⁺ T cells. It is worth noting that T cells primed in the presence of HIV‐1 suppressed priming of other naïve T cells in a contact‐dependent manner. We identified PD‐1, CTLA‐4, and TRAIL pathways as responsible for this suppresion, as blocking these negative molecules restored T‐cell proliferation to a higher degree. In conclusion, the presence of HIV‐1 during DC priming produced cells with inhibitory effects on T‐cell activation and proliferation, i.e. suppressor T cells, a mechanism that could contribute to the enhancement of HIV‐1 pathogenesis.     

Quantitative assessment of the functional plasticity of memory CD8+ T cells

While the functional plasticity of memory CD4⁺ T cells has been studied extensively, less is known about this property in memory CD8⁺ T cells. Here, we report the direct measurement of plasticity by paired daughter analysis of effector and memory OT‐I CD8⁺ T cells primed in vivo with ovalbumin. Naïve, effector, and memory OT‐I cells were isolated and activated in single‐cell culture; then, after the first division, their daughter cells were transferred to new cultures with and without IL‐4; expression of IFN‐γ and IL‐4 mRNAs was measured 5 days later in the resultant subclones. Approximately 40% of clonogenic memory CD8⁺ T cells were bipotential in this assay, giving rise to an IL‐4^? subclone in the absence of IL‐4 and an IL‐4⁺ subclone in the presence of IL‐4. The frequency of bipotential cells was lower among memory cells than naïve cells but markedly higher than among 8‐day effectors. Separation based on high or low expression of CD62L, CD122, CD127, or Ly6C did not identify a phenotypic marker of the bipotential cells. Functional plasticity in memory CD8⁺ T‐cell populations can therefore reflect modulation at the level of a single memory cell and its progeny.     

Cardiolipin activates antigen‐presenting cells via TLR2‐PI3K‐PKN1‐AKT/p38‐NF‐kB signaling to prime antigen‐specific naïve T cells in mice

Mitochondrial defects and antimitochondrial cardiolipin (CL) antibodies are frequently detected in autoimmune disease patients. CL from dysregulated mitochondria activates various pattern recognition receptors, such as NLRP3. However, the mechanism by which mitochondrial CL activates APCs as a damage‐associated molecular pattern to prime antigen‐specific naïve T cells, which is crucial for T‐cell‐dependent anticardiolipin IgG antibody production in autoimmune diseases is unelucidated. Here, we show that CL increases the expression of costimulatory molecules in CD11c⁺ APCs both in vitro and in vivo. CL activates CD11c⁺ APCs via TLR2‐PI3K‐PKN1‐AKT/p38MAPK‐NF‐κB signaling. CD11c⁺ APCs that have been activated by CL are sufficient to prime H‐Y peptide‐specific naïve CD4⁺ T cells and OVA‐specific naïve CD8⁺ T cells. TLR2 is necessary for anti‐CL IgG antibody responses in vivo. Intraperitoneal injection of CL does not activate CD11c⁺ APCs in CD14 KO mice to the same extent as in wild‐type mice. CL binds to CD14 (Kd = 7 × 10^?7 M). CD14, but not MD2, plays a role in NF‐kB activation by CL, suggesting that CD14⁺ macrophages contribute to recognizing CL. In summary, CL activates signaling pathways in CD11c⁺ APCs through a mechanism similar to gram (+) bacteria and plays a crucial role in priming antigen‐specific naïve T cells.     

TLR9 stimulation drives naïve B cells to proliferate and to attain enhanced antigen presenting function

Mechanisms that regulate naïve B cell proliferation and function are incompletely defined. In this study, we test the hypothesis that naïve B cell expansion, survival and ability to present antigen to T lymphocytes can be directly modulated by Toll‐like receptor (TLR) agonists. In the absence of B cell receptor stimulation, CpG oligonucleotide, a TLR9 agonist, was particularly efficient in inducing naïve B cell proliferation and survival. Although the expanded naïve B cells did not mature into CD27⁺ or IgG⁺ memory B cells, these cells did differentiate into IgM‐secreting cells with increased surface expression of HLA‐DR, CD40 and CD80. This was associated with an increased potential for these B cells to activate allogeneic T cells. We propose that the activation and expansion of naïve B cells induced by TLR9 agonists could enhance the potential of these cells to interact with cognate antigens and facilitate cell‐mediated immune responses.     

Interleukin‐4 downregulates CD127 expression and activity on human thymocytes and mature CD8+ T cells

Signaling via the IL‐7 receptor complex (IL‐7Rα/CD127 and IL‐2Rγ/CD132) is required for T‐cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL‐2Rγ‐sharing (γ_C) cytokines decrease CD127 expression on CD4⁺ and CD8⁺ T cells in mice (IL‐2, IL‐4, IL‐7, IL‐15) and in humans (IL‐2, IL‐7), suggesting a common function. IL‐4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL‐4 in regulating CD127 expression and IL‐7 activity in human thymocytes and mature CD8⁺ T cells is unknown and was therefore investigated. IL‐4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA⁺) CD8⁺ T cells, without altering membrane‐bound CD127 mRNA expression. Pre‐treatment of thymocytes or CD8⁺ T cells with IL‐4 inhibited IL‐7‐mediated phosphorylation of STAT5 and decreased proliferation of CD8⁺ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8⁺ T cells, IL‐4 is a potential contributor to impaired CD8⁺ T‐cell function in some anti‐viral and anti‐tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL‐7 activity is impaired and IL‐4 concentrations are elevated.     

