High Functional Avidity Research Articles

Synergistic enhancement of CD8+ T cell-mediated tumor vaccine efficacy by an anti-transforming growth factor-beta monoclonal antibody.

Transforming growth factor-beta (TGF-beta) is an immunosuppressive cytokine, having direct suppressive activity against conventional CD4(+) and CD8(+)T cells and natural killer cells, thereby inhibiting tumor immunosurveillance. Here, we investigated possible synergy between anti-TGF-beta (1D11) and a peptide vaccine on induction of antitumor immunity, and the mechanisms accounting for synergistic efficacy. The effect of combination treatment with a peptide vaccine and anti-TGF-beta was examined in a subcutaneous TC1 tumor model, as well as the mechanisms of protection induced by this treatment. Anti-TGF-beta significantly and synergistically improved vaccine efficacy as measured by reduction in primary tumor growth, although anti-TGF-beta alone had no impact. The number of tumor antigen-specific CTL with high functional avidity as measured by IFN-gamma production and lytic activity was significantly increased in vaccinated mice by TGF-beta neutralization. Although TGF-beta is known to play a critical role in CD4(+)Foxp3(+) Treg cells, Treg depletion/suppression by an anti-CD25 monoclonal antibody (PC61) before tumor challenge did not enhance vaccine efficacy, and adding anti-TGF-beta did not affect Treg numbers in lymph nodes or tumors or their function. Also, TGF-beta neutralization had no effect on interleukin-17-producing T cells, which are induced by TGF-beta and interleukin-6. Absence of type II NKT cells, which induce myeloid cells to produce TGF-beta, was not sufficient to eliminate all sources of suppressive TGF-beta. Finally, the synergistic protection induced by anti-TGF-beta vaccine augmentation was mediated by CD8(+) T cells since anti-CD8 treatment completely abrogated the effect. These results suggest that TGF-beta blockade may be useful for enhancing cancer vaccine efficacy.

Dendritic cells pulsed with RNA encoding allogeneic MHC and antigen induce T cells with superior antitumor activity and higher TCR functional avidity

AbstractAdoptive transfer of T cells expressing transgenic T-cell receptors (TCRs) with antitumor function is a hopeful new therapy for patients with advanced tumors; however, there is a critical bottleneck in identifying high-affinity TCR specificities needed to treat different malignancies. We have developed a strategy using autologous dendritic cells cotransfected with RNA encoding an allogeneic major histocompatibility complex molecule and a tumor-associated antigen to obtain allo-restricted peptide-specific T cells having superior capacity to recognize tumor cells and higher functional avidity. This approach provides maximum flexibility because any major histocompatibility complex molecule and any tumor-associated antigen can be combined in the dendritic cells used for priming of autologous T cells. TCRs of allo-restricted T cells, when expressed as transgenes in activated peripheral blood lymphocytes, transferred superior function compared with self-restricted TCR. This approach allows high-avidity T cells and TCR specific for tumor-associated self-peptides to be easily obtained for direct adoptive T-cell therapy or for isolation of therapeutic transgenic TCR sequences.

Differential TCR signal transduction pathways involved in high and low avidity CD8+ T-cell responsiveness (35.26)

Abstract CTLs that display high functional avidity are known to be optimal for pathogen clearance. However, the molecular mechanism that allows high avidity CTLs to respond at very low levels of peptide antigen remains to be elucidated. To investigate this question, we compared TCR signal transduction events in high and low avidity CD8+ T-cells generated from OT-I TCR transgenic mice. Upon stimulation with APCs preloaded with a range of Ova257-264 peptide concentrations (10^-6, 10^-9 and 10^-12 M), we found that high avidity CD8+ T-cells induced higher levels of MAPK ERK-1/2 phosphorylation as well as higher levels of the dephosphorylated form of NFAT at each peptide concentration used for stimulation. We next determined whether the observed divergence in NFAT activation was reflected in differences in calcium mobilization. We found efficient mobilization of Ca2+ in high avidity cells even when the lowest concentration of peptide was used for stimulation, a level of peptide at which no increase in calcium was observed in low avidity cells. The decreased peptide requirement for calcium mobilization in high avidity cells correlated with a decreased peptide requirement for ZAP-70 activation and CD3ζ phosphorylation, the latter of which was higher and prolonged in high versus low avidity cells. Thus the functional avidity of the CD8+ effector cells studies here is controlled by differential regulation of the most membrane proximal event, phosphorylation of CD3ζ. Studies are currently underway to determine the mechanism by which high avidity cells acheive CD3ζ phosphorylation at limiting peptide levels.

