June Kan-Mitchell

Human Immunodeficiency Virus Type 1 Vpr Impairs Dendritic Cell Maturation and T-Cell Activation: Implications for Viral Immune Escape

ABSTRACT Antigen presentation and T-cell activation are dynamic processes involving signaling molecules present in both APCs and T cells. Effective APC function and T-cell activation can be compromised by viral immune evasion strategies, including those of human immunodeficiency virus type 1 (HIV-1). In this study, we determined the effects of HIV-1 Vpr on one of the initial target of the virus, dendritic cells (DC), by investigating DC maturation, cytokine profiling, and CD8-specific T-cell stimulation function followed by a second signal. Vpr impaired the expression of CD80, CD83, and CD86 at the transcriptional level without altering normal cellular transcription. Cytokine profiling indicated that the presence of Vpr inhibited production of interleukin 12 (IL-12) and upregulated IL-10, whereas IL-6 and IL-1β were unaltered. Furthermore, DC infected with HIV-1 vpr+ significantly reduced the activation of antigen-specific memory and recall cytotoxic-T-lymphocyte responses. Taken together, these results indicate that HIV-1 Vpr may in part be responsible for HIV-1 immune evasion by inhibiting the maturation of costimulatory molecules and cytokines essential for immune activation.

Phase I Trial of Adoptive Immunotherapy With Cytolytic T Lymphocytes Immunized Against a Tyrosinase Epitope

PURPOSEnTo study distribution and toxicity of cytolytic T lymphocytes (CTLs) against a single melanoma epitope.nnnPATIENTS AND METHODSnCD8(+) T cells obtained by leukapheresis from 10 patients with disseminated HLA-A2.1(+), tyrosinase-positive melanomas were immunized in vitro against tyrosinase(369-377) (YMNGTMSQV). Drosophila cells transduced with HLA-A2.1, CD80, and CD54 (intracellular adhesion molecule-1) were used for priming, followed by two rounds of immunization with mononuclear cells as antigen-presenting cells. 1 x 10(8) CTL were infused intravenously (IV) on day 1. CTL frequency was measured by limiting dilutions in five patients. (111)In labeling and scintigraphy measured distribution of CTL in next five. Five days later, 1 x 10(8) CTLs were infused on 4 successive days to both groups. Immunohistology of response was judged by biopsies.nnnRESULTSnInfusions were nontoxic. CTLs were undetectable in the blood, going to lungs within 5 minutes. At 4, 24, and 72 hours, they were found in liver and spleen. Lesions were visualized by scintiscans in one responding patient where two subcutaneous nodules were noted at 4 and 24 hours. A second patient had a partial response and remains alive with disease 2 years later. CD8(+) T cells were found in lesions of responders, associated with the presence of HLA-A2 molecules and tyrosinase. Two nonresponders without tyrosinase and HLA-A2 molecules had a paucity of CD8(+) T cells in their lesions. Whether the CD8(+) T cells in lesions of responders were those we had reinfused is uncertain.nnnCONCLUSIONnCTLs immunized against a single melanoma epitope were nontoxic but did not specifically localize to tumor sites. Nevertheless, two patients had disease regression. Additional therapeutic studies with specifically immunized CTL seem justified.

The HIV-1 HLA-A2-SLYNTVATL Is a Help-Independent CTL Epitope

The CTL response to the HLA-A*0201-restricted, HIV-1 p17 Gag77–85 epitope (SLYNTVATL; SL9) has been extensively studied in patients. Although this reactivity is exceptionally prominent in chronically infected patients and inversely correlated to viral load, SL9-specific CTLs (SL9-CTLs) are rarely detected in acute infection. To explore the cellular basis for this unusual manifestation, SL9-CTLs primed ex vivo from naive circulating CD8+ T cells of healthy, seronegative donors were generated and characterized. SL9 appeared to differ from other well-studied A*0201-restricted epitopes in several significant respects. In contrast to published reports for influenza and melanoma peptides and the HIV gag IV9 epitope studied here in parallel, SL9-CTLs were primed by immature but not mature autologous dendritic cells. Highly activated SL9-CTLs produce sufficient autocrine mediators to sustain clonal expansion and CTL differentiation for months without CD4+ T cells or exogenous IL-2. Moreover, SL9-CTLs were sensitive to paracrine IL-2-induced apoptosis. IL-2 independence and sensitivity to paracrine IL-2 were also characteristic of SL9-CTLs immunized by dendritic cells transduced by a nonreplicating lentiviral vector encoding full-length Gag. In vitro-primed SL9-CTLs resembled those derived from patients in degeneracy of recognition and functional avidities for both SL9 and its natural mutations. Together, these data show that SL9 is a highly immunogenic, help-independent HIV epitope. The scarcity of SL9-CTLs in acute infection may result from cytokine-induced apoptosis with the intense activation of the innate immunity. In contrast, SL9-CTLs that constitutively produce autocrine help would predominate during CD4-diminished chronic infection.

