CD4+ T cell help improves CD8+ T cell memory by retained CD27 expression

CD4+ T cell help during the priming of CD8+ T lymphocytes imprints the capacity for optimal secondary expansion upon re-encounter with antigen. Helped memory CD8+ T cells rapidly expand in response to a secondary antigen exposure, even in the absence of T cell help and, are most efficient in protection against a re-infection. In contrast, helpless memory CTL can mediate effector function, but secondary expansion is reduced. How CD4+ T cells instruct CD8+ memory T cells during priming to undergo efficient secondary expansion has not been resolved in detail. Here, we show that memory CTL after infection with lymphocytic choriomeningitis virus are CD27(high) whereas memory CTL primed in the absence of CD4+ T cell have a reduced expression of CD27. Helpless memory CTL produced low amounts of IL-2 and did not efficiently expand after restimulation with peptide in vitro. Blocking experiments with monoclonal antibodies and the use of CD27(-/-) memory CTL revealed that CD27 ligation during restimulation increased autocrine IL-2 production and secondary expansion. Therefore, regulating CD27 expression on memory CTL is a novel mechanism how CD4+ T cells control CTL memory.

Quantitative multiparameter assays to measure the effect of adjuvants on human antigen-specific CD8 T-cell responses

Large numbers and functionally competent T cells are required to protect from diseases for which antibody-based vaccines have consistently failed (1), which is the case for many chronic viral infections and solid tumors. Therefore, therapeutic vaccines aim at the induction of strong antigen-specific T-cell responses. Novel adjuvants have considerably improved the capacity of synthetic vaccines to activate T cells, but more research is necessary to identify optimal compositions of potent vaccine formulations. Consequently, there is a great need to develop accurate methods for the efficient identification of antigen-specific T cells and the assessment of their functional characteristics directly ex vivo. In this regard, hundreds of clinical vaccination trials have been implemented during the last 15 years, and monitoring techniques become more and more standardized.

Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity

Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8(+) T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.

L-selectin-negative CCR7- effector and memory CD8+ T cells enter reactive lymph nodes and kill dendritic cells

T lymphocytes lacking the lymph node-homing receptors L-selectin and CCR7 do not migrate to lymph nodes in the steady state. Instead, we found here that lymph nodes draining sites of mature dendritic cells or adjuvant inoculation recruited L-selectin-negative CCR7- effector and memory CD8+ T cells. This recruitment required CXCR3 expression on T cells and occurred through high endothelial venules in concert with lumenal expression of the CXCR3 ligand CXCL9. In reactive lymph nodes, recruited T cells established stable interactions with and killed antigen-bearing dendritic cells, limiting the ability of these dendritic cells to activate naive CD4+ and CD8+ T cells. The inducible recruitment of blood-borne effector and memory T cells to lymph nodes may represent a mechanism for terminating primary and limiting secondary immune responses.

HLA-B⁎27 subtype specificity determines targeting and viral evolution of a hepatitis C virus-specific CD8+ T cell epitope

Background & Aims: HLA-B⁄27 is associated with spontaneous HCV genotype 1 clearance. HLA-B⁄27-restricted CD8+ T cells target three NS5B epitopes. Two of these epitopes are dominantly targeted in the majority of HLA-B⁄27+ patients. In chronic infection, viral escape occurs consistently in these two epitopes. The third epitope (NS5B2820) was dominantly targeted in an acutely infected patient. This was in contrast, however, to the lack of recognition and viral escape in the large majority of HLA-B⁄27+ patients. Here, we set out to determine the host factors contributing to selective targeting of this epitope. Methods: Four-digit HLA class I typing and viral sequence analyses were performed in 78 HLA-B⁄27+ patients with chronic HCV genotype 1 infection. CD8+ T cell analyses were performed in a subset of patients. In addition, HLA/peptide affinity was compared for HLA-B⁄27:02 and 05. Results: The NS5B2820 epitope is only restricted by the HLA-B⁄27 subtype HLA-B⁄27:02 (that is frequent in Mediterranean populations), but not by the prototype HLA-B⁄27 subtype B⁄27:05. Indeed, the epitope is very dominant in HLA-B⁄27:02+ patients and is associated with viral escape mutations at the anchor position for HLA-binding in 12 out of 13 HLA-B⁄27:02+ chronically infected patients. Conclusions: The NS5B2820 epitope is immunodominant in the context of HLA-B⁄27:02, but is not restricted by other HLA-B⁄27 subtypes. This finding suggests an important role of HLA subtypes in the restriction of HCV-specific CD8+ responses. With minor HLA subtypes covering up to 39% of specific populations, these findings may have important implications for the selection of epitopes for global vaccines.

