Optimal induction of T helper 17 cells in humans requires T cell receptor ligation in the context of Toll-like receptor-activated monocytes

Recently, a new lineage of CD4⁺ T cells has been described in the mouse that specifically secretes IL-17 [T helper (Th) 17]. This discovery has led to a revision of the hypothesis that many autoimmune diseases are predominantly a Th1 phenomenon and may instead be critically dependent on the presence of Th17 cells. Murine Th17 cells differentiate from naïve T cell precursors in the presence of TGF-β and IL-6 or IL-21. However, given their putative importance in human autoimmunity, very little is known about the pathways that control the expression of IL-17 in humans. Here we show that the factors that determine the expression of IL-17 in human CD4⁺ T cells are completely different from mice. IL-6 and IL-21 were unable to induce IL-17 expression in either naïve or effector T cells, and TGF-β actually inhibited IL-17 expression. The expression of IL-17 was maximally induced from precommitted precursors present in human peripheral blood by cell–cell contact with Toll-like receptor-activated monocytes in the context of T cell receptor ligation. Furthermore, unlike IFN-γ, IL-17 expression was not suppressed by the presence of FOXP3⁺ regulatory CD4⁺ T cells. Taken together, these data indicate that human and mouse Th17 cells have important biological differences that may be of critical importance in the development of therapeutic interventions in diseases characterized by aberrant T cell polarization.     

Na?ve and memory CD8 T cell pool homeostasis in advanced aging: impact of age and of antigen-specific responses to cytomegalovirus

Alterations in the circulating CD8+ T cell pool, with a loss of naïve and accumulation of effector/effector memory cells, are pronounced in older adults. However, homeostatic forces that dictate such changes remain incompletely understood. This observational cross-sectional study explored the basis for variability of CD8+ T cell number and composition of its main subsets: naïve, central memory and effector memory T cells, in 131 cytomegalovirus (CMV) seropositive subjects aged over 60 years. We found great heterogeneity of CD8+ T cell numbers, which was mainly due to variability of the CD8 + CD28− T cell subset regardless of age. Analysis, by multiple regression, of distinct factors revealed that age was a predictor for the loss in absolute number of naïve T cells, but was not associated with changes in central or effector memory CD8+ T cell subsets. By contrast, the size of CD8+ T cells specific to pp65 and IE-1 antigens of CMV, predicted CD28 − CD8+ T cell, antigen-experienced CD8+ T cell, and even total CD8+ T cell numbers, but not naïve CD8+ T cell loss. These results indicate a clear dichotomy between the homeostasis of naïve and antigen-experienced subsets of CD8+ T cells which are independently affected, in human later life, by age and antigen-specific responses to CMV, respectively.

Electronic supplementary material

The online version of this article (doi:10.1007/s11357-013-9594-z) contains supplementary material, which is available to authorized users.     

Ablation of thymic export causes accelerated decay of naive CD4 T cells in the periphery because of activation by environmental antigen

A model of chemical thymectomy by inducible Rag ablation was used to study peripheral T cell homeostasis. Induction of Rag ablation was efficient and complete, leading to cessation of thymic T cell production within 3–4 weeks. The decay of peripheral T cells became apparent with a delay of an additional 2–3 weeks and was entirely accounted for by loss of naïve T cells, whereas numbers of memory phenotype and regulatory T cells were not decreased. Naïve CD4 T cells decayed with an average half-life of 50 days, whereas naïve CD8 T cells exhibited a considerably longer half-life. The rapid decay of naïve CD4 T cells was not caused by intrinsic survival differences compared with naïve CD8 T cells, but was caused by changes in the lymphopenic environment resulting in higher microbial load and consequential activation. This finding suggests that in lymphopenic conditions involving compromised thymic function replenishment and survival of a naïve CD4 T cell repertoire may be severely curtailed because of chronic activation. Such a scenario might play a role in the aging immune system and chronic viral infection, such as HIV infection, and contribute to loss of CD4 T cells and impaired immune function. As our data show, continued replenishment with cells from the thymus seems to be required to maintain efficient gut mucosal defense.     