Phase I Trial of Large Multivalent Immunogen Derived from Melanoma Lysates in Patients with Disseminated Melanoma

Purpose: The purpose of this research was to determine the toxicity and immunological activity of large multivalent immunogen (LMI), a preparation of tumor cell membranes affixed to amorphous silica microbeads, in patients with melanoma. Experimental Design: Nineteen patients with metastatic (stage IV) melanoma were entered into the study, of whom 15 received the full 3 months of treatment with LMI. LMI was administered without adjuvant, one-half intradermally (i.d.) and the other half s.c. Because we expected little toxicity, we first treated 2 patients at each dose level, 10-, 30-, or 100-million tumor cell equivalents on weeks 0, 4, and 8, and subsequently randomized the remaining 13 patients to receive treatment with one of those dosage schedules, for a total of 19 patients. Two patients who were registered were found to be ineligible because of brain metastases, and 2 others did not complete the course of treatment for reasons other than toxicity. Thus, 15 patients were fully evaluable. Patients with evidence of a clinical response (at least stable disease at the 12-week checkpoint) had the option of continuing treatment at 4-week intervals. Frequencies of cytolytic T cell precursors against HLA-A2 matched melanoma cells, and delayed-type hypersensitivity to a melanoma cell membrane preparation from a component melanoma cell line were performed to measure immunological efficacy, and serum chemistries and complete blood counts were performed every 2 weeks throughout the study to measure possible toxicity. Computed tomography scans were performed pretreatment and at week 12 to measure possible beneficial effects on known lesions. Results: Eight of the 15 evaluable patients had an increase in cytolytic T-cell precursors during the course of therapy, usually by day 42. No patient had demonstrable delayed-type hypersensitivity to a melanoma membrane preparation before or after treatment. No toxicity of any kind was observed. A degree of clinical effectiveness of LMI was suggested by the elicitation of stable disease in 5 patients at 12 weeks. One patient had >50% regression of a lung nodule but progression of disease to the brain, whereas a second patient had a bona fide partial remission of a 3-cm diameter solitary lung nodule. Conclusions: LMI was nontoxic, improved immunological reactivity to melanoma cells, and showed evidence of clinical effectiveness (shrinkage of tumor) in 1 patient. Additional studies with LMI with added adjuvant materials, in melanoma and other cancers, appear warranted.

Degeneracy and Repertoire of the Human HIV-1 Gag p1777–85 CTL Response

CD8+ CTL responses are important for the control of HIV-1 infection. The immunodominant HLA-A2-restricted Gag epitope, SLYNTVATL (SL9), is considered to be a poor immunogen because reactivity to it is rare in acute infection despite its paradoxical dominance in patients with chronic infection. We have previously reported SL9 to be a help-independent epitope in that it primes highly activated CTLs ex vivo from CD8+ T cells of seronegative healthy donors. These CTLs produce sufficient cytokines for extended autocrine proliferation but are sensitive to activation-induced cell death, which may cause them to be eliminated by a proinflammatory cytokine storm. Here we identified an agonist variant of the SL9 peptide, p41 (SLYNTVAAL), by screening a large synthetic combinatorial nonapeptide library with ex vivo-primed SL9-specific T cells. p41 invariably immunized SL9-cross-reactive CTLs from other donors ex vivo and H-2Db β2m double knockout mice expressing a chimeric HLA-A*0201/H2-Db MHC class I molecule. Parallel human T cell cultures showed p41-specific CTLs to be less fastidious than SL9-CTLs in the level of costimulation required from APCs and the need for exogenous IL-2 to proliferate (help dependent). TCR sequencing revealed that the same clonotype can develop into either help-independent or help-dependent CTLs depending on the peptide used to activate the precursor CD8+ T cells. Although Ag-experienced SL9-T cells from two patients were also sensitive to IL-2-mediated cell death upon restimulation in vitro, the loss of SL9 T cells was minimized with p41. This study suggests that agonist sequences can replace aberrantly immunogenic native epitopes for the rational design of vaccines targeting HIV-1.

Enhanced induction of HIV-specific cytotoxic T lymphocytes by dendritic cell-targeted delivery of SOCS-1 siRNA.

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8(+) T cells from healthy donors. Finally, stimulation of CD8(+) T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8+ T cells from healthy donors. Finally, stimulation of CD8+ T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.