Generation of cytotoxicity against hepatitis delta virus genotypes and quasispecies by epitope modification

Quasispecies are likely responsible for virus escape from host immune surveillance. The aim of this study was to enhance the immune response against varied sequences within the HDV quasispecies in an attempt to control chronic delta hepatitis. The HLA-A2 restricted peptides spanning aa 43-51 of HDAg and three variant peptides bearing single amino acid substitutions were synthesized. Their immunogenicity and capacity to induce effective CTL responses were studied in HHD-2 mice. Native HDV epitope produced limited cytotoxic immune response. Two modified HDV peptides (HDV 43-51 1Y; tyrosine substitution in positive 1, and 43-51 3A; alanine substitution in position 3) could enhance not only the binding affinity with HLA-A2.1 molecules but also the immunogenicity. Ex vivo interferon-gamma ELISPOT and CTL assays revealed that the two modified epitopes-induced CTLs had a higher functional avidity and produced stronger cytotoxicity to lyse constitutively HDAg-expressing Hep-G2 cells. Interestingly, the spectrums of the T cell receptor (TCR) cross-reactivity are broadened and response to multiple HDV variants by the enhanced epitopes immunization. The modified HDV peptides can enhance the immunogenicity and the induced CTLs can cross-react with multiple HDV variants. Combination with the two enhanced epitopes might be a potential immunotherapeutic agent to control HDV quasispecies in HLA-A2 chronic hepatitis D patients.

Relationship between CD8-dependent antigen recognition, T cell functional avidity, and tumor cell recognition

Effective immunotherapy using T cell receptor (TCR) gene-modified T cells requires an understanding of the relationship between TCR affinity and functional avidity of T cells. In this study, we evaluate the relative affinity of two TCRs isolated from HLA-A2-restricted, gp100-reactive T cell clones with extremely high functional avidity. Furthermore, one of these T cell clones, was CD4- CD8- indicating that antigen recognition by this clone was CD8 independent. However, when these TCRs were expressed in CD8- Jurkat cells, the resulting Jurkat cells recognized gp100:209-217 peptide loaded T2 cells and had high functional avidity, but could not recognize HLA-A2+ melanoma cells expressing gp100. Tumor cell recognition by Jurkat cells expressing these TCRs could not be induced by exogenously loading the tumor cells with the native gp100:209-217 peptide. These results indicate that functional avidity of a T cell does not necessarily correlate with TCR affinity and CD8-independent antigen recognition by a T cell does not always mean its TCR will transfer CD8-independence to other effector cells. The implications of these findings are that T cells can modulate their functional avidity independent of the affinity of their TCRs.

CD4+CD25− T Cells Transduced to Express MHC Class I-Restricted Epitope-Specific TCR Synthesize Th1 Cytokines and Exhibit MHC Class I-Restricted Cytolytic Effector Function in a Human Melanoma Model

Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.

Cutting Edge: Dendritic Cells Prime a High Avidity CTL Response Independent of the Level of Presented Antigen

CTL that possess a high functional avidity are known to be optimal for the clearance of pathogens in vivo. We have shown that the amount of peptide encountered by a CD8+ CTL determines its functional avidity. Notably, in these studies nonprofessional APC were used. However, it is mature dendritic cells (DC) that are predominantly responsible for the activation of naive T cells in vivo. Whether DC also direct dose dependent-differences in avidity is unknown. In this work we examined the ability of mature DC presenting a high vs low level of peptide to generate CTL of distinct avidities. In contrast to what was observed with nonprofessional APC, CTL generated by stimulation with mature DC were of high avidity regardless of the amount of peptide presented. This DC property may promote generation of highly effective CTL that retain plasticity, which would allow the tuning of avidity in the periphery to promote optimal pathogen recognition and clearance.

Capture of Tumor Cell Membranes by Trogocytosis Facilitates Detection and Isolation of Tumor-Specific Functional CTLs

The success of adoptive cell transfer in the treatment of metastatic cancer in humans is dependent on the selection of highly active tumor-specific cytotoxic T cells. We report here that CTLs capture membrane fragments from their targets while exerting cytotoxic activity and thus gain a detectable functional signature by which they can be identified. Fluorochrome labeling or biotinylation was used to tag tumor cells. CD8(+) T cells were coincubated with the tagged targets, sorted, and functionally evaluated. Our results show that membrane capture by CD8(+) lymphocytes is T-cell receptor dependent, epitope specific, and preferentially associated with highly cytotoxic clonal subsets. CTLs that captured membranes from unmodified melanoma exhibited enhanced cytotoxic activity against tumor cell lines and autologous melanoma. In a human melanoma in vivo model, adoptive transfer of membrane-capturing, peptide-specific T cells, but not noncapturing or bulk CD8(+) T cells, inhibits tumor progression. Membrane capture is therefore a signature of antigen-specific CTLs endowed with high functional avidity and may have direct relevance in the clinical application of adoptive immunotherapy.