VLA-4 integrin concentrates at the peripheral supramolecular activation complex of the immune synapse and drives T helper 1 responses

The integrin α4β1 (VLA-4) not only mediates the adhesion and transendothelial migration of leukocytes, but also provides costimulatory signals that contribute to the activation of T lymphocytes. However, the behavior of α4β1 during the formation of the immune synapse is currently unknown. Here, we show that α4β1 is recruited to both human and murine antigen-dependent immune synapses, when the antigen-presenting cell is a B lymphocyte or a dendritic cell, colocalizing with LFA-1 at the peripheral supramolecular activation complex. However, when conjugates are formed in the presence of anti-α4 antibodies, VLA-4 colocalizes with the CD3-ζ chain at the center of the synapse. In addition, antibody engagement of α4 integrin promotes polarization toward a T helper 1 (Th1) response in human in vitro models of CD4⁺ T cell differentiation and naïve T cell priming by dendritic cells. The in vivo administration of anti-α4 integrin antibodies also induces an immune deviation to Th1 response that dampens a Th2-driven autoimmune nephritis in Brown Norway rats. These data reveal a regulatory role of α4 integrins on T lymphocyte-antigen presenting cell cognate immune interactions.After the recognition of antigens (Ag) presented by dendritic cells (DCs), naïve T lymphocytes proliferate and differentiate into T helper (Th) 1 or 2 effector cells. These effector lymphocytes are characterized by distinct patterns of cytokine production and homing behavior. Th1 cells mainly produce IFN-γ and IL-2 and have a key role in the cellular immune responses. Conversely, Th2 cells produce IL-4, IL-5, IL-6, and IL-10 and promote the humoral immune responses (1). DCs are the only Ag-presenting cells (APCs) involved in the priming of naïve Th cells and their polarization toward Th1 or Th2 differentiation. To acquire this capacity, DCs must undergo a maturation process characterized by the loss of their Ag-capturing capacity and the increase of their expression of costimulatory and adhesion molecules, including α4β1 integrin (2). However, other APCs (e.g., B lymphocytes) are also involved in regulating the cytokine profiles of Th cell responses, indicating the importance of postpriming events (3).The interaction between T cells and APCs plays an important role in directing Th cell polarization. The strength of antigenic stimulation, the duration of T cell receptor engagement, the presence of different cytokines, and the participation of distinct costimulatory molecules are critical in determining the phenotype of differentiated T cells. The cytokine IL-12, high doses of Ag, and CD28/B7–1 interaction promote Th1 differentiation, whereas an environment enriched in IL-4, low doses of Ag, and CD28/B7–2 or inducible costimulator (ICOS)/ICOS ligand participation promote Th2 responses (4).Integrins are a large family of αβ heterodimeric transmembrane proteins that mediate cell–cell and cell–extracellular matrix adhesion. Several integrins, lymphocyte function-associated (LFA-1; α_Lβ₂), very late activation antigen-4 (VLA-4; α4β1), and VLA-1 (α1β1) have been involved also in the transduction of costimulatory signals in T cells (5). However, whereas the involvement of α_Lβ2 during Ag presentation is well known, the role of α4β1 has not been addressed. The α_Lβ2 integrin mediates T cell adhesion to APCs, facilitating the formation of the immunological synapse (IS) (6). The pair α_Lβ₂/intracellular adhesion molecule-1 (ICAM-1) forms an adhesion ring that is called the peripheral supramolecular activation complex (pSMAC), that surrounds the T cell receptor–peptide–MHC complexes localized at the central SMAC of the IS (7, 8). Several studies in mouse models revealed that α_Lβ₂/ICAM-1 interaction could be important for driving Th1 polarization (9, 10).The α4β1 integrin is predominantly expressed on hematopoietic cells and serves as a receptor for fibronectin and vascular cell adhesion molecule 1 (VCAM-1). In addition to mediating leukocyte adhesion to endothelium and extracellular matrix, α4β1 has been implicated in T cell costimulation (11–13). The dual role of α4β1 as an adhesion and costimulatory molecule suggests that this integrin could be involved in the modulation of the T cell response during Ag presentation. However, the behavior of α4β1 and its possible function during the establishment of an IS has not been examined. Here, we show that α4β1 is recruited to the pSMAC of IS colocalizing with LFA-1 integrin. We also demonstrate the functional involvement of this integrin in the priming of T lymphocytes toward a Th1 response in vitro and in vivo.     