Loss of tapasin correlates with diminished CD8(+) T-cell immunity and prognosis in colorectal cancer.

BACKGROUND Tapasin is a crucial component of the major histocompatibility (MHC) class I antigen presentation pathway. Defects in this pathway can lead to tumor immune evasion. The aim of this study was to test whether tapasin expression correlates with CD8(+) cytotoxic T lymphocyte (CTL) infiltration of colorectal cancer (CRC) and overall survival. METHODS A next-generation tissue microarray (ngTMA) of 198 CRC patients with full clinicopathological information was included in this study. TMA slides were immunostained for tapasin, MHC I and CD8. Marker expression was analyzed with immune-cell infiltration, patient survival and TNM-staging. RESULTS A reduction of tapasin expression strongly correlated with venous invasion (AUC 0.682, OR 2.7, p = 0.002; 95% CI 1.7-5.0), lymphatic invasion (AUC 0.620, OR 2.0, p = 0.005; 95 % CI 1.3-3.3), distant metastasis (AUC 0.727, OR 2.9, p = 0.004; 95% CI 1.4-5.9) and an infiltrative tumor border configuration (AUC 0.621, OR 2.2, p = 0.017; 95% CI 1.2-4.4). Further, tapasin expression was associated with CD8(+) CTL infiltration (AUC 0.729, OR 5.4, p < 0.001; 95% CI 2.6-11), and favorable overall survival (p = 0.004, HR 0.6, 95% CI 0.42-0.85). CONCLUSIONS Consistent with published functional data showing that tapasin promotes antigen presentation, as well as tumor immune recognition and destruction by CD8(+) CTLs, a reduction in tapasin expression is associated with tumor progression in CRC.

Single-cell gene-expression profiling reveals qualitatively distinct CD8 T cells elicited by different gene-based vaccines

CD8 T cells play a key role in mediating protective immunity against selected pathogens after vaccination. Understanding the mechanism of this protection is dependent upon definition of the heterogeneity and complexity of cellular immune responses generated by different vaccines. Here, we identify previously unrecognized subsets of CD8 T cells based upon analysis of gene-expression patterns within single cells and show that they are differentially induced by different vaccines. Three prime-boost vector combinations encoding HIV Env stimulated antigen-specific CD8 T-cell populations of similar magnitude, phenotype, and functionality. Remarkably, however, analysis of single-cell gene-expression profiles enabled discrimination of a majority of central memory (CM) and effector memory (EM) CD8 T cells elicited by the three vaccines. Subsets of T cells could be defined based on their expression of Eomes, Cxcr3, and Ccr7, or Klrk1, Klrg1, and Ccr5 in CM and EM cells, respectively. Of CM cells elicited by DNA prime-recombinant adenoviral (rAd) boost vectors, 67% were Eomes(-) Ccr7(+) Cxcr3(-), in contrast to only 7% and 2% stimulated by rAd5-rAd5 or rAd-LCMV, respectively. Of EM cells elicited by DNA-rAd, 74% were Klrk1(-) Klrg1(-)Ccr5(-) compared with only 26% and 20% for rAd5-rAd5 or rAd5-LCMV. Definition by single-cell gene profiling of specific CM and EM CD8 T-cell subsets that are differentially induced by different gene-based vaccines will facilitate the design and evaluation of vaccines, as well as enable our understanding of mechanisms of protective immunity.

Plasmodium berghei sporozoite challenge of vaccinated BALB/c mice leads to the induction of humoral immunity and improved function of CD8(+) memory T cells.

Protection against malaria can be achieved by induction of a strong CD8(+) T-cell response against the Plasmodium circumsporozoite protein (CSP), but most subunit vaccines suffer from insufficient memory responses. In the present study, we analyzed the impact of postimmunization sporozoite challenge on the development of long-lasting immunity. BALB/c mice were immunized by a heterologous prime/boost regimen against Plasmodium berghei CSP that induces a strong CD8(+) T-cell response and sterile protection, which is short-lived. Here, we show that protective immunity is prolonged by a sporozoite challenge after immunization. Repeated challenges induced sporozoite-specific antibodies that showed protective capacity. The numbers of CSP-specific CD8(+) T cells were not substantially enhanced by sporozoite infections; however, CSP-specific memory CD8(+) T cells of challenged mice displayed a higher cytotoxic activity than memory T cells of immunized-only mice. CD4(+) T cells contributed to protection as well; but CD8(+) memory T cells were found to be the central mediator of sterile protection. Based on these data, we suggest that prolonged protective immunity observed after immunization and infection is composed of different antiparasitic mechanisms including CD8(+) effector-memory T cells with increased cytotoxic activity as well as CD4(+) memory T cells and neutralizing antibodies.

