Isolation of CD4and CD8T-Cell Clones from Mice Immunized with Synthetic Peptides on Splenic Dendritic Cells

Splenic dendritic cells (DC) express high levels of MHC, co-stimulator, and adhesion molecules and have been shown to be extremely potent antigen-presenting cells for both CD4⁺and CD8⁺T-cell responses. Previous studies have shown that murine DC can be loaded with exogenous antigens and used to prime CD4⁺, Class II-restricted T-cell responsesin vivo.This article describes protocols for immunization using DC loaded with peptides bound to Class I or Class II molecules and for the derivation and characterization of CD4⁺and CD8⁺antigen-specific T-cell lines and clones. The rationale for using DC as antigen-presenting cells is discussed, together with the advantages and disadvantages of these cells compared with more conventional methods of immunization.     

IL-21-treated naive CD45RA<Superscript>+</Superscript> CD8<Superscript>+</Superscript> T cells represent a reliable source for producing leukemia-reactive cytotoxic T lymphocytes with high proliferative potential and early differentiation phenotype

Clinical tumor remissions after adoptive T-cell therapy are frequently not durable due to limited survival and homing of transfused tumor-reactive T cells, what can be mainly attributed to the long-term culture necessary for in vitro expansion. Here, we introduce an approach allowing the reliable in vitro generation of leukemia-reactive cytotoxic T lymphocytes (CTLs) from naive CD8⁺ T cells of healthy donors, leading to high cell numbers within a relatively short culture period. The protocol includes the stimulation of purified CD45RA⁺ CD8⁺ T cells with primary acute myeloid leukemia blasts of patient origin in HLA-class I-matched allogeneic mixed lymphocyte-leukemia cultures. The procedure allowed the isolation of a large diversity of HLA-A/-B/-C-restricted leukemia-reactive CTL clones and oligoclonal lines. CTLs showed reactivity to either leukemia blasts exclusively, or to leukemia blasts as well as patient-derived B lymphoblastoid-cell lines (LCLs). In contrast, LCLs of donor origin were not lysed. This reactivity pattern suggested that CTLs recognized leukemia-associated antigens or hematopoietic minor histocompatibility antigens. Consistent with this hypothesis, most CTLs did not react with patient-derived fibroblasts. The efficiency of the protocol could be further increased by addition of interleukin-21 during primary in vitro stimulation. Most importantly, leukemia-reactive CTLs retained the expression of early T-cell differentiation markers CD27, CD28, CD62L and CD127 for several weeks during culture. The effective in vitro expansion of leukemia-reactive CD8⁺ CTLs from naive CD45RA⁺ precursors of healthy donors can accelerate the molecular definition of candidate leukemia antigens and might be of potential use for the development of adoptive CTL therapy in leukemia.     

Regulation of CD4 and CD8 surface expression on human thymocyte subpopulations by triggering through CD2 and the CD3-T cell receptor

Human thymocytes bearing the CD4 and/or CD8 antigens can be fractionated into cells with an immature and more mature phenotype based on their quantitative expression of the CD3 Ag (J. Immunol. 138:3108; J. Immunol. 139:1065). We show that the expression of CD4 and CD8 on thymocyte subpopulations with low CD3 (CD3L) and high CD3 (CD3H) is regulated by activation through the CD2 molecule and perturbation of the CD3-T cell receptor complex (CD3-Ti). Similar to its previously reported effects on peripheral T cells, PMA was able to induce the down-regulation of surface CD4, but not CD8, on thymocyte subpopulations. PMA could induce CD4 and CD8 phosphorylation in both CD3L and CD3H fractions. These results suggest that if changes in phosphorylation represent the mechanism by which CD4 and CD8 are able to transmit signals, this mechanism is operative in both CD3L and CD3H subpopulations. Treatment with anti-T11(2) and anti-T11(3) antibodies (CD2 activation pathway) resulted in partial down-regulation of CD4 but not CD8 surface expression on both CD3L and CD3H thymocytes. Similar treatment had no detectable effect on peripheral T cells. The down-regulation of surface CD4 induced by activation via CD2 could be inhibited by treatment of thymocytes with anti-CD3 antibodies. Treatment of thymocytes with anti-CD3 alone or following CD2 activation induced the selective down-regulation of surface CD8 within 15 minutes. These results suggest that CD2 and CD3-Ti triggering may regulate CD4 and CD8 surface expression on thymocytes. Furthermore, these results suggest that "cross-talk" between the CD2 and CD3-Ti pathway of activation may involve CD4 and CD8 molecules.     

