Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.     

Heterogeneity of CD4+ memory T cells: Functional modules for tailored immunity

Phenotypic and functional heterogeneity is the hallmark of effector and memory T cells. Upon antigenic stimulation, naïve CD4⁺ T cells make choices to become effector Th1, Th2 or Th17 cells, or even Treg. In addition to differences in cytokine repertoire, effector CD4⁺ T cells exhibit diversity in homing, such as migration to lymph node follicles to help B cells versus migration to inflamed tissues. Upon clearance of the antigen, two major types of memory T cells remain: central memory cells, which patrol lymphoid organs, and effector memory cells that act as sentinels in peripheral tissues such as the skin and the gut. Here, we review our current understanding of CD4⁺ T‐cell lineage heterogeneity and flexibility, with emphasis on the human system, and propose an organization of effector and memory T cells based on distinct functional modules.     

Differential requirements of CD4+ T‐cell signals for effector cytotoxic T‐lymphocyte (CTL) priming and functional memory CTL development at higher CD8+ T‐cell precursor frequency

Increased CD8⁺ T‐cell precursor frequency (PF) precludes the requirement of CD4⁺ helper T (Th) cells for primary CD8⁺ cytotoxic T‐lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) ‐pulsed dendritic cells (DC_OVA) derived from C57BL/6, CD40 knockout (CD40^?/?) or CD40 ligand knockout (CD40L^?/?) mice were used to immunize C57BL/6, Ia^b?/?, CD40^?/? or CD40L^?/? mice, whose PF was previously increased with transfer of 1 × 10⁶ CD8⁺ T cells derived from OVA‐specific T‐cell receptor (TCR) transgenic OTI, OTI(CD40^?/?) or OTI(CD40L^?/?) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4⁺ T‐cell help and Th‐provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA‐expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4⁺ T‐cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4⁺ T‐cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4⁺ T‐cell functions.     

Retention of Ag‐specific memory CD4+ T cells in the draining lymph node indicates lymphoid tissue resident memory populations

Several different memory T‐cell populations have now been described based upon surface receptor expression and migratory capabilities. Here we have assessed murine endogenous memory CD4⁺ T cells generated within a draining lymph node and their subsequent migration to other secondary lymphoid tissues. Having established a model response targeting a specific peripheral lymph node, we temporally labelled all the cells within draining lymph node using photoconversion. Tracking of photoconverted and non‐photoconverted Ag‐specific CD4⁺ T cells revealed the rapid establishment of a circulating memory population in all lymph nodes within days of immunisation. Strikingly, a resident memory CD4⁺ T cell population became established in the draining lymph node and persisted for several months in the absence of detectable migration to other lymphoid tissue. These cells most closely resembled effector memory T cells, usually associated with circulation through non‐lymphoid tissue, but here, these cells were retained in the draining lymph node. These data indicate that lymphoid tissue resident memory CD4⁺ T‐cell populations are generated in peripheral lymph nodes following immunisation.     

Monitoring the efficacy of dendritic cell vaccination by early detection of 99mTc‐HMPAO‐labelled CD4+ T cells

DC vaccines have been used to induce tumour‐specific cytotoxic T cells 1 . However, this approach to cancer immunotherapy has had limited success. To be successful, injected DCs need to migrate to the LNs where they can stimulate effector T cells 1 . We and others have previously demonstrated by MRI that tumour antigen‐pulsed‐DCs labelled ex vivo with superparamagnetic iron oxide nanoparticles migrated to the draining LNs and are capable of activating antigen‐specific T cells 2 , 3 . The results from our study demonstrated that ex vivo superparamagnetic iron oxide nanoparticles‐labelled and OVA‐pulsed DCs prime cytotoxic CD8⁺ T‐cell responses to protect against a B16‐OVA tumour challenge. In the clinic, a possible noninvasive surrogate marker for efficacy of DC vaccination is to image the specific migration and accumulation of T cells following DC vaccination.     