CD8/CD45RO T-cell infiltration in endoscopic biopsies of colorectal cancer predicts nodal metastasis and survival

BACKGROUND AND AIMS: Reliable prognostic markers based on biopsy specimens of colorectal cancer (CRC) are currently missing. We hypothesize that assessment of T-cell infiltration in biopsies of CRC may predict patient survival and TNM-stage before surgery. METHODS: Pre-operative biopsies and matched resection specimens from 130 CRC patients treated from 2002-2011 were included in this study. Whole tissue sections of biopsy material and primary tumors were immunostained for pancytokeratin and CD8 or CD45RO. Stromal (s) and intraepithelial (i) T-cell infiltrates were analyzed for prediction of patient survival as well as clinical and pathological TNM-stage of the primary tumor. RESULTS: CD8 T-cell infiltration in the preoperative biopsy was significantly associated with favorable overall survival (CD8i p = 0.0026; CD8s p = 0.0053) in patients with primary CRC independently of TNM-stage and postoperative therapy (HR [CD8i] = 0.55 (95% CI: 0.36-0.82), p = 0.0038; HR [CD8s] = 0.72 (95% CI: 0.57-0.9), p = 0.0049). High numbers of CD8i in the biopsy predicted earlier pT-stage (p < 0.0001) as well as absence of nodal metastasis (p = 0.0015), tumor deposits (p = 0.0117), lymphatic (p = 0.008) and venous invasion (p = 0.0433) in the primary tumor. Infiltration by CD45ROs cells was independently associated with longer survival (HR = 0.76 (95% CI: 0.61-0.96), p = 0.0231) and predicted absence of venous invasion (p = 0.0025). CD8 counts were positively correlated between biopsies and the primary tumor (r = 0.42; p < 0.0001) and were reproducible between observers (ICC [CD8i] = 0.95, ICC [CD8s] = 0.75). For CD45RO, reproducibility was poor to moderate (ICC [CD45i] = 0.16, ICC [CD45s] = 0.49) and correlation with immune infiltration in the primary tumor was fair and non-significant (r[CD45s] = 0.16; p = 0.2864). For both markers, no significant relationship was observed with radiographic T-stage, N-stage or M-stage, indicating that assessment of T-cells in biopsy material can add additional information to clinical staging in the pre-operative setting. CONCLUSIONS: T-cell infiltration in pre-operative biopsy specimens of CRC is an independent favorable prognostic factor and strongly correlates with absence of nodal metastasis in the resection specimen. Quantification of CD8i is highly reproducible and allows superior prediction of clinicopathological features as compared to CD45RO. The assessment of CD8i infiltration in biopsies is recommended for prospective investigation.

Chronic myelogenous leukemia maintains specific CD8(+) T cells through IL-7 signaling

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease of hematopoietic stem cells. The disease progresses after several years from an initial chronic phase to a blast phase. Leukemia-specific T cells are regularly detected in CML patients and may be involved in the immunological control of the disease. Here, we analyzed the role of leukemia-specific CD8(+) T cells in CML disease control and the mechanism that maintains CD8(+) T-cell immunosurveillance in a retroviral-induced murine CML model. To study antigen-specific immune responses, the glycoprotein of the lymphocytic choriomeningitis virus was used as model leukemia antigen. Leukemia-specific CTL activity was detectable in vivo in CML mice and depletion of CD8(+) T cells rapidly led to disease progression. CML-specific CTL were characterized by the expression of the IL-7 receptor -chain. In addition, leukemia cells produced IL-7 that was crucial for the maintenance of leukemia-specific CTL and for disease control. Therefore, CML cells maintain the specific CD8(+) T-cell-mediated immune control by IL-7 secretion. This results in prolonged control of disease and probably contributes to the characteristic chronic phase of the disease.

Virus-like particles induce robust human T-helper cell responses

Among synthetic vaccines, virus-like particles (VLPs) are used for their ability to induce strong humoral responses. Very little is reported on VLP-based-vaccine-induced CD4(+) T-cell responses, despite the requirement of helper T cells for antibody isotype switching. Further knowledge on helper T cells is also needed for optimization of CD8(+) T-cell vaccination. Here, we analysed human CD4(+) T-cell responses to vaccination with MelQbG10, which is a Qβ-VLP covalently linked to a long peptide derived from the melanoma self-antigen Melan-A. In all analysed patients, we found strong antibody responses of mainly IgG1 and IgG3 isotypes, and concomitant Th1-biased CD4(+) T-cell responses specific for Qβ. Although less strong, comparable B- and CD4(+) T-cell responses were also found specific for the Melan-A cargo peptide. Further optimization is required to shift the response more towards the cargo peptide. Nevertheless, the data demonstrate the high potential of VLPs for inducing humoral and cellular immune responses by mounting powerful CD4(+) T-cell help.