Th Cells Promote CTL Survival and Memory via Acquired pMHC-I and Endogenous IL-2 and CD40L Signaling and by Modulating Apoptosis-Controlling Pathways

Involvement of CD4⁺ helper T (Th) cells is crucial for CD8⁺ cytotoxic T lymphocyte (CTL)-mediated immunity. However, CD4⁺ Th’s signals that govern CTL survival and functional memory are still not completely understood. In this study, we assessed the role of CD4⁺ Th cells with acquired antigen-presenting machineries in determining CTL fates. We utilized an adoptive co-transfer into CD4⁺ T cell-sufficient or -deficient mice of OTI CTLs and OTII Th cells or Th cells with various gene deficiencies pre-stimulated in vitro by ovalbumin (OVA)-pulsed dendritic cell (DCova). CTL survival was kinetically assessed in these mice using FITC-anti-CD8 and PE-H-2K^b/OVA_257-264 tetramer staining by flow cytometry. We show that by acting via endogenous CD40L and IL-2, and acquired peptide-MHC-I (pMHC-I) complex signaling, CD4⁺ Th cells enhance survival of transferred effector CTLs and their differentiation into the functional memory CTLs capable of protecting against highly-metastasizing tumor challenge. Moreover, RT-PCR, flow cytometry and Western blot analysis demonstrate that increased survival of CD4⁺ Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of TRAIL, and altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (Bcl-10, Casp-3, Casp-4, Casp-7) molecules. Taken together, our results reveal a previously unexplored mechanistic role for CD4⁺ Th cells in programming CTL survival and memory recall responses. This knowledge could also aid in the development of efficient adoptive CTL cancer therapy.     

Isolation of the CD7 gene from the DNA of transfected L cells

Using DNA from L cells which expressed high levels of the CD7 (Leu-9 or HuLy-m2) antigen obtained after two cycles of transfection, a genomic library was constructed in the lambda phage Charon 4A. Recombinant clones containing the gene coding for this antigen were identified by first screening the library with both the HSVtk gene and a probe detecting the human repetitive (Alu) sequences. DNA from 10 tk⁺ and 12 Alu⁺ recombinant clones was used to transfect L cells which were analyzed for the cell-surface expression of CD7 either early (48–72 h posttransfection) or later when hypoxanthine aminopterin thymidine-resistant colonies were obtained. Transfection with either Alu⁺ or tk⁺ recombinant phages led to transient early expression of CD7, and stable CD7⁺ transfectants were also established. Thus the CD7 gene has been isolated in a number of clones in association with either the Alu repetitive sequence or with the HSV-tk gene; the insert size in one of the genomic clones was 13.5 kb.     