CD4 T cell help is required for primary CD8 T cell responses to vesicular antigen delivered to dendritic cells in vivo

Insight into the mechanisms by which dendritic cells (DC) present exogenous antigen to T cells is of major importance in the design of vaccines. We examined the effectiveness of free antigen as well as antigen with lipopolysaccharide, emulsified in complete Freund's adjuvant, and antigen encapsulated in liposomes in activating adoptively transferred antigen-specific CD4 and CD8 T cells. When contained in liposomes, 100- to 1000-fold lower antigen amounts were as efficient in inducing proliferation and effector functions of CD4 and CD8 T cells in draining lymph nodes as other antigen forms. CD11c(+)/CD11b(+)/CD205(mod)/CD8alpha(-) DC that captured liposomes were activated and presented this form of antigen in an MHC class I- and class II-restricted manner. CD4 T cells differentiated into Th1 and Th2 effector cells. Primary expansion and cytotoxic activity of CD8 T cells were CD4 T cell-dependent and required the transporter associated with antigen processing (TAP). Finally, adoptively transferred CD4 and CD8 T cells were not deleted after primary immunization and rapidly responded to a secondary immunization with antigen-containing liposomes. In conclusion, encapsulation of antigen in liposomes is an efficient way of delivering antigen to DC for priming of both CD4 and CD8 T cell responses. Importantly, primary CD8 T cell responses were CD4 T cell-dependent.     

In vivo depletion of DC impairs the anti‐tumor effect of agonistic anti‐CD137 mAb

Anti‐CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti‐tumor effect involves co‐stimulation of tumor‐specific CD8⁺ T cells. Whether antigen cross‐presenting DC are required for the efficacy of anti‐CD137 mAb treatment has never been examined. Here we show that the administration of anti‐CD137 mAb eradicates EG7‐OVA tumors by a strictly CD8β⁺ T‐cell‐dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross‐presentation revealed that CD11c⁺ cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor‐draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti‐OVA CTL induction. Using CD11c diphtheria toxin receptor‐green fluorescent protein→C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross‐presentation in CTL induction against OVA_257–264 epitope and in the antitumor efficacy induced by anti‐CD137 mAb.     

Induction of Th1 and Th2 CD4+ T cell responses by oral or parenteral immunization with ISCOMS

We examined the ability of oral or parenteral immunization with immune stimulating complexes containing ovalbumin (ISCOMS-OVA) to prime T cell proliferative and cytokine responses. A single subcutaneous immunization with ISCOMS-OVA primed potent antigen-specific proliferative responses in the draining popliteal lymph node, which were entirely dependent on the presence of CD4⁺ T cells. CD8⁺ T cells did not proliferate in vitro even in the presence of the appropriate peptide epitope and exogenous interleukin (IL)-2. Primed popliteal lymph node cells produced IL-2, IL-5 and interferon (IFN)-γ, but not IL-4 when restimulated with OVA in vitro. Serum antigen-specific IgG1 and IgG2a antibody responses were also primed by subcutaneous immunization with ISCOMS-OVA, confirming the stimulation of both Th1 and Th2 cells in vivo. Spleen cells from subcutaneously primed mice produced a similar pattern of cytokines, indicating that disseminated priming had occurred. Oral immunization with ISCOMS-OVA also primed local antigen-specific proliferative responses in the mesenteric lymph node and primed an identical pattern of systemic cytokine responses in the spleen. The ability of ISCOMS to prime both Th1 and Th2 CD4⁺ T cell responses may be central to their potent adjuvant activities and confirm the potential of ISCOMS as future oral vaccine vectors.     

Th1 and Th2 cells help CD8 T-cell responses

Help from CD4 T cells is often important for the establishment of primary and memory CD8 T-cell responses. However, it has yet to be determined whether T helper polarization affects the delivery of help and/or whether responding CD8 T cells helped by Th1 or Th2 cells express distinct effector properties. To address these issues, we compared CD8 T-cell responses in the context of Th1 or Th2 help by injecting dendritic cells copulsed with the major histocompatibility complex class I-restricted OVA peptide plus, respectively, bacterial or helminth antigens. We found that Th2 cells, like Th1 cells, can help primary and long-lived memory CD8 T-cell responses. Experiments in interleukin-12 (IL-12)-/- and IL-4-/- mice, in which polarized Th1 or Th2 responses, respectively, fail to develop, indicate that the underlying basis of CD4 help is independent of attributes acquired as a response to polarization.     