Competition for self-peptide-MHC complexes and cytokines between naive and memory CD8+ T cells expressing the same or different T cell receptors

To study competition between naïve and memory T cells, we examined proliferation of adoptively transferred naïve CD8⁺ T cells in lymphopenic recipients or recipients containing a clonal population of CD8⁺ T cells. We find a hierarchy in the extent of T cell proliferation that appears to correlate with the strength of T cell receptor (TCR)-self-peptide-MHC (pepMHC) interactions. CD8⁺ T cells also proliferate in recipients containing a full complement of CD8⁺ cells with a different TCR if the transferred T cells experience stronger TCR-self-pepMHC interactions than the resident T cells. Furthermore, CD8⁺ T cells proliferate in recipients that contain memory CD8⁺ cells with a different TCR, but in this case the relative strengths of TCR-self-pepMHC interactions are not as critical. In contrast, CD8⁺ T cells do not proliferate significantly in recipients harboring naïve or memory CD8⁺ cells that bear the same TCR as the transferred cells. These results suggest that, among naïve T cells and between naïve and memory T cells, CD8⁺ cells having the same TCR compete for both self-pepMHC and cytokines, whereas TCR-different CD8⁺ cells compete for cytokines. These competitive relationships probably help maintain the size and TCR diversity of naïve and memory T cell populations required for optimal immune responses.In a normal adult individual, peripheral T cells include both naïve cells that have not yet encountered antigen and antigen-experienced memory T cells. Both the naïve and memory cell compartments are composed of large numbers of T cells that express diverse antigen-specific T cell receptors (TCRs). Naïve T cells are critical for initiating immune responses to novel antigens, whereas memory T cells mount more rapid and vigorous responses on reencountering the same antigens. Because the total number of peripheral T cells is maintained at a relatively constant level (homeostasis) (1, 2), a proper size and sufficient diversity of naïve and memory T cell compartments are critical for the immune system''s ability to respond to unpredictable encounters with an enormous number of different antigens (3). The molecular mechanisms that regulate the size and diversity of naïve and memory T cell compartments are under active investigation but are still not well understood.The number of naïve T cells in the periphery is determined by their production in the thymus and by their survival, proliferation, and differentiation in peripheral lymphoid organs. Studies have shown that the number of T cells remains constant irrespective of whether the pool is oversupplied or undersupplied (4), indicating mechanisms of regulation in the periphery. Indeed, survival of naïve CD4⁺ and CD8⁺ T cells requires both soluble factors, such as IL-7 (5, 6), and interactions between T cells'' TCR and self-peptide-MHC complexes (pepMHC) (MHC class I for CD8⁺ T cells and MHC class II for CD4⁺ T cells) (7-11). The numbers of memory T cells are similarly regulated by their generation, survival, proliferation, and differentiation. Unlike naïve T cells, survival of memory CD4⁺ and CD8⁺ T cells does not appear to require interactions between TCR and self-pepMHC complexes (12-14). Maintenance of a stable number of memory CD8⁺ T cells requires IL-15 (or IL-7 in the absence of IL-15), which stimulates memory CD8⁺ T cells to proliferate and replenish those lost by attrition (6, 15-17). However, neither IL-15 nor IL-7 (or other factors) is known to be required for survival or proliferation of memory CD4⁺ T cells (17).Naïve T cells do not proliferate significantly in normal adults in the absence of cognate foreign antigen (pepMHC complexes, usually with self-MHC plus peptides derived from foreign proteins). However, after severe T cell depletion induced by ionizing radiation, chemotherapy, or virus infection, the residual T cells undergo expansion in the absence of foreign antigens (18, 19). Similarly, proliferation occurs when small numbers of naïve T cells are adoptively transferred into syngeneic lymphopenic hosts, such as mice deficient in recombination activating gene (RAG). The spontaneous T cell proliferation under lymphopenic conditions is often referred to as homeostatic proliferation because it increases cell number, and it also requires IL-7 and interactions between T cells'' TCR with self-pepMHC complexes (5-9, 11, 12). After proliferation in lymphopenic mice, the persisting T cells acquire surface markers and functions characteristic of antigen-induced memory T cells (20-23). Yet, after proliferation, the total T cell numbers in lymphopenic