Coreceptor function of CD4 in response to the MHC class I molecule

The specificity of the T-cell receptor (TCR) and its interaction with coreceptors play a crucial role in T-cell passing through developmental checkpoints and, eventually, determine the efficiency of adaptive immunity. The genes for the α and β chains of TCR were cloned from T-cell hybridoma 1D1, which was obtained by fusion of BWZ.36CD8α cells with CD8⁺ memory cells specific for the H-2K^b MHC class I molecule. Retroviral transduction of the 1D1 TCR genes and the CD4 and CD8 coreceptor genes was used to obtain 4G4 thymoma variants that exposed the CD3/TCR complex together with CD4, CD8, or both of the coreceptors on their surface. Although the main function of CD4 is to stabilize the interaction of TCR with MHC class II molecules, CD4 was found to mediate the activation of transfected cells via TCR specific for the H-2K^b MHC class I molecule. Moreover, CD4 proved to dominate over CD8, since the response of CD4⁺CD8⁺ transfectants was suppressed by antibodies against CD4 and the A^b MHC class II molecule but not to CD8. The response of CD4⁺ transfectants was not due to a cross-reaction of 1D1 TCR with MHC class II molecules, because the transfectants did not respond to splenocytes of H-2^b knockout mice, which were defective in the assembly of the MHC class I molecule/β2 microglobulin/peptide complex and did not expose the complex on cell surface. The domination was not due to sequestration of p56^lck kinase, since CD4 devoid of the kinase-binding site was functional in 4G4 thymoma cells. The results were used to explain some features of intrathymic cell selection and assumed to provide an experimental basis for developing new methods of anticancer gene therapy.     

Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering

The function of the T cell differentiation antigens CD4 (Leu-3/T4) and CD8 (Leu-2/T8) on human cytotoxic T lymphocytes (CTL) is presently seen only in conjugate formation between CTL and target cell via class II or class I MHC antigens rather than in the later killing steps. In this study, human CD4+ and CD8+ CTL clones were used to investigate the effects of monoclonal antibodies against these differentiation antigens on nonspecific triggering of cytotoxicity. Cytotoxicity was induced either by antibodies against the CD3 (T3) antigen or by the lectins Con A and PHA. Anti-CD4 or anti-CD8 antibodies specifically inhibited all types of cytotoxicity of CD4+ or CD8+ CTL, respectively, regardless of the specificity of the CTL for class I or class II HLA antigens and regardless of whether target cells expressed class I or class II antigens. These results are incompatible with an exclusive role of the CD4 and CD8 molecules in MHC class recognition and are discussed with respect to a function as negative signal receptors for these molecules on CTL.     

Tim-3-Expressing CD4+ and CD8+ T Cells in Human Tuberculosis (TB) Exhibit Polarized Effector Memory Phenotypes and Stronger Anti-TB Effector Functions

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4⁺ and CD8⁺ T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3⁻ counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3⁺CD4⁺ and Tim-3⁺CD8⁺ T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4⁺ and CD8⁺ T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.     

Correlation between Direct Binding Ability of SyntheticT. gondiiSAG1 Peptides to HLA-A2 Measured by a Sensor for Surface Plasmon Resonance and Antigenicity of the Peptides forT. gondii-Infected Cell-Specific CTL

Toxoplasma gondii(T. gondii) -infected B lymphoma cells presentT. gondiiantigens in the context of major histocompatibility complex molecules toT. gondii-specific CD8⁺cytotoxic T cells (CTL). HLA-A2 molecules ofT. gondii-infected human cells have been shown to be used in presenting T. gondiiantigens to CD8⁺CTL. SAG1, one of the major antigenic molecules ofT. gondii,is an antigen forT. gondii-specific CTL, and represents a possible basis for vaccines. The direct binding of nonamer SAG1 peptides to HLA-A2 was assayed here using an automated biosensor system with a sensor for surface plasmon resonance detection. The antigenicity of synthetic SAG1 peptides toT. gondii-specific CD8⁺CTL also was assayed. The present study found a high correlation between the binding ability of synthetic SAG1 peptides to HLA-A2 and the antigenicity of peptides toT. gondii-infected cell-specific CD8⁺CTL.     

Generation of protective immunity against an immunogenic carcinoma requires CD40/CD40L and B7/CD28 interactions but not CD4+ T cells

Interactions between CD40 and CD40L play a central role in the regulation of both humoral and cellular immunity. Recently, interactions between these molecules have also been implicated in the generation of protective cell-mediated tumor immunity. We have generated a tumor model in which a well-understood and clearly immunostimulatory antigen, influenza hemagglutinin has been transfected into the BALB/c-derived, MHC-class-I-positive, B7-deficient murine mammary carcinoma, MT901. In this model, expression of the influenza hemagglutinin antigen does not alter tumorigenicity in naïve but serves as a tumor-rejection target in immunized mice. T-cell-depletion experiments indicate that successful tumor protection can occur following immunization in mice depleted of CD4⁺ but not CD8⁺ T cells, suggesting that tumor protection is largely CD8-mediated and CD4-independent. Interestingly, despite the ability of tumor protection to be generated in the absence of CD4⁺ T cells, effective immunization was clearly dependent on CD40/CD40L as well as CD28/B7 interactions.     