Memory B cells from a subset of treatment‐naïve relapsing‐remitting multiple sclerosis patients elicit CD4+ T‐cell proliferation and IFN‐γ production in response to myelin basic protein and myelin oligodendrocyte glycoprotein

Recent evidence suggests that B‐ and T‐cell interactions may be paramount in relapsing‐remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B‐cell pools from RRMS patients may specifically harbor a subset of potent neuro‐APC that support neuro‐Ag reactive T‐cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA‐DR expression, IL‐10 and lymphotoxin‐α secretion, neuro‐Ag binding capacity, and neuro‐Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4⁺ T‐cell proliferation and IFN‐γ secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4⁺ T‐cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B‐cell pool in RRMS harbors neuro‐Ag specific B cells that can activate T cells.     

Atopic asthma: differential activation phenotypes among memory T helper cells

Background T cells have been implicated in the pathogenesis of atopic asthma. We have previously shown that memory T helper cells (CD4⁺CD45RO⁺) are preferentially activated relative to naïve T helper cells (CD4⁺CD45RA⁺) after bronchial allergen challenge. However, specific T helper subpopulations that are activated in atopy and/or asthma remain undefined. Objective To determine the T helper subpopulations and activation phenotypes relevant to acute and stable asthma that may be common with or distinct from atopy. Methods Two groups of atopic asthmatics (ten acute and nine stable asthmatics) and two non‐asthmatic groups (14 non‐asthmatic atopics and eight normal non‐atopic controls) were analysed. Ten acute asthmatics were assessed in the emergency room during an acute episode (FEV₁ 43.6% ± 18.4). Nine stable asthmatics were assessed during a symptom‐free period (FEV₁ 85% ± 6). Using multiple colour flow cytometry we analysed T cell subpopulations and the expression of IL‐2‐receptor (IL‐2R) and MHC‐class II antigens (MHC II) on naïve and memory T helper cells in the peripheral blood of asthmatic and non‐asthmatic groups. Results Atopic asthmatics (acute and stable) had an increased percentage of memory T helper cells expressing IL‐2R compared with normal non‐atopics (mean SD 16.1 ± 6%, 12.4 ± 2% and 7.7 ± 1.8%, P < 0.05) but not compared with non‐asthmatic atopics (10 ± 3.5%). Naïve T helper cells had low expression of IL‐2R and MHC II in all four groups. MHC II antigen expression was increased in memory T helper cells of asthmatics (acute and stable) compared with normal non‐atopics (13.9 ± 7.5, 10.6 ± 5 and 4.9 ± 2.5, P < 0.05) but not compared with non‐asthmatic atopics (7.92 4). A novel finding was that IL‐2R and the MHC II molecules were mainly expressed in non‐overlapping populations and coexpression was found predominantly on memory T helper cells. Asthmatics (acute and stable) had higher proportion of double positive memory T helper cells (IL‐2R⁺MHC II⁺) compared with both non‐asthmatic groups (P < 0.05). Conclusions We demonstrate a differential expression of IL‐2R⁺ and MCH II⁺ on CD45RO⁺ T helper cells that would suggest that there are three subsets of activated memory T helper cells in asthmatics. Two non‐overlapping IL‐2R⁺ or MHC II⁺ CD45RO⁺ T helper cells and a third subpopulation of activated cells that coexpress IL‐2R and MHC II (double positives). This latter subpopulation is significantly higher in asthmatics (acute or stable) compared with both non‐asthmatic groups, suggesting a specific T helper activation phenotype distinct to atopic asthmatics as compared with atopic non‐asthmatics.     

Freeze-thaw lysates of Plasmodium falciparum-infected red blood cells induce differentiation of functionally competent regulatory T cells from memory T cells

In addition to naturally occurring regulatory T (nTreg) cells derived from the thymus, functionally competent Treg cells can be induced in vitro from peripheral blood lymphocytes in response to TCR stimulation with cytokine costimulation. Using these artificial stimulation conditions, both naïve as well as memory CD4⁺ T cells can be converted into induced Treg (iTreg) cells, but the cellular origin of such iTreg cells in vivo or in response to more physiologic stimulation with pathogen‐derived antigens is less clear. Here, we demonstrate that a freeze/thaw lysate of Plasmodium falciparum schizont extract (PfSE) can induce functionally competent Treg cells from peripheral lymphocytes in a time‐ and dose‐dependent manner without the addition of exogenous costimulatory factors. The PfSE‐mediated induction of Treg cells required the presence of nTreg cells in the starting culture. Further experiments mixing either memory or naïve T cells with antigen presenting cells and CFSE‐labeled Treg cells identified CD4⁺CD45RO⁺CD25^? memory T cells rather than Treg cells as the primary source of PfSE‐induced Treg cells. Taken together, these data suggest that in the presence of nTreg cells, PfSE induces memory T cells to convert into iTreg cells that subsequently expand alongside PfSE‐induced effector T cells.     

In situ imaging reveals different responses by naïve and memory CD8 T cells to late antigen presentation by lymph node DC after influenza virus infection

Pulmonary influenza infection causes prolonged lymph node hypertrophy while processed viral antigens continue to be presented to virus‐specific CD8 T cells. We show that naïve, but not central/memory, nucleoprotein (NP)‐specific CD8 T cells recognized antigen‐bearing CD11b⁺ DC in the draining lymph nodes more than 30 days after infection. After these late transfers, the naïve CD8 T cells underwent an abortive proliferative response in the mediastinal lymph node (MLN), where large clusters of partially activated cells remained in the paracortex until at least a week after transfer. A majority of the endogenous NP‐specific CD8 T cells that were in the MLN between 30 and 50 days after infection also showed signs of a continuing response to antigen stimulation. A high frequency of endogenous NP‐specific CD8 T cells in the MLN indicates that late antigen presentation may help shape the epitope dominance hierarchy during reinfection.