Andrew K. Sewell

Transmission and accumulation of CTL escape variants drive negative associations between HIV polymorphisms and HLA

Human immunodeficiency virus (HIV)-1 amino acid sequence polymorphisms associated with expression of specific human histocompatibility leukocyte antigen (HLA) class I alleles suggest sites of cytotoxic T lymphocyte (CTL)-mediated selection pressure and immune escape. The associations most frequently observed are between expression of an HLA class I molecule and variation from the consensus sequence. However, a substantial number of sites have been identified in which particular HLA class I allele expression is associated with preservation of the consensus sequence. The mechanism behind this is so far unexplained. The current studies, focusing on two examples of “negatively associated” or apparently preserved epitopes, suggest an explanation for this phenomenon: negative associations can arise as a result of positive selection of an escape mutation, which is stable on transmission and therefore accumulates in the population to the point at which it defines the consensus sequence. Such negative associations may only be in evidence transiently, because the statistical power to detect them diminishes as the mutations accumulate. If an escape variant reaches fixation in the population, the epitope will be lost as a potential target to the immune system. These data help to explain how HIV is evolving at a population level. Understanding the direction of HIV evolution has important implications for vaccine development.

Interaction between the CD8 Coreceptor and Major Histocompatibility Complex Class I Stabilizes T Cell Receptor-Antigen Complexes at the Cell Surface

The off-rate (koff) of the T cell receptor (TCR)/peptide-major histocompatibility complex class I (pMHCI) interaction, and hence its half-life, is the principal kinetic feature that determines the biological outcome of TCR ligation. However, it is unclear whether the CD8 coreceptor, which binds pMHCI at a distinct site, influences this parameter. Although biophysical studies with soluble proteins show that TCR and CD8 do not bind cooperatively to pMHCI, accumulating evidence suggests that TCR associates with CD8 on the T cell surface. Here, we titrated and quantified the contribution of CD8 to TCR/pMHCI dissociation in membrane-constrained interactions using a panel of engineered pMHCI mutants that retain faithful TCR interactions but exhibit a spectrum of affinities for CD8 of >1,000-fold. Data modeling generates a “stabilization factor” that preferentially increases the predicted TCR triggering rate for low affinity pMHCI ligands, thereby suggesting an important role for CD8 in the phenomenon of T cell cross-reactivity.

T-cell responses directed against multiple HLA-A*0201-restricted epitopes derived from Wilms' tumor 1 protein in patients with leukemia and healthy donors: Identification, quantification, and characterization

Purpose: Antigens derived from the Wilms tumor (WT1) protein, which is overexpressed in leukemias, are attractive targets for immunotherapy. Four HLA-A*0201-restricted WT1-derived epitopes have been identified: WT37, WT126, WT187, and WT235. We determined the natural immunogenecity of these antigens in patients with hematologic malignancies and healthy donor. Experimental Design: To detect very low frequencies of WT1-specific CD8+ T cells, we used quantitative reverse transcription-PCR to measure IFN-γ mRNA production by WT1 peptide–pulsed CD8+ T cells from 12 healthy donors, 8 patients with chronic myelogenous leukemia, 6 patients with acute myelogenous leukemia, and 8 patients with acute lymphoblastic leukemia. Results: Responses were detected in 5 of 8 chronic myelogenous leukemia patients, 4 of 6 patients with acute myelogenous leukemia, and 7 of 12 healthy donors. No responses were detected in patients with acute lymphoblastic leukemia. The magnitude and extent of these CD8+ T-cell responses was greater in patients with myeloid leukemias than in healthy donors. Clonotypic analysis of WT1-specific CD8+ T cells directly ex vivo in one case showed that this naturally occurring population was oligoclonal. Using fluorescent peptide-MHC class I tetramers incorporating mutations in the α3 domain (D227K/T228A) that abrogate binding to the CD8 coreceptor, we were able to confirm the presence of high-avidity T-cell clones within the antigen-specific repertoire. Conclusion: The natural occurrence of high-avidity WT1-specific CD8+ T cells in the periphery could facilitate vaccination strategies to expand immune responses against myeloid leukemias.

Cytotoxic T lymphocyte responses to human immunodeficiency virus: control and escape.

Effective preventive and therapeutic intervention in individuals exposed to or infected with human immunodeficiency virus (HIV) depends, in part, on a clear understanding of the interactions between the virus and those elements of the host immune response which control viral replication. Recent advances have provided compelling evidence that cytotoxic T lymphocytes (CTLs) constitute an essential component of protective antiretroviral immunity. Here, we review briefly the significance of this work in the context of previous studies, and outline the mechanisms through which HIV evades CTL activity.

Ultrasensitive Detection and Phenotyping of CD4+ T Cells with Optimized HLA Class II Tetramer Staining

HLA class I tetramers have revolutionized the study of Ag-specific CD8+ T cell responses. Technical problems and the rarity of Ag-specific CD4+ Th cells have not allowed the potential of HLA class II tetramers to be fully realized. Here, we optimize HLA class II tetramer staining methods through the use of a comprehensive panel of HIV-, influenza-, CMV-, and tetanus toxoid-specific tetramers. We find rapid and efficient staining of DR1- and DR4-restricted CD4+ cell lines and clones and show that TCR internalization is not a requirement for immunological staining. We combine tetramer staining with magnetic bead enrichment to detect rare Ag-specific CD4+ T cells with frequencies as low as 1 in 250,000 (0.0004% of CD4+ cells) in human PBLs analyzed directly ex vivo. This ultrasensitive detection allowed phenotypic analysis of rare CD4+ T lymphocytes that had experienced diverse exposure to Ag during the course of viral infections. These cells would not be detectable with normal flow-cytometric techniques.