Vaccination-induced functional competence of circulating human tumor-specific CD8 T-cells

T-cells specific for foreign (e.g., viral) antigens can give rise to strong protective immune responses, whereas self/tumor antigen-specific T-cells are thought to be less powerful. However, synthetic T-cell vaccines composed of Melan-A/MART-1 peptide, CpG and IFA can induce high frequencies of tumor-specific CD8 T-cells in PBMC of melanoma patients. Here we analyzed the functionality of these T-cells directly ex vivo, by multiparameter flow cytometry. The production of multiple cytokines (IFNγ, TNFα, IL-2) and upregulation of LAMP-1 (CD107a) by tumor (Melan-A/MART-1) specific T-cells was comparable to virus (EBV-BMLF1) specific CD8 T-cells. Furthermore, phosphorylation of STAT1, STAT5 and ERK1/2, and expression of CD3 zeta chain were similar in tumor- and virus-specific T-cells, demonstrating functional signaling pathways. Interestingly, high frequencies of functionally competent T-cells were induced irrespective of patient's age or gender. Finally, CD8 T-cell function correlated with disease-free survival. However, this result is preliminary since the study was a Phase I clinical trial. We conclude that human tumor-specific CD8 T-cells can reach functional competence in vivo, encouraging further development and Phase III trials assessing the clinical efficacy of robust vaccination strategies.

HLA haplotype determines hapten or p-i T cell reactivity to flucloxacillin

Drug-induced liver injury (DILI) is a main cause of drug withdrawal. A particularly interesting example is flucloxacillin (FLUX)-DILI, which is associated with the HLA-B*57:01 allele. At present, the mechanism of FLUX-DILI is not understood, but the HLA association suggests a role for activated T cells in the pathomechanism of liver damage. To understand the interaction among FLUX, HLA molecules, and T cells, we generated FLUX-reacting T cells from FLUX-naive HLA-B*57:01(+) and HLA-B*57:01(-) healthy donors and investigated the mechanism of T cell stimulation. We found that FLUX stimulates CD8(+) T cells in two distinct manners. On one hand, FLUX was stably presented on various HLA molecules, resistant to extensive washing and dependent on proteasomal processing, suggesting a hapten mechanism. On the other hand, in HLA-B*57:01(+) individuals, we observed a pharmacological interaction with immune receptors (p-i)-based T cell reactivity. FLUX was presented in a labile manner that was further characterized by independence of proteasomal processing and immediate T cell clone activation upon stimulation with FLUX in solution. This p-i-based T cell stimulation was restricted to the HLA-B*57:01 allele. We conclude that the presence of HLA-B*57:01 drives CD8(+) T cell responses to the penicillin-derivative FLUX toward nonhapten mechanism.

T cell-mediated hypersensitivity to quinolones: mechanisms and cross-reactivity

BACKGROUND: Quinolones are widely used, broad spectrum antibiotics that can induce immediate- and delayed-type hypersensitivity reactions, presumably either IgE or T cell mediated, in about 2-3% of treated patients. OBJECTIVE: To better understand how T cells interact with quinolones, we analysed six patients with delayed hypersensitivity reactions to ciprofloxacin (CPFX), norfloxacin (NRFX) or moxifloxacin (MXFX). METHODS: We confirmed the involvement of T cells in vivo by patch test and in vitro by means of the lymphocyte proliferation test (LTT). The nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones were investigated through the generation and analysis (flow cytometry and proliferation assays) of quinolone-specific T cell clones (TCC). RESULTS: The LTT confirmed the involvement of T cells because peripheral blood mononuclear cells (PBMC) mounted an enhanced in vitro proliferative response to CPFX and/or NRFX or MXFX in all patients. Patch tests were positive after 24 and 48 h in three out of the six patients. From two patients, CPFX- and MXFX-specific CD4(+)/CD8(+) T cell receptor (TCR) alphabeta(+) TCC were generated to investigate the nature of the drug-T cell interaction as well as the cross-reactivity with other quinolones. The use of eight different quinolones as antigens (Ag) revealed three patterns of cross-reactivity: clones exclusively reacting with the eliciting drug, clones with a limited cross-reactivity and clones showing a broad cross-reactivity. The TCC recognized quinolones directly without need of processing and without covalent association with the major histocompatability complex (MHC)-peptide complex, as glutaraldehyde-fixed Ag-presenting cells (APC) could present the drug and washing quinolone-pulsed APC removed the drug, abrogating the reactivity of quinolone-specific TCC. CONCLUSION: Our data show that T cells are involved in delayed immune reactions to quinolones and that cross-reactivity among the different quinolones is frequent.