Antigen-specific CD4 T cell clonal expansion and differentiation in the aged lymphoid microenvironment. II. The memory T cell response is diminished

We investigated the ability of the aged host environment to support a memory CD4 T cell response to a secondary immunization with a specific antigen. Using an adoptive transfer model, we previously reported that young CD4 T cells, transferred into the young or the aged lymphoid microenvironment, clonally expand and differentiate in a similar fashion after primary immunization with a specific antigen. In this report, we have monitored the clonal expansion of the donor-derived memory CD4 T population following a secondary challenge with the specific antigen. We show that antigen-specific memory T cell clonal expansion is diminished in the aged hosts. However, upon in vitro re-stimulation, antigen-specific CD4 T cells isolated from young and from aged hosts were equally responsive to antigen, thus indicating an inhibitory effect in the aged host environment. We provide evidence that factors specifically present in previously immunized aged hosts must be responsible for the diminished response of otherwise functional, antigen-specific memory CD4 T cells. Our results suggest that the decline in immunologic memory to foreign antigens may be due not only to intrinsic defects in aged T cells, but also to age-related differences in the lymphoid microenvironment of previously immunized hosts.     

The role of B cell antigen presentation in the initiation of CD4+ T cell response

B cells have been known for their ability to present antigens to T cells for almost 40 years. However, the precise roles of B cell antigen presentation in various immune responses are not completely understood. The term “professional” antigen-presenting cells (APCs) was proposed to distinguish APCs that are required for initiating the immune responses from those use antigen presentation to enhance their own effector functions. Unlike dendritic cells, which are defined as professional APCs for their well-established functions in activating naive T cells, B cells have been shown in the past to mostly present antigens to activated CD4+ T cells mainly to seek help from T helper cells. However, recent evidence suggested that B cells can act as professional APCs under infectious conditions or conditions mimicking viral infections. B cell antigen receptors (BCRs) and the innate receptor Toll-like receptors are activated synergistically in response to pathogens or virus-like particles, under which conditions B cells are not only potent but also the predominant APCs to turn naive CD4+ T cells into T follicular helper cells. The discovery of B cells as professional APCs to initiate CD4+ T cell response provides a new insight for both autoimmune diseases and vaccine development.     

乙肝疫苗无应答者CD4~+T细胞TCR Vβ基因克隆化特征的研究

目的:研究乙肝疫苗接种者CD4+T细胞TCR Vβ基因克隆化特征,分析乙肝疫苗有应答者和无应答者TCR Vβ基因单克隆改变的差异性。方法:采用多引物PCR技术扩增80例乙肝疫苗接种者CD4+T细胞TCR Vβ基因22个家族的CDR3区基因片段,对PCR产物分别应用琼脂糖凝胶电泳和基因扫描两种方法检测。结果:80例乙肝疫苗接种者中有58例产生抗体,有22例未产生抗体。TCR Vβ基因单克隆改变主要集中在Vβ2、Vβ8、Vβ9、Vβ11和Vβ17五个家族上,乙肝疫苗无应答组这五个Vβ基因单克隆改变频率明显低于乙肝疫苗有应答组(P<0.01或P<0.05)。结论:CD4+T细胞TCR Vβ基因克隆化改变是影响机体对乙肝疫苗免疫应答效果的重要因素之一。     

Homotypic interaction of the heat-stable antigen is not responsible for its co-stimulatory activity for T cell clonal expansion

The heat-stable antigen (HSA) is an important co-stimulatory molecule on antigen-presenting cells (APC). However, the receptor on T cells that receives the co-stimulatory signal from HSA has not been identified. Because the HSA is transiently expressed on T cells after the T cell receptor/CD3 complex is engaged, and because it can bind to itself in a homotypic fashion, it has been proposed that homotypic interaction of HSA is responsible for its co-stimulatory activity. Here we test this hypothesis using mice that have a targeted mutation of the HSA gene, as well as novel transgenic mice that constitutively express HSA on T cells. We show that HSA-deficient T cells remain responsive to co-stimulation by HSA. Furthermore, constitutive expression of HSA does not enhance T cell response to co-stimulatory by HSA. Taken together, our results demonstrate that homotypic interaction of HSA is not responsible for co-stimulation mediated by HSA expressed on APC.     