Antigen Conjugated to Anti‐CD23 Antibodies is Rapidly Transported to Splenic Follicles by Recirculating B Cells

IgE–antigen complexes, administered intravenously to mice, induce a several 100‐fold higher specific antibody response than antigen alone. Additionally, in vivo activation and proliferation of specific CD4⁺ T cells is enhanced. The mechanism behind these effects is thought to be that peripheral B cells capture IgE–antigen complexes via their low‐affinity receptor for IgE, CD23, and rapidly transport them to splenic B cell follicles where an immune response is initiated. Here, we demonstrate that ovalbumin, covalently coupled to anti‐CD23 antibodies and administered intravenously to mice, is also transported to splenic follicles and induces an enhanced primary antibody response. These effects are absent in CD23‐deficient mice. No enhanced induction of immunological memory was observed. These findings extend previous observations regarding the in vivo role of CD23 and emphasize that recirculating B cells play an important role in antigen transport to the spleen.     

Dendritic cells activated by an anti‐inflammatory agent induce CD4+ T helper type 2 responses without impairing CD8+ memory and effector cytotoxic T‐lymphocyte responses

Prevalence of pro‐inflammatory diseases is rising in developed country populations. The increase in these diseases has fuelled the search for new, immune suppressive, anti‐inflammatory therapies, which do not impact, or minimally impact, CD4⁺ and/or CD8⁺ T‐cell‐mediated immunity. The goal of this study was to determine if antigen‐presenting cells (APCs) activated by the anti‐inflammatory oligosaccharide, lacto‐N‐fucopentaose III (LNFPIII), would have an impaired ability to drive CD4⁺ T helper (Th) or CD8⁺ memory and effector T‐cell responses. To investigate this we activated splenic dendritic cells (SDCs) with LNFPIII and examined their ability to drive antigen‐specific CD4⁺ Th, and CD8⁺ memory and cytotoxic T‐cell (CTL) responses compared with lipopolysaccharide (LPS) ‐stimulated SDCs. The LNFPIII‐activated SDCs had altered co‐stimulatory molecule expression compared with LPS‐stimulated SDCs, while the levels of SDC chemokines following activation by either compound were similar. LNFPIII‐activated SDCs produced significantly lower levels of interleukin‐12 but surprisingly higher levels of interleukin‐6 than LPS‐activated SDCs. Similar to previous studies using bone‐marrow‐derived DCs, LNFPIII‐activated SDCs induced strong Th2 responses in vivo and ex vivo. LNFPIII activation of APCs was independent of the Toll‐interleukin‐1 receptor adaptor myeloid differentiating factor 88. Importantly, LNFPIII‐matured DCs induced CD8⁺ memory and effector CTL responses similar to those driven by LPS‐matured DCs, including the frequency of interferon‐γ‐producing CD8⁺ T cells and induction of CTL effectors. Treatment of APCs by the anti‐inflammatory glycan LNFPIII did not impair their ability to drive CD8⁺ effector and memory cell‐mediated immunity.     

CXCR5+ CCR7- CD8 T cells are early effector memory cells that infiltrate tonsil B cell follicles

Naive and central memory CD8 T cells use CCR7 to recirculate through T cell zones of secondary lymphoid organs where they can encounter antigen. Here we describe a subset of human CD8 T cells expressing CXCR5 which enables homing in response to CXCL13 produced within B cell follicles. CXCR5+ CD8 T cells were found in tonsil B cell follicles, and isolated cells migrated towards CXCL13 in vitro. They expressed CD27, CD28, CD45RO, CD69, and were CD7low, and produced IFN-gamma and granzyme A but lacked perforin, a functional profile suggesting that these cells are early effector memory cells in the context of contemporary T cell differentiation models. Receptors important in the interaction with B cells, including CD70, OX40 and ICOS, were induced upon activation, and CXCR5+ CD8 T cells could to some extent support survival and IgG production in tonsil B cells. Furthermore, CXCR5+ CD8 T cells expressed CCR5 but no CCR7, suggesting a migration pattern distinct from that of follicular CD4 T cells. The finding that a subset of early effector memory CD8 T cells use CXCR5 to locate to B cell follicles indicates that MHC class I-restricted CD8 T cells are part of the follicular T cell population.     