Rapid CD8+ T Cell Repertoire Focusing and Selection of High-Affinity Clones into Memory Following Primary Infection with a Persistent Human Virus: Human Cytomegalovirus

To investigate the mechanism of selection of individual human CD8+ T cell clones into long-term memory following primary infection with a persistent human virus (human CMV (HCMV)), we undertook a longitudinal analysis of the diversity of T cell clones directed toward an immunodominant viral epitope: we followed this longitudinally from early T cell expansion through the contraction phase and selection into the memory pool. We show that following initial HCMV infection, the early primary response against a defined epitope was composed of diverse clones possessing many different TCR Vbeta segments. Longitudinal analysis showed that this usage rapidly focused predominantly on a single TCR Vbeta segment within which dominant clones frequently had public TCR usage, in contrast to subdominant or contracted clones. Longitudinal clonotypic analysis showed evidence of disproportionate contraction of certain clones that were abundant in the primary response, and late expansion of clones that were subdominant in the primary response. All dominant clones selected into memory showed similar high functional avidity of their TCR, whereas two clones that greatly contracted showed substantially lower avidity. Expression of the IL-7R is required for survival of murine effector CD8+ T cells into memory, but in primary HCMV infection IL-7R was not detected on circulating Ag-specific cells until memory had been established. Thus, the oligoclonal T cell repertoire against an immunodominant persistent viral epitope is established early in primary infection by the rapid selection of public clonotypes, rather than being a stochastic process.

Dose‐dependent modulation of CD8 and functional avidity as a result of peptide encounter

The generation of an optimal CD8(+) cytotoxic T lymphocyte (CTL) response is critical for the clearance of many intracellular pathogens. Previous studies suggest that one contributor to an optimal immune response is the presence of CD8(+) cells exhibiting high functional avidity. In this regard, CD8 expression has been shown to contribute to peptide sensitivity. Here, we investigated the ability of naive splenocytes to modulate CD8 expression according to the concentration of stimulatory peptide antigen. Our results showed that the level of CD8 expressed was inversely correlated with the amount of peptide used for the primary stimulation, with higher concentrations of antigen resulting in lower expression of both CD8alpha and CD8beta. Importantly the ensuing CD8(low) and CD8(high) CTL populations were not the result of the selective outgrowth of naive CD8(+) T-cell subpopulations expressing distinct levels of CD8. Subsequent encounter with peptide antigen resulted in continued modulation of both the absolute level and the isoform of CD8 expressed and in the functional avidity of the responding cells. We propose that CD8 cell surface expression is not a static property, but can be modulated to 'fine tune' the sensitivity of responding CTL to a defined concentration of antigen.

HLA-A2-restricted protection against lethal lymphocytic choriomeningitis.

The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8(+) T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides-nucleoprotein NP(69-77), glycoprotein precursor GPC(10-18), GPC(447-455), and zinc-binding protein Z(49-58)-that displayed high-affinity binding (< or =275 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC(447-455)), after peptide immunization of HLA-A*0201 mice, induced CD8(+) T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV.

Dendritic cells transfected with interleukin-12 and tumor-associated antigen messenger RNA induce high avidity cytotoxic T cells

Dendritic cells (DC) transfected with messenger RNA (mRNA) encoding tumor-associated antigens (TAA) are able to induce potent tumor-specific T-cell responses directed to a broad spectrum of tumor-associated epitopes. The in vitro generation of DC possessing all the features crucial for the induction of type 1 immune responses, such as mature state, migratory potential and interleukin-12 (IL-12p70) production is complicated. Particularly migratory potential is inversely correlated with IL-12p70 production after maturation with prostaglandin E2 (PGE2), which is included in maturation cocktails currently used in most vaccination trials. Here, we show that transfection of PGE2 matured DC with a single mRNA strain encoding for ubiquitin followed by a TAA which was linked to IL-12 by a self-cleaving 2A sequence, produced biological active IL-12p70 and were able to present the transfected TAA up to 72 h after transfection. Furthermore, use of the anti-reverse cap analog for in vitro transcription of the IL-12 mRNA enabled constitutive IL-12p70 production for up to 5 days. These transfected mature DC migrated efficiently towards lymph node derived chemokines. DCs constitutively expressing IL-12p70, generate TAA-specific cytotoxic T cells with an high functional avidity, independent of CD4+ T-cell help.