Availability of a diversely Avid CD8+ T cell repertoire specific for the subdominant HLA-A2-restricted HIV-1 gag p2419-27 epitope

HLA-A2-restricted CTL responses to immunodominant HIV-1 epitopes do not appear to be very effective in the control of viral replication in vivo. In this study, we studied human CD8+ T cell responses to the subdominant HLA-A2-restricted epitope TV9 (Gag p2419–27, TLNAWVKVV) to explore the possibility of increasing its immune recognition. We confirmed in a cohort of 313 patients, infected by clade B or clade C viruses, that TV9 is rarely recognized. Of interest, the functional sensitivity of the TV9 response can be relatively high. The potential T cell repertoires for TV9 and the characteristics of constituent clonotypes were assessed by ex vivo priming of circulating CD8+ T cells from healthy seronegative donors. TV9-specific CTLs capable of suppressing viral replication in vitro were readily generated, suggesting that the cognate T cell repertoire is not limiting. However, these cultures contained multiple discrete populations with a range of binding avidities for the TV9 tetramer and correspondingly distinct functional dependencies on the CD8 coreceptor. The lack of dominant clonotypes was not affected by the stage of maturation of the priming dendritic cells. Cultures primed by dendritic cells transduced to present endogenous TV9 were also incapable of clonal maturation. Thus, a diffuse TCR repertoire appeared to be an intrinsic characteristic of TV9-specific responses. These data indicate that subdominance is not a function of poor immunogenicity, cognate TCR repertoire availability, or the potential avidity properties thereof, but rather suggest that useful responses to this epitope are suppressed by competing CD8+ T cell populations during HIV-1 infection.

The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope, M1.2.

The mucin MUC1 molecule is overexpressed on a variety of adenocarcinomas and is thus, a potential target for immunotherapy. Of the MUC1 peptides that bind to HLA-A*0201(A2), M1.2 (LLLLTVLTV) from the signal sequence appears to be the most immunogenic in humans. Here we have shown that large numbers (109) of tetramer-binding M1.2-specific cytotoxic T lymphocytes (CTL) can be generated ex vivo from circulating precursors, derived from healthy adults. However, there was significant interpersonal variation in the level of co-stimulatory signal required. Tetramer-binding cells also required maturation in culture to become proficient killers of the HLA-A2+ MUC1+ MCF7 cell line, known to express a low number of endogenously processed M1.2. The functional avidity of M1.2-specific CTL, however, was low as compared to CTL specific for an HIV-1 epitope. Despite the low avidity, M1.2-specific CTL were polyfunctional, secreting multiple cytokines upon degranulation with antigen recognition. To identify potential agonist peptides that may be superior immunogens, an M1.2-specific CTL culture was used to scan a large nonameric combinatorial peptide library. Of 54 predicted peptides, 4 were “consensus” agonists because they were recognized by CTL from two other donors. Two agonists, p29 (LLPWTVLTV) and p15 (VLLWTVLTV), were equally stimulatory when loaded onto C1R target cells transfected with wild-type HLA-A2. Both agonists induced IL-2, TNF-α, IFN-γ, and degranulation with M1.2-specific CTL. In contrast, production of these cytokines, which are tightly regulated by specific activation through the T cell receptor, was restricted when the CTL were stimulated with peptides loaded onto C1R cells that were transfected with an HLA-A2 molecule bearing a mutation that abrogates binding to the CD8 co-receptor. Thus, activation by both M1.2 and its agonists was dependent upon CD8, showing that compensation by the co-receptor was necessary for the human T cell response to M1.2.

The Nucleocapsid Protein of Rift Valley Fever Virus Is a Potent Human CD8+ T Cell Antigen and Elicits Memory Responses

There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV), a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N) protein as a CD8+ T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2)-restricted epitopic determinants were identified with N-specific CD8+ T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs) across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N121–129) and IL9 (ILDAHSLYL, N165–173), were defined. VT9- and IL9-specific CD8+ T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8+ T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8+ T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8+ T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.

Generation of robust CD8+ T-cell responses against subdominant epitopes in conserved regions of HIV-1 by repertoire mining with mimotopes.

HLA‐A*0201‐restricted virus‐specific CD8+ CTL do not appear to control HIV effectively in vivo. To enhance the immunogenicity of a highly conserved subdominant epitope, TV9 (TLNAWVKVV, p24 Gag19–27), mimotopes were designed by screening a large combinatorial nonapeptide library with TV9‐specific CTL primed in vitro from healthy donors. A mimic peptide with a low binding affinity to HLA‐A*0201, TV9p6 (KINAWIKVV), was studied further. Parallel cultures of in vitro‐primed CTL showed that TV9p6 consistently activated cross‐reactive and equally functional CTL as measured by cytotoxicity, cytokine production and suppression of HIV replication in vitro. Comparison of TCRB gene usage between CTL primed from the same donors with TV9 or TV9p6 revealed a degree of clonal overlap in some cases and an example of a conserved TCRB sequence encoded distinctly at the nucleotide level between individuals (a “public” TCR); however, in the main, distinct clonotypes were recruited by each peptide antigen. These findings indicate that mimotopes can mobilize functional cross‐reactive clonotypes that are less readily recruited from the naïve T‐cell pool by the corresponding WT epitope. Mimotope‐induced repertoire diversification could potentially override subdominance under certain circumstances and enhance vaccine‐induced responses to conserved but poorly immunogenic determinants within the HIV proteome.