TCR-Independent Induction of Low Responsiveness by Chemically Fixed Cells in Alloreactive CTL Clones and Its Prevention through Cognate Cell-Cell Interaction

We established BALB/c-derived CD8⁺ CTL clones D2-22 (V_β 6⁺), D2-23 (V_β 8⁺) and D2-24 (V_β 8⁺) specific for B10.D2 minor H antigen. D2-22 and D2-23 proliferated without producing IL-2 in response to X-ray-irradiated antigenic cells, Con A, aCD3, PMA and IL-2. Paraformaldehyde-fixed antigenic spleen cells neither induced proliferation in the presence of costimulatory cells nor inhibited responses to irradiated antigenic cells added simultaneously. Unlike the previously reported results with IL-2-producing CTL clones and Th1 clones, the fixed antigenic cells failed to induce antigen-specific unresponsiveness in these IL-2-nonproducing CTL clones. Instead, the responsiveness of these clones to fresh stimulation was found to be reduced severely after 2 days in the culture added with either antigenic or syngeneic fixed cells. Induction of their antigen-nonspecific low responsiveness by the fixed cells was prevented by adding irradiated syngeneic cells into the culture or even by increasing the concentration of responder D2-23 cells. Close contact of D2-23 and irradiated syngeneic cells was required to prevent the reduction of the responsiveness, although this cognate cell-cell interaction could be replaced by exogenously added IL-2 or PMA. Cytolytic and tumor cell growth inhibitory activities of D2-23 were also reduced by incubation with the fixed cells, which was prevented by the addition of irradiated syngeneic cells. These findings showed the unique properties of IL-2-nonproducing CTL clones in signal requirements for maintaining normal responsiveness for proliferation and cytolytic activity.     

CTLA-4 blockade increases antigen-specific CD8+ T cells in prevaccinated patients with melanoma: three cases

Background

Anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) antibodies, such as ipilimumab, have generated measurable immune responses to Melan-A, NY-ESO-1, and gp100 antigens in metastatic melanoma. Vaccination against such targets has potential for immunogenicity and may produce an effector-memory T-cell response.

Methods

To determine the effect of CTLA-4 blockade on antigen-specific responses following vaccination, in-depth immune monitoring was performed on three ipilimumab-treated patients prevaccinated with gp100 DNA (IMF-24), gp100^209?C217 and tyrosinase peptides plus GM-CSF DNA (IMF-32), or NY-ESO-1 protein plus imiquimod (IMF-11); peripheral blood mononuclear cells were analyzed by tetramer and/or intracellular cytokine staining following 10-day culture with HLA-A*0201-restricted gp100^209?C217 (ITDQVPFSV), tyrosinase^369?C377 (YMDGTMSQV), or 20-mer NY-ESO-1 overlapping peptides, respectively. Tumors from IMF-32 were analyzed by immunohistochemistry to help elucidate mechanism(s) underlying tumor escape.

Results

Following vaccination, patients generated weak to no CD4⁺ or CD8⁺ T-cell response specific to the vaccine antigen but demonstrated increases in effector-memory (CCR7^loCD45RA^lo) tetramer⁺CD8⁺ T cells. After ipilimumab induction, patients experienced a robust, although sometimes transient, antigen-specific response for gp100 (IMF-32 and IMF-24) or NY-ESO-1 (IMF-11) and produced polyfunctional intracellular cytokines. Primary and metastatic tumors expressed tyrosinase but not gp100 or class I/II MHC molecules.