recipients are not restored to the levels found in normal adult mice (24, 25). Hence, we prefer to call this process lymphopenia-induced proliferation and differentiation. Lymphopenia-induced T cell proliferation and differentiation also occurs under normal physiological conditions, as in neonates (26-28), and the process appears to play a role in generating and/or maintaining an appropriate size and diversity of the memory T cell compartment.Adoptive transfer of naïve T cells into lymphopenic mice has been the model of choice to investigate factors that regulate the size and diversity of T cell compartments, because it requires the same factors (IL-7 and cognate self-pepMHC complexes) as those essential for naïve T cell survival. Using this model, two recent studies examined competition among naïve T cells that express different TCRs. In one study, a clonal population of naïve CD4⁺ T cells was adoptively transferred into either RAG^- recipients or transgenic mice expressing a different TCR on CD4⁺ T cells. The transferred CD4⁺ T cells were reported to proliferate to the same extent in the two recipients, although one had no T cells and the other had a relatively normal number of CD4⁺ T cells (29). The transferred CD4⁺ T cells presumably proliferated in the CD4⁺ TCR transgenic mice because the two clonal populations of T cells did not compete for the same self-pepMHC complexes. Because lymphopenia-induced T cell proliferation also requires IL-7, these results suggest that IL-7 was sufficient for proliferation of the introduced CD4⁺ T cells but not the resident CD4⁺ T cells. In the other study, when naïve CD8⁺ T cells expressing one TCR were transferred into CD8⁺ transgenic mice expressing another TCR, or vice versa, the transferred CD8⁺ T cells proliferated to the same extent (30). Although the level of IL-7 in the transgenic hosts does not support the spontaneous proliferation of the resident CD8⁺ T cells, it is apparently sufficient to support proliferation of the transferred CD8⁺ T cells. Based on the assumption that the amount of IL-7 in normal, RAG^-, and TCR transgenic mice is finite and that endogenous naïve T cells compete for IL-7, these results are puzzling and warrant further examination.There is also little known about the factors that regulate the relative size of naïve and memory T cell compartments. Several studies have concluded that naïve and memory CD8⁺ T cell compartments are regulated independently (12, 31, 32). If this conclusion were correct, a mechanism would be required to keep naïve and memory T cells with the same TCR from competing for the same cognate self-pepMHC complexes. That there may be such a mechanism is suggested by the finding that survival of memory T cells does not require interactions between TCR and self-pepMHC (12-14). However, in the absence of TCR-self-pepMHC interactions, memory CD4⁺ T cells lose many of their functional properties (33), suggesting that these interactions normally occur and are essential. Nevertheless, whether naïve and memory T cells with the same TCR compete for self-pepMHC complexes has not been unequivocally demonstrated. Furthermore, memory CD8⁺ T cells express receptors for both IL-7 and IL-15 and can use IL-7 for their survival and proliferation in the absence of IL-15 (6, 17). The purported independent regulation of naïve and memory CD8⁺ T cell compartments would therefore require mechanisms to prevent competition for IL-7 between naïve and memory CD8⁺ T cells. To date, no such mechanism has been found. Thus, whether naïve and memory T cell compartments are regulated independently needs to be reevaluated.In the present study, we investigated competition among naïve CD8⁺ T cells and between naïve and memory CD8⁺ T cells expressing the same or different TCRs. We show that naïve CD8⁺ T cells can undergo limited proliferation when transferred into hosts whose naïve CD8⁺ T cells express a different TCR; whether the transferred T cells proliferate or not depends on the relative strengths of the TCR-self-pepMHC interactions of the two T cell populations. We also show that naïve CD8⁺ T cells proliferate in hosts that harbor memory CD8⁺ T cells with a different TCR, but, in this case, the relative strengths of TCR-self-pepMHC interactions are not as critical. In contrast, naïve CD8⁺ T cells do not proliferate significantly when transferred into hosts with naïve or memory CD8⁺ T cells that bear the same TCR as the transferred cells. Thus, within the naïve T cell compartment and between naïve and memory T cell compartments, CD8⁺ T cells with the same TCR likely compete for cognate self-pepMHC complexes and cytokines, whereas CD8⁺ T cells having different TCR compete for cytokines.     