Fluorescence-Activated Cell Sorting and Cloning of Bona Fide CD8+ CTL with Reversible MHC-Peptide and Antibody Fab′ Conjugates

The isolation of subsets of Ag-specific T cells for in vitro and in vivo studies by FACS is compromised by the fact that the soluble MHC-peptide complexes and Abs used for staining, especially when combined, induce unwanted T cell activation and eventually apoptosis. This is especially a problem for CD8+ CTL, which are susceptible to activation-dependent cell death. In this study, we show that reversible MHC-peptide complexes (tetramers) can be prepared by conjugating MHC-peptide monomers with desthiobiotin (DTB; also called dethiobiotin) and multimerization by reaction with fluorescent streptavidin. While in the cold these reagents are stable and allow good staining, they rapidly dissociate in monomers at elevated temperatures, especially in the presence of free biotin. FACS cloning of Melan-A (MART-1)-specific CTL from a melanoma-infiltrated lymph node with reversible HLA-A2 Melan-A26-35 multimers yielded over two times more clones than when using the conventional biotin-containing multimers. CTL clones obtained by means of reversible multimers killed Melan-A-positive tumor cells more efficiently as compared with clones obtained with the stable multimers. Among the CTL obtained with the reversible multimers, but much less among those obtained with the stable multimers, a high proportion of clones exhibited high functional and physical avidity and died upon incubation with soluble MHC-peptide complexes. Finally, we show that Fab' of an anti-CD8 Ab can be converted in reversible DTB streptavidin conjugates the same way. These DTB reagents efficiently and reversibly stained murine and human CTL without affecting their viability.

Identification of protective Lassa virus epitopes that are restricted by HLA-A2.

Recovery from Lassa virus (LASV) infection usually precedes the appearance of neutralizing antibodies, indicating that cellular immunity plays a primary role in viral clearance. To date, the role of LASV-specific CD8(+) T cells has not been evaluated in humans. To facilitate such studies, we utilized a predictive algorithm to identify candidate HLA-A2 supertype epitopes from the LASV nucleoprotein and glycoprotein precursor (GPC) genes. We identified three peptides (GPC(42-50), GLVGLVTFL; GPC(60-68), SLYKGVYEL; and GPC(441-449), YLISIFLHL) that displayed high-affinity binding (< or =98 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LASV GPC in human HLA-A*0201-positive target cells. HLA-A*0201 mice immunized with either GPC(42-50) or GPC(60-68) were protected against challenge with a recombinant vaccinia virus that expressed LASV GPC. The epitopes identified in this study represent potential diagnostic reagents and candidates for inclusion in epitope-based vaccine constructs. Our approach is applicable to any pathogen with existing sequence data, does not require manipulation of the actual pathogen or access to immune human donors, and should therefore be generally applicable to category A through C agents and other emerging pathogens.

High-avidity antitumor T-cell generation by toll receptor 8–primed, myeloid- derived dendritic cells is mediated by IL-12 production

High-level production of heterodimeric p70 interleukin (IL)-12 by myeloid-derived dendritic cells (DCs) requires 2 signals: interferon gamma (IFN-gamma) and a maturation signal provided by CD40 ligation (CD40L) or lipopolysaccharide (LPS). In the current study we demonstrate that signaling through toll-like receptor (TLR) 8, but not TLR3, TLR2, or TLR4, provides a priming signal to myeloid-derived DC for high IL-12 p70 heterodimer production. All the TLR agonists induced maturation of DC as evidenced by increased expression of CD83, CD80, and CD86. Both IFN-gamma and TLR7/8 agonist R848 increased expression of TLR8 in immature monocyte-derived DCs. The combination of TLR7/8 agonist R848 and maturation signals LPS or CD40L induced high-level expression of IL-12p35 and p40 similar to that induced by IFN-gamma plus LPS. In contrast, receptor agonists specific for TLR7 did not prime for IL-12 production. The p70 IL-12 produced by the TLR8-primed DC polarized CD4+ T for Th1 cytokine production and induced CD8+ T cells, displaying high functional avidity with enhanced tumor cell recognition. The data suggest that toll 8 receptor agonists are useful for inducing type-1 polarized DCs for vaccine design in treating cancer and infectious disease.