Conclusion

Vaccination induced a measurable antigen-specific T-cell response that increased following CTLA-4 blockade, potentially ??boosting?? the vaccine-primed response. Tumor escape may be related to antigen loss or lack of MHC expression necessary for immune activity. These results in a limited number of patients support the need for further research into combining vaccination with ipilimumab and provide insight into mechanisms underlying tumor escape.     

Interferon-α and -γ Differentially Reduce Rapid Immature T-Cell Death by Contact with HIV-1 Carrier Cell Clones In Vitro

The non-antigen specific rapid cytotoxic (CT) death of immature TdT⁺CD4⁺CD8⁺ T cells due to contact with HIV-1 carrier T-cell clones we have found recently is a novel phenomenon. The effects of interferons (IFN) on this CT reaction were studied in vitro. Treatment of the HIV-1 carrier clones, referred to as “effectors,” with IFN-α but not IFN-γ, or of the susceptible immature TdT⁺CD4⁺CD8⁺ T cells, referred to as “targets,” with IFN-γ but not IFN-α, for 24 hr prior to CT testing was found to reduce the CT reaction. Simultaneously, a down-regulated CD8 expression and an up-regulated antigen expression of both major histocompatibility antigen complex class I (MHC-I) and HIV-1 gp120/gp160 in the IFN-α treated effector (gp120⁺CD8⁺ HPB-ALL/HIV), and/or simultaneously up-regulated antigen expression of both CD8 and MHC-I in the IFN-γ treated target (CD4⁺CD8⁺ HPB-ALL) were found to be associated with reduced CT reaction. However, altered antigen expression in the IFN-γ treated effectors or IFN-α treated targets did not affect the ultimate degree of CT reaction. This study thus suggests a possible therapeutic efficacy of IFN by reducing the direct elimination of the T-cell precursors in HIV-1 infection.     

Regulatory B Cells Inhibit Cytotoxic T Lymphocyte (CTL) Activity and Elimination of Infected CD4 T Cells after In Vitro Reactivation of HIV Latent Reservoirs

During HIV infection, IL-10/IL-10 receptor and programmed death-1 (PD-1)/programmed death-1-ligand (PD-L1) interactions have been implicated in the impairment of cytotoxic T lymphocyte (CTL) activity. Despite antiretroviral therapy (ART), attenuated anti-HIV CTL functions present a major hurdle towards curative measures requiring viral eradication. Therefore, deeper understanding of the mechanisms underlying impaired CTL is crucial before HIV viral eradication is viable. The generation of robust CTL activity necessitates interactions between antigen-presenting cells (APC), CD4⁺ and CD8⁺ T cells. We have shown that in vitro, IL-10^hiPD-L1^hi regulatory B cells (Bregs) directly attenuate HIV-specific CD8⁺-mediated CTL activity. Bregs also modulate APC and CD4⁺ T cell function; herein we characterize the Breg compartment in uninfected (HIV_NEG), HIV-infected “elite controllers” (HIV_EC), ART-treated (HIV_ART), and viremic (HIV_vir), subjects, and in vitro, assess the impact of Bregs on anti-HIV CTL generation and activity after reactivation of HIV latent reservoirs using suberoylanilide hydroxamic acid (SAHA). We find that Bregs from HIV_EC and HIV_ART subjects exhibit comparable IL-10 expression levels significantly higher than HIV_NEG subjects, but significantly lower than HIV_VIR subjects. Bregs from HIV_EC and HIV_ART subjects exhibit comparable PD-L1 expression, significantly higher than in HIV_VIR and HIV_NEG subjects. SAHA-treated Breg-depleted PBMC from HIV_EC and HIV_ART subjects, displayed enhanced CD4⁺ T-cell proliferation, significant upregulation of antigen-presentation molecules, increased frequency of CD107a⁺ and HIV-specific CD8⁺ T cells, associated with efficient elimination of infected CD4⁺ T cells, and reduction in integrated viral DNA. Finally, IL-10-R and PD-1 antibody blockade partially reversed Breg-mediated inhibition of CD4⁺ T-cell proliferation. Our data suggest that, possibly, via an IL-10 and PD-L1 synergistic mechanism; Bregs likely inhibit APC function and CD4⁺ T-cell proliferation, leading to anti-HIV CTL attenuation, hindering viral eradication.     