Activation of naïve CD4 T cells by anti-CD3 reveals an important role for Fyn in Lck-mediated signaling

Although there was no impairment in IL-2 secretion and proliferation of Fyn-deficient naïve CD4 cells after stimulation with antigen and antigen-presenting cells, stimulation of these cells with anti-CD3 and anti-CD28 revealed profound defects. Crosslinking of purified wild-type naïve CD4 cells with anti-CD3 activated Lck and initiated the signaling cascade downstream of Lck, including phosphorylation of ZAP-70, LAT, and PLC-γ1; calcium flux; and dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT)p. All of these signaling events were diminished severely in Fyn-deficient naïve cells activated by CD3 crosslinking. Coaggregation of CD3 and CD4 reconstituted this Lck-dependent signaling pathway in Fyn^-/- T cells. These results suggest that when signaling of naïve T cells is restricted to the T cell antigen receptor, Fyn plays an essential role by positive regulation of Lck activity.Signaling through the T cell antigen receptor (TCR) is initiated by the activity of the Src family tyrosine kinases, Fyn and Lck (1). It has been shown in numerous studies using Lck-deficient cell lines and mice whose peripheral T cells are deficient in Lck that this enzyme plays a critical upstream role in the signaling cascade leading to T cell development, activation, and differentiation (2). In contrast to Lck, the role of Fyn kinase in T cell activation and development is less well defined. With the exception of natural killer T (NKT) cells (3, 4), Fyn deficiency has little or no effect on T cell development in the thymus, and peripheral T cells from Fyn-deficient mice, to the extent they have been studied, show variable and incomplete defects in mounting immune responses (5-7). These findings are consistent with the concept that, for the most part, Fyn is a redundant Src kinase without a unique function in the signaling of T cells. The finding that some of the substrates for Lck [such as the immunoreceptor tyrosine-based activation motifs (ITAMs) of TCRζ as well as CD3γδε] can also serve as substrates for Fyn (8) supports this view. Furthermore, some substrates of ZAP-70, such as Vav (9) and SLP 76 (8), are also substrates of Fyn. However, other studies have shown that Fyn has its own unique substrates, some of which play significant roles in T cell activation. These include ADAP (SLAP-130/FYB) (10), Pyk2 (11), WASp (12), and CBP (13). Other indications that Fyn plays some unique role in TCR-mediated signaling come from our previous studies that have shown that stimulation of T cells with low affinity ligands that function as TCR antagonists leads to preferential activation of Fyn in the absence of any detectable changes in the activity of Lck or ZAP-70 (14, 15). Also, Fyn-deficient T cells are particularly inefficient at responding to weak TCR agonists (7).Because of these more recent findings that suggest a unique role for Fyn in T cell activation, we have reinvestigated the capacity of Fyn-deficient T cells to respond to TCR-mediated signaling in this study. We compared naïve CD4 T cells from Fyn-deficient and wild-type mice that contained a TCR transgene for their capacity to respond to either antigen presented by antigen-presenting cells (APCs) or to respond to antibody-mediated CD3 crosslinking. These experiments demonstrated that the relative importance of Fyn depended highly on the mode of stimulation and the activation status of T cells; stimulation of Fyn-deficient T cells by antigen-pulsed APCs led to a robust proliferative and IL-2 response, whereas anti-CD3-mediated stimulation revealed a profound signaling defect in Fyn-deficient T cells. This defect could be corrected by the cocrosslinking of CD3 with CD4. Furthermore, the signaling defect of Fyn-deficient T cells stimulated with anti-CD3 was restricted to naïve T cells and was not observed when Fyn-deficient effector T cells were analyzed. Biochemical experiments on anti-CD3 stimulated cells strongly suggest that the presence of Fyn was necessary for an effective recruitment and/or activation of Lck in the region of the TCR.     

Blinatumomab induces autologous T-cell killing of chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia is an incurable B-cell malignancy that is associated with tumor cell-mediated T-cell dysfunction. It therefore represents a challenging disease for T-cell immunotherapeutics. The CD19/CD3 bi-specific antibody construct blinatumomab (AMG103 or MT103) has been tested clinically in non-Hodgkin’s lymphoma and acute lymphoblastic leukemia but has not been assessed in chronic lymphocytic leukemia. We investigated whether blinatumomab could overcome T-cell dysfunction in chronic lymphocytic leukemia in vitro. Blinatumomab was tested on peripheral blood mononuclear cells from 28 patients (treatment naïve and previously treated). T-cell activation and function, as well as cytotoxicity against leukemic tumor cells were measured. Blinatumomab induced T-cell activation, proliferation, cytokine secretion and granzyme B release in a manner similar to that occurring with stimulation with anti-CD3/anti-CD28 beads. However, only blinatumomab was able to induce tumor cell death and this was found to require blinatumomab-mediated conjugate formation between T cells and tumor cells. Cytotoxicity of tumor cells was observed at very low T-cell:tumor cell ratios. A three-dimensional model based on confocal microscopy suggested that up to 11 tumor cells could cluster round each T cell. Importantly, blinatumomab induced cytotoxicity against tumor cells in samples from both treatment-naïve and treated patients, and in the presence of co-culture pro-survival signals. The potent cytotoxic action of blinatumomab on tumor cells appears to involve conjugation of T cells with tumor cells at both the activation and effector stages. The efficacy of blinatumomab in vitro suggests that the bi-specific antibody approach may be a powerful immunotherapeutic strategy in chronic lymphocytic leukemia.     