CD40 expression in renal cell carcinoma is associated with tumor apoptosis,CD8 T cell frequency and patient survival

The co-stimulatory molecule, CD40, is expressed in renal cell carcinoma (RCC) and a variety of inflammatory diseases in the kidney. We investigated the relationship between tumor-associated CD40 expression, immune milieu of the tumor microenvironment, tumor stage and survival of patients with RCC. The expression of CD40, TUNEL and CD8 in human renal cell carcinomas was analyzed by immunohistochemistry performed on tissue samples obtained at the time of surgery. Computer-assisted quantitation of protein expression was used to analyze results in connection with patient survival and tumor stage. We show for the first time that tumor-associated CD40 expression is associated with prolonged survival in RCC patients. Tumor apoptosis (TUNEL) and CD8 immunostaining were also associated with patient survival. No relation was observed between CD40 expression and tumor stage. Our results suggest CD40 may be a prognostic biomarker indicative of prolonged RCC patient survival. Strategies that up-regulate CD40 expression in some RCC patients may thus improve survival, supporting further studies of agonistic CD40 antibodies in RCC.     

Altered naive CD4 and CD8 T cell homeostasis in patients with relapsing-remitting multiple sclerosis: thymic versus peripheral (non-thymic) mechanisms

We have reported previously that naive T cells from relapsing-remitting multiple sclerosis (RRMS) patients have T cell receptor (TCR) repertoire shifts, but the basis of these TCR repertoire shifts was uncertain. Here, we questioned whether RRMS patients have altered naive CD4 and CD8 T cell homeostasis by studying homeostatic proliferation and thymic production in RRMS patients and healthy controls. We measured thymic production by quantifying signal joint T cell receptor excision circles (sjTRECs). Both naive T subsets from controls showed an age-associated decrease in sjTRECs, i.e. evidence of progressive thymic involution, but we detected no age-associated decrease in sjTRECs in RRMS patients. Instead, naive CD8 T cells from patients had lower sjTRECs (P = 0.012) and higher Ki-67 proliferation levels (P = 0.04) than controls. Naive CD4 T cell sjTRECs did not differ between patients and controls. However, in RRMS these sjTRECs correlated strongly with CD31, a marker expressed by newly generated CD4 T cells but not by naive CD4 T cells that have undergone homeostatic proliferation. HLA-DR2 positivity correlated negatively with naive CD4 T cell CD31 expression in RRMS (P = 0.002). We conclude in RRMS that naive T subsets have homeostatic abnormalities due probably to peripheral (non-thymic) mechanisms. These abnormalities could have relevance for MS pathogenesis, as naive T cell changes may precede MS onset.     

Low-dose antigen-experienced CD4+ T cells display reduced clonal expansion but facilitate an effective memory pool in response to secondary exposure

The strength and duration of an antigenic signal at the time of initial stimulation were assumed to affect the development and response of effectors and memory cells to secondary stimulation with the same antigen. To test this assumption, we used T-cell receptor (TCR)-transgenic CD4⁺ T cells that were stimulated in vitro with various antigen doses. The primary effector CD4⁺ T cells generated in response to low-dose antigen in vitro exhibited reduced clonal expansion upon secondary antigenic exposure after adoptive transfer to hosts. However, the magnitude of their contraction was much smaller than both those generated by high-dose antigen stimulation and by naïve CD4⁺ T cells, resulting in higher numbers of antigen-specific CD4⁺ T cells remaining until the memory stage. Moreover, secondary effectors and memory cells developed by secondary antigen exposure were not functionally impaired. In hosts given the low-dose antigen-experienced CD4⁺ T cells, we also observed accelerated recall responses upon injection of antigen-bearing antigen-presenting cells. These results suggest that primary TCR stimulation is important for developing optimal effectors during initial antigen exposure to confer long-lasting memory CD4⁺ T cells in response to secondary exposure.     

Impaired in vivo CD4+ T cell expansion and differentiation in aged mice is not solely due to T cell defects: decreased stimulation by aged dendritic cells

CD4+ T cells regulate humoral and cell-mediated immune responses, which are progressively impaired in aging, resulting in susceptibility to infections and cancer. Dendritic cells (DCs) are major activators of T cells, providing signals that drive their expansion and differentiation. In this study, we asked if decreased CD4+ T cell responses were influenced by the age of DCs rather than being exclusively due to T cell defects. Old T cells transferred to young recipients expanded and differentiated similarly to young T cells. However, aged recipients were poor stimulators of both old and young T cells, which failed to acquire CD44 expression and produce interferon gamma (IFN-γ). DCs in aged hosts expressed fewer MHC-peptide complexes. The CD86 expression in the DCs of both hosts was similar; however, CD40 levels were reduced in old DCs. Finally, old DCs failed to produce inflammatory cytokines in response to LPS. Our results indicate that the impairment of aged CD4+ T cell function is intimately related to multiple alterations in aged DCs, rather than being caused solely by intrinsic T cell defects, suggesting that the function of aged T cells may be partially rescued in vivo when appropriate stimulation is applied. These findings are relevant to vaccination design for elderly populations.     