HLA-A2-restricted peripheral blood cytolytic T lymphocyte response to HPV type 16 proteins E6 and E7 from patients with neoplastic cervical lesions

 The DNA from human papillomavirus (HPV) can be detected in 90% of cervical carcinomas. To address whether patients infected with HPV can mount efficient T cell responses to this pathogen we examined the cytotoxic T lymphocyte (CTL) response of peripheral blood mononuclear cells (PBMC) from patients with abnormal genital epithelial cells. PBMC from 11 HLA-A2⁺ patients were stimulated with CaSki, a cervical carcinoma cell line that is HPV 16⁺ and HLA-A2⁺. The CTL were screened for reactivity to the cervical carcinoma cell line C33A (HPV^–, HLA-A2⁺) transfected with the HPV 16 E6 or E7 genes or the plasmid without insert. The CTL of 1 patient showed particularly strong CaSki and HPV E6 or E7 protein-specific cytotoxicity in a HLA-A2-restricted fashion. In contrast, these CTL lysed neither a vector-only transfectant, the natural killer cell (NK) target, K562 nor the lymphokine-activated killer cell (LAK) target, Daudi. HLA-A2 restriction was demonstrated by the lack of recognition of a HLA-A2^– CaSki cell line developed in our laboratory. The CTL line was cloned and 99 clones were harvested and screened; 51 clones lysed CaSki, of which 17 did not lyse the A2^– CaSki. Of these HLA-A2^– restricted clones, 8 did not lyse C33A transfectants, 6 lysed all C33A transfectants, 3 lysed C33A-E7 only and none lysed C33A-E6 only. These data imply that, within the bulk CTL line, HLA-A2-restricted recognition of antigens was restricted to CaSki antigens, antigens common to cervical carcinoma (CaSki plus C33A), or HPV-16-E7-derived antigen on the clonal level. The E7-restricted clones were negative for recognition of known HLA-A2-binding peptides from E7. Received: 16 November 1995 / Accepted: 15 January 1996     

Concanamycin A, a vacuolar type H+-ATPase inhibitor, induces cell death in activated CD8+ CTL

Concanamycin A (CMA) and concanamycin B (CMB) are specific inhibitors of vacuolar type H⁺-ATPase (V-ATPase). In our previous studies, intraperitoneal injection of CMB was shown to suppress the increase in CD8⁺ CTL population, but not to affect CD4⁺ and B220⁺ populations, in mice immunized with allogeneic tumors. To clarify the molecular basis of the selective decrease in the CD8⁺ CTL population by CMB, we have performed a series of in vitro experiments with use of CMA. Cell viability of the CD8⁺ population prepared from the immunized mice was preferentially decreased by CMA treatment. Moreover, in the CD8⁺ CTL clone, CMA induced a marked DNA fragmentation and nuclear condensation characteristic of apoptosis. Anti-CD3 or phorbol ester accelerated the CMA-induced reduction in cell viability of the CD8⁺ CTL clone, but not CD4⁺ T cell clones. However, this rapid cell death was not accompanied by DNA fragmentation and nuclear condensation. Perforin and granzyme B were unlikely to be involved in such cell death. Thus, our data suggest that V-ATPase activity is essential for survival of CD8⁺ CTL especially when activated. This revised version was published online in June 2006 with corrections to the Cover Date.     