Dramatic increase in naive T cell turnover is linked to loss of naive T cells from old primates

The loss of naïve T cells is a hallmark of immune aging. Although thymic involution is a primary driver of this naïve T cell loss, less is known about the contribution of other mechanisms to the depletion of naïve T cells in aging primates. We examined the role of homeostatic cycling and proliferative expansion in different T cell subsets of aging rhesus macaques (RM). BrdU incorporation and the expression of the G₁-M marker Ki-67 were elevated in peripheral naïve CD4 and even more markedly in the naïve CD8 T cells of old, but not young adult, RM. Proliferating naïve cells did not accumulate in old animals. Rather, the relative size of the naïve CD8 T cell compartment correlated inversely to its proliferation rate. Likewise, T cell receptor diversity decreased in individuals with elevated naïve CD8 T cell proliferation. This apparent contradiction was explained by a significant increase in turnover concomitant with the naïve pool loss. The turnover increased exponentially when the naïve CD8 T cell pool decreased below 4% of total blood CD8 cells. These results link the shrinking naïve T cell pool with a dramatic increase in homeostatic turnover, which has the potential to exacerbate the progressive exhaustion of the naïve pool and constrict the T cell repertoire. Thus, homeostatic T cell proliferation exhibits temporal antagonistic pleiotropy, being beneficial to T cell maintenance in adulthood but detrimental to the long-term T cell maintenance in aging individuals.     

Age-associated increase in lifespan of na?ve CD4 T cells contributes to T-cell homeostasis but facilitates development of functional defects

With age, T-cell generation from the thymus is much reduced, yet a substantial naïve T-cell pool is maintained even in aged animals, suggesting that naïve T cells either persist longer or turn over faster to maintain T-cell homeostasis. We found that with age, naïve CD4 T cells became progressively longer-lived. Their longer lifespan did not depend on recognition of self-peptide/class II. Newly generated naïve T cells derived from aged stem cells had a shorter lifespan, like that of young naïve T cells. Conversely, naïve CD4 T cells derived from middle-aged thymectomized mice were longer-lived in vivo, and their development of functional defects was accelerated. These observations suggest that naïve T cells develop their longer lifespan during their sojourn in the periphery. Increased longevity of naïve CD4 T cells correlated well with reduced expression of proapoptotic molecule Bim. We suggest that the intrinsic increase in longevity helps maintain naïve T-cell homeostasis but facilitates the development of functional defects in mice.     

The CD69 activation pathway in rheumatoid arthritis synovial fluid t cells

Objective. To study the CD69 activation pathway in synovial fluid (SF) T lymphocytes from patients with rheumatoid arthritis (RA). Methods. Peripheral blood mononuclear cells (PBMC) or SF mononuclear cells (SFMC) were used in proliferation assays with anti-CD69, anti-CD28, anti-CD3, phorbol myristate acetate (PMA), and/or recombinant interleukin-2 (IL-2). CD69+, CD69—, and resting SF T cells were also proliferated. CD25 expression and production of IL-2 after CD69 activation were assessed by flow cytometry and in a bioassay with the IL-2-dependent cell line CTLL-2. Results. RA SFMC did not proliferate either in the presence of anti-CD69 monoclonal antibodies alone or with concomitant PMA activation, when compared with paired or control PBMC. Similar low proliferative responses via the CD3 or CD28 pathway with PMA were observed. This defective proliferation of RA SFMC after stimulation through the CD69 molecule was explained in part by a failure to express CD25 and to produce IL-2. SF CD69- T cells and resting SF T cells had higher rates of proliferation through the alternative costimulatory pathway CD28 than did SF CD69+ T cells or freshly isolated SF T cells. Conclusion. Freshly isolated SF T cells present a profound state of hyporesponsiveness through the CD69 and CD28 costimulatory pathways. This state appears to be dependent on the activation status of SF T cells, since CD69— and resting SF T cells showed recovery of the ability to proliferate through the CD28 activation pathway.     

CD4 cell-secreted, posttranslationally modified cytokine GIF suppresses Th2 responses by inhibiting the initiation of IL-4 production