Evidence for a selective and multi-step model of T cell differentiation: CD4+CD8low thymocytes selected by a transgenic T cell receptor on major histocompatibility complex class I molecules

We have characterized a prominent (15-20 %) thymocyte population expressing CD4 at a high and CD8 at a low level “CD4⁺8^lo” in mice transgenic for a T cell receptor “TCR” restricted by major histocompatibility complex “MHC” class I molecules. The results demonstrate that the CD4⁺8^lo population is an intermediate stage between immature CD4⁺8⁺ and end-stage CD4⁺8^- thymocytes and that the survival of these cells crucially depends on the successful interaction of the transgenic TCR with self MHC class I molecules. In addition we demonstrate that the avidity of the interaction between TCR and self MHC class I molecules determines whether CD4⁺8^lo thymocytes are found in significant numbers in this transgenic model. Our findings support a selective and multi-step model of T cell differentiation in the thymus.     

T cell receptor repertoire and mitotic responses of lamina propria T lymphocytes in inflammatory bowel disease.

Human intestinal lamina propria T lymphocytes (LPL-T) physiologically exhibit minimal proliferation in response to antigen receptor stimulation in vitro. This is thought to occur as a consequence of regulatory influences which are exerted by the mucosal microenvironment. The present study is aimed at investigating whether proliferative responses of intestinal LPL-T to antigen receptor stimulation are altered in patients with inflammatory bowel disease. Accordingly, proliferative responses of LPL-T in patients with Crohn's disease and ulcerative colitis to stimulation with CD3 MoAb plus IL-2 were examined and compared with controls. In addition, T cell receptor (TCR) repertoires of LPL-T and peripheral blood T lymphocytes were determined by indirect immunofluorescence using a panel of 11 TCR V beta specific antibodies. In most patients with inflammatory bowel disease, LPL-T showed enhanced proliferation to antigen receptor stimulation compared with controls. Moreover, perhaps as a consequence, an enhanced frequency of in vivo preactivated T cells was seen as judged from an increased spontaneous proliferative response to low concentrations of exogenous IL-2. LPL-T and peripheral blood T lymphocytes exhibited similar percentages of TCR V beta gene usage both in controls and in patients. In summary, polyclonal activation of LPL-T due to impairment of local adjustment, i.e. insufficient down-regulation of TCR/CD3-dependent signalling processes, may contribute to the pathogenesis of inflammatory bowel disease.     

Antigen-specific CD8+ T cell subset distribution in lymph nodes draining the site of herpes simplex virus infection

Inoculation with replicating virus leads to an increase in T cell numbers within lymph nodes that drain the site of infection. This increase has been associated with a nonspecific proliferation of bystander cells, with only a minority thought to be directed to the infectious agent. Such an assumption is largely based on precursor cytotoxic T lymphocyte (CTL) estimations using limiting dilution analysis. Recently, studies using more advanced molecular approaches have suggested that such functionally derived precursor frequencies considerably underestimate the proportion of T cells specific for the antigen under investigation. We have defined T cell receptor sequences characteristic of CTL populations directed to a dominant determinant of the herpes simplex virus (HSV) glycoprotein B (gB). In this investigation, we used this receptor signature as a probe to directly monitor changes occurring within lymph nodes draining the sites of active infection with HSV. We found that although lymph node CD8⁺ T cell numbers increase as a consequence of HSV infection, the majority of these cells are small resting cells that are not enriched for gB-specific receptors. In contrast, a significant proportion of activated T cells are highly enriched for CTL bearing gB-specific receptors. Our results are therefore consistent with a nonspecific migration of CTL precursors into the lymph nodes draining the site of infection, followed by the activation and proliferation of the antigen-specific subset that normally makes up a small proportion of the naive T cell repertoire.     