CD160-Associated CD8 T-Cell Functional Impairment Is Independent of PD-1 Expression

Expression of co-inhibitory molecules is generally associated with T-cell dysfunction in chronic viral infections such as HIV or HCV. However, their relative contribution in the T-cell impairment remains unclear. In the present study, we have evaluated the impact of the expression of co-inhibitory molecules such as 2B4, PD-1 and CD160 on the functions of CD8 T-cells specific to influenza, EBV and CMV. We show that CD8 T-cell populations expressing CD160, but not PD-1, had reduced proliferation capacity and perforin expression, thus indicating that the functional impairment in CD160⁺ CD8 T cells may be independent of PD-1 expression. The blockade of CD160/CD160-ligand interaction restored CD8 T-cell proliferation capacity, and the extent of restoration directly correlated with the ex vivo proportion of CD160⁺ CD8 T cells suggesting that CD160 negatively regulates TCR-mediated signaling. Furthermore, CD160 expression was not up-regulated upon T-cell activation or proliferation as compared to PD-1. Taken together, these results provide evidence that CD160-associated CD8 T-cell functional impairment is independent of PD-1 expression.     

T-cell Receptor-optimized Peptide Skewing of the T-cell Repertoire Can Enhance Antigen Targeting

Altered peptide antigens that enhance T-cell immunogenicity have been used to improve peptide-based vaccination for a range of diseases. Although this strategy can prime T-cell responses of greater magnitude, the efficacy of constituent T-cell clonotypes within the primed population can be poor. To overcome this limitation, we isolated a CD8⁺ T-cell clone (MEL5) with an enhanced ability to recognize the HLA A*0201-Melan A_27–35 (HLA A*0201-AAGIGILTV) antigen expressed on the surface of malignant melanoma cells. We used combinatorial peptide library screening to design an optimal peptide sequence that enhanced functional activation of the MEL5 clone, but not other CD8⁺ T-cell clones that recognized HLA A*0201-AAGIGILTV poorly. Structural analysis revealed the potential for new contacts between the MEL5 T-cell receptor and the optimized peptide. Furthermore, the optimized peptide was able to prime CD8⁺ T-cell populations in peripheral blood mononuclear cell isolates from multiple HLA A*0201⁺ individuals that were capable of efficient HLA A*0201⁺ melanoma cell destruction. This proof-of-concept study demonstrates that it is possible to design altered peptide antigens for the selection of superior T-cell clonotypes with enhanced antigen recognition properties.     

CD154 and IL-2 Signaling of CD4+ T Cells Play a Critical Role in Multiple Phases of CD8+ CTL Responses Following Adenovirus Vaccination

Adenoviral (AdV) vectors represent most commonly utilized viral vaccines in clinical studies. While the role of CD8⁺ cytotoxic T lymphocyte (CTL) responses in mediating AdV-induced protection is well understood, the involvement of CD4⁺ T cell-provided signals in the development of functional CD8⁺ CTL responses remain unclear. To explore CD4⁺ T helper signals required for AdVova-stimulated CTL responses, we established an adoptive transfer system by transferring CD4⁺ T cells derived from various knock out and transgenic mice into wild-type and/or CD4-deficient animals, followed by immunizing with recombinant ovalbumin (OVA)-expressing AdVova vector. Without CD4⁺ T help, both primary and memory CTL responses were greatly reduced in this model, and were associated with increased PD-1 expression. The provision of OVA-specific CD4⁺ T help in CD4⁺ T cell-deficient mice restored AdVova-induced primary CTL responses, and supported survival and recall responses of AdVova-stimulated memory CTLs. These effects were specifically mediated by CD4⁺ T cell-produced IL-2 and CD154 signals. Adoptive transfer of “helped” or “unhelped” effector and memory CTLs into naïve CD4⁺ T cell-deficient or -sufficient mice also revealed an additional role for polyclonal CD4⁺ T cell environment in the survival of AdVova-stimulated CTLs, partially explaining the extension of CTL contraction phase. Finally, during recall responses, CD4⁺ T cell environment, particularly involving memory CD4⁺ T cells, greatly enhanced expansion of memory CTLs. Collectively, our data strongly suggest a critical role for CD4⁺ T help in multiple phases of AdV-stimulated CTL responses, and could partially explain certain failures in AdV-based immunization trials targeting malignant tumors and chronic diseases that are often associated with compromised CD4⁺ T cell population and function.