Naive Tumour‐Specific CD4+ T Cells were Efficiently Primed in Acute Lymphoblastic Leukaemia

The recognition and neutralization of tumour cells is one of the big challenges in immunity. The immune system has to recognize syngeneic tumour cells and has to be primed and respond in an adequate manner. Priming of a leukaemia‐specific immune response is a crucial step in tumour immunology that can mislead to tumour tolerance either by T cell ignorance, deletion or Treg induction. To resemble the situation of acute lymphoblastic leukaemia (ALL) in patients, we used the murine BALB/c model with syngeneic BM185 tumour cells. We established a tumour cell line that expresses the neo‐antigen ovalbumin (BM185‐OVA/GFP) to allow the application of T cell receptor transgenic, antigen‐specific CD4⁺ T cells. Here, we demonstrate that effective anti‐ALL immunity can be established by in vivo priming of CD4⁺ T cells that is sufficient to differentiate into effector cells. Yet they failed to control tumour alone, but initiated a Th1 response. An efficient tumour clearance was dependent on both antigen‐specific CD4⁺ T cells and CD8⁺ effector T cells from the endogenous repertoire. The tolerogeneic milieu was characterized by increased Tregs numbers and elevated IL‐10 level. Tregs hamper effective antitumour immune response, but their depletion did not result in reduced tumour growth. In contrast, neutralization of IL‐10 improved median mouse survival. Future therapies should focus on establishing a strong CD4+ T cells response, either by adjuvant or by adoptive transfer.     

Transient Treg‐cell depletion in adult mice results in persistent self‐reactive CD4+ T‐cell responses

Depletion of Foxp3⁺CD4⁺ regulatory T cells (Treg) in adults results in chronic inflammation and autoimmune disease. However, the impact of transient Treg‐cell depletion on self‐reactive responses is poorly defined. Here, we studied the effect of transient depletion of Treg cells on CD4⁺ T‐cell responses to endogenous self‐antigens. Short‐term ablation of Treg cells in mice resulted in rapid activation of CD4⁺ T cells, increased percentage of IFN‐γ⁺ and Th17 cells in lymphoid organs, and development of autoimmune gastritis. To track self‐reactive responses, we analyzed the activation of naïve gastric‐specific CD4⁺ T cells. There was a dramatic increase in proliferation and acquisition of effector function of gastric‐specific T cells in the stomach draining LNs of Treg‐cell‐depleted mice, compared with untreated mice, either during Treg‐cell depletion or after Treg‐cell reconstitution. Moreover, the hyperproliferation of gastric‐specific T cells in the Treg‐cell‐ablated mice was predominantly antigen‐dependent. Transient depletion of Treg cells resulted in a shift in the ratio of peripheral:thymic Treg cells in the reemerged Treg‐cell population, indicating an altered composition of Treg cells. These findings indicate that transient Treg‐cell depletion results in ongoing antigen‐driven self‐reactive T‐cell responses and emphasize the continual requirement for an intact Treg‐cell population.     

抗原特异性初始CD4+T细胞的体内分化及特性

为了探讨抗原特异性CD4+T细胞在体内的分裂、表型、Th1细胞因子的产生和组织器官的分布。将CFSE标记的抗原特异性初始CD4+T细胞静脉被动输给小鼠后,进行免疫,3d后处死小鼠取其脾脏、淋巴结和肺组织,分离单个核细胞,利用流式细胞计数仪在单个细胞水平上,观察细胞的分裂、表型、Th1细胞因子的产生和组织分布。结果显示在没有抗原刺激的情况下,未见初始CD4+T细胞分裂,其主要分布于淋巴结和脾脏。当受到抗原刺激后,CD4+T细胞分裂1～5次,主要分布于脾脏和肺组织,CD25的表达增加,CD62L的表达随着细胞分裂次数的增加而减少。IL-12促进CD25的表达和细胞的分裂。促进Th1细胞的分化和IFN-γ的表达。研究的结果提示,在体内,当CD4+T细胞活化后,主要分布于脾和非淋巴组织发挥其免疫效应。     

Pathways of memory CD8+ T‐cell development

CD8⁺ T‐cell responses must have at least two components, a replicative cell type that proliferates in the secondary lymphoid tissue and that is responsible for clonal expansion, and cytotoxic cells with effector functions that mediate the resolution of the infection in the peripheral tissues. To confer memory, the response must also generate replication‐competent T cells that persist in the absence of antigen after the primary infection is cleared. The current models of memory differentiation differ in regards to whether or not memory CD8⁺ T cells acquire effector functions during their development. In this review we discuss the existing models for memory development and the consequences that the recent finding that memory CD8⁺ T cells may express granzyme B during their development has for them. We propose that memory CD8⁺ T cells represent a self‐renewing population of T cells that may acquire effector functions but that do not lose the naïve‐like attributes of lymphoid homing, antigen‐independent persistence or the capacity for self‐renewal.     