T helper 2 (Th2) cells are critical to the induction of IgE antibody and allergic inflammation, but how the pathological pathways are controlled in nonallergic individuals remains unclear. Here we report that glycosylation-inhibiting factor (GIF) suppresses Th2 effector generation. GIF is a cytokine encoded by the same gene that codes for macrophage migration inhibitory factor (MIF). GIF-deficient mice demonstrated enhanced T-dependent antibody formation especially of IgE isotype and allergic airway inflammation with the generation of regulatory T cells unaffected. GIF-deficient macrophages and dendritic cells revealed normal responsiveness to toll-like receptor (TLR) ligands. GIF undergoes a unique posttranslational modification, cysteinylation. The modified GIF, mainly secreted by activated T cells derived from CD4⁺CD25⁻ cells, inhibited IL-4 production by the same cells whereas the unmodified GIF showed no effect. Bone marrow chimera experiment demonstrated that T cell-derived GIF suppressed the generation of Th effectors that secrete IL-4. During the first 24 h of CD3/CD28 stimulation in vitro, GIF secreted from naïve CD4 cells acted on the same cells, maintained nuclear factor of activated T cells (NFAT)c2 in the nucleus, and repressed IL-4 mRNA levels. Thus, GIF represents a self-regulatory mechanism of Th2 cell generation from naïve CD4 cells, in which the posttranslational modification plays a crucial role.     

Secretion of IFN-gamma and not IL-2 by anergic human T cells correlates with assembly of an immature immune synapse

We report differences in the supramolecular organization of the immunologic synapse (IS) formed by resting and anergic human T cells with agonist peptide-loaded antigen-presenting cells (APCs). T cells reactive to influenza A hemagglutinin peptide or Fel d 1 peptide 4 were rendered both anergic and regulatory by incubation with high doses of agonist peptide in the absence of APCs. At the IS between resting T cells and peptide-loaded APCs, both CD3epsilon and CD3zeta initially accumulate within a ring or arc before redistributing within 30 minutes to single or multiple foci more central to the contact. In contrast, at synapses formed by anergized T cells, CD3epsilon and CD3zeta remained organized within an arc or ring and failed to redistribute centrally. However, intercellular communication between anergic human T cells and agonist peptide-loaded APCs was not a null event, since it triggered secretion of T-cell interferon gamma (IFN-gamma) but not, for example, interleukin 2 (IL-2). Thus, distinct organizations of CD3 at the T-cell IS correlate with different cytokine profiles; the mature IS formed by resting T cells correlates with their production of both IFN-gamma and IL-2, whereas the immature IS formed by anergic T cells seems able to facilitate IFN-gamma but not IL-2 production.     

Regulation of memory CD4 T-cell pool size and function by natural killer T cells in vivo

To develop more effective vaccines and strategies to regulate chronic inflammatory diseases, it is important to understand the mechanisms of immunological memory. Factors regulating memory CD4⁺ T helper (Th)-cell pool size and function remain unclear, however. We show that activation of type I invariant natural killer T (iNKT) cells with glycolipid ligands and activation of type II natural killer T (NKT) cells with the endogenous ligand sulfatide induced dramatic proliferation and expansion of memory, but not naïve, CD4 T cells. NKT cell-induced proliferation of memory Th1 and Th2 cells was dependent largely on the production of IL-2, with Th2-cell proliferation also affected by loss of IL-4. Type II NKT cells were also required for efficient maintenance of memory CD4 T cells in vivo. Activation of iNKT cells resulted in up-regulation of IFN-γ expression by memory Th2 cells. These IFN-γ–producing memory Th2 cells showed a decreased capability to induce Th2 cytokines and eosinophilic airway inflammation. Thus, activated NKT cells directly regulate memory CD4 T-cell pool size and function via the production of cytokines in vivo.     

Alloreactive and leukemia-reactive T cells are preferentially derived from naive precursors in healthy donors: implications for immunotherapy with memory T cells

Background

HLA mismatch antigens are major targets of alloreactive T cells in HLA-incompatible stem-cell transplantation, which can trigger severe graft-versus-host disease and reduce survival in transplant recipients. Our objective was to identify T-cell subsets with reduced in vitro reactivity to allogeneic HLA antigens.

Design and Methods

We sorted CD4 and CD8 T-cell subsets from peripheral blood by flow cytometry according to their expression of naive and memory markers CD45RA, CD45RO, CD62L, and CCR7. Subsets were defined by a single marker to facilitate future establishment of a clinical-grade procedure for reducing alloreactive T-cell precursors and graft-versus-host disease. T cells were stimulated in mixed lymphocyte reactions against HLA-deficient K562 cells transfected with single HLA-A/-B/-C/-DR/-DQ mismatch alleles. Alloreactivity was measured by interferon-γ spot production and cell proliferation.