T cell receptor (TCR) BV gene repertoires and clonal expansions of CD4 cells in patients with HIV infections

Despite extensive investigation, the pathogenesis of T cell depletions that characterize AIDS has not been elucidated. To study this process further, we evaluated T cell antigen receptor β-chain variable gene (TCRBV) repertoires in peripheral blood lymphocytes (PBL) of 23 HIV-infected patients. Expression levels of 28 TCRBV were determined by multiprobe RNase protection assay after polymerase chain reaction (PCR) amplifications. Abnormal expansions (>2 s.d. from mean normal values) were frequent in HIV CD4, accounting for 26% of total measured TCRBV in this population. The number and magnitude of abnormalities among individuals were inversely proportional to their CD4 counts (P<0.012 and P<0.01, respectively). While abnormalities were not randomly distributed among TCRBV subfamilies, no particular genes were expanded or contracted among all patients. Only 14% of CD8 TCRBV were proportionally expanded (P<0.01 compared with CD4), and there were limited concordances between paired CD8 and CD4 repertoires among individuals. CDR3 length analyses and TCRBV sequencing showed that most CD4 expansions comprised clonal or oligoclonal populations. Thus, T cell responses in HIV patients are characterized by severe TCRBV biases and clonal expansions among CD4 subsets, and these processes are exaggerated with disease progression. The heterogeneity and oligoclonality of the TCRBV expansions are consistent with responses to HIV-encoded or other conventional antigens rather than superantigenic effects. The presence of CD4 clonal proliferations in these patients may be important in the pathogenesis of HIV, and the absence or reduction of many T cell specificities due to oligoclonal expansions may increase susceptibility to opportunistic infections.     

Differential response of CD4+ V7+ and CD4+ V7− T cells to T cell receptor-dependent signals: CD4+ V7+ T cells are co-stimulation independent and anti-V7 antibody blocks the induction of anergy by bacterial superantigen

V7 is a novel cell surface glycoprotein that is expressed on 25% of circulating T lymphocytes. This molecule appears to play a critical role in T cell activation based on the observation that a monoclonal anti-V7 antibody inhibits T cell receptor (TCR)-dependent interleukin-2 (IL-2) production and proliferation of T cells. In the current study, CD4⁺ V7+ and CD4⁺ V7? T cells were separated from one another and their response to various stimuli analyzed. Although there were only minor differences between the two subsets in the expression of activation/differentiation markers, including CD45RA and R0 isotypes, when exposed to immobilized anti-CD3 or anti-TCR antibodies in the absence of APC, CD4⁺ V7+ T cells alone produced IL-2 and proliferated vigorously. By contrast, CD4⁺ V7? cells responded poorly to such stimuli, but they recovered their capacity to respond if antigen-presenting cells (APC) or anti-CD28 antibody were added to the cultures. The enhancement of the V7? T cell response by APC appears to be related to augmentation of TCR signals because the effect could be blocked by antibodies against molecules on APC [major histocompatibility (MHC) class II, CD86] that are known to up-regulate such signals through their interaction with counter-receptors on T cells. To assess the role of V7 in a system independent of co-stimulation, CD4⁺ T cells were stimulated with the bacterial superantigens, staphylococcal enterotoxins A and B. The cells responded by proliferating and then becoming anergic. Addition of anti-V7 antibody at the initiation of culture with superantigen did not inhibit cellular proliferation but prevented T cells from becoming anergic, while addition of anti-CD28 antibody had no effect on either proliferation or anergy induction. These results indicate that V7 and CD28 mediate distinct intracellular signals and suggest that V7 functions to preserve T cell reactivity whether the stimulus is mitogenic or anergizing.     

CD4+ T helper 2 cells--microbial triggers, differentiation requirements and effector functions

Over the past 10 years we have made great strides in our understanding of T helper cell differentiation, expansion and effector functions. Within the context of T helper type 2 (Th2) cell development, novel innate‐like cells with the capacity to secrete large amounts of interleukin‐5 (IL‐5), IL‐13 and IL‐9 as well as IL‐4‐producing and antigen‐processing basophils have (re)‐emerged onto the type 2 scene. To what extent these new players influence αβ⁺ CD4⁺ Th2 cell differentiation is discussed throughout this appraisal of the current literature. We highlight the unique features of Th2 cell development, highlighting the three necessary signals, T‐cell receptor ligation, co‐stimulation and cytokine receptor ligation. Finally, putting these into context, microbial and allergenic properties that trigger Th2 cell differentiation and how these influence Th2 effector function are discussed and questioned.     