The Hypervariable Immunodominant NP418-426Epitope from the Influenza A Virus Nucleoprotein Is Recognized by Cytotoxic T Lymphocytes with High Functional Avidity

Recently it was shown that influenza A viruses can accumulate mutations in epitopes associated with escape from recognition by human virus-specific cytotoxic T lymphocytes (CTL). It is unclear what drives diversification of CTL epitopes and why certain epitopes are variable and others remain conserved. It has been shown that simian immunodeficiency virus-specific CTL that recognize their epitope with high functional avidity eliminate virus-infected cells efficiently and drive diversification of CTL epitopes. T-cell functional avidity is defined by the density of major histocompatibility complex class I peptide complexes required to activate specific CTL. We hypothesized that functional avidity of CTL contributes to epitope diversification and escape from CTL also for influenza viruses. To test this hypothesis, the functional avidity of polyclonal CTL populations specific for nine individual epitopes was determined. To this end, peripheral blood mononuclear cells from HLA-A- and -B-genotyped individuals were stimulated in vitro with influenza virus-infected cells to allow expansion of virus-specific CTL, which were used to determine the functional avidity of CTL specific for nine individual epitopes in enzyme-linked immunospot assays. We found that the functional avidity for the respective epitopes varied widely. Furthermore, the functional avidity of CTL specific for the hypervariable NP(418-426) epitope was significantly higher than that of CTL recognizing other epitopes (P < 0.01). It was speculated that the high functional avidity of NP(418-426)-specific CTL was responsible for the diversification of this influenza A virus CTL epitope.

Impact of HLA-B Alleles, Epitope Binding Affinity, Functional Avidity, and Viral Coinfection on the Immunodominance of Virus-Specific CTL Responses

Immunodominance is variably used to describe either the most frequently detectable response among tested individuals or the strongest response within a single individual, yet factors determining either inter- or intraindividual immunodominance are still poorly understood. More than 90 individuals were tested against 184 HIV- and 92 EBV-derived, previously defined CTL epitopes. The data show that HLA-B-restricted epitopes were significantly more frequently recognized than HLA-A- or HLA-C-restricted epitopes. HLA-B-restricted epitopes also induced responses of higher magnitude than did either HLA-A- or HLA-C-restricted epitopes, although this comparison only reached statistical significance for EBV epitopes. For both viruses, the magnitude and frequency of recognition were correlated with each other, but not with the epitope binding affinity to the restricting HLA allele. The presence or absence of HIV coinfection did not impact EBV epitope immunodominance patterns significantly. Peptide titration studies showed that the magnitude of responses was associated with high functional avidity, requiring low concentration of cognate peptide to respond in in vitro assays. The data support the important role of HLA-B alleles in antiviral immunity and afford a better understanding of the factors contributing to inter- and intraindividual immunodominance.

Evaluation of high functional avidity CTL to Gag epitope clusters in EIAV carrier horses

Cytotoxic T lymphocytes (CTL) are critical for lentivirus control including EIAV. Since CTL from most EIAV carrier horses recognize Gag epitope clusters (EC), the hypothesis that carrier horses would have high functional avidity CTL to optimal epitopes in Gag EC was tested. Twenty-two optimal EC epitopes were identified; two in EC1, six in EC2, and seven each in EC3 and 4. However, only five of nine horses had high functional avidity CTL (≤11 nM) recognizing six epitopes in EC; four in relatively conserved EC3; and one each in EC1 and 2. Horses with high functional avidity CTL had significantly more days since the last clinical episode than horses with low avidity CTL, and this was not explained by analyzing duration of infection. Furthermore, there was a significant inverse correlation between the CTL functional avidity of the nine horses and the days since the last clinical episode. Gag CTL epitope escape variants were found in three horses, but only one of these was recognized by high functional avidity CTL. Thus, not all carrier horses had high functional avidity CTL to Gag EC, but those that did had longer periods without disease episodes.

Transfer of Specific T Cell Clones with Graft-Versus-Leukemia (GVL) Activity from Donor to Recipient in Stem Cell Transplantation: Identification, Quantification and Characterization.