Results

We observed that allogeneic HLA-reactivity was preferentially derived from subsets enriched for naïve T cells rather than memory T cells in healthy donors, irrespective of the HLA mismatch allele. This separation was most efficient if CD45RA (versus other markers) was used for sorting. The numbers of allogeneic HLA-reactive effector cells were in median 7.2-fold and 16.6-fold lower in CD45RA^neg memory CD8 and CD4 T cells than in entire CD8 and CD4 T cells, respectively. In contrast, proliferation of memory T cells in response to allogeneic HLA was more variably reduced (CD8) or equivalent (CD4) when compared to that of naïve T cells. We also demonstrated in HLA-matched donor-patient pairs that leukemia-reactive CD8 cytotoxic T-lymphocytes were mainly derived from subsets enriched for naïve T cells compared to memory T cells.

Conclusions

Memory T-cell subsets of most healthy individuals showed decreased allogeneic HLA-reactivity, but lacked significant anti-leukemia responses in vitro. The clinical use of memory or naïve-depleted T cells might be beneficial for HLA-mismatched patients at high risk of graft-versus-host disease and low risk of leukemia relapse. Preferred allografts are those which contain leukemia-reactive memory T cells. Alternatively, replenishment with leukemia-reactive T cells isolated from naïve subsets is desirable.     

Inhibition of synovial fluid T cell proliferation by anti-CD5 monoclonal antibodies. A potential mechanism for their immunotherapeutic action in vivo

Objective. Monoclonal antibodies (MAb) directed against the T cell surface molecule CD5 are able to provide accessory stimulatory signals to resting T cells. The potential role of CD5 as an immunoregulatory molecule in inflammatory synovitis was examined. Methods. Synovial fluid and peripheral blood T cells of patients with active rheumatoid arthritis (RA) were purified and stimulated with interleukin-2 (IL-2), and the effect of MAb directed against CD5 on IL-2 responsiveness was examined. Results. IL-2—induced proliferation of synovial fluid T cells was strongly inhibited by anti-CD5 MAb, but not by anti-CD28 or anti-CD3 MAb. In RA peripheral blood T cells, MAb directed against CD5, CD3, and CD28 induced IL-2—dependent T cell growth, similar to findings in healthy controls. The difference in activity of anti-CD5 MAb on synovial fluid T cells compared with peripheral blood T cells was not due to different surface expression of CD5. Conclusion. Anti-CD5 has an inhibitory effect on in vivo—activated synovial fluid T cells. The disease-ameliorative effects of anti-CD5 immunotoxin treatment of RA may be partly due to “switching-off” of T cell activation in the joints.     

Persistent loss of IL-27 responsiveness in CD8+ memory T cells abrogates IL-10 expression in a recall response

CD8+ T cells are central to the eradication of intracellular pathogens, but they can also act to limit inflammation and immunopathology. During primary respiratory viral infection CD8+ effector T cells release the immunosuppressive cytokine IL-10, which is essential for host survival. Here we report that CD8+ T-cell–derived IL-10 is absent in a recall response. We show in mice that the lack of IL-10 is due to a persistent loss of IL-27 responsiveness in CD8+ memory T cells, caused by down-regulation of the common cytokine receptor, glycoprotein 130. CD8+ memory T cells secreted less IL-10 when activated in the presence of IL-27 than did naïve controls, and retroviral expression of glycoprotein 130 restored IL-10 and reduced IFN-γ production upon restimulation. We demonstrate that human CD8+ memory cells are also characterized by impaired IL-27 responsiveness. Our data suggest that CD8+ T-cell activation involves a persistent loss of specific cytokine receptors that determines the functional potential of these cells during rechallenge infection.     

CD4+ T cells support cytotoxic T lymphocyte priming by controlling lymph node input

Rapid induction of CD8⁺ cytotoxic T lymphocyte (CTL) responses is critical to combat acute infection with intracellular pathogens. CD4⁺ T cells help prime antigen-specific CTLs in secondary lymphoid organs after infection in the periphery. Although the frequency of naïve precursors is very low, the immune system is able to efficiently screen for cognate CTLs through mechanisms that are not well understood. Here we examine the role of CD4⁺ T cells in early phases of the immune response. We show that CD4⁺ T cells help optimal CTL expansion by facilitating entry of naïve polyclonal CD8⁺ T cells into the draining lymph node (dLN) early after infection or immunization. CD4⁺ T cells also facilitate input of naïve B cells into reactive LNs. Such “help” involves expansion of the arteriole feeding the dLN and enlargement of the dLN through activation of dendritic cells. In an antigen- and CD40-dependent manner, CD4⁺ T cells activate dendritic cells to support naïve lymphocyte recruitment to the dLN. Our results reveal a previously unappreciated mode of CD4⁺ T-cell help, whereby they increase the input of naïve lymphocytes to the relevant LN for efficient screening of cognate CD8⁺ T cells.