The peripheral CD8 T cell repertoire is largely independent of the presence of intestinal flora

While numerous studies have analyzed the shaping of T cell repertoires by self or foreign peptides, little is known on the influence of commensal self peptides derived from the intestinal flora (IF). Here, we have analyzed naive and immune repertoires in mice devoid of IF [germ-free (GF) mice]. First, by means of an extensive CDR3beta sequencing strategy, we show that the naive peripheral CD8 T cell repertoire does not exhibit a major imprint of IF antigens. Second, using MHC-peptide tetramers, CDR3beta length distribution analyses and TCR sequencing, we show that cytotoxic T lymphocyte (CTL) responses specific for two distinct epitopes are quasi-identical in normal and GF mice. Our findings indicate that, in general, peptides derived from the intestinal microflora have little if any influence on CTL responses in the mouse.     

Activation requirements and responses to TLR ligands in human CD4 T cells: Comparison of two T cell isolation techniques

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4⁺ T cells isolated either by IMACS (IMACS-CD4⁺) or by IMACS followed by FACS (IMACS/FACS-CD4⁺). As expected, IMACS-CD4⁺ were less pure than IMACS/FACS-CD4⁺ (92.5% ± 1.4% versus 99.7% ± 0.2%, respectively). Consequently, IMACS-CD4⁺ proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4⁺. In addition IMACS-CD4⁺ but not IMACS/FACS-CD4⁺ responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4⁺ and highly purified IMACS-/FACS-CD4⁺. Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.     

Preservation of clonal heterogeneity of the Pneumocystis carinii-specific CD4 T cell repertoire in HIV infected, asymptomatic individuals

The loss of CD4 lymphocytes in HIV disease associates with opportunistic infections. Since diverse CD4 T cell clones respond to an opportunistic pathogen, we asked whether CD4 depletion deletes selected clones in the repertoire (vertical depletion) or it affects all clones by reducing the cell number in each progeny without affecting the overall number of clones (horizontal depletion). Understanding this point may help explain the mode of CD4 depletion and the mode of immunoreconstitution after therapy. Therefore we examined the CD4 T cell repertoire specific for Pneumocystis carinii, a relevant opportunistic pathogen in AIDS, in HIV-infected, asymptomatic individuals. We identified two patients of 36 asymptomatics for lack of proliferation to P. carinii, suggesting selective depletion of specific CD4 cells. To investigate clonal heterogeneity of P. carinii-responsive CD4 lymphocytes, specific CD4 T cell lines were generated and studied by TCR BV gene family usage and CDR3 length analysis (spectratyping). Clonal heterogeneity was similar in antigen-specific CD4 lines generated from P. carinii non-responding HIV seropositives and from controls. Thus, despite undetectable response to the pathogen, residual specific cells probably prevent overt infection and, when expanded in vitro, exhibit a clonal diversity similar to normal controls. These findings suggest a horizontal, rather than vertical, depletion in these asymptomatic patients.     

The acquisition of CD4 and CD8 during the differentiation of early thymocytes in short-term culture

Subpopulations of thymocytes known to represent early stages of T cell development were isolated from the adult mouse thymus, and their ability to differentiate during short periods of culture was assessed by their acquisition of surface CD4 and CD8. Virtually all cells of the most mature of the CD4-CD8- thymocyte subpopulations (other surface markers CD3- HSA++ IL-2R-Pgp-1-) and of the immature CD4-CD8+ thymocyte subpopulation (other surface markers also CD3- HSA++ IL-2R- Pgp-1-) became CD4+CD8+ in less than 1 day of culture without added stimuli or growth factors. This suggested they had already received signals initiating CD4 and CD8 acquisition. However, stimulation of these precursor cells with phorbyl ester and ionomycin prevented this acquisition of CD4 and CD8. No distinct CD4-CD8+ intermediate was detected as the CD4-CD8- cells became CD4+CD8+ in the non-stimulated cultures, thus questioning the assumption that these three groups of cells are sequential steps in one lineage. In contrast to this pre-programmed acquisition of CD4 and CD8, the less mature CD4-CD8- IL-2R+ subpopulation did not progress to the CD4+CD8+ stage in culture, although it is able to develop further on intrathymic transfer. It is likely that this subpopulation represents a control point requiring specific differentiation signals for further development.