Antigen–specific release of β-chemokines by anti-HIV-1 cytotoxic T lymphocytes

Abstract A major advance in understanding human immunodeficiency virus (HIV) biology was the discovery that the β -chemokines MIP-1 α (macrophage inflammatory protein-1 α ), MIP-1 β (macrophage inflammatory protein-1 β ) and RANTES (regulated on activation, normal T-cell expressed and secreted) inhibit entry of HIV-1 into CD4 + cells by blocking the critical interaction between the CCR5 coreceptor and the V3 domain of the viral envelope glycoprotein gp120 [1,2]. CD8 + lymphocytes are a major source of β -chemokines [3], but the stimulus for chemokine release has not been well defined. Here, we have shown that engagement of CD8 + cytotoxic T lymphocytes (CTLs) with HIV-1-encoded human leukocyte antigen (HLA) class I-restricted peptide antigens caused rapid and specific release of these β -chemokines. This release paralleled cytolytic activity and could be attenuated by naturally occurring amino acid variation within the HLA class I-restricted peptide sequence. Epitope variants that bound to appropriate HLA class I molecules but failed to stimulate cytolytic activity in CTLs also failed to stimulate chemokine release. We conclude that signalling through the T-cell receptor (TCR) following binding of antigen results in β -chemokine release from CTLs in addition to cytolytic activity, and that both responses can be abolished by epitope mutation. These results suggest that antigenic variation within HIV-1 might not only allow the host cell to escape lysis, but might also contribute to the propagation of infection by failing to activate β -chemokine-mediated inhibition of HIV-1 entry.

Cytotoxic T lymphocytes, chemokines and antiviral immunity

Evidence that CD8+ CTLs produce chemokines following engagement of viral antigens, and that MIP-1alpha is required for an inflammatory response to virus challenge, suggests that these molecules are key elements in the generation of effective antiviral immunity. Here, David Price and colleagues argue that the antigen-dependent release of chemokines by CTLs provides an elegant mechanism linking localization, amplification and coordination of the antiviral immune response to specific recognition of infected host cells beyond the confines of the lymphoid system.

HLA-A*0201 presents TAP-dependent peptide epitopes to cytotoxic T lymphocytes in the absence of tapasin

Tapasin is a 48‐kDa endoplasmic reticulum (ER)‐resident glycoprotein that binds to the transporter associated with antigen processing (TAP) and mediates an interaction between TAP and newly synthesized MHC class I molecules. It is also essential for the proper antigen presenting function of HLA‐A*0101 (HLA‐A1), HLA‐A*0801 (HLA‐B8) and HLA‐B*4402 (HLA‐B4402). We show here that while tapasin is required for HLA‐A*0201 (HLA‐A2) molecules to bind to TAP, its absence does not block the presentation of HLA‐A2‐restricted TAP‐dependent epitopes to cytotoxic T lymphocytes indicating that, unlike HLA‐A1, HLA‐B8 and HLA‐B4402, HLA‐A2 has access to the TAP‐dependent peptide pool even in the absence of tapasin. Nevertheless, the overall efficiency with which HLA‐A2 was loaded with optimal, stabilizing peptides was impaired in the cell line .220, resulting in a significant increase in the fraction of HLA‐A2 molecules being released from the ER in a “peptide‐receptive” state.

CD8+ T cell epitope-flanking mutations disrupt proteasomal processing of HIV-1 Nef

CTL play a critical role in the control of HIV and SIV. However, intrinsic genetic instability enables these immunodeficiency viruses to evade detection by CTL through mutation of targeted antigenic sites. These mutations can impair binding of viral epitopes to the presenting MHC class I molecule or disrupt TCR-mediated recognition. In certain regions of the virus, functional constraints are likely to limit the capacity for variation within epitopes. Mutations elsewhere in the protein, however, might still enable immune escape through effects on Ag processing. In this study, we describe the coincident emergence of three mutations in a highly conserved region of Nef during primary HIV-1 infection. These mutations (R69K, A81G, and H87R) flank the HLA B*35-restricted VY8 epitope and persisted to fixation as the early CTL response to this Ag waned. The variant form of Nef showed a reduced capacity to activate VY8-specific CTL, although protein stability and expression levels were unchanged. This effect was associated with altered processing by the proteasome that caused partial destruction of the VY8 epitope. Our data demonstrate that a variant HIV genotype can significantly impair proteasomal epitope processing and substantiate the concept of immune evasion through diminished Ag generation. These observations also indicate that the scale of viral escape may be significantly underestimated if only intraepitope variation is evaluated.

Antagonism of cytotoxic T-lymphocyte activation by soluble CD8

The CD8 co-receptor is important in the differentiation and selection of class I MHC-restricted T cells during thymic development, and in the activation of mature T lymphocytes in response to antigen. Here we show that soluble CD8αα receptor, despite an extremely low affinity for MHC, inhibits activation of cytotoxic lymphocytes by obstructing CD3 ζ-chain phosphorylation. We propose a model for this effect that involves interference of productive receptor multimerization at the T-cell surface. These results provide new insights into the mechanism of T-cell activation and evidence that CD8 function is exquisitely sensitive to disruption, an effect that might be exploited by molecular therapeutics.