The self-antigens PR1 and WT1 that are aberrantly expressed on malignant cells may be important target antigens for GVL effects from donor-derived anti-leukemia T cells. It is now clear that T cells recognizing these antigens circulate in transplant recipients and can be detected in small numbers in healthy individuals. To determine whether the same T cell clones in the donor are transferred to the recipient and induce GVL effects we sought for presence of leukemia-reactive T cell clones in healthy donors and their transfer into the patient after transplant and following DLI. We identified CD8+ T cell clones specific for PR1 and WT1 from 2 healthy donors. The HLA-A2/PR1-binding and HLA-A2/WT1-binding CD8+ T cells were purified by flow cytometric cell sorting and analyzed for their T cell receptor (TCR) usage by template switch anchored RT-PCR. This showed an oligoclonal population of WT1-specific CD8+ T cells and a polyclonal population of PR1-specific CD8+ T cells. In addition, using a fluorescent peptide/MHC class I multimeric complex incorporating mutations in the a3 domain that abrogate binding to the CD8 coreceptor, we selectively isolated WT1-specific CD8+ T cells of high functional avidity and demonstrated that high avidity T cells comprise a single clonotype. One patient with CML received an alloSCT from the donor in whom PR1-specific CD8+ T cell clones were detected. Using quantitative real-time PCR for IFN-g production and HLA-A2/PR1 tetrameric complexes, we showed the emergence of PR1-specific CD8+ T cells in the blood of the recipient 10 weeks after SCT and again 8 weeks post-DLI given to treat a molecular relapse of CML. HLA-A2/PR1 tetramer-positive CD8+ T cells were sorted by flow cytometry post-alloSCT and again following DLI. By comparing TCRb CDR3 sequences, we confirmed direct transfer and expansion of PR1-specific CD8+ T cell clones from the donor into the recipient and the reemergence of the same PR1-specific clones following DLI. The appearance of these HLA-A2/PR1 tetramer-positive CD8+ T cells was followed by complete molecular remission of CML by sensitive PCR for BCR/ABL The PR1-specific CD8+ T cells in the donor were of memory phenotype and expanded in the recipient after both alloSCT and DLI. During the early phase post-transfer in the recipient, the majority of PR1-specific CD8+ T cells had an effector memory phenotype (CD45RO+ CD57+). There was a shift towards a central memory phenotype (CD45 RO+ CD57−) during the course of the GVL effect. This is the first direct demonstration of the transfer of leukaemia-reactive specific T cell clones from a healthy donor to a patient with leukemia. Further, the identification and monitoring of T cell clones that mediate the GVL effect as described here can be undertaken before stem cell transplantation and could aid donor selection.

Persistent recognition of autologous virus by high-avidity CD8 T cells in chronic, progressive human immunodeficiency virus type 1 infection.

CD8 T-cell responses are thought to be crucial for control of viremia in human immunodeficiency virus (HIV) infection but ultimately fail to control viremia in most infected persons. Studies in acute infection have demonstrated strong CD8-mediated selection pressure and evolution of mutations conferring escape from recognition, but the ability of CD8 T-cell responses that persist in late-stage infection to recognize viruses present in vivo has not been determined. Therefore, we studied 24 subjects with advanced HIV disease (median viral load = 142,000 copies/ml; median CD4 count = 71/ micro l) and determined HIV-1-specific CD8 T-cell responses to all expressed viral proteins using overlapping peptides by gamma interferon Elispot assay. Chronic-stage virus was sequenced to evaluate autologous sequences within Gag epitopes, and functional avidity of detected responses was determined. In these subjects, the median number of epitopic regions targeted was 13 (range, 2 to 39) and the median cumulative magnitude of CD8 T-cell responses was 5,760 spot-forming cells/10(6) peripheral blood mononuclear cells (range, 185 to 24,700). On average six (range, one to 8) proteins were targeted. For 89% of evaluated CD8 T-cell responses, the autologous viral sequence was predicted to be well recognized by these responses and the majority of analyzed optimal epitopes were recognized with medium to high functional avidity by the contemporary CD8 T cells. Withdrawal of antigen by highly active antiretroviral therapy led to a significant decline both in breadth (P = 0.032) and magnitude (P = 0.0098) of these CD8 T-cell responses, providing further evidence that these responses had been driven by recognition of autologous virus. These results indicate that strong, broadly directed, and high-avidity gamma-interferon-positive CD8 T-cells directed at autologous virus persist in late disease stages, and the absence of mutations within viral epitopes indicates a lack of strong selection pressure mediated by these responses. These data imply functional impairment of CD8 T-cell responses in late-stage infection that may not be reflected by gamma interferon-based screening techniques.