Antigen-bearing dendritic cells from the sublingual mucosa recirculate to distant systemic lymphoid organs to prime mucosal CD8 T cells

Effector T cells are described to be primed in the lymph nodes draining the site of immunization and to recirculate to effector sites. Sublingual immunization generates effector T cells able to disseminate to the genital tract. Herein, we report an alternative mechanism that involves the recirculation of antigen-bearing dendritic cells (DCs) in remote lymphoid organs to prime T cells. Sublingual immunization with a muco-adhesive model antigen unable to diffuse through lymphatic or blood vessels induced genital CD8 T cells. The sublingual draining lymph nodes were not mandatory to generate these lymphocytes, and antigen-bearing DCs from distant lymph nodes and spleen were able to prime specific CD8 T cells in a time- and dose-dependent manner. This study demonstrates, for the first time, that antigen-bearing DCs originating from the site of immunization recirculate to distant lymphoid organs and provides insights into the mechanism of distant CD8 T-cell generation by sublingual immunization.     

ICOS is required for the generation of both central and effector CD4+ memory T‐cell populations following acute bacterial infection

Interactions between ICOS and ICOS ligand (ICOSL) are essential for the development of T follicular helper (Tfh) cells and thus the formation and maintenance of GC reactions. Given the conflicting reports on the requirement of other CD4⁺ T‐cell populations for ICOS signals, we have employed a range of in vivo approaches to dissect requirements for ICOS signals in mice during an endogenous CD4⁺ T‐cell response and contrasted this with CD28 signals. Genetic absence of ICOSL only modestly reduced the total number of antigen‐specific CD4⁺ T cells at the peak of the primary response, but resulted in a severely diminished number of both T central memory and T effector memory cells. Treatment with blocking anti‐ICOS mAb during the primary response recapitulated these effects and caused a more substantial reduction than blocking CD28 signals with CTLA4Ig. During the memory phase of the response further signals through ICOS or CD28 were not required for survival. However, upon secondary challenge only Tfh cell expansion remained heavily ICOS‐dependent, while CD28 signals were required for optimal expansion of all subsets. These data demonstrate the importance of ICOS signals specifically for memory CD4⁺ T‐cell formation, while highlighting the potential of therapeutically targeting this pathway.     

Nano‐particle vaccination combined with TLR‐7 and ‐9 ligands triggers memory and effector CD8+ T‐cell responses in melanoma patients

Optimal vaccine strategies must be identified for improving T‐cell vaccination against infectious and malignant diseases. MelQbG10 is a virus‐like nano‐particle loaded with A‐type CpG‐oligonucleotides (CpG‐ODN) and coupled to peptide_16–35 derived from Melan‐A/MART‐1. In this phase IIa clinical study, four groups of stage III‐IV melanoma patients were vaccinated with MelQbG10, given (i) with IFA (Montanide) s.c.; (ii) with IFA s.c. and topical Imiquimod; (iii) i.d. with topical Imiquimod; or (iv) as intralymph node injection. In total, 16/21 (76%) patients generated ex vivo detectable Melan‐A/MART‐1‐specific T‐cell responses. T‐cell frequencies were significantly higher when IFA was used as adjuvant, resulting in detectable T‐cell responses in all (11/11) patients, with predominant generation of effector‐memory‐phenotype cells. In turn, Imiquimod induced higher proportions of central‐memory‐phenotype cells and increased percentages of CD127⁺ (IL‐7R) T cells. Direct injection of MelQbG10 into lymph nodes resulted in lower T‐cell frequencies, associated with lower proportions of memory and effector‐phenotype T cells. Swelling of vaccine site draining lymph nodes, and increased glucose uptake at PET/CT was observed in 13/15 (87%) of evaluable patients, reflecting vaccine triggered immune reactions in lymph nodes. We conclude that the simultaneous use of both Imiquimod and CpG‐ODN induced combined memory and effector CD8⁺ T